U.S. patent application number 15/586196 was filed with the patent office on 2017-11-02 for inhibition and enhancement of reprogramming by chromatin modifying enzymes.
This patent application is currently assigned to Children's Medical Center Corporation. The applicant listed for this patent is Children's Medical Center Corporation. Invention is credited to George Q. Daley, Tamer T. Onder.
Application Number | 20170313989 15/586196 |
Document ID | / |
Family ID | 48082254 |
Filed Date | 2017-11-02 |
United States Patent
Application |
20170313989 |
Kind Code |
A1 |
Onder; Tamer T. ; et
al. |
November 2, 2017 |
INHIBITION AND ENHANCEMENT OF REPROGRAMMING BY CHROMATIN MODIFYING
ENZYMES
Abstract
In one aspect, the disclosure provides methods and compositions
for the production of stem cells. In one aspect, the disclosure
provides methods and uses of stem cells. In some embodiments, the
stem cells are induced pluripotent stem cells.
Inventors: |
Onder; Tamer T.; (Newton,
MA) ; Daley; George Q.; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Medical Center Corporation |
Boston |
MA |
US |
|
|
Assignee: |
Children's Medical Center
Corporation
Boston
MA
|
Family ID: |
48082254 |
Appl. No.: |
15/586196 |
Filed: |
May 3, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14351717 |
Apr 14, 2014 |
9670463 |
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PCT/US2012/000514 |
Oct 14, 2012 |
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15586196 |
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61547404 |
Oct 14, 2011 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0696 20130101;
C12N 2501/065 20130101 |
International
Class: |
C12N 5/074 20100101
C12N005/074 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. R01 DK70055 from the National Institutes of Health. The
Government has certain rights in this invention.
Claims
1. A method of producing induced pluripotent stem cells, the method
comprising inhibiting Dot1L in a differentiated cell and culturing
the differentiated cell under reprogramming conditions to produce
induced pluripotent stem cells.
2. The method of claim 1, wherein inhibiting Dot1L comprises
inhibiting the methyltransferase activity of Dot1L.
3. The method of claim 1, wherein Dot1L is inhibited by contacting
the differentiated cell with a composition comprising a compound of
formula I, II, III, or IV.
4. The method of claim 1, wherein Dot1L is inhibited by contacting
the differentiated cell with a composition comprising a compound of
formula: ##STR00174##
5. The method of claim 1, wherein Dot1L is inhibited by contacting
the differentiated cell with an shRNA that knocks down Dot1L
expression.
6. The method of claim 1, wherein the reprogramming conditions
comprise a reprogramming cocktail comprising a transcription
factor.
7. The method of claim 6, wherein the reprogramming cocktail
comprises Oct4 and Sox2.
8. (canceled)
9. The method of claim 6, wherein the reprogramming cocktail does
not include Klf4 or c-Myc.
10. The method of claim 6, wherein Dot1L is inhibited when the cell
is cultured with the reprogramming cocktail.
11. The method of claim 1, wherein the differentiated cell is a
fibroblast.
12. (canceled)
13. The method of claim 1, wherein the differentiated cell is a
human cell.
14. The method of claim 13, wherein the differentiated human cell
is dH1fs, IMR-90 or MRC-5.
15. The method of claim 1, wherein the differentiated cell is a
mouse cell.
16-19. (canceled)
20. A method of accelerating the production of induced pluripotent
stem cells, the method comprising inhibiting Dot1L in a
differentiated cell and culturing the differentiated cell under
reprogramming conditions to accelerate the production of induced
pluripotent stem cells, wherein the production of induced
pluripotent stem cells is accelerated compared to a differentiated
cell in which Dot1L is not inhibited.
21. A method of producing induced pluripotent stem cells, the
method comprising upregulating the expression of Nanog and Lin28 in
a differentiated cell and culturing the differentiated cell under
reprogramming conditions to produce induced pluripotent stem
cells.
22. The method of claim 21, wherein the expression of Nanog and
Lin28 is upregulated by inhibiting Dot1L.
23. A method of producing induced pluripotent stem cells, the
method comprising inhibiting SUV39H1 in a differentiated cell and
culturing the differentiated cell under reprogramming conditions to
produce induced pluripotent stem cells.
24. A method of producing induced pluripotent stem cells, the
method comprising inhibiting YY1 in a differentiated cell and
culturing the differentiated cell under reprogramming conditions to
produce induced pluripotent stem cells.
25. A composition comprising a population of human induced
pluripotent stem cells produced according to the method of claim
1.
26. (canceled)
27. A method of treatment comprising administering to a person in
need thereof the composition of claim 25.
28. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of U.S. provisional application Ser. No. 61/547,404
filed Oct. 14, 2011, the disclosure of which is incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The field of invention relates to methods and compositions
for the production of stem cells.
BACKGROUND OF THE INVENTION
[0004] A variety of methods are available for reprogramming adult
cells to obtain induced pluripotent stem cells. However, the
methods currently available suffer from low efficiency and
incomplete programming. In addition, some of the methods currently
available result in the upregulation of oncogenes, thereby
increasing the risk of tumor formation. New methods for the
production of induced pluripotent stem cells are needed
therefore.
SUMMARY OF THE INVENTION
[0005] Aspects of the disclosure provide compositions and methods
for reprogramming differentiated cells to produce pluripotent cells
(e.g., stem cells). In some embodiments, certain enzyme inhibitors
enhance cellular reprogramming by accelerating the reprogramming
process and/or by simplifying the process (for example, by reducing
the number and type of factors required for cellular
reprogramming). For example, in some embodiments, methods and
compositions described herein can be used to reprogram
differentiated cells without using transcription factors that can
have unwanted side effects such as oncogenesis. In some
embodiments, inhibiting one or more of Dot1L (a histone H3
methyltransferase), YY1 (a transcriptional repressor protein),
and/or SUV39H1 (a histone-lysine N-methyltransferase) helps promote
the reprogramming of differentiated cells. One or more of these
enzymes can be inhibited using any appropriate technique,
including, for example, by inhibiting expression (e.g.,
transcription, translation, and/or modification) and/or activity of
the enzyme(s).
[0006] Pluripotent (e.g., induced pluripotent stems cells--iPSCs)
produced methods and compositions described herein are useful for
therapeutic and research applications.
[0007] Accordingly, in some embodiments the disclosure provides
methods and compositions for the production of stem cells. In
certain embodiments, the disclosure provides stem cells (for
example induced pluripotent stem cells) and methods and
compositions for their use.
[0008] In some embodiments, a method of producing induced
pluripotent stem cells includes inhibiting of Dot1L, YY1, and/or
SUV39H1 in a differentiated cell (e.g., in a preparation containing
one or more differentiated cell types). In some embodiments, the
method also includes culturing the differentiated cell(s) under one
or more reprogramming conditions. In some embodiments, the
inhibition of Dot1L, YY1, and/or SUV39H1 occurs under the cell
reprogramming conditions. However, in some embodiments, inhibition
of Dot1L, YY1, and/or SUV39H1 is initiated prior to exposing the
cell(s) to reprogramming conditions.
[0009] In some embodiments, the act of inhibiting includes
inhibiting the activity of Dot1L, YY1, and/or SUV39H1 (e.g.,
inhibiting the methyltransferase activity of Dot1L). In some
embodiments, the activity is inhibited by contacting a
differentiated cell with a composition comprising one or more
enzyme inhibitors (e.g., one or more enzyme inhibitors specific for
Dot1L, YY1, and/or SUV39H1).
[0010] In some embodiments, the act of inhibiting includes knocking
down the expression of Dot1L, YY1, and/or SUV39H1 (e.g., by
inhibiting transcription and/or translation of the Dot1L, YY1,
and/or SUV39H1 gene). In some embodiments, a differentiated cell is
contacted with one or more RNAi (e.g., shRNA) molecules that
specifically inhibit Dot1L, YY1, and/or SUV39H1 expression.
[0011] In some embodiments, the reprogramming conditions include
using a cocktail containing one or more reprogramming factors
(e.g., one or more reprogramming transcription factors). In some
embodiments, the reprogramming cocktail includes Oct4 and/or Sox2.
In some embodiments, the reprogramming cocktail consists
essentially of Oct4 and Sox2. In some embodiments, the
reprogramming cocktail includes Klf4 and/or c-Myc (e.g., in
addition to Oct4 and/or Sox2). However, in some embodiments, the
reprogramming cocktail does not include Klf4 or c-Myc. In some
embodiments, an inhibitor (e.g., a Dot1L inhibitor) is added to the
transcription factor(s) in the reprogramming cocktail.
[0012] In some embodiments, the differentiated cell is a somatic
cell (e.g., a somatic cell obtained from a subject) or a cultured
cell. In some embodiments, the differentiated cell is a fibroblast
(e.g., an adult fibroblast). In some embodiments, the
differentiated cell is a human cell (e.g., dH1fs, IMR-90 or MRC-5),
or a mouse cell.
[0013] In some embodiments, the presence of induced pluripotent
stem cells is determined (e.g., by detecting one or more markers
characteristic of an induced pluripotent stem cell) after cellular
reprogramming. In some embodiments; the presence of induced
pluripotent stem cells is determined by evaluating the presence of
one or more markers selected from the group consisting of SSEA4,
SSEA3, Tra-1-81, Oct4, Sox2 and Nanog. In some embodiments, induced
pluripotent stem cells are isolated after reprogramming of one or
more differentiated cell types.
[0014] In some embodiments, the production of induced pluripotent
stem cells is accelerated by inhibiting Dot1L, YY1, and/or SUV39H1
in a differentiated cell. In some embodiments, the production of
induced pluripotent stem cells is more efficient when Dot1L, YY1,
and/or SUV39H1 are inhibited in a differentiated cell. Inhibition
can occur (e.g., be initiated) before, during, or after culturing
the differentiated cell under reprogramming conditions. A reduction
in time and/or increase in efficiency can be obtained relative to a
reprogramming of the differentiated cell in which Dot1L, YY1,
and/or SUV39H1 are not inhibited.
[0015] In some embodiments, a method of producing induced
pluripotent stem cells includes upregulating the expression of
Nanog and/or Lin28 in a differentiated cell. In some embodiments,
these-cells are cultured (e.g., before, concurrently, and/or
subsequently) under reprogramming conditions to produce induced
pluripotent stem cells. In some embodiments, the expression of
Nanog and Lin28 is upregulated by inhibiting Dot1L.
[0016] In some embodiments, a preparation of induced pluripotent
stem cells is provided wherein the stem cells were produced as
described herein. In some embodiments, a preparation of induced
pluripotent stem cells includes one or more inhibitors of Dot1L,
YY1, and/or SUV39H1 (e.g., one or more expression and/or activity
inhibitors). In some embodiments, the inhibitors are present in
trace amounts. In some embodiments, the induced pluripotent stem
cells are human cells.
[0017] In some embodiments, the induced pluripotent stems cell can
differentiate (e.g., be differentiated using appropriate factors
and/or conditions) into ectoderm, mesoderm and/or endoderm
cells.
[0018] In some embodiments, a preparation of induced pluripotent
stem cells is administered to a subject. In some embodiments, a
preparation of differentiated stem cells (e.g., induced pluripotent
stem cells that were differentiated ex vivo) is administered to a
subject. In some embodiments, the subject is a patient (e.g., a
human or animal patient) in need of treatment with pluripotent stem
cells and/or differentiated cells. In some embodiments, a subject
in need of treatment is patient having brain damage (e.g.,
associated with a neurodegenerative disorder such as Parkinson's
disease), cancer, spinal cord injury, heart damage, baldness,
deafness, diabetes, neuronal defects, blindness, amyotrophic
lateral sclerosis, a genetic disorder, infertility, and/or unhealed
wounds. It should be appreciated that the pluripotent and
differentiated cell populations described herein may be used in a
variety of in vivo methods including but not limited to therapeutic
or cosmetic applications.
[0019] In some embodiments, one or more inhibitors of Dot1L, YY1,
and/or SUV39H1 is administered to a subject to promote stem cell
growth or development (e.g., locally at or near the site of local
administration, or systemically). In some embodiments, one or more
inhibitors may be administered in combination with one or more
other factors, including but not limited to, one or more
transcription factors (e.g., 2, 3, 4 or more transcription factors
such as those described herein), for example in the form of a
transcription factor expressing vector (e.g., for inducing local
stem cell populations).
[0020] In some aspects, the disclosure provides kits, compositions,
and methods for identifying enhancers and/or inhibitors of cell
reprogramming. In some aspects, the disclosure provides kits,
compositions, and methods for producing or isolating induced
pluripotent stem cells and/or differentiated cells obtained from
the stem cells.
[0021] These and other aspects and embodiments of the invention are
described in greater detail below.
[0022] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention.
[0023] This invention is not limited in its application to the
details of construction and the arrangement of components set forth
in the following description or illustrated in the drawings. The
invention is capable of other embodiments and of being practiced or
of being carried out in various ways. Also, the phraseology and
terminology used herein is for the purpose of description and
should not be regarded as limiting. The use of "including",
"comprising", or "having", "containing", "involving", and
variations thereof herein, is meant to encompass the items listed
thereafter and equivalents thereof as well as additional items.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The figures are illustrative only and are not required for
enablement of the invention disclosed herein.
[0025] FIG. 1 illustrates a non-limiting example of a screen for
inhibitors and enhancers of cellular reprogramming;
[0026] FIG. 2 shows a non-limiting example of cellular
reprogramming enhanced by Dot1L inhibition;
[0027] FIG. 3 shows a non-limiting example of Dot1L inhibition
using a small molecule;
[0028] FIG. 4 shows a non-limiting example of Nanog and Lin28
associated with reprogramming by Dot1L inhibition;
[0029] FIG. 5 shows a non-limiting example of H3K79me2 marks during
reprogramming;
[0030] FIG. 6 shows a non-limiting example of knockdown efficiency
by shRNA in dH1f cells;
[0031] FIG. 7 shows a non-limiting example of cell proliferation
upon PRC1/2 knockdown;
[0032] FIG. 8 shows a non-limiting example of Dot1L mRNA and total
H3K79me2 levels under knock-down conditions;
[0033] FIG. 9 shows a non-limiting example of same-well
reprogramming of admixed control and Dot1L inhibited cell
populations;
[0034] FIG. 10 shows a non-limiting characterization of shDot1L iPS
cells;
[0035] FIG. 11 shows a non-limiting example of Dot1L knockdown
dynamics during reprogramming;
[0036] FIG. 12 shows a non-limiting example of the Growth dynamics
of shDot1L cells pre- and post-OSKM transduction;
[0037] FIG. 13 shows a non-limiting example apoptosis and cell
cycle profile of Dot1L-inhibited cells;
[0038] FIG. 14 shows a non-limiting example of the kinetics of iPSC
colony formation upon Dot1L knockdown;
[0039] FIG. 15 shows a non-limiting example of increased
reprogramming efficiency using small molecule inhibitor of
Dot1L;
[0040] FIG. 16 shows a non-limiting example of reprogramming of
Dot1L conditional knockout tail-tip fibroblasts TTFs;
[0041] FIG. 17 shows a non-limiting example of knockdown efficiency
of Nanog and Lin28 during 2-factor reprogramming;
[0042] FIG. 18 illustrates a non-limiting example of Chip-seq
experimental design;
[0043] FIG. 19 shows a non-limiting example of the relationship
between H3K79me2 and H3K27me3; and,
[0044] FIG. 20 shows a non-limiting example of genes marked with
K79me2 specifically in fibroblasts, in ESCs and in both cell
types.
DETAILED DESCRIPTION OF THE INVENTION
[0045] In one aspect, the disclosure provides methods and
compositions that are useful in the production of stem cells (e.g.,
induced pluripotent stem cells--iPSC) from differentiated cells. In
some embodiments, the inhibition of Dot1L, SUV39H1, and/or YY1
during at least a portion of a cell reprogramming technique
increases the efficiency of and/or accelerates the reprogramming
process. In some embodiments, the inhibition of one or more of
these proteins also can be used to reduce the number of
transcription factors required for a cell reprogramming
procedure.
[0046] Stem cells produced using compositions and methods described
herein are useful in therapeutic and/or research applications. In
addition, methods and compositions of promoting stems cell
production can be used for therapeutic and/or research
purposes.
[0047] Methods and compositions described herein can simplify the
process of reprogramming a somatic cell. In general, the production
of induced pluripotent stem cells (iPSCs) from differentiated cell
types by somatic cell reprogramming involves resetting the
epigenetic states of the differentiated cells. Typical
reprogramming techniques involve exposing cells to several
transcription factors, some of which can have undesirable
side-effects (e.g., they can be oncogenic). Surprisingly,
inhibiting one or more of Dot1L, SUV39H1, and/or YY1 during at
least a portion of a reprogramming technique allows for the use of
fewer transcription factors, thereby reducing the risks associated
with certain transcription factors, in addition to enhancing the
efficiency and speed of the overall reprogramming procedure.
[0048] In some aspects, the disclosure also provides compositions
and methods for identifying genes and proteins that are negative or
positive regulators in the reprogramming of differentiated cells.
While several proteins are known to regulate chromatin marks
associated with the distinct epigenetic states of cells before and
after reprogramming, how chromatin-modifying proteins influence the
reprogramming process remains largely unknown. By identifying
negative or positive regulators of iPSC generation, techniques for
controlling the process of iPSC generation can be further developed
or refined by inhibiting or increasing the expression and/or
activity of one or more of the negative or positive regulators.
[0049] In some embodiments, inhibition of the core components of
the polycomb repressive complex 1 and 2, including the histone 3
lysine 27 methyltransferase Ezh2, reduced reprogramming efficiency.
However, surprisingly, inhibition of SUV39H1, YY1, and Dot1L,
increased reprogramming. In contrast to genes whose functions
appear to be required for reprogramming, inhibition of these three
genes enhanced reprogramming (see FIG. 1D). YY1 is a transcription
factor that activates or represses transcription in a
context-dependent manner.sup.10,11, whereas Suv39H1 is a histone
H3K9 methyltransferase implicated in heterochromatin
formation.sup.12, and Dot1L is a H3K79 histone
methyl-transferase.
[0050] In some embodiments, inhibition of Dot1L, the H3K79 histone
methyl-transferase, either by RNAi or by a small molecule inhibitor
accelerated reprogramming, significantly increased the yield of
iPSC colonies, and substituted for Klf4 and c-Myc in a
reprogramming cocktail. In some embodiments, inhibition of Dot1L
functions early in the reprogramming process can be used to
markedly induce two alternative reprogramming factors, Nanog and
Lin28. Furthermore, in loss-of-function experiments, it was shown
that Nanog and Lin28 play essential functional roles in the
enhancement of reprogramming by Dot1L-inhibition in some
embodiments.
[0051] As shown herein, suppression of Dot1L expression using
shRNAs or inhibition of its catalytic activity using a small
molecule both accelerates and increases the yield of iPSCs and
substitutes for both Klf4 and Myc in the reprogramming process.
These effects are primarily mediated through induction of two key
pluripotency factors, Nanog and Lin28, whose activation normally
occurs during the later stages of reprogramming.sup.20,21,22,23.
Accordingly, in some embodiments, cell reprogramming can be
promoted (e.g., ex vivo) by inhibiting Dot1L and/or stimulating
Nanog and/or Lin28 expression and/or activity.
[0052] Genome-wide analysis of K79me2 distribution revealed that
fibroblast-specific, epithelial to mesenchymal
transition-associated genes start to lose K79me2 in the initial
phases of reprogramming and Dot1L inhibition facilitates the loss
of this mark from such genes that eventually get repressed in the
pluripotent state. Accordingly, in some embodiments these marks can
be used to evaluate and/or monitor the effectiveness of Dot1L
inhibition in a cell reprogramming procedure that is used to
generate induced pluripotent stems cells.
[0053] It should be appreciated that in some embodiments, stem
cells can be generated from cultured somatic cell lines. However,
in some embodiments, methods and compositions described herein can
be used to generate "personalized" stem cells (e.g., for autologous
cell therapy) by obtaining one or more somatic cells from a subject
and reprogramming the cell(s) ex vivo as described herein. These
personalized stem cells can be useful to produce cell preparations
(e.g., containing undifferentiated stem cells and/or differentiated
stem cells) that can be reintroduced to the subject (e.g., to treat
a disease or disorder) with a low risk of host rejection of the
reimplanted cells.
[0054] In some embodiments, inhibition of Dot1L can be used to
prepare cells for a reprogramming procedure by inhibiting Dot1L
prior to exposing the cells to a reprogramming cocktail (e.g.,
containing one or more transcription factors that are useful to
promote reprogramming). However, in some embodiments, Dot1L
inhibition can be initiated during reprogramming. In some
embodiments, one or more Dot1L inhibitors are provided along with
one or more transcription factors in a reprogramming cocktail. In
some embodiments, one or more Dot1L inhibitors are added to a
reprogramming cocktail that contains one or more transcription
factors. Accordingly, it should be appreciated that cells being
reprogrammed can be contacted with (e.g., incubated with) one or
more Dot1L inhibitors (and/or one or more YY1 inhibitors and/or one
or more SUV39H1 inhibitors) prior to, along with, and/or after
incubation with one or more transcription factors in an incubation
cocktail. The presence of the inhibitor(s) can be useful to
increase the efficiency of the reprogramming, accelerate the
reprogramming (e.g., reduce the incubation time with the
reprogramming cocktail), and/or reduce the number of transcription
factors required in the incubation cocktail.
[0055] Stem cells described herein can be used for therapeutic and
research applications. In some embodiments, stem cells can be
administered to a subject (e.g., a human subject). In some
embodiments, stem cells can be differentiated into one or more
somatic cell types of interest prior to administration to a
subject. In some embodiments, stem cells can be used to produce
artificial tissue or organs ex vivo (e.g., for organ
transplantation purposes). Techniques for differentiating stem
cells into different types of cell types (e.g., prior to
administration to a subject or for use in generating artificial
tissue or organs) are known in the art.
[0056] Although a cell reprogramming technique typically occurs ex
vivo using isolated cells that are obtained from a cell bank or a
subject, methods and compositions described herein also can be used
in vivo to promote stem cell production and or maintenance in a
subject. For example, in some embodiments one or more inhibitors
described herein can be administered to a subject (e.g., using any
suitable route) to promote or maintain stem cell populations in the
subject. In some embodiments, the inhibitor(s) may be administered
systemically. In some embodiments, the inhibitor(s) may be
administered locally (e.g., to provide local stimulation or
maintenance of a stem cell population). In some embodiments, one or
more inhibitors may be administered in combination with one or more
other factors, including but not limited to, one or more
transcription factors (e.g., 2, 3, 4 or more transcription factors
such as those described herein). In some embodiments, the
transcription factors are provided in the form of a transcription
factor expressing vector that expresses one or more transcription
factors in vivo after administration. In some embodiments, a vector
can include one or more expression regulators to limit expression
of the transcription factor(s) to particular tissue or cell types.
In some embodiments, the inhibitor(s) and transcription factor(s)
are administered locally. Accordingly, methods and compositions
described herein can be used to induce local stem cell
populations.
[0057] In some embodiments, one or more small molecule inhibitors
of Dot1L, YY1 and/or SUV39H1 are provided to cells (e.g., ex vivo,
or in vivo) in an amount sufficient to inhibit protein function. In
some embodiments, the cells are exposed to 0.01 uM, 0.1 uM, 1 uM or
10 uM of inhibitor for a time sufficient to inhibit the protein
during a reprogramming procedure. In some embodiments, the cells
are exposed to the inhibitor for several hours or several days
(e.g., 1-10 days, for example about 5 days) before, during, or
after incubation with a reprogramming cocktail.
[0058] In some embodiments, one or more of the inhibitors described
herein (for example one or more inhibitors of Formula I, II, III,
or IV) may be used to inhibit Dot1L in vivo or in vitro. In some
embodiments, an inhibitor of structure:
##STR00001##
pharmaceutically acceptable salt thereof
##STR00002##
may be used to inhibit Dot1L in vitro (e.g., contacted to
differentiated cells in vitro, for example alone or in combination
with one or more transcription factors or other agents described
herein, to promote iPSC generation in vitro) or in vivo (e.g.,
administered to a subject in vivo, for example alone or in
combination with one or more transcription factors or other agents
described herein, to promote iPSC generation in vivo, for example
to promote local stem cell production or to support local stem cell
growth or maintenance).
[0059] In some embodiments, Dot1L, YY1 and/or SUV39H1 is inhibited
by contacting a differentiated cell with one or more nucleic acids
that prevent production of the protein(s). In some embodiments,
Dot1L, YY1 and/or SUV39H1 is inhibited by an shRNA that knocks down
expression. Non-limiting embodiments of shRNAs that knock down
expression are provided in the Examples section.
[0060] Nucleic acids that prevent Dot1L, YY1 and/or SUV39H1
expression can be provided to the cells in a variety of formats
including dsRNA, siRNA and shRNA. "RNA interference (RNAi)" is an
evolutionally conserved process whereby the expression or
introduction of RNA of a sequence that is identical or highly
similar to a target gene results in the sequence specific
degradation or specific post-transcriptional gene silencing (PTGS)
of messenger RNA (mRNA) transcribed from that targeted gene thereby
inhibiting expression of the target gene. In one embodiment, the
RNA is double stranded RNA (dsRNA). This process has been described
in plants, invertebrates, and mammalian cells. In nature, RNAi is
initiated by the dsRNA-specific endonuclease Dicer, which promotes
processive cleavage of long dsRNA into double-stranded fragments
termed siRNAs. "Short interfering RNA" (siRNA), also referred to
herein as "small interfering RNA" is defined as a nucleic
acid-comprising agent which functions to inhibit expression of a
target gene, by RNAi. An siRNA may be chemically synthesized, may
be produced by in vitro transcription, or may be produced within a
host cell. In one embodiment, siRNA is a double stranded RNA
(dsRNA) molecule of about 15 to about 40 nucleotides in length,
preferably about 15 to about 28 nucleotides, more preferably about
19 to about 25 nucleotides in length, and more preferably about 19,
20, 21, 22, or 23 nucleotides in length, and may contain a 3'
and/or 5' overhang on each strand having a length of about 0, 1, 2,
3, 4, or 5 nucleotides. The length of the overhang is independent
between the two strands, i.e., the length of the overhang on one
strand is not dependent on the length of the overhang on the second
strand. Preferably the siRNA is capable of promoting RNA
interference through degradation or specific post-transcriptional
gene silencing (PTGS) of the target messenger RNA (mRNA). siRNAs
also include small hairpin (also called stem loop) RNAs (shRNAs).
In one embodiment, these shRNAs are composed of a short (e.g.,
about 19 to about 25 nucleotide) antisense strand, followed by a
nucleotide loop of about 5 to about 9 nucleotides, and the
analogous sense strand. Alternatively, the sense strand may precede
the nucleotide loop structure and the antisense strand may follow.
These shRNAs may be encoded by plasmids, retroviruses, and
lentiviruses and expressed from, for example, the pol III U6
promoter, or another promoter (see, e.g., Stewart, et al., RNA
April; 9(4):493-501 (2003), incorporated by reference herein in its
entirety).
[0061] It is envisioned that inhibitors of Dot1L, YY1 and/or
SUV39H1 are used in effective amounts. In some embodiments, an
effective amount of Dot1L inhibitor (e.g., small molecule or RNAi
molecule) is an amount sufficient to inhibit the transferase
activity (e.g., by at least 25%, at least 50%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%,
or more) in the targeted cells (e.g., cells growing in vitro, or
targeted cells types in a subject). In some embodiments, an
effective amount of YY1 inhibitor is an amount sufficient to
inhibit the activity (e.g., by at least 25%, at least 50%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99%, or more) in the targeted cells (e.g., cells growing in
vitro, or targeted cells types in a subject). In some embodiments,
an effective amount of SUV39H1 inhibitor is an amount sufficient to
inhibit the activity (e.g., by at least 25%, at least 50%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99%, or more) in the targeted cells (e.g., cells growing in
vitro, or targeted cells types in a subject).
[0062] In some embodiments, a subject may be evaluated to determine
whether Dot1L is overexpressed. Certain genetic abnormalities
associated with Dot1L (e.g., the Dot1L regulatory pathway) result
in Dot1L overexpression. Assays for detecting genetic abnormalities
associated with Dot1L overexpression are known in the art and can
include, but are not limited to, molecular assays (e.g.,
hybridization or sequencing) or in situ or chromosomal assays
(e.g., FISH) or other chromosomal assays. Depending on the level of
Dot1L in the subject (or in cells obtained from the subject) the
amount of Dot1L inhibitor that is used (e.g., in vivo or in vitro)
may be adjusted. One of ordinary skill can perform assays to
determine suitable levels of Dot1L inhibitor to use in order to
inhibit Dot1L levels that are present in a subject (for example in
vivo or in vitro in cells isolated from the subject).
Reprogramming
[0063] In some embodiments, inhibiting Dot1L, YY1 and/or SUV39H1
can be used to promote cellular reprogramming (e.g., ex vivo or in
vivo). During development, individual cells become specified to
adopt distinct fates through the activation of lineage-specific
gene expression programs. Under normal circumstances, these gene
expression programs are stably inherited throughout mitotic
divisions, thereby maintaining cell identity. At the molecular
level, this cellular identity is largely controlled at the
chromatin level whereby genes appropriate for a given lineage are
embedded in a chromatin structure that supports their
transcriptional activity, whereas genes specifying other lineages
are sequestered in repressive chromatin structures resulting in
transcriptional silencing.sup.1. Differentiated cells such as
fibroblasts can be converted into induced pluripotent stem cells
(iPSCs) upon overexpression of critical transcriptional regulators
of embryonic stem cells (ESCs).sup.2,3,4.
[0064] In some embodiments, the effect of inhibition of one or more
proteins on reprogramming was evaluated in dH1fs cells. The dH1fs
cells were transfected with shRNA pools (at high multiplicity of
infection to ensure all cells received an shRNA vector) followed by
super-infection with reprogramming vectors expressing Oct4, Sox2,
Klf4 and c-Myc (OSKM), and the resulting iPSCs were identified by
Tra-1-60 staining.
[0065] In one aspect, the disclosure provides a method of producing
induced pluripotent stem cells, by inhibiting Dot1L (and/or YY1
and/or SUV39H1) in a differentiated cell and culturing the
differentiated cell under reprogramming conditions to produce
induced pluripotent stem cells. In some embodiments, the
reprogramming conditions comprise the presence of a reprogramming
cocktail. In some embodiments, the reprogramming cocktail comprises
Oct4 and/or Sox2. In some embodiments, the reprogramming cocktail
includes Klf4 and/or c-Myc. In some embodiments, the reprogramming
cocktail does not include Klf4 and/or c-Myc. In some embodiments,
the reprogramming cocktail consists essentially of Oct4 and Sox2.
In some embodiments, Dot1L (and/or YY1 and/or SUV39H1) is inhibited
at the same time as providing the reprogramming cocktail.
[0066] Reprogramming, when relating to cells, generally refers to
the process of changing a cell from a first phenotype to a second
phenotype. In particular, and as used herein, reprogramming refers
to the process of changing a differentiated cell into an induced
pluripotent stem cell.
[0067] Reprogramming conditions as used herein refers to the
conditions that allow a differentiated cell to transform into an
induced pluripotent stem cell. Reprogramming conditions include
culture media, culture components (e.g., buffer, pH, salt) and the
duration of the culturing of the cells.
[0068] In some embodiments, the reprogramming conditions comprise
the presence of a reprogramming cocktail. A reprogramming cocktail
as used herein refers to the combination of specific agents (e.g.,
Sox2, Oct 4, Klf4, c-Myc, or a combination of any 2 or 3 thereof,
or all 4, and/or one or more additional transcription factors) that
is required to reprogram a cell from a differentiated cell into an
induced pluripotent stem cell. In some embodiments, the
reprogramming cocktail comprises Oct4 and Sox2. In some
embodiments, the reprogramming cocktail consists essentially of
Oct4 and Sox2. A reprogramming cocktail that consists essentially
of Oct4 and Sox2, refers to a reprogramming cocktail that does not
include any other specific agents (e.g., transcription factors)
that could be used to reprogram a differentiated cell. However, in
some embodiments, a reprogramming cocktail (e.g., a cocktail that
consists essentially of specified transcription factors) may
include buffers, salts, sugars, and other components that may be
useful to support the growth and reprogramming of the
differentiated cells.
[0069] The production of induced pluripotent stem cells is
generally achieved by the introduction of nucleic acid sequences
encoding stem cell-associated genes into a somatic cell. In
general, these nucleic acids are introduced using retroviral
vectors and expression of the gene products results in cells that
are morphologically and biochemically similar to pluripotent stem
cells (e.g., embryonic stem cells). This process of altering a cell
phenotype from a somatic cell or progenitor cell phenotype to a
stem cell-like phenotype is termed "reprogramming".
[0070] It was surprisingly found herein that inhibiting DotL1
provides an alternative route for reprogramming cell, eliminating
the need for one or more cell transforming factors (e.g., c-Myc)
that are associated with oncogenesis.
[0071] In some embodiments, reprogramming can be achieved by
introducing a combination of stem cell-associated genes including,
for example Oct3/4 (Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18,
NANOG, Klf1, Klf2, Klf4, Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In
general, successful reprogramming is accomplished by introducing
Oct-3/4, a member of the Sox family, a member of the Klf family,
and a member of the Myc family to a somatic or progenitor cell. In
some embodiments, the nucleic acid sequences of Oct-4, Sox2, c-MYC,
and Klf4 are delivered using a viral vector, such as an adenoviral
vector, a lentiviral vector or a retroviral vector. However, while
it is understood that reprogramming is usually accomplished by
viral delivery of stem-cell associated genes, it is also
contemplated herein that reprogramming can be induced using other
delivery methods.
[0072] In one aspect, the disclosure provides a method of
accelerating the production of induced pluripotent stem cells, the
method comprising inhibiting Dot1L (and/or YY1 and/or SUV39H1) in a
differentiated cell and culturing the differentiated cell under
reprogramming conditions to accelerate the production of induced
pluripotent stem cells, wherein the production of induced
pluripotent stem cells is accelerated compared to a differentiated
cell in which Dot1L is not inhibited. Thus, as provided herein, in
some embodiments, a differentiated cell can be reprogrammed into an
induced pluripotent stem cells by using a known combination of
cell-associated genes (e.g., Oct-4, Sox2, c-MYC, and Klf4 or a
subset thereof). It was unexpectedly shown herein that the addition
of Dot1L to the combination of cell-associated genes resulted in
the acceleration of the production of induced pluripotent stem
cells.
[0073] The efficiency of reprogramming (e.g., the number of
reprogrammed cells) can be enhanced by the addition of various
small molecules as shown by Shi, Y., et al (2008) Cell-Stem Cell.
2:525-528, Huangfu, D., et al (2008) Nature Biotechnology
26(7):795-797, Marson, A., et al (2008) Cell-Stem Cell 3:132-135,
which are incorporated herein by reference in their entirety. It is
contemplated that the methods described herein can also be used in
combination with a single small molecule (or a combination of small
molecules) that enhances the efficiency of induced pluripotent stem
cell production. Some non-limiting examples of agents that enhance
reprogramming efficiency include soluble Wnt, Wnt conditioned
media, BIX-01294 (a G9a histone methyltransferase), PD0325901 (a
MEK inhibitor), DNA methyltransferase inhibitors, histone
deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine,
dexamethasone, suberoylanilide, hydroxamic acid (SAHA),
trichostatin (TSA), and inhibitors of the TGF-.beta. signaling
pathway, among others. It is also contemplated herein that
inhibitors can be used alone or in combination with other small
molecule(s) to replace one or more of the reprogramming factors
used for the production of induced pluripotent stem cells.
[0074] In some embodiments, a method of producing pluripotent stem
cells includes upregulating the expression of Nanog and/or Lin28 in
a differentiated cell and culturing the differentiated cell under
reprogramming conditions to produce induced pluripotent stem cells.
In some embodiments, Nanog and/or Lin28 are upregulated by
inhibiting Dot1L. In some embodiments, Nanog and/or Lin28 are
upregulated by introducing agents that stimulate expression of
Nanog and/or Lin28, and/or by introducing agents that suppress
removal of Nanog and/or Lin28 from the cell (e.g., by protease
action). In some embodiments, promoters are introduced into the
genome of the cell that result in the upregulation of the
expression of Nanog and/or Lin28.
[0075] In some embodiments, a method of producing pluripotent stem
cells includes inhibiting SUV39H1 in a differentiated cell and
culturing the differentiated cell under reprogramming conditions to
produce induced pluripotent stem cells. Histone-lysine
N-methyltransferase SUV39H1 is a member of the suppressor of
variegation 3-9 homolog family and encodes a protein with a
chromodomain and a C-terminal SET domain. This nuclear protein
moves to the centromeres during mitosis and functions as a histone
methyltransferase, methylating Lys-9 of histone H3 Histone-lysine
N-methyltransferase SUV39H1. It was surprisingly shown herein that
inhibiting SUV39H1 provides a novel pathway for producing induced
pluripotent stem cells. In some embodiments, SUV39H1 is inhibited
by providing the cell with an siRNA, shRNA (or other RNAi) against
SUV39H1. In some embodiments, SUV39H1 is inhibited by providing the
cell with an SUV39H1 inhibitor (e.g., a small molecule inhibitor
such as Chaetocin, see Greiner et al. Nat Chem Biol. 2005 August;
1(3):143-5).
[0076] In some embodiments, a method of producing pluripotent stem
cells includes inhibiting YY1 in a differentiated cell and
culturing the differentiated cell under reprogramming conditions to
produce induced pluripotent stem cells. Transcriptional repressor
protein YY1 is a ubiquitously distributed transcription factor
belonging to the GLI-Kruppel class of zinc finger proteins. The
protein is involved in repressing and activating a diverse number
of promoters. YY1 may direct histone deacetylases and histone
acetyltransferases to a promoter in order to activate or repress
the promoter, thus implicating histone modification in the function
of YY1. It was surprisingly shown herein that inhibiting YY1
provides a novel pathway for producing induced pluripotent stem
cells. In some embodiments, YY1 is inhibited by providing the cell
with an siRNA, shRNA (or other RNAi) against YY1. In some
embodiments, YY1 is inhibited by providing the cell with an YY1
inhibitor (e.g., a small molecule inhibitor).
Differentiated Cells
[0077] Methods described herein embrace the use of any
differentiated cell (e.g., somatic cell). In some embodiments, the
differentiated cell is a fibroblast, epithelial, endothelial,
neuronal, adipose, cardiac, skeletal muscle, immune cells, hepatic,
splenic, lung, circulating blood, gastrointestinal, renal, bone
marrow, or pancreatic cell. The differentiated cell can be a
primary cell isolated from any somatic tissue including, but not
limited to brain, liver, lung, gut, stomach, intestine, fat,
muscle, uterus, skin, spleen, endocrine organ, bone, etc., Methods
for isolating differentiated cells from a body are known in the
art.
[0078] In some embodiments, the differentiated cell is a
fibroblast. In some embodiments, the differentiated cell is an
adult fibroblast.
[0079] The differentiated cell used according to the methods
provided herein can be from any mammalian species, with
non-limiting examples including a murine, bovine, simian, porcine,
equine, ovine, or human cell.
[0080] In some embodiments, the differentiated cell is a human
cell. In some embodiments, the differentiated cell is dH1fs, IMR-90
or MRC-5.
[0081] In some embodiments, the differentiated cell is a mouse
cell.
[0082] The differentiated cells used in the methods provided
herein, and prior to being reprogrammed, can be maintained under in
vitro conditions using conventional tissue culture.
[0083] In some embodiments, one or more differentiated cells are
obtained from a subject that will subsequently be treated by
reimplanting induced pluripotent stem cells obtained from the
differentiated cells and/or by reimplanting redifferentiated cells,
tissue, or organs obtained from the stem cells.
Induced Pluripotent Stem Cells
[0084] In one aspect, the disclosure provides methods for producing
stem cells. In some embodiments, the cells are induced pluripotent
stem cells. The term "pluripotent," as used in the context of
cells, describes the developmental capacity of a cell or cell
population and refers to cells that are capable of self-renewal and
have the capacity to differentiate into cells of any of the three
germ layers (endoderm, mesoderm, and ectoderm). Examples of
pluripotent cells are embryonic stem cells (ES-cells), embryonic
carcinoma cells (EC cells), and induced pluripotent stem cells (iPS
cells). A pluripotent cell has the potential to give rise to any
fetal or adult cell type. However, pluripotent cells cannot
contribute to extraembryonic tissue, such as, the placenta. This
distinguishes pluripotent cells from totipotent cells, which can
give rise to all fetal, adult, and extraembryonic cell types.
[0085] In one aspect of the methods provided herein, the presence
of induced pluripotent stem cells is determined. In some
embodiments, the presence of induced pluripotent stem cells is
determined by evaluating the presence of one or more markers
selected from the group consisting of SSEA4, SSEA3, Tra-1-81, Oct4,
Sox2 and Nanog.
[0086] In humans and mice; the transcription factors Oct-4, Sox2,
and Nanog are biomarkers that are specific for pluripotent cells
(see, e.g., Looijenga, L. H. et al. (2003) Cancer Res 63, 2244-50;
Peske, M. and Scholer, H. R. (2001) Stem Cells 19, 271-8; Pan, G.
and Thomson, J. A. (2007) Cell Res 17, 42-9; the entire contents of
each of which are incorporated herein by reference). Additional
biomarkers specific for human pluripotent cells include stage
specific embryonic antigens SSEA3 and SSEA4, as well as the
podocalyxin antigens TRA 1-81 and TRA 1-60 (see, e.g., Schopperle
et al. (2007) Stem Cells 25(3):723-30. Epub 2006 Nov. 22;
Henderson, J. K. et al. (2002) Stem Cells 20, 329-37; and Draper,
J. S. et al. (2002) J Anat 200, 249-58; the entire contents of each
of which are incorporated herein by reference). Additional
biomarkers for mouse pluripotent stem cells include SSEA1, which is
specific for pluripotent cells, and the less stringent marker
alkaline phosphatase, which is indicative, but not specific for
pluripotent cells (see, e.g., Brambrink, T. et al. (2008) Cell Stem
Cell 2(2):151-9; the entire contents of which are incorporated
herein by reference). Expression of these pluripotency markers
decreases and is eventually lost during differentiation,
corresponding to a restriction in developmental potential and,
thus, a loss of pluripotency (see, e.g., Schopperle, W. M. and
DeWolf, W. C. (2007) Stem Cells 25, 723-30; the entire contents of
which are incorporated herein by reference). Table 1 below lists
some additional biomarkers useful for the identification of
pluripotent stem cells from mouse, human, and other mammals, as
well as their expression spectrum among pluripotent cell types.
TABLE-US-00001 TABLE 1 stem cell markers Biomarker Expressed in
Remarks Alkaline ES, EC Elevated expression of this enzyme is
associated phosphatase with undifferentiated pluripotent stem cell
(PSC) Cluster ES, EC Surface receptor molecule found specifically
on designation 30 PSC (CD30) Cripto (TDGF-1) ES, Growth factor
expressed by ES cells, primitive cardiomyocyte ectoderm, and
developing cardiomyocyte GCTM-2 ES, EC Extracellular-matrix antigen
that is synthesized by undifferentiated PSCs Genesis ES, EC
Transcription factor expressed by PSCs Germ cell nuclear ES, EC
Transcription factor expressed by PSCs factor OCT4/POU5F1 ES, EC
Transcription factor unique to PSCs Stage-specific ES, EC
Glycoprotein specifically expressed in early embryonic embryonic
development and by undifferentiated PSCs antigen-3 (SSEA-3)
Stage-specific ES, EC Glycoprotein specifically expressed in early
embryonic embryonic development and by undifferentiated PSCs
antigen-4 (SSEA-4) Stem cell factor ES, EC, HSC, Membrane protein
that enhances proliferation of ES (SCF or c-Kit MSC and EC cells,
hematopoietic stem cell (HSCs), and ligand) mesenchymal stem cells
(MSCs); binds the receptor c-Kit Telomerase ES, EC An enzyme
uniquely associated with immortal cell lines; useful for
identifying undifferentiated PSCs TRA-1-60 ES, EC Antibody to a
specific extracellular matrix molecule is synthesized by
undifferentiated PSCs TRA-1-81 ES, EC Antibody to a specific
extracellular matrix molecule normally synthesized by
undifferentiated PSCs
[0087] In some embodiments, methods provided herein include a step
of isolating the produced induced pluripotent stem cells. In some
embodiments, the induced pluripotent stem cells are isolated from a
mixed population cells comprising pluripotent cells and cells that
have not yet or only partially been reprogrammed. Methods for
isolating induced pluripotent stem cells are known in the art and
are generally based on moieties that bind to markers that are
uniquely expressed on induced pluripotent stem cells (e.g.,
SSEA-1).
[0088] In some embodiments, methods described herein include a step
of evaluating the developmental capacity of the induced pluripotent
stem cells. In some embodiments, the compositions of induced
pluripotent stem cells provided herein can be evaluated for their
developmental capacity. In some embodiments, the induced
pluripotent stem cells are evaluated for their capacity to
differentiate into ectoderm, mesoderm and endoderm cells. In some
embodiments, the developmental capacity of the induced pluripotent
stem cells is evaluated in a teratoma assay.
[0089] The developmental capacity of a cell or a cell population
can be tested by assays well known to those of skill in the art.
One test for pluripotency of a mouse cell or a population of mouse
cells, is diploid blastocyst complementation, in which one or more
of the cells in question are injected into a host blastocyst, which
is then transferred to a foster mouse. If the injected cell(s) are
pluripotent, contribution to all three germ layers, and to the germ
line, will be observed in the resulting pup, which will typically
be a chimera comprising cells derived from the host blastocyst and
from the injected cell(s). The most stringent test for the
developmental capacity of a pluripotent mouse cell, e.g., an ES or
iPS cell is the tetraploid blastocyst complementation assay, which
is well known to those of skill in the art (see, e.g., Eggan et
al., PNAS, 2001 (May) 6209-6214; and Li et al., Reproduction, 2005
130:53-59; the entire contents of which are incorporated herein by
reference). In this assay, a cell or a plurality of cells is
injected into a tetraploid host blastocyst. The cells of the
tetraploid host blastocyst can contribute to extra-embryonic
tissues, e.g., the placenta, but cannot give rise to fetal or adult
cell types. Accordingly, a tetraploid blastocyst alone cannot give
rise to a live pup at birth, because it cannot generate the
required fetal tissues. However, if a pluripotent cell or a
plurality of such cells is injected into a tetraploid blastocyst,
thus creating a complemented tetraploid blastocyst, the pluripotent
cell(s) can give rise to the cell types required for embryonic
development, if the injected cells exhibit a high developmental
capacity. Tetraploid blastocyst complementation is a more stringent
test for developmental capacity than the derivation of chimeric
mice after diploid blastocyst injection, because any defect in
developmental capacity of the injected cell(s) cannot be
compensated by cells of the host blastocyst. A pup resulting from a
complemented tetraploid blastocyst typically consists of cells
derived from the injected cell(s).
[0090] Additional tests for developmental capacity or pluripotency
include teratoma formation assays, also sometimes referred to as
teratocarcinoma assays (see. e.g., Wesselschmidt, R. L. (2011)
Methods Mol Biol. 767:231-41, the entire contents of which are
incorporated herein by reference). Typically, a population of cells
to be tested is injected subcutaneously into an immunocompromised
host animal, e.g., a SCID mouse. If the cell population comprises
pluripotent cells, a solid tumor will form at the site of
injection. Teratocarcinomas derived from pluripotent cells will
contain differentiated cell types of all three germ layers. This
assay is an in vivo assay for differentiation capacity that can be
used for all pluripotent cells, including cells that cannot be
tested by blastocyst complementation (e.g., human cells).
[0091] In some embodiments, methods described herein include a step
of promoting the development and/or differentiation one or more of
the induced pluripotent stem cells.
[0092] In some embodiments, the iPSCs produced as described herein
may be differentiated in vitro, partially or completely. Methods of
promoting the development or differentiation of stem cells are
known in the art. For example, differentiation protocols are known
in the art and include those described in U.S. Pat. No. 7,326,572
(endoderm differentiation), U.S. Pat. No. 7,282,366 (hepatocyte
differentiation), U.S. Pat. No. 7,250,294 (neural differentiation),
and U.S. Pat. No. 7,033,831 (islet cell differentiation). In some
embodiments, Embryoid Body differentiation, or in vitro directed
differentiation using cytokines, growth factors, and/or co-culture
with mature cell types may be used. A variety of differentiation
factors that can act on pluripotent stem cells and their precursor
progeny are known in the art. For example, members of the BMP
family of factors have been used to differentiate pluripotent stem
cells such. These include the use of BMP-4 and BMP-7 to generate
endoderm-like differentiation. (Xu et al. Nat Biotechnol
20:1261-1264, 2002; Pera et al. J Cell Sci 117:1269-1280, 2004.)
Activin A can be used to differentiate pluripotent stem cells into
definitive endoderm using monolayers or three dimensional (e.g.,
EB) culture systems. (D'Amour et al. Nat Biotechnol 23:1534-1541,
2005.) Nervous system cells have been observed as a result of
culture with epidermal growth factor and fibroblast growth factor
(resulting in the generation of neurospheres that comprise neural
stem cells), subsequent removal of these factors (resulting in the
generation of astrocyte-like cells) or supplementation with nerve
growth factor (resulting in the generation of neurons and glial
cells). (Kim et al. Nature 418:50-6, 2002; Lee et al. Nat
Biotechnol 18:675-9, 2000.) Dopaminergic neurons, useful in
Parkinson's disease, may be formed through culture or contact with
FGF20 and FGF2. Bjorklund et al. (PNAS 2002, 99:2344-2349) provides
additional methods for differentiating ES cells into dopaminergic
neurons. Hepatic cell differentiation may be induced through
contact and/or culture with an insulin, dexamethasone, and collagen
type I (via EB formation) combination; a sodium butyrate and DMSO
combination; an FGF4, HGF and collagen type I combination; an aFGF,
HGF, oncostatin M, dexamethasone and collagen type I combination;
and a bFGF, variant HGF, DMSO and dexamethasone combination in the
presence of poly-amino-urethane coated non-woven
polytetrafluoroethylene fabric. (Shirahashi et al. Cell Transplant
13:197-211, 2004; Rambhatla et al. Cell Transplant 12:1-11, 2003;
Schwartz et al. Stem Cells Dev 14:643-655, 2005; Baharvand et al.
Int J Dev Biol 50:645-652, 2006; Soto-Gutierrez et al. Cell
Transplant 15:335-341, 2006.) Hepatic differentiation may also
occur spontaneously. (Lavon et al. Differentiation 72:230-238,
2004.) Pancreatic differentiation, including differentiation
towards beta-islet cells, can be induced using Activin A, retinoic
acid, FGF2 and FGFIO, betacellulin, HGF, Exendin 4, DKK1 and DKK3.
(Gu et al. Mech Dev 120:35-43, 2003; Grapin-Botton et al. Trends
Genet 16:124-130, 2000; D'Amour et al. Nat Biotechnol 23:1534-1541,
2005a; D'Amour et al. published US application US2005-0266554A1.)
Endothelial differentiation may be induced in the presence of ECM
proteins such as collagen type IV, optionally in the presence of
VEGF and bFGF. (Gerecht-Nir et al. Lab Invest 83:1811-1820, 2003.)
Further reference may be made to published PCT application
WO2009/007852 for a review of various differentiative procedures
known in the art and applicable to the differentiation of the
immature and precursor cells of the invention. Such teachings, and
in particular those found on pages 57-61 (under the subheading
"Cell Differentiation") of WO2009/007852, are incorporated by
reference herein. Still other references include West and Daley,
2004, Curr Opin Cell Biol 16:688-692; U.S. Pat. No. 6,534,052 B1;
Kehat and Gepstein, 2003, 8:229-236; Nir et al., 2003, 58:313-323;
and U.S. Pat. Nos. 6,613,568 and 6,833,269.
[0093] In some embodiments, the disclosure provides compositions
comprising a population of induced pluripotent stem cells (e.g.,
human induced pluripotent stem cells) produced according to the
methods provided herein. In some embodiments, the disclosure
provides compositions comprising a population of differentiated
stem cells (e.g., human differentiated stem cells) obtained from
stem cells produced according to the methods provided herein. In
some embodiments, a composition, in addition to the population of
induced pluripotent stem cells and/or differentiated stem cells,
may include cell culture and cell culture components needed for
cell viability. In some embodiments, the compositions include
pharmaceutical excipients allowing for the administration of the
induced pluripotent stem cells.
[0094] It should be appreciated that differentiated cells (e.g.,
obtained from a subject), induced pluripotent stem cells, and/or
differentiated iPSCs referred to herein may be isolated cells
(e.g., in the form of preparations of isolated cells). As used
herein, isolated cells are cells which have been physically
separated from their environment. If the cells are naturally
occurring, then isolation implies that the cells are physically
separated from the naturally occurring environment from which they
derive. In some instances, isolated cells are additionally or
alternatively physically separated, in whole or in part, from an in
vitro environment. Thus, as used herein, the term isolated means
that a molecule, cell, cell population and the like is physically
separated from an environment in which it normally exists, or in
which it originally or previously existed. Isolation may refer to
physical separation of cells from a culture condition (e.g., a
differentiation culture), from a naturally occurring environment or
source, and the like. A differentiated cell population may be
isolated from a differentiation culture condition, for example, by
harvesting the cells and removing the culture medium (e.g., by
centrifugation). Isolating may also involve washing the cells.
Typically, the cells are resuspended in fresh medium. Isolation of
the differentiated cell population from the differentiation culture
therefore can serve to remove factors or stimuli used to
differentiated the pluripotent stem cells towards one or more
lineages.
[0095] In some embodiments, cells described herein (e.g., original
differentiated cells, iPSCs, and or subsequently differentiated
cells) may be genetically manipulated (e.g., they may be
transfected) or they may not be genetically manipulated.
Transfection refers to genetic manipulation of cells to introduce
and typically express an exogenous nucleic acid. The exogenous
nucleic acid may be a reporter such as green fluorescent protein
(GFP) or it may be a selection marker such as thymidine kinase or
it may be a transcription factor or other factor that is used to
promote reprogramming or subsequent redifferentiation. It will be
understood that in some instances reporters such as GFP may also
serve as selection markers, particularly if their expression is
controlled by pluripotent gene promoters such as an Oct4
promoter.
[0096] It should be appreciated that techniques described herein
may be used with cells obtained from different species, including
for example, human, and various animal species including household
species such as dogs and cats, agricultural species such as cows,
pigs, and horses, laboratory species such as mice and rats, and the
like, or other species.
In Vivo Uses
[0097] In some embodiments, the disclosure provides methods of
treatment using the induced pluripotent stem cells or
differentiated cells (e.g., obtained from iPSCs) provided herein.
In some embodiments, the methods of treatment comprise
administering to a person in need thereof a composition comprising
induced pluripotent stem cells and/or differentiated stems cells
produced according to the methods provided herein.
[0098] In some embodiments, a person in need of treatment with a
composition comprising induced pluripotent stem cells or
differentiated cells is a person having brain damage, cancer,
spinal cord injury, heart damage, baldness, deafness, diabetes,
neuronal defects, blindness, amyotrophic lateral sclerosis, a
genetic disorder, infertility, or unhealed wounds. In some
embodiments, cells obtained as described herein also may be used to
treat hematological malignancies, immunodeficiencies, age related
macular degeneration, and other conditions. For example cell
populations (e.g., differentiated cell populations) can be used in
transplant settings in the treatment or prevention of various
conditions including but not limited to Parkinson's disease
(dopaminergic neurons), Alzheimer's disease (neural precursors),
Huntington's disease (GABAergic neurons), blood disorders such as
leukemia, lymphoma myeloma and anemia (hematopoietic cells),
side-effects of radiation e.g., in transplant patients
(hematopoietic precursors), cardiovascular disease, myocardial
infarction, ischemic cardiac tissue or heart-failure (partially- or
fully-differentiated cardiomyocytes), muscular dystrophy (skeletal
muscle cells), liver cirrhosis or failure (hepatocytes), chronic
hepatitis (hepatocytes), diabetes including type I diabetes
(insulin-producing cells such as islet cells), ischemic brain
damage (neurons), spinal cord injury (glial progenitor cells and
motor neurons), amyotrophic lateral sclerosis (ALS) (motor
neurons), orthopedic tissue injury (osteoblasts), kidney disease
(kidney cells), corneal scarring (corneal stem cells), cartilage
damage (chondrocytes), bone damage (osteogenic cells including
osteocytes), osteoarthritis (chondrocytes), myelination disorders
such as Pelizaeus-Merzbacher disease, multiple sclerosis,
adenoleukodystrophies, neuritis and neuropathies
(oligodendrocytes), and hair loss.
[0099] Pluripotent or differentiated cell populations may be
provided as pharmaceutical compositions that are sterile and
appropriate for in vivo use, optionally together with a
pharmaceutically acceptable carrier. As used herein, a
pharmaceutically-acceptable carrier means a non-toxic material that
does not interfere with the effectiveness of the biological
activity of the active ingredients. Pharmaceutically acceptable
carriers include diluents, fillers, salts, buffers, stabilizers,
solubilizers and other materials which are well-known in the art.
Such preparations may routinely contain salt, buffering agents,
preservatives, compatible carriers, and optionally other
therapeutic agents. Cell populations may be formulated for local or
systemic administration including as part of an implant. Cells may
be used alone or together with another agent, whether active or
inactive, including but not limited to a scaffold, a matrix, and
the like. These cells may further be included in a kit that
additionally comprises at a minimum instructions for use of the
cells, and optionally comprises one or more other agents whether
active or inactive. The cells may be provided as a frozen aliquot
of cells, a culture of cells, or a liquid suspension of cells.
[0100] Cells may be administered in numbers effective to produce a
desired result, including but not limited to a short-term or
long-term therapeutic result. Such result may include an
improvement in or complete eradication of symptoms associated with
a particular condition. The cell numbers to be administered will
depend on a number of factors including the weight and age of the
subject, the type of condition being treated, the desired effect
(e.g., short-term or long-term), and the like. Some treatments
therefore may require as few as 10.sup.3 cells, while others may
require 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9
or more cells.
In Vitro Uses
[0101] Cell populations produced as described herein may be studied
for gene expression profiles and/or responses to various external
stimuli in order to understand differentiation or other processes
more fully. In some embodiments, disease-specific iPS cells may be
used to model diseases (e.g., ALS or other diseases). In some
embodiments, cells may be used in methods for screening and/or
identifying agents (e.g., therapeutic agents such as inhibitors)
being used or to be used clinically. These assays may measure the
therapeutic efficacy and/or toxicity of the candidate agent, among
other things. The readouts from such in vitro assays are
correlative of the in vivo toxicity or efficacy such agents would
exhibit in subjects. Thus, the effect of the agent on the
differentiated cells generated according to the invention in vitro
is a form of surrogate marker or readout for how the agent will
function in vivo in a subject. The agents to be tested include
those used clinically as well as experimental agents. In some more
common embodiments, such testing will focus on the toxicity of
agents including drugs in particular differentiated progeny.
Accordingly, in these assays, the readout would be cell death (or
conversely cell viability). These in vitro assays may employ
suspensions of pluripotent or differentiated cells, adherent
populations of pluripotent or differentiated cells, or three
dimensional structures comprised of pluripotent or differentiated
cells (e.g., in vitro organ tissues, matrices and
architectures).
Administration of Cell Preparations
[0102] In one aspect, the disclosure provides methods for
administering induced pluripotent stem cells or differentiated
cells produced according to methods provided herein.
[0103] Cells can be administered to hosts by a variety of methods
as discussed elsewhere herein. In certain embodiments the cells are
administered by injection, such as by intravenous injection. In
some embodiments cells are encapsulated for administration. In some
embodiments the cells are administered in situ. In some embodiments
of the invention, cells are administered in doses measured by the
ratio of cells to body mass (weight). Alternatively, iPSCs can be
administered in doses of a fixed number of cells.
[0104] In some embodiments the purity of cells for administration
to a subject is about 100%. In other embodiments it is 95% to 100%.
In some embodiments it is 85% to 95%. Particularly in the case of
admixtures with other cells, the percentage of IPSCs or
differentiated cells can be 25%-30%, 30%-35%, 35%-40%, 40%-45%,
45%-50%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%.
[0105] The number of iPSCs or differentiated cells in a given
volume can be determined by well known and routine procedures and
instrumentation. The percentage of iPSCs or differentiated cells in
a given volume of a mixture of cells can be determined by much the
same procedures. Cells can be readily counted manually or by using
an automatic cell counter. Specific cells can be determined in a
given volume using specific staining and visual examination and by
automated methods using specific binding reagent, typically
antibodies, fluorescent tags, and a fluorescence activated cell
sorter.
[0106] The choice of formulation for administering cells for a
given application will depend on a variety of factors. Prominent
among these will be the species of subject, the nature of the
disorder, dysfunction, or disease being treated and its state and
distribution in the subject, the nature of other therapies and
agents that are being administered, the optimum route for
administration of the cells, survivability of cells via the route,
the dosing regimen, and other factors that will be apparent to
those skilled in the art. In particular, for instance, the choice
of suitable carriers and other additives will depend on the exact
route of administration and the nature of the particular dosage
form, for example, liquid dosage form (e.g., whether the
composition is to be formulated into a solution, a suspension, gel
or another liquid form, such as a time release form or
liquid-filled form).
[0107] Examples of compositions comprising iPSCs and/or
differentiated cells include liquid preparations, including
suspensions and preparations for intramuscular or intravenous
administration (e.g., injectable administration), such as sterile
suspensions or emulsions. Such compositions may comprise an
admixture of cells with a suitable carrier, diluent, or excipient
such as sterile water, physiological saline, glucose, dextrose, or
the like. The compositions can also be lyophilized. The
compositions can contain auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, gelling or viscosity
enhancing additives, preservatives, flavoring agents, colors, and
the like, depending upon the route of administration and the
preparation desired. Standard texts, such as "REMINGTON'S
PHARMACEUTICAL SCIENCE," 17th edition, 1985, incorporated herein by
reference, may be consulted to prepare suitable preparations,
without undue experimentation.
[0108] Typically, the compositions will be isotonic, for example,
they will have the same osmotic pressure as blood and lacrimal
fluid when properly prepared for administration. The desired
isotonicity of the compositions of this invention may be
accomplished using sodium chloride, or other pharmaceutically
acceptable agents such as dextrose, boric acid, sodium tartrate,
propylene glycol, or other inorganic or organic solutes. Sodium
chloride is preferred particularly for buffers containing sodium
ions.
[0109] A pharmaceutically acceptable preservative or cell
stabilizer can be employed to increase the life of cellular
compositions. If such preservatives are included, it is well within
the purview of the skilled artisan to select compositions that will
not affect the viability or efficacy of the cells.
[0110] Sterile injectable solutions can be prepared by
incorporating the cells utilized in practicing the present
invention in the required amount of the appropriate solvent with
various amounts of the other ingredients, as desired.
[0111] For any composition to be administered to an animal or
human, and for any particular method of administration, it is
preferred to determine therefore: toxicity, such as by determining
the lethal dose (LD) and LD50 in a suitable animal model, e.g.,
rodent such as mouse or rat; and, the dosage of the composition(s),
concentration of components therein, and timing of administering
the composition(s), which elicit a suitable response. Such
determinations do not require undue experimentation from the
knowledge of the skilled artisan, this disclosure, and the
documents cited herein. And, the time for sequential
administrations can be ascertained without undue
experimentation.
[0112] Order of administration, formulations, doses, frequency of
dosing, and routes of administration of factors (such as the
cytokines discussed herein) and IPSCs generally will vary with the
disorder or disease being treated, its severity, the subject, other
therapies that are being administered, the stage of the disorder or
disease, and prognostic factors, among others. General regimens
that have been established for other treatments provide a framework
for determining appropriate dosing in cell-mediated direct or
adjunctive therapy. These, together with the additional information
provided herein, will enable the skilled artisan to determine
appropriate administration procedures in accordance with
embodiments of the invention, without undue experimentation.
[0113] It should be appreciated that iPSCs or differentiated cells
described herein can be administered to a subject by any of a
variety of routes known to those skilled in the art that may be
used to administer cells to a subject.
[0114] Among methods that may be used in this regard in embodiments
of the invention are methods for administering cells by a
parenteral route. Parenteral routes of administration useful in
various embodiments of the invention include, among others,
administration by intravenous, intraarterial, intracardiac,
intraspinal, intrathecal, intraosseous, intraarticular,
intrasynovial, intracutaneous, intradermal, subcutaneous, and/or
intramuscular injection. In some embodiments intravenous,
intraarterial, intracutaneous, intradermal, subcutaneous and/or
intramuscular injection are used. In some embodiments intravenous,
intraarterial, intracutaneous, subcutaneous, and/or intramuscular
injection are used.
[0115] Cells may be administered to the subject through a
hypodermic needle by a syringe in some embodiments of the
invention. In various embodiments, cells are administered to the
subject through a catheter. In a variety of embodiments, cells are
administered by surgical implantation. Further in this regard, in
various embodiments, cells are administered to the subject by
implantation using an arthroscopic procedure. In some embodiments
cells are administered to the subject in or on a solid support,
such as a polymer or gel. In various embodiments, cells are
administered to the subject in an encapsulated form.
[0116] In additional embodiments of the invention, cells are
suitably formulated for oral, rectal, epicutaneous, ocular, nasal,
and/or pulmonary delivery and are administered accordingly.
[0117] Compositions can be administered in dosages and by
techniques well known to those skilled in the medical and
veterinary arts taking into consideration such factors as the age,
sex, weight, and condition of the particular patient, and the
formulation that will be administered (e.g., solid vs. liquid).
Doses for humans or other mammals can be determined without undue
experimentation by the skilled artisan, from this disclosure, the
documents cited herein, and the knowledge in the art.
Kits
[0118] In some embodiments, the disclosure provides kits for the
production of induced pluripotent stem cells. In some embodiment,
the kits include a Dot1L inhibitor. In some embodiments, the kits
include a Dot1L inhibitor and a reprogramming cocktail (e.g., Sox2
and/or Oct4). In some embodiments, the kits include instructions
for the production of induced pluripotent stem cells. In some
embodiment, the kits include a YY1 inhibitor. In some embodiment,
the kits include an SUV39H1 inhibitor.
[0119] In some embodiments, the disclosure provides a kit for the
production of induced pluripotent stem cells. In some embodiments,
the kit includes one or more Dot1L inhibitors. In some embodiments,
the Dot1L inhibitor is a compound of Formula I, II, III or IV (for
example one or more of the embodiments described herein), or a
pharmaceutically acceptable salt thereof. In some embodiments, the
Dot1L inhibitor is an expression inhibitor such as an RNAi
inhibitor, for example one or more of the shRNA molecules provided
herein.
[0120] In some embodiments, the kit includes one or more YY1
inhibitors and/or one or more SUV39H1 inhibitors. In some
embodiments, the YY1 and/or SUV39H1 inhibitors are small molecule
inhibitors or expression inhibitors such as RNAi inhibitors, for
example one or more of the shRNA molecules provided herein.
[0121] In some embodiments, the kits include a reprogramming
cocktail, or one or more moieties that can be used to generate a
reprogramming cocktail. In some embodiments, the kit includes
nucleic acids for the introduction of one or more stem
cell-associated genes into cells, including, for example Oct3/4
(Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,
Klf4, Klf5, c-Myc, 1-Myc, n-Myc and LIN28. In some embodiments, the
kit includes nucleic acids for the introduction of Oct-4, Sox2,
c-MYC, and Klf4 into a cell. In some embodiments, the kit includes
nucleic acids for the introduction of Oct-4, and Sox2 into a cell.
In some embodiments, the nucleic acids are in delivery vehicle such
as a viral vector, such as an adenoviral vector, a lentiviral
vector or a retroviral vector.
[0122] In some embodiments, this kit includes one or more agents
that enhance reprogramming efficiency including, for example,
soluble Wnt, Wnt conditioned media, BIX-01294 (a G9a histone
methyltransferase), PD0325901 (a MEK inhibitor), DNA
methyltransferase inhibitors, histone deacetylase (HDAC)
inhibitors, valproic acid, 5'-azacytidine, dexamethasone,
suberoylanilide, hydroxamic acid (SAHA), trichostatin (TSA), and
inhibitors of the TGF-.beta. signaling pathway.
[0123] In some embodiments, this kit includes one or more agents
that can bind markers to detect the presence of induced pluripotent
stem cells. Such markers include SSEA4, SSEA3, Tra-1-81, Oct4, Sox2
and Nanog. Agents that bind such markers include antibodies and
compounds that selectively bind the markers.
[0124] In some embodiments, the kit includes one or more elements
useful in establishing a reprogramming cocktail, including buffers,
salts, sugars, and other components that may be useful to support
the growth and reprogramming of the differentiated cells.
[0125] In some embodiments, the kit includes one or more components
for administering the induced pluripotent stem cells or
differentiated cells. These components include pharmaceutical
carriers for systemic and/or local administration of the cells.
[0126] In some embodiments, the kit includes separate containers.
Such containers include small glass containers, plastic containers
or strips of plastic or paper. Such containers allow the efficient
transfer of reagents from one compartment to another compartment
such that the samples and reagents are not cross-contaminated and
the agents or solutions of each container can be added in a
quantitative fashion from one compartment to another.
Dot1L
[0127] In one aspect, the disclosure provides methods of promoting
the production of induced pluripotent stem cells. In some
embodiments, the methods of producing induced pluripotent stem
cells include inhibiting Dot1L in a differentiated cell. In some
embodiments, the differentiated cell is cultured under
reprogramming conditions to produce induced pluripotent stem cells.
In some embodiments, the methods of producing induced pluripotent
stem comprise inhibiting the methyltransferase activity of
Dot1L.
[0128] In some embodiments, the methods of producing induced
pluripotent stem comprise inhibiting Dot1L in a differentiated
cell. Dot1L is a histone methyl transferase (HMT) known to
methylate lysine 79 of histone H3 ("H3K79") in viveo (Feng et al.
(2002) Curr. Biol. 12: 1052-1058). Similar to other HMTs, Dot1L
contains a 5-adenosylmethionine (SAM) binding site and uses SAM as
a methyl donor. Dot1L nucleic acid and polypeptides have previously
been described (see, e.g., U.S. Patent Application Publication No.
2005-0048634 A1 (incorporated by reference); Feng et al. (2002)
Curr. Biol. 12: 1052-1058; and Okada et al. (2005) Cell 121:
167-78). The human Dot1L homolog has been cloned, isolated, and has
been designated as hDot1L (human Dot1-like protein). The sequences
of the human nucleic acid and protein have been deposited under
GenBank Accession No. AF509504, while the mouse homolog is GenBank
Accession No. XP125730). Additional hDot1L homologs are known as
well (See e.g., WO2012075500). The 2.5 angstrom resolution
structure of a fragment of the hDot1L protein containing the
catalytic domain (amino acids 1-416) has been solved; and the
atomic coordinates for amino acids 1-416 of hDot1L have been
determined and deposited in the RCSB database under ID code 1NW3
and described in the scientific literature (see Min, et al. (2003)
Cell 112:711-723).
[0129] In some embodiments, Dot1L is inhibited by contacting Dot1l,
or a cell expressing Dot1L with one or more of the compounds
provided herein. In some embodiments, Dot1l is inhibited by
contacting a cell expressing Dot1l with a nuclei acid that "knocks
down" Dot1L, thereby inhibiting Dot1L. It should be appreciated
that inhibiting Dot1L activity as used herein included both
complete (i.e., about 100%) and partial inhibition. In some
embodiments, partial inhibition results in 90% or less, 80% or
less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or
less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or
less up to complete inhibition of Dot1L. Dot1L inhibition in a cell
can be achieved both by suppressing one more biological functions
of Dot1L (e.g., by contacting the cell with a compound that binds
the active site of Dot1L), or by "knocking down" Dot1L (e.g., by
contacting the cell with a nucleic acid that prevents production of
Dot1L in the cell. In some embodiments, Dot1L is inhibited by
inhibiting the catalytic function of Dot1L.
[0130] In one aspect, the disclosure provides methods of producing
induced pluripotent stem comprise inhibiting Dot1L in a
differentiated cell. In some embodiments inhibiting Dot1L comprises
inhibiting the methyltransferase activity of Dot1L. In some
embodiments, Dot1L is inhibited by contacting the cell with one or
more small molecules that inhibit Dot1L activity. Small molecules
that inhibit Dot1L are described for instance in WO2012/075500,
WO2012/082436, WO2012/075381, and WO2012/075492.
[0131] In some embodiments, Dot1L is inhibited by contacting the
differentiated cell with a composition comprising a compound of
formula I:
##STR00003##
or a pharmaceutically acceptable salt, hydrate, enantiomer or
stereoisomer thereof, wherein independently for each
occurrence,
[0132] X is
##STR00004##
[0133] R.sup.1 is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl,
alkylaryl, haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,
##STR00005##
or (C.sub.2-C.sub.4) alkyl substituted with
##STR00006##
except that when X is
##STR00007##
R.sup.1 is not
##STR00008##
[0135] R.sup.10 is hydrogen or alkyl;
[0136] R.sup.11a is hydrogen, alkyl, or alkyl-cycloalkyl;
[0137] R.sup.11b is hydrogen or alkyl; or taken together with
R.sup.11a and the nitrogen to which it is attached forms a 4- to
8-membered heterocyclyl comprising 0 or 1 additional
heteroatoms;
[0138] R.sup.13 is hydrogen, alkyl, aryl, heteroaryl, aralkyl,
heteroaralkyl, or silyl;
[0139] R.sup.14 is hydrogen, alkyl, aryl, heteroaryl, aralkyl, or
heteroaralkyl;
[0140] R.sup.15 is alkyl, cycloalkyl, or cycloalkylalkyl;
[0141] R.sup.20 is hydrogen, alkyl, cycloalkyl, or
cycloalkylalkyl;
##STR00009##
[0142] R.sup.2 is
##STR00010##
[0143] Y is --NH--, --N(alkyl)-, --O--, or --CR.sup.6.sub.2--;
[0144] R.sup.22a is aryl, heteroaryl, aralkyl, heteroaralkyl, fused
bicyclyl, biaryl, aryloxyaryl, heteroaryloxyaryl,
aryloxyheteroaryl, or heteroaryloxyheteroaryl;
[0145] R.sup.22b is hydrogen or alkyl;
[0146] R.sup.24 is hydrogen or alkyl;
[0147] R.sup.25a, R.sup.25b, R.sup.25c, and R.sup.25d independently
are -M.sub.2-T.sub.2, in which M.sub.2 is a bond, SO.sub.2, SO, S,
CO, CO.sub.2, O, O--C.sub.1-C.sub.4 alkyl linker, C.sub.1-C.sub.4
alkyl linker, NH, or N(R.sub.t), R.sub.t being C.sub.1-C.sub.6
alkyl, and T.sub.2 is H, halo, or R.sub.S4, R.sub.S4 being
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
8-membered heterocycloalkyl, or 5 to 10-membered heteroaryl, and
each of O--C.sub.1-C.sub.4 alkyl linker, C.sub.1-C.sub.4 alkyl
linker, R.sub.t, and Rs.sub.4 being optionally substituted with one
or more substituents selected from the group consisting of halo,
hydroxyl, carboxyl, cyano, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl;
[0148] R.sup.3 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy,
alkylcarbonyloxy, or silyloxy;
[0149] R.sup.4 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy,
alkylcarbonyloxy, or silyloxy;
[0150] R.sup.41 is hydrogen, alkyl, or alkynyl;
[0151] Z is hydrogen or
##STR00011##
[0152] R.sup.5a is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl,
carbocyclyl, heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl,
alkynylalkyl, carbocyclylalkyl, heterocyclylalkyl, aralkyl,
heteroaralkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,
aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,
aralkylthioalkyl, or heteroaralkylthioalkyl; or alkyl substituted
with 1, 2 or 3 substituents independently selected from the group
consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy,
nitro, amino, amido, aryl, and heteroaryl;
[0153] R.sup.5b is hydrogen, alkyl, haloalkyl, alkenyl, alkynyl,
carbocyclyl, heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl,
alkynylalkyl, carbocyclylalkyl, heterocyclylalkyl, aralkyl,
heteroaralkyl, alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,
aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,
aralkylthioalkyl, or heteroaralkylthioalkyl; or alkyl substituted
with 1, 2 or 3 substituents independently selected from the group
consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy,
nitro, amino, amido, aryl, and heteroaryl; or taken together with
R.sup.5a and the nitrogen to which it is attached forms a 4- to
8-membered heterocyclyl comprising 0 or 1 additional
heteroatoms;
[0154] R.sup.6 is hydrogen, alkyl, or halo; or two geminal R.sup.6
taken together are ethylene, propylene, or butylene;
[0155] R.sup.7a is hydrogen, lower alkyl, lower haloalkyl, cyano,
halo, lower alkoxy, or C.sub.3-C.sub.5 cycloalkyl, optionally
substituted with 1, 2, or 3 substituents independently selected
from the group consisting of cyano, lower alkoxy, and halo;
[0156] R.sup.7b is hydrogen, lower alkyl, lower haloalkyl, cyano,
halo, lower alkoxy, or C.sub.3-C.sub.5 cycloalkyl, optionally
substituted with 1, 2, or 3 substituents independently selected
from the group consisting of cyano, lower alkoxy, and halo; and
[0157] R.sup.7c is hydrogen, lower alkyl, lower haloalkyl, cyano,
halo, lower alkoxy, or C.sub.3-C.sub.5 cycloalkyl, optionally
substituted with 1, 2, or 3 substituents independently selected
from the group consisting of cyano, lower alkoxy, and halo.
[0158] Compounds of formula I, methods of making thereof, and
methods of use thereof can be found in WO 2012/075500, which is
incorporated herein by reference.
[0159] In certain embodiments, X is
##STR00012##
In certain embodiments, X is
##STR00013##
[0160] In certain embodiments,
##STR00014##
In certain embodiments, R.sup.2 is
##STR00015##
[0161] In certain embodiments, R.sup.24 is hydrogen or alkyl. In
certain embodiments, R.sup.24 is hydrogen.
[0162] In certain embodiments, R.sup.25a is hydrogen, alkyl,
--O-alkyl, halogen, trifluoroalkyl, --O-- trifluoromethyl, or
--SO.sub.2-trifluoromethyl. In certain embodiments, R.sup.25b is
hydrogen, alkyl, halogen, or trifluoroalkyl. In certain
embodiments, R.sup.25c is hydrogen, alkyl, or halogen. In certain
embodiments, R.sup.25c is hydrogen or halogen.
[0163] In certain embodiments, R.sup.2 is
##STR00016##
[0164] In certain embodiments, Y is --NH-- or --N(alkyl)-. In
certain embodiments, Y is --NH--.
[0165] In certain embodiments, Y is --N(CH.sub.3)--. In certain
embodiments Y is --O--. In certain embodiments, Y is
--CH.sub.2--.
[0166] In certain embodiments, R.sup.22a is aryl or aralkyl. In
certain embodiments, R.sup.22a is substituted phenyl or substituted
benzyl. In certain embodiments, R.sup.22a is one of the
following:
##STR00017##
In certain embodiments, R.sup.22a is one of the following:
##STR00018##
[0167] In certain embodiments, R.sup.22b is hydrogen. In certain
embodiments, R.sup.22b is methyl.
[0168] In certain embodiments, R.sup.1 is hydrogen. In certain
embodiments, R.sup.1 is alkyl. In certain embodiments, R.sup.1 is
--CH.sub.3, --CH.sub.2CH.sub.3, --CH.sub.2CH(CH.sub.3).sub.2 or
--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2. In certain embodiments,
R.sup.1 is C.sub.3-C.sub.7 cycloalkyl. In certain embodiments,
R.sup.1 is cyclopropyl, cyclopropylmethyl, 2-cyclopropylethyl,
cyclobutyl, cyclobutylmethyl, 2-cyclobutylethyl, cyclopentyl,
cyclopentylmethyl, or 2-cyclopentylethyl. In certain embodiments,
R.sup.1 is
##STR00019##
In certain embodiments, R.sup.1 is --CH.sub.2CF.sub.3. In certain
embodiments, R.sup.1 is --CH.sub.2Ph. In certain embodiments,
R.sup.1 is --C(.dbd.O)H. In certain embodiments, R.sup.1 is
--C(.dbd.O)CH.sub.3. In certain embodiments, R.sup.1 is
heterocyclyl or heterocyclylalkyl. In certain embodiments, R.sup.1
is
##STR00020##
In certain embodiments, R.sup.1 is
##STR00021##
In certain embodiments, R.sup.15 is alkyl. In certain embodiments,
R.sup.15 is cycloalkyl. In certain embodiments, R.sup.15 is
cycloalkylalkyl.
[0169] In certain embodiments, R.sup.1 is (C.sub.2-C.sub.4) alkyl
substituted with
##STR00022##
In certain embodiments, R.sup.1 is
##STR00023##
In certain embodiments, R.sup.11a is hydrogen, alkyl, or
alkyl-cycloalkyl. In certain embodiments, R.sup.11a is hydrogen,
methyl, or i-propyl. In certain embodiments, R.sup.1 is
##STR00024##
In certain embodiments, R.sup.13 is hydrogen.
[0170] In certain embodiments, R.sup.1 is
##STR00025##
[0171] In certain embodiments, A is
##STR00026##
In certain embodiments, A is
##STR00027##
In certain embodiments, A is
##STR00028##
In certain embodiments, A is
##STR00029##
In certain embodiments, A is
##STR00030##
In certain embodiments, A is
##STR00031##
In certain embodiments, A is
##STR00032##
In certain embodiments, A is
##STR00033##
In certain embodiments, A is
##STR00034##
In certain embodiments, A is
##STR00035##
and R.sup.6 is alkyl. In certain embodiments, A is
##STR00036##
and R.sup.6 is methyl, ethyl, or isopropyl.
[0172] In certain embodiments, R.sup.3 is hydroxyl. In certain
embodiments, R.sup.3 is hydrogen. In certain embodiments, R.sup.4
is hydroxyl. In certain embodiments, R is hydrogen. In certain
embodiments, R.sup.41 is hydrogen. In certain embodiments, R.sup.41
is methyl. In certain embodiments, R.sup.3 is hydroxyl; and R is
hydroxyl. In certain embodiments, R.sup.3 is hydroxyl; R.sup.4 is
hydroxyl; and R.sup.41 is hydrogen. In certain embodiments, R.sup.3
is hydroxyl; R.sup.4 is hydroxyl; and R.sup.41 is methyl. In
certain embodiments, R.sup.3 is hydrogen; and R.sup.4 is hydroxyl.
In certain embodiments, R.sup.3 is hydrogen; R.sup.4 is hydroxyl;
and R.sup.41 is hydrogen. In certain embodiments, R.sup.3 is
hydrogen; R.sup.4 is hydroxyl; and R.sup.41 is methyl. In certain
embodiments, R.sup.3 is hydroxyl; and R.sup.4 is hydrogen. In
certain embodiments, R.sup.3 is hydroxyl; R.sup.4 is hydrogen; and
R.sup.4 is hydrogen. In certain embodiments R.sup.3 is hydroxyl;
R.sup.4 is hydrogen; and R.sup.41 is methyl.
[0173] In certain embodiments, Z is hydrogen or
##STR00037##
In certain embodiments, Z is hydrogen. In certain embodiments, Z
is
##STR00038##
In certain embodiments, R.sup.5a is hydrogen, alkyl, carbocyclyl,
heterocyclyl, aryl, heteroaryl, carbocyclylalkyl,
heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain
embodiments, R.sup.5a is hydrogen, aralkyloxyalkyl, alkyl, aryl,
aralkyl, aminoalkyl or hydroalkyl. In certain embodiments, R.sup.5a
is --H, --CH.sub.2CH.sub.2OCH.sub.2Ph, --CH.sub.2CH.sub.3,
--CH(CH.sub.3).sub.2, -Ph, --CH.sub.2CH(CH.sub.3), --CH.sub.3,
--CH.sub.2Ph, --CH.sub.2CH.sub.2NH.sub.2, --CH.sub.2(cyclohexyl) or
--CH.sub.2CH.sub.2OH. In certain embodiments, R.sup.5b is hydrogen,
alkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl,
carbocyclylalkyl, heterocyclylalkyl, aralkyl, or heteroaralkyl. In
certain embodiments, R.sup.5b is hydrogen, aralkyloxyalkyl, alkyl,
aryl, aralkyl, aminoalkyl or hydroalkyl. In certain embodiments,
R.sup.5b is hydrogen. In certain embodiments, R.sup.5a is --H,
--CH.sub.2CH.sub.2OCH.sub.2Ph, --CH.sub.2CH.sub.3,
--CH(CH.sub.3).sub.2, -Ph, --CH.sub.2CH(CH.sub.3), --CH.sub.3,
--CH.sub.2Ph, --CH.sub.2CH.sub.2NH.sub.2, --CH.sub.2(cyclohexyl) or
--CH.sub.2CH.sub.2OH; and R.sup.5b is --H.
[0174] In certain embodiments, R.sup.7a is hydrogen or lower alkyl.
In certain embodiments, R.sup.7a is hydrogen. In certain
embodiments, R.sup.7b is hydrogen or lower alkyl. In certain
embodiments, R.sup.7b is hydrogen. In certain embodiments, R.sup.7c
is hydrogen or lower alkyl. In certain embodiments, R.sup.7c is
hydrogen.
[0175] In some embodiments, Dot1L is inhibited by contacting the
differentiated cell with a composition comprising a compound of
formula II:
##STR00039##
or a pharmaceutically acceptable salt, hydrate, enantiomer or
stereoisomer thereof, wherein independently for each
occurrence,
[0176] X is
##STR00040##
[0177] R.sup.1 is hydrogen, alkyl, cycloalkyl, alkylcycloalkyl,
alkylaryl, haloalkyl, formyl, heterocyclyl, heterocyclylalkyl,
##STR00041##
or (C.sub.2-C.sub.4)alkyl substituted with
##STR00042##
except that when X is
##STR00043##
R.sup.1 is not
##STR00044##
[0179] R.sup.10 is hydrogen or alkyl;
[0180] R.sup.11a is hydrogen, alkyl, or alkyl-cycloalkyl;
[0181] R.sup.11b is hydrogen or alkyl; or taken together with R and
the nitrogen to which it is attached forms a 4- to 8-membered
heterocyclyl comprising 0 or 1 additional heteroatoms;
[0182] R.sup.13 is hydrogen, alkyl, aryl, heteroaryl, aralkyl,
heteroaralkyl or silyl;
[0183] R.sup.14 is hydrogen, alkyl, aryl, heteroaryl, aralkyl or
heteroaralkyl;
[0184] R.sup.15 is alkyl, cycloalkyl or cycloalkylalkyl;
[0185] R.sup.20 is hydrogen, alkyl, cycloalkyl or
cycloalkylalkyl;
[0186] A is
##STR00045##
[0187] R.sup.2 is
##STR00046##
[0188] Y is --NH--, --N(alkyl)-, --O--, or --CR.sup.6.sub.2;
[0189] R.sup.22a is aryl, heteroaryl, aralkyl, heteroaralkyl, fused
bicyclyl, biaryl, aryloxyaryl, heteroaryloxyaryl, aryloxyheteroaryl
or heteroaryloxyheteroaryl;
[0190] R.sup.22b is hydrogen or alkyl;
[0191] R.sup.24 is hydrogen or alkyl;
[0192] R.sup.25a, R.sup.25b, R.sup.25c, R.sup.25d are independently
-M.sub.2-T.sub.2, in which M.sub.2 is a bond, SO.sub.2, SO, S, CO,
CO.sub.2, O, O--C.sub.1-C.sub.4 alkyl linker, C.sub.1-C.sub.4 alkyl
linker, NH, or N(R.sub.t), R.sub.t being C.sub.1-C.sub.6 alkyl, and
T.sub.2 is H, halo, or Rs.sub.4, Rs.sub.4 being C.sub.1-C.sub.6
alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, biaryl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 8-membered
heterocycloalkyl, or 5 to 10-membered heteroaryl, and each of
O--C.sub.1-C.sub.4 alkyl linker, C.sub.1-C.sub.4 alkyl linker,
R.sub.t, and Rs.sub.4 being optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
carboxyl, cyano, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1--C alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl;
[0193] R.sup.3 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy,
alkylcarbonyloxy or silyloxy;
[0194] R.sup.4 is hydrogen, halogen, hydroxy, alkyloxy, aralkyloxy,
alkylcarbonyloxy or silyloxy;
[0195] R.sup.41 is hydrogen, alkyl or alkynyl;
[0196] Z is hydrogen or
##STR00047##
[0197] R.sup.5a is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl,
heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,
carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,
alkylcarbonylarhinoalkyl, arylcarbonylaminoalkyl,
aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,
aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted
with 1, 2 or 3 substituents independently selected from the group
consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy,
nitro, amino, amido, aryl and heteroaryl;
[0198] R.sup.5b is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl,
heterocyclyl, aryl, heteroaryl, biaryl, alkenylalkyl, alkynylalkyl,
carbocyclylalkyl, heterocyclylalkyl, aralkyl, heteroaralkyl,
alkylcarbonylaminoalkyl, arylcarbonylaminoalkyl,
aralkylcarbonylaminoalkyl, arylsulfonylaminoalkyl, alkylthioalkyl,
aralkylthioalkyl or heteroaralkylthioalkyl; or alkyl substituted
with 1, 2 or 3 substituents independently selected from the group
consisting of hydroxy, halo, carboxy, alkyoxy, aryloxy, aralkyloxy,
nitro, amino, amido, aryl and heteroaryl; or taken together with
R.sup.5a and the nitrogen to which it is attached forms a 4- to
8-membered heterocyclyl comprising 0 or 1 additional
heteroatoms;
[0199] R.sup.6 is hydrogen, alkyl or halo; or two geminal R.sup.6
taken together are ethylene, propylene or butylene; and
[0200] R.sup.7a is hydrogen, lower alkyl, lower haloalkyl, cyano,
halo, lower alkoxy, or C.sub.3-C.sub.5 cycloalkyl, optionally
substituted with 1, 2, or 3 substituents independently selected
from the group consisting of cyano, lower alkoxy and halo; and
[0201] R.sup.7b is hydrogen, lower alkyl, lower haloalkyl, cyano,
halo, lower alkoxy, or C.sub.3-C.sub.5 cycloalkyl, optionally
substituted with 1, 2, or 3 substituents independently selected
from the group consisting of cyano, lower alkoxy and halo.
[0202] Compounds of formula II, methods of making thereof, and
methods of use thereof can be found in WO 2012/082436, which is
incorporated herein by reference.
[0203] In certain embodiments, X is
##STR00048##
In certain embodiments, X is
##STR00049##
[0204] In certain embodiments, R.sup.2 is
##STR00050##
In certain embodiments, R.sup.2 is
##STR00051##
In certain embodiments, R.sup.24 is hydrogen or alkyl. In certain
embodiments, R.sup.24 is hydrogen. In certain embodiments,
R.sup.25a is hydrogen, alkyl, --O-alkyl, halogen, trifluoroalkyl,
--O-trifluoromethyl, or --SO.sub.2-trifluoromethyl. In certain
embodiments, R.sup.25b is hydrogen, alkyl, halogen, or
trifluoroalkyl. In certain embodiments, R.sup.25c is hydrogen,
alkyl, or halogen. In certain embodiments, R.sup.25c is hydrogen or
halogen.
[0205] In certain embodiments, R.sup.2 is
##STR00052##
In certain embodiments, Y is --NH-- or --N(alkyl)-. In certain
embodiments, Y is --NH--. In certain embodiments, Y is
--N(CH.sub.3)--. In certain embodiments, Y is --O--. In certain
embodiments, Y is --CH.sub.2--. In certain embodiments, R.sup.22a
is aryl or aralkyl. In certain embodiments, R.sup.22a is
substituted phenyl or substituted benzyl. In certain embodiments,
R.sup.22a is one of the following:
##STR00053##
[0206] In certain embodiments, R.sup.22a is one of the
following:
##STR00054##
[0207] In certain embodiments, R.sup.22b is hydrogen. In certain
embodiments, R.sup.22b is methyl.
[0208] In certain embodiments, R.sup.1 is hydrogen. In certain
embodiments, R.sup.1 is alkyl. In certain embodiments, R.sup.1 is
--CH.sub.3, --CH.sub.2CH.sub.3, --CH.sub.2CH(CH.sub.3).sub.2 or
--CH.sub.2CH.sub.2CH(CH.sub.3).sub.2. In certain embodiments,
R.sup.1 is
##STR00055##
In certain embodiments, R is --CH.sub.2CF.sub.3. In certain
embodiments, R.sup.1 is --CH.sub.2Ph. In certain embodiments,
R.sup.1 is --C(.dbd.O)H. In certain embodiments, R.sup.1 is
--C(.dbd.O)CH.sub.3. In certain embodiments, R.sup.1 is
heterocyclyl or heterocyclylalkyl. In certain embodiments, R.sup.1
is
##STR00056##
In certain embodiments, R.sup.1 is
##STR00057##
In certain embodiments, R.sup.1 is
##STR00058##
In certain embodiments, R.sup.1 is
##STR00059##
In certain embodiments, R.sup.15 is alkyl. In certain embodiments,
R.sup.15 is methyl. In certain embodiments, R.sup.15 is cycloalkyl.
In certain embodiments, R.sup.15 is cycloalkylalkyl.
[0209] In certain embodiments, R.sup.1 is (C.sub.2-C.sub.4)alkyl
substituted with
##STR00060##
In certain embodiments, R.sup.1 is
##STR00061##
In certain embodiments, R.sup.11a is hydrogen, alkyl, or
alkyl-cycloalkyl. In certain embodiments, R.sup.11a is hydrogen,
methyl, or i-propyl. In certain embodiments, R.sup.22a is
heteroaryl. In certain embodiments, R.sup.22a is substituted
phenyloxyphenyl, substituted 4-(phenyl)phenyl or optionally
substituted 4-(heteroaryl)phenyl.
[0210] In certain embodiments, R.sup.22a is one of the
following:
##STR00062##
In certain embodiments, R.sup.22a is one of the following:
##STR00063##
[0211] In certain embodiments, R.sup.11a is hydrogen. In certain
embodiments, R.sup.11b is hydrogen. In certain embodiments,
R.sup.11b is methyl.
[0212] In certain embodiments, R.sup.1 is
##STR00064##
In certain embodiments, R.sup.1 is
##STR00065##
In certain embodiments, R.sup.13 is hydrogen.
[0213] In certain embodiments, R.sup.1 is
##STR00066##
[0214] In certain embodiments, A is
##STR00067##
[0215] In certain embodiments, A is
##STR00068##
In certain embodiments, A is
##STR00069##
In certain embodiments, A is
##STR00070##
In certain embodiments, A is
##STR00071##
In certain embodiments, A is
##STR00072##
In certain embodiments, A is
##STR00073##
In certain embodiments, A is
##STR00074##
In certain embodiments, A is
##STR00075##
In certain embodiments, A is
##STR00076##
and R.sup.6 is alkyl. In certain embodiments, A is
##STR00077##
and R.sup.6 is methyl, ethyl, or isopropyl.
[0216] In certain embodiments, R.sup.3 is hydroxyl. In certain
embodiments, R.sup.3 is hydrogen. In certain embodiments, R.sup.4
is hydroxyl. In certain embodiments, R.sup.4 is hydrogen. In
certain embodiments, R.sup.41 is hydrogen. In certain embodiments,
R.sup.41 is methyl. In certain embodiments, R.sup.3 is hydroxyl;
and R.sup.4 is hydroxyl. In certain embodiments, R.sup.3 is
hydroxyl; R.sup.4 is hydroxyl; and R.sup.41 is hydrogen. In certain
embodiments, R.sup.3 is hydroxyl; R.sup.4 is hydroxyl; and R.sup.41
is methyl. In certain embodiments, R.sup.3 is hydrogen; and R.sup.4
is hydroxyl. In certain embodiments, R.sup.3 is hydrogen; R.sup.4
is hydroxyl; and R.sup.41 is hydrogen. In certain embodiments
R.sup.3 is hydrogen; R.sup.4 is hydroxyl; and R.sup.41 is methyl.
In certain embodiments, R.sup.3 is hydroxyl; and R.sup.4 is
hydrogen. In certain embodiments, R.sup.3 is hydroxyl; R.sup.4 is
hydrogen; and R.sup.41 is hydrogen. In certain embodiments, R.sup.3
is hydroxyl; R.sup.4 is hydrogen; and R.sup.41 is methyl.
In certain embodiments, Z is hydrogen or
##STR00078##
In certain embodiments, Z is hydrogen. In certain embodiments, Z
is
##STR00079##
In certain embodiments, R.sup.5a is hydrogen, alkyl, carbocyclyl,
heterocyclyl, aryl, heteroaryl, carbocyclylalkyl,
heterocyclylalkyl, aralkyl, or heteroaralkyl. In certain
embodiments, R.sup.5a is hydrogen, aralkyloxyalkyl, alkyl, aryl,
aralkyl, aminoalkyl or hydroxyalkyl. In certain embodiments,
R.sup.5a is --H, --CH.sub.2CH.sub.2OCH.sub.2Ph, --CH.sub.2CH.sub.3,
--CH(CH.sub.3).sub.2, -Ph, --CH.sub.2CH(CH.sub.3), --CH.sub.3,
--CH.sub.2Ph, --CH.sub.2CH.sub.2NH.sub.2, --CH.sub.2(cyclohexyl) or
--CH.sub.2CH.sub.2OH. In certain embodiments, R.sup.5b is hydrogen,
alkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl,
carbocyclylalkyl, heterocyclylalkyl, aralkyl, or heteroaralkyl. In
certain embodiments, R.sup.5b is hydrogen, aralkyloxyalkyl, alkyl,
aryl, aralkyl, aminoalkyl or hydroalkyl. In certain embodiments,
R.sup.5b is hydrogen. In certain embodiments, R.sup.5a is --H,
--CH.sub.2CH.sub.2OCH.sub.2Ph, --CH.sub.2CH.sub.3,
--CH(CH.sub.3).sub.2, -Ph, --CH.sub.2CH(CH.sub.3), --CH.sub.3,
--CH.sub.2Ph, --CH.sub.2CH.sub.2NH.sub.2, --CH.sub.2(cyclohexyl) or
--CH.sub.2CH.sub.2OH; and R.sup.5b is --H.
[0217] In certain embodiments, R.sup.7a is hydrogen or lower alkyl.
In certain embodiments, R.sup.7a is hydrogen. In certain
embodiments, R.sup.7b is hydrogen or lower alkyl. In certain
embodiments, R.sup.7b is hydrogen.
[0218] In some embodiments, Dot1L is inhibited by contacting the
differentiated cell with a composition comprising a compound of
formula III:
##STR00080##
or pharmaceutically acceptable salt or ester thereof, wherein:
[0219] A is O or CH.sub.2;
[0220] each of G and J, independently, is H, halo, C(O)OH,
C(O)O--C.sub.1-C.sub.6 alkyl or OR.sub.a, R.sub.a being H,
C.sub.1-C.sub.6 alkyl or C(O)--C.sub.1-C.sub.6 alkyl, wherein
C(O)O--C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl or
C(O)--C.sub.1-C.sub.6 alkyl is optionally substituted with one or
more substituents selected from the group consisting of halo,
cyano, hydroxyl, carboxyl, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino, and
C.sub.3-C.sub.8 cycloalkyl;
[0221] Q is H, NH.sub.2, NHR.sub.b, NR.sub.bR.sub.c, R.sub.b, or
OR.sub.b, in which each of R.sub.b and R.sub.c independently is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, 5 to 10-membered heteroaryl, or
-M.sub.1-T.sub.1 in which M.sub.1 is a bond or C.sub.1-C.sub.6
alkyl linker optionally substituted with halo, cyano, hydroxyl or
C.sub.1-C.sub.6 alkoxyl and T.sub.1 is C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
10-membered heteroaryl, or R.sub.b and R.sub.c, together with the N
atom to which they attach, form 4 to 7-membered heterocycloalkyl
having 0 or 1 additional heteroatoms to the N atom optionally
substituted with C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH,
C(O)O--C.sub.1-C.sub.6 alkyl, OC(O)--C.sub.1-C.sub.6 alkyl, cyano,
C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino,
di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
6-membered heteroaryl, and each of R.sub.b, R.sub.c, and T.sub.1 is
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, cyano,
C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino,
di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to
6-membered heteroaryl;
[0222] X is N or CR.sub.X, in which R.sub.x is H, halo, hydroxyl,
carboxyl, cyano, or Rs.sub.1, Rs.sub.1 being amino, C.sub.1-C.sub.6
alkoxyl, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
6-membered heteroaryl, and R.sub.S1 being optionally substituted
with one or more substituents selected from the group consisting of
halo, hydroxyl, carboxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl;
[0223] L.sub.1 is N(Y), S, SO, or SO.sub.2;
[0224] L.sub.2 is CO or absent when L.sub.1 is N(Y) or is absent
when L.sub.1 is S, SO, or SO.sub.2, in which Y is H, R.sub.d,
SO.sub.2R.sub.d, or COR.sub.d when L.sub.2 is absent, or Y is H or
R.sub.d when L.sub.2 is CO, R.sub.d being C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, or 5 to 6-membered heteroaryl, and R.sub.d being
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, cyano,
C.sub.1-C.sub.6 alkoxyl, C.sub.1-C.sub.6 alkylsulfonyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl and with
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, or 5 to 6-membered heteroaryl further optionally
substituted with C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH,
C(O)O--C.sub.1-C.sub.6 alkyl, OC(O)--C.sub.1-C.sub.6 alkyl, cyano,
C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino,
di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
6-membered heteroaryl;
[0225] each of R.sub.1, R.sub.2, R.sub.3, R.sup.4, R.sub.5,
R.sub.6, and R.sub.7, independently, is H, halo, hydroxyl,
carboxyl, cyano, Rs.sub.2, Rs.sub.2 being amino, C.sub.1-C.sub.6
alkoxyl, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.6 alkynyl, and each Rs.sub.2 being optionally
substituted with one or more substituents selected from the group
consisting of halo, hydroxyl, carboxyl, cyano, C.sub.1-C.sub.6
alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl;
[0226] R.sub.8 is H, halo or Rs.sub.3, Rs.sub.3 being
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl, and Rs.sub.3 being optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
carboxyl, cyano amino, C.sub.1-C.sub.6 alkoxyl,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino, and
C.sub.3-C.sub.8 cycloalkyl;
[0227] R.sup.9 is
##STR00081##
in which each of R.sub.c, R.sub.f, R.sub.g, and R.sub.h,
independently is -M.sub.2-T.sub.2, in which M.sub.2 is a bond,
SO.sub.2, SO, S, CO CO.sub.2, O, O--C.sub.1-C.sub.4 alkyl linker,
C.sub.1-C.sub.4 alkyl linker, NH, or N(R.sub.t), R.sub.t being
C.sub.1-C.sub.6 alkyl, and T.sub.2 is H, halo, or R.sub.S4,
Rs.sub.4 being C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 8-membered heterocycloalkyl, or 5 to
10-membered heteroaryl, and each of O--C.sub.1-C.sub.4 alkyl
linker, C.sub.1-C.sub.4 alkyl linker, R.sub.t, and R.sub.S4 being
optionally substituted with one or more substituents selected from
the group consisting of halo, hydroxyl, carboxyl, cyano,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl, R.sub.i is H or
C.sub.1-C.sub.6 alkyl optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
carboxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl, D is O, NR.sub.j,
or CR.sub.jR.sub.k, each of R.sub.j and R.sub.k independently being
H or C.sub.1-C.sub.6 alkyl, or R.sub.j and R.sub.k taken together,
with the carbon atom to which they are attached, form a
C.sub.3-C.sub.10 cycloalkyl ring, and E is -M.sub.3-T.sub.3,
M.sub.3 being a bond or C.sub.1-C.sub.6 alkyl linker optionally
substituted with halo or cyano, T.sub.3 being C.sub.3-C.sub.10
cycloalkyl, C.sub.6-C.sub.10 aryl, 5 to 10-membered heteroaryl, or
4 to 10-membered heterocycloalkyl, and T.sub.3 being optionally
substituted with one or more substituents selected from the group
consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.1-C.sub.6 alkoxyl, C.sub.1-C.sub.6 haloalkyl,
C.sub.1-C.sub.6 haloalkoxyl, C.sub.1-C.sub.6 alkylthio,
C.sub.1-C.sub.6 alkylsulfonyl, C.sub.1-C.sub.6 haloalkylsulfonyl,
C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkoxycarbonyl, oxo,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.12
alkylcycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10 aryloxyl,
C.sub.7-C.sub.14 alkylaryl, C.sub.6-C.sub.10 aminoaryloxyl,
C.sub.6-C.sub.10 arylthio, 4 to 6-membered heterocycloalkyl
optionally substituted with halo, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 haloalkyl, 5 to 6-membered heteroaryl optionally
substituted with halo, C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.6
alkyl that is substituted with hydroxy, halo, C.sub.1-C.sub.6
alkoxycarbonyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl,
4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl
optionally further substituted with halo, hydroxyl, or
C.sub.1-C.sub.6 alkoxyl;
[0228] q is 0, 1, 2, 3, or 4;
[0229] m is 0, 1, or 2; and
[0230] n is 0, 1, or 2.
[0231] Compounds of formula III, methods of making thereof, and
methods of use thereof can be found in WO 2012/075500, which is
incorporated herein by reference.
[0232] In certain embodiments, the sum of m and n is at least 1. In
certain embodiments, m is 1 or 2 and n is 0. In certain
embodiments, m is 2 and n is 0.
[0233] In certain embodiments, A is CH.sub.2. In certain
embodiments, A is O.
[0234] In certain embodiments, L.sub.1 is N(Y). In certain
embodiments, L.sub.1 is SO or SO.sub.2.
[0235] In certain embodiments, Y is Rj.
[0236] In certain embodiments, R.sub.d is C.sub.1-C.sub.6
alkyl.
[0237] In certain embodiments, L.sub.2 is absent.
[0238] In certain embodiments, each of G and J independently is OR.
In certain embodiments, R.sub.a is hydrogen.
[0239] In certain embodiments, R.sub.9 is
##STR00082##
In certain embodiments, R.sub.9 is
##STR00083##
In certain embodiments, at least one of R.sub.e, R.sub.f, R.sub.g,
and R.sub.h is halo (such as F, CI, and Br), C.sub.1-C.sub.6
alkoxyl optionally substituted with one or more halo (such as
OCH.sub.3, OCH.sub.2CH.sub.3, O-iPr, and OCF.sub.3),
C.sub.1-C.sub.6 alkylsulfonyl optionally substituted with one or
more halo (such as SO.sub.2CF.sub.3), or C.sub.1-C.sub.6 alkyl
optionally substituted with one or more halo (such as CH.sub.3,
i-propyl, n-butyl, and CF.sub.3). In certain embodiments, Rj is H
or C.sub.1-C.sub.6 alkyl (e.g., methyl, ethyl, n-propyl, i-propyl,
n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl).
[0240] In certain embodiments,
##STR00084##
is unsubstituted benzimidazolyl or one of the following groups:
##STR00085## ##STR00086## ##STR00087##
[0241] In certain embodiments, R.sup.9 is
##STR00088##
In certain embodiments, D is O. In certain embodiments, D is
NR.sub.j. In certain embodiments, R.sub.j is H. In certain
embodiments, D is CRjR.sub.k. In certain embodiments, each of
R.sub.j and R.sub.k is hydrogen. In certain embodiments, E is
-M.sub.3-T.sub.3, in which M.sub.3 is a bond or C.sub.1-C.sub.3
alkyl linker, T.sub.3 is phenyl, naphthyl, thienyl, cyclopropyl, or
cyclohexyl, and T.sub.3 is optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
thiol, carboxyl, cyano, nitro, C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6
alkoxyl, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 haloalkoxyl,
C.sub.1-C.sub.6 alkylthio, C.sub.1-C.sub.6 alkylsulfonyl,
C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkoxycarbonyl, oxo,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.12
alkylcycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10 aryloxyl,
C.sub.7-C.sub.14 alkylaryl, C.sub.6-C.sub.10 aminoaryloxyl,
C.sub.6-C.sub.10 arylthio, 4 to 6-membered heterocycloalkyl
optionally substituted with C.sub.1-C.sub.4 alkyl, 5 to 6-membered
heteroaryl optionally substituted with C.sub.1-C.sub.4 alkyl, and
C.sub.1-C.sub.6 alkyl that is substituted with hydroxy,
C.sub.1-C.sub.6 alkoxycarbonyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
6-membered heteroaryl. In certain embodiments, T.sub.3 is phenyl
optionally substituted with one or more substituents selected from
the group consisting of halo, hydroxyl, carboxyl, cyano, nitro,
C.sub.1-C.sub.6 alkyl (e.g. methyl, ethyl, n-propyl, i-propyl,
n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl and n-hexyl),
C.sub.1-C.sub.6 alkoxyl, C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6
haloalkoxyl, C.sub.1-C.sub.6 alkylsulfonyl, C.sub.6-C.sub.10 aryl
(e.g., phenyl or naphthyl), and C.sub.6-C.sub.10 aryloxyl, and
C.sub.7-C.sub.14 alkylaryl.
[0242] In certain embodiments, E is
##STR00089## ##STR00090## ##STR00091##
[0243] In certain embodiments, X is N. In certain embodiments, X is
CR.sub.X. In certain embodiments, X is CH.
[0244] In certain embodiments, Q is NH.sub.2 or NHR.sub.b, in which
R.sub.b is -M.sub.1-T.sub.1, M.sub.1 being a bond or
C.sub.1-C.sub.6 alkyl linker and T.sub.1 being C.sub.3-C.sub.8
cycloalkyl. In certain embodiments, Q is H.
[0245] In certain embodiments, R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, and R.sub.8 are each H.
[0246] In certain embodiments, when R.sub.8 is halo and is attached
to the same carbon atom as J, then J is not hydroxyl. In certain
embodiments, when R.sub.8 is halo and is attached to the same
carbon atom as G, then G is not hydroxyl. In certain embodiments,
T.sub.2 is not halo when M.sub.2 is SO.sub.2, SO, S, CO or O.
[0247] In certain embodiments, T.sub.2 is a 4-8 membered
heterocycloalkyl which is bound to M.sub.2 via a heteroatom. In
certain embodiments, T.sub.2 is a 4-8 membered heterocycloalkyl
which is bound to M.sub.2 via a N atom. In certain embodiments,
T.sub.2 is a 4-8 membered heterocycloalkyl which is bound to
M.sub.2 via a C atom.
[0248] In certain embodiments, the compound is of formula
III-a:
##STR00092##
or pharmaceutically acceptable salt or ester thereof, wherein A, Q,
X, L.sub.I, L.sub.2, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7, R.sub.8, R.sub.9, q, m, and n are as described
herein for formula III.
[0249] In certain embodiments, the compound is of formula III-b or
III-c:
##STR00093##
or pharmaceutically acceptable salt or ester thereof, wherein A, Q,
X, L.sub.1, L.sub.2, R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sup.6, R.sub.7, R.sub.8, R.sub.e, R.sub.f, R.sub.g, R.sub.h,
R.sub.i, q, m, and n are as described herein for formula III.
[0250] In certain embodiments, the compound is of formula
III-d:
##STR00094##
or pharmaceutically acceptable salt or ester thereof, wherein A, Q,
X, G, J, L.sub.1, L.sub.2, R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, D, E, q, m, and n are as
described herein for formula III.
[0251] In certain embodiments, the compound is of formula
III-e:
##STR00095##
or its N-oxide, or a pharmaceutically acceptable salt or ester
thereof, wherein A, Q, X, Y, R.sub.1, R.sub.2, R.sub.e, R.sub.f,
R.sub.g, R.sub.h, and m are as described herein for formula
III.
[0252] In certain embodiments, A is O. In certain embodiments, A is
O and m is 2. In certain embodiments, X is N. In certain
embodiments, Q is NH.sub.2 or NHR.sub.b, in which R.sub.b is
-M.sub.1-T, M.sub.1 being a bond or C.sub.1-C.sub.6 alkyl linker
and T.sub.1 being C.sub.3-C.sub.8 cycloalkyl. In certain
embodiments, R.sub.1 and R.sub.2 are each H. In certain
embodiments, Y is R.sub.d. In certain embodiments, R.sub.d is
C.sub.1-C.sub.6 alkyl optionally substituted with C.sub.3-C.sub.8
cycloalkyl or halo. In certain embodiments, R.sub.d is
C.sub.3-C.sub.8 cycloalkyl optionally substituted with
C.sub.1-C.sub.6 alkyl or halo. In certain embodiments, at least one
of R.sub.e, R.sub.f, R.sub.g, and R is halo, C.sub.1-C.sub.6
alkoxyl optionally substituted with one or more halo;
C.sub.1-C.sub.6 alkylsulfonyl optionally substituted with one or
more halo; C.sub.1-C.sub.6 alkyl optionally substituted with one or
more substituents selected from CN, halo, C.sub.3-C.sub.8
cycloalkyl, hydroxy, and C.sub.1-C.sub.6 alkoxyl; C.sub.3-C.sub.8
cycloalkyl optionally substituted with one or more C.sub.1-C.sub.6
alkyl or CN; or 4 to 8-membered heterocycloalkyl optionally
substituted with one or more substituents selected from CN, halo,
hydroxy, C.sub.1-C.sub.6 alkyl and C.sub.1-C.sub.6 alkoxyl. In
certain embodiments, at least one of R.sub.e, R.sub.f, R.sub.g, and
R.sub.h is selected from F; Cl; Br; CF.sub.3; OCF.sub.3;
SO.sub.2CF.sub.3; oxetanyl optionally substituted with one or more
substituents selected from CN, halo, hydroxy, C.sub.1-C.sub.6 alkyl
and C.sub.1-C.sub.6 alkoxyl; C.sub.3-C.sub.8 cycloalkyl optionally
substituted with one or more substituents selected from
C.sub.1-C.sub.4 alkyl; and C.sub.1-C.sub.4 alkyl optionally
substituted with one or more substituents selected from halo,
C.sub.3-C.sub.8 cycloalkyl, hydroxy and C.sub.1-C.sub.6 alkoxyl. In
certain embodiments, at least one of R.sub.f and R.sub.g is alkyl,
optionally substituted with hydroxyl. In certain embodiments, at
least one of R.sub.f and R.sub.g is i-butyl substituted with
hydroxyl.
[0253] In some embodiments, Dot1L is inhibited by contacting the
differentiated cell with a composition comprising a compound of
formula IV:
##STR00096##
or a pharmaceutically acceptable salt or ester thereof,
wherein:
[0254] each of G and J, independently, is H, halo, C(O)OH,
C(O)O--C.sub.1-C.sub.6 alkyl or OR.sub.a, R.sub.a, being H,
C.sub.1-C.sub.6 alkyl or C(O)--C.sub.1-C.sub.6 alkyl, wherein
C(O)O--C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl or
C(O)--C.sub.1-C.sub.6 alkyl is optionally substituted with one or
more substituents selected from the group consisting of halo, cyano
hydroxyl, carboxyl, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino, and
C.sub.3-C.sub.8 cycloalkyl;
[0255] Q is H, NH.sub.2, NHR.sub.b, NR.sub.bR.sub.c, R.sub.b, or
OR.sub.b, in which each of R.sub.b and R.sub.c independently is
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to
7-membered heterocycloalkyl, 5 to 10-membered heteroaryl, or
-M.sub.1-T.sub.1 in which M.sub.1 is a bond or C.sub.1-C.sub.6
alkyl linker optionally substituted with halo, cyano, hydroxyl or
C.sub.1-C.sub.6 alkoxyl and T.sub.t is C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
10-membered heteroaryl, or R.sub.b and R.sub.c, together with the N
atom to which they attach, form 4 to 7-membered heterocycloalkyl
having 0 or 1 additional heteroatoms to the N atom optionally
substituted with C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH,
C(O)O--C.sub.1-C.sub.6 alkyl, OC(O)--C.sub.1-C.sub.6 alkyl, cyano,
C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino,
di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
6-membered heteroaryl, and each of R.sub.b, R.sub.c, and T.sub.1 is
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, cyano,
C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino,
di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, and 5 to
6-membered heteroaryl;
[0256] X is N or CR.sub.X, in which R.sub.x is H, halo, hydroxyl,
carboxyl, cyano, or R.sub.S1, Rs.sub.1 being amino, C.sub.1-C.sub.6
alkoxyl, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
6-membered heteroaryl, and Rs.sub.1 being optionally substituted
with one or more substituents selected from the group consisting of
halo, hydroxyl, carboxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl;
[0257] L.sub.1 is N(Y), S, SO, or SO.sub.2;
[0258] L.sub.2 is CO or absent when L.sub.1 is N(Y) or is absent
when L.sub.1 is S, SO, or SO.sub.2, in which Y is H, R.sub.d,
SO.sub.2R.sub.d, or COR.sub.d when L.sub.2 is absent, or Y is H or
R.sub.d when L.sub.2 is CO, R.sub.d being C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, or 5 to 6-membered heteroaryl, and R.sub.d being
optionally substituted with one or more substituents selected from
the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6
alkenyl, C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, cyano,
C.sub.1-C.sub.6 alkoxyl, C.sub.1-C.sub.6 alkylsulfonyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl and with
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, or 5 to 6-membered heteroaryl further optionally
substituted with C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, halo, hydroxyl, carboxyl, C(O)OH,
C(O)O--C.sub.1-C.sub.6 alkyl, OC(O)--C.sub.1-C.sub.6 alkyl, cyano,
C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6 alkylamino,
di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 6-membered heterocycloalkyl, or 5 to
6-membered heteroaryl;
[0259] each of R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sup.6, and
R.sub.7, independently, is H, halo, hydroxyl, carboxyl, cyano,
R.sub.S2, Rs.sub.2 being amino, C.sub.1-C.sub.6 alkoxyl,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl, and each Rs.sub.2 being optionally substituted with one or
more substituents selected from the group consisting of halo,
hydroxyl, carboxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl;
[0260] R.sub.8 is H, halo or Rs.sub.3, Rs.sub.3 being
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.6
alkynyl, and Rs.sub.3 being optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
carboxyl, cyano amino, C.sub.1-C.sub.6 alkoxyl,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino, and
C.sub.3-C.sub.8 cycloalkyl;
[0261] R.sub.9 is
##STR00097##
in which each of R.sub.e, R.sub.f, R.sub.g, and R.sub.h,
independently is -M.sub.2-T.sub.2, in which M.sub.2 is a bond,
SO.sub.2, SO, S, CO, CO.sub.2, O, O--C.sub.1-C.sub.4 alkyl linker,
C.sub.1-C.sub.4 alkyl linker, NH, or N(R.sub.t), R being
C.sub.1-C.sub.6 alkyl, and T.sub.2 is H, halo, or R.sub.S4,
Rs.sub.4 being C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.10 aryl, 4 to 8-membered heterocycloalkyl, or 5 to
10-membered heteroaryl, and each of O--C.sub.1-C.sub.4 alkyl
linker, C.sub.1-C.sub.4 alkyl linker, R.sub.t, and R.sub.S4 being
optionally substituted with one or more substituents selected from
the group consisting of halo, hydroxyl, carboxyl, cyano,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.1-C.sub.6 alkoxyl, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl, R.sub.1 is H or
C.sub.1-C.sub.6 alkyl optionally substituted with one or more
substituents selected from the group consisting of halo, hydroxyl,
carboxyl, cyano, C.sub.1-C.sub.6 alkoxyl, amino,
mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6 alkylamino,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, and 5 to 6-membered heteroaryl; D is O, NR.sub.j,
or CR.sub.jR.sub.k, each of R.sub.j and R.sub.k independently being
H or C.sub.1-C.sub.6 alkyl, or R.sub.j and R.sub.k taken together,
with the carbon atom to which they are attached, form a
C.sub.3-C.sub.10 cycloalkyl ring, and E is-M.sub.3-T.sub.3, M.sub.3
being a bond or C.sub.1-C.sub.6 alkyl linker optionally substituted
with halo or cyano, T.sub.3 being C.sub.3-C.sub.10 cycloalkyl,
C.sub.6-C.sub.10 aryl, 5 to 10-membered heteroaryl, or 4 to
10-membered heterocycloalkyl, and T.sub.3 being optionally
substituted with one or more substituents selected from the group
consisting of halo, hydroxyl, thiol, carboxyl, cyano, nitro,
C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6
alkynyl, C.sub.1-C.sub.6 alkoxyl, C.sub.1-C.sub.6 haloalkyl,
C.sub.1-C.sub.6 haloalkoxyl, C.sub.1-C.sub.6 alkylthio,
C.sub.1-C.sub.6 alkylsulfonyl, C.sub.1-C.sub.6 haloalkylsulfonyl,
C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkoxycarbonyl, oxo,
amino, mono-C.sub.1-C.sub.6 alkylamino, di-C.sub.1-C.sub.6
alkylamino, C.sub.3-C.sub.8 cycloalkyl, C.sub.4-C.sub.12
alkylcycloalkyl, C.sub.6-C.sub.10 aryl, C.sub.6-C.sub.10 aryloxyl,
C.sub.7-C.sub.14 alkylaryl, C.sub.6-C.sub.10 aminoaryloxyl,
C.sub.6-C.sub.10 arylthio, 4 to 6-membered heterocycloalkyl
optionally substituted with halo, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 haloalkyl, 5 to 6-membered heteroaryl optionally
substituted with halo, C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.6
alkyl that is substituted with hydroxy, halo, C.sub.1-C.sub.6
alkoxycarbonyl, C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl,
4 to 6-membered heterocycloalkyl, or 5 to 6-membered heteroaryl
optionally further substituted with halo, hydroxyl, or
C.sub.1-C.sub.6 alkoxyl;
[0262] m is 1 or 2; and
[0263] n is 1 or 2.
[0264] Compounds of formula IV, methods of making thereof, and
methods of use thereof can be found in WO 2012/075492, which is
incorporated herein by reference.
[0265] In certain embodiments, at least one of m and n is 2.
[0266] In certain embodiments, each of G and J independently is
OR.sub.a. In certain embodiments, each of G and J is OH.
[0267] In certain embodiments, L.sub.1 is N(Y). In certain
embodiments, L, is SO or SO.sub.2.
[0268] In certain embodiments, Y is R.sub.d. In certain
embodiments, R.sub.d is C.sub.1-C.sub.6 alkyl.
[0269] In certain embodiments, L.sub.2 is absent.
[0270] In certain embodiments, R.sub.9 is
##STR00098##
In certain embodiments, R.sup.9 is
##STR00099##
In certain embodiments, R.sub.i is H or C.sub.1-C.sub.6 alkyl
(e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl,
t-butyl, n-pentyl, s-pentyl and n-hexyl).
[0271] In certain embodiments, at least one of R.sub.e, R.sub.f,
R.sub.g, and R.sub.h is halo (such as F, CI, and Br),
C.sub.1-C.sub.6 alkoxyl optionally substituted with one or more
halo (such as OCH.sub.3, OCH.sub.2CH.sub.3, O-iPr, and OCF.sub.3),
C.sub.1-C.sub.6 alkylsulfonyl optionally substituted with one or
more halo (such as SO.sub.2CF.sub.3), or C.sub.1-C.sub.6 alkyl
optionally substituted with one or more halo (such as CH.sub.3,
i-propyl, n-butyl, and CF.sub.3). In certain embodiments, at least
one of R.sub.e, R.sub.f, R.sub.g, and R.sub.h is selected from the
group consisting of F, Cl, CF.sub.3, OCF.sub.3, C.sub.1-C.sub.4
alkyl, and C.sub.1-C.sub.4 alkoxyl.
In certain embodiments,
##STR00100##
is unsubstituted benzimidazolyl or one of the following groups:
##STR00101## ##STR00102## ##STR00103##
In certain embodiments, R.sub.9 is
##STR00104##
In certain embodiments, R.sup.9 is hydrogen.
[0272] In certain embodiments, D is O. In certain embodiments, D is
NRj. In certain embodiments, R.sub.j is H. In certain embodiments,
D is CR.sub.jR.sub.k. In certain embodiments, each of R.sub.j and
R.sub.k is H.
[0273] In certain embodiments, E is -M.sub.3-T.sub.3, in which
M.sub.3 is a bond or C.sub.1-C.sub.3 alkyl linker, T.sub.3 is
phenyl, naphthyl, thienyl, cyclopropyl, or cyclohexyl, and T.sub.3
is optionally substituted with one or more substituents selected
from the group consisting of halo, hydroxyl, thiol, carboxyl,
cyano, nitro, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl,
C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, C.sub.1-C.sub.6
haloalkyl, C.sub.1-C.sub.6 haloalkoxyl, C.sub.1-C.sub.6 alkylthio,
C.sub.1-C.sub.6 alkylsulfonyl, C.sub.1-C.sub.6 alkylcarbonyl,
C.sub.1-C.sub.6 alkoxycarbonyl, oxo, amino, mono-C.sub.1-C.sub.6
alkylamino, di-C.sub.1-C.sub.6 alkylamino, C.sub.3-C.sub.8
cycloalkyl, C.sub.4-C.sub.12 alkylcycloalkyl, C.sub.6-C.sub.10
aryl, C.sub.6-C.sub.10 aryloxyl, C.sub.7-C.sub.14 alkylaryl,
C.sub.6-C.sub.10 aminoaryloxyl, C.sub.6-C.sub.10 arylthio, 4 to
6-membered heterocycloalkyl optionally substituted with
C.sub.1-C.sub.4 alkyl, 5 to 6-membered heteroaryl optionally
substituted with C.sub.1-C.sub.4 alkyl, and C.sub.1-C.sub.6 alkyl
that is substituted with hydroxy, C.sub.1-C.sub.6 alkoxycarbonyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.10 aryl, 4 to 6-membered
heterocycloalkyl, or 5 to 6-membered heteroaryl. In certain
embodiments, T.sub.3 is phenyl optionally substituted with one or
more substituents selected from the group consisting of halo,
hydroxyl, carboxyl, cyano, nitro, C.sub.1-C.sub.6 alkyl (e.g.,
methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl,
n-pentyl, s-pentyl and n-hexyl), C.sub.1-C.sub.6 alkoxyl,
C.sub.1-C.sub.6 haloalkyl, C.sub.1-C.sub.6 haloalkoxyl,
C.sub.1-C.sub.6 alkylsulfonyl, C.sub.6-C.sub.10 aryl (e.g., phenyl
or naphthyl), and C.sub.6-C.sub.10 aryloxyl, and C.sub.7-C.sub.14
alkylaryl. In certain embodiments, E is
##STR00105## ##STR00106## ##STR00107## ##STR00108##
[0274] In certain embodiments, X is N. In certain embodiments, X is
CR.sub.X. In certain embodiments, X is CH.
[0275] In certain embodiments, Q is NH.sub.2 or NHR.sub.b, in which
R.sub.b is -M.sub.1-T.sub.1, M.sub.1 being a bond or
C.sub.1-C.sub.6 alkyl linker and T.sub.1 being C.sub.3-C.sub.8
cycloalkyl. In certain embodiments, Q is H.
[0276] In certain embodiments, R.sub.1, R.sub.2, R.sup.4, R.sub.5,
R.sup.6, R.sub.7, and R.sub.8 are each H.
[0277] In certain embodiments, when R.sup.8 is halo and is attached
to the same carbon atom as J, then J is not hydroxyl. In certain
embodiments, when R.sub.8 is halo and is attached to the same
carbon atom as G, then G is not hydroxyl. In certain embodiments,
T.sub.2 is not halo when M.sub.2 is SO.sub.2, SO, S, CO or O.
[0278] In certain embodiments, T.sub.2 is a 4-8 membered
heterocycloalkyl which is bound to M.sub.2 via a heteroatom. In
certain embodiments, T.sub.2 is a 4-8 membered heterocycloalkyl
which is bound to M.sub.2 via a N atom. In certain embodiments,
T.sub.2 is a 4-8 membered heterocycloalkyl which is bound to
M.sub.2 via a C atom.
[0279] In certain embodiments, the compound is of formula
III-a:
##STR00109##
or a pharmaceutically acceptable salt or ester thereof, wherein G,
J, Q, X, L.sub.1, L.sub.2, R.sub.1, R.sub.2, R.sub.4, R.sub.5,
R.sub.6 R.sup.7, R.sub.8, R.sub.9, m, and n are as described herein
for compounds of formula IV.
[0280] In certain embodiments, the compound is of the formula IV-b
or IV-c:
##STR00110##
pharmaceutically acceptable salt or ester thereof, wherein G, J, Q,
X, L.sub.1, L.sub.2, R.sub.1, R.sub.2, R.sup.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8, R.sub.e, R.sub.f, R.sub.g, R.sub.h, R.sub.i, m
and n are as described herein for compounds of formula IV. In
certain embodiments, the compound is of formula IV-d:
##STR00111##
pharmaceutically acceptable salt or ester thereof, wherein G, J, Q,
X, L.sub.1, L.sub.2, R.sub.1, R.sub.2, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8, D, E, m, and n are as described herein for
formula IV.
[0281] Exemplary compound useful in methods described include:
##STR00112## ##STR00113## ##STR00114## ##STR00115## ##STR00116##
##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121##
##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126##
##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131##
##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136##
##STR00137## ##STR00138## ##STR00139##
##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144##
##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149##
##STR00150## ##STR00151## ##STR00152## ##STR00153## ##STR00154##
##STR00155## ##STR00156## ##STR00157## ##STR00158## ##STR00159##
##STR00160## ##STR00161## ##STR00162## ##STR00163## ##STR00164##
##STR00165## ##STR00166## ##STR00167## ##STR00168##
##STR00169##
or pharmaceutically acceptable salts thereof.
[0282] In some embodiments, Dot1L is inhibited by contacting the
differentiated cell with a composition comprising a compound of
formula:
##STR00170##
or a pharmaceutically acceptable salt thereof.
[0283] In some embodiments, Dot1L is inhibited by contacting the
differentiated cell with a composition comprising a compound of
formula:
##STR00171##
[0284] As used herein, "alkyl", "C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5 or C.sub.6 alkyl" or "C.sub.1-C.sub.6 alkyl" is
intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 or
C.sub.6 straight chain (linear) saturated aliphatic hydrocarbon
groups and C.sub.3, C.sub.4, C.sub.5 or C.sub.6 branched saturated
aliphatic hydrocarbon groups. For example, C.sub.1-C.sub.6 alkyl is
intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 and
C.sub.6 alkyl groups. Examples of alkyl include moieties having
from one to six carbon atoms, such as, but not limited to, methyl,
ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, n-pentyl,
s-pentyl or n-hexyl. In certain embodiments, a straight chain or
branched alkyl has six or fewer carbon atoms (e.g., C.sub.1-C.sub.6
for straight chain, C.sub.3-C.sub.6 for branched chain), and in
another embodiment, a straight chain or branched alkyl has four or
fewer carbon atoms.
[0285] As used herein, the term "cycloalkyl" refers to a saturated
or unsaturated nonaromatic hydrocarbon mono- or multi-ring system
having 3 to 30 carbon atoms (e.g., C.sub.3-C.sub.10). Examples of
cycloalkyl include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,
cyclopentenyl, cyclohexenyl, cycloheptenyl, and adamantyl. The term
"heterocycloalkyl" refers to a saturated or unsaturated nonaromatic
5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered
tricyclic ring system having one or more heteroatoms (such as O, N,
S, or Se). Examples of heterocycloalkyl groups include, but are not
limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, and
tetrahydrofuranyl.
[0286] The term "optionally substituted alkyl" refers to
unsubstituted alkyl or alkyl having designated substituents
replacing one or more hydrogen atoms on one or more carbons of the
hydrocarbon backbone. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0287] An "arylalkyl" or an "aralkyl" moiety is an alkyl
substituted with an aryl (e.g., phenylmethyl (benzyl)). An
"alkylaryl" moiety is an aryl substituted with an alkyl (e.g.,
methylphenyl).
[0288] As used herein, "alkyl linker" is intended to include
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 or C.sub.6 straight
chain (linear) saturated divalent aliphatic hydrocarbon groups and
C.sub.3, C.sub.4, C.sub.5 or C.sub.6 branched saturated aliphatic
hydrocarbon groups. For example, C.sub.1-C.sub.6 alkyl linker is
intended to include C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5 and
C.sub.6 alkyl linker groups. Examples of alkyl linker include,
moieties having from one to six carbon atoms, such as, but not
limited to, methyl (--CH.sub.2--), ethyl (--CH.sub.2CH.sub.2--),
n-propyl (--CH.sub.2CH.sub.2CH.sub.2--), i-propyl
(--CHCH.sub.3CH.sub.2--), n-butyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), s-butyl
(--CHCH.sub.3CH.sub.2CH.sub.2--), i-butyl
(--C(CH.sub.3).sub.2CH.sub.2--), n-pentyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), s-pentyl
(--CHCH.sub.3CH.sub.2CH.sub.2CH.sub.2--) or n-hexyl
(--CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2--).
[0289] "Alkenyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
that contain at least one double bond. For example, the term
"alkenyl" includes straight chain alkenyl groups (e.g., ethenyl,
propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,
decenyl), and branched alkenyl groups. In certain embodiments, a
straight chain or branched alkenyl group has six or fewer carbon
atoms in its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). The term "C.sub.2-C.sub.6"
includes alkenyl groups containing two to six carbon atoms. The
term "C.sub.3-C.sub.6" includes alkenyl groups containing three to
six carbon atoms.
[0290] The term "optionally substituted alkenyl" refers to
unsubstituted alkenyl or alkenyl having designated substituents
replacing one or more hydrogen atoms on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl; sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, heterocyclyl, alkylaryl, or an aromatic or
heteroaromatic moiety.
[0291] "Alkynyl" includes unsaturated aliphatic groups analogous in
length and possible substitution to the alkyls described above, but
which contain at least one triple bond. For example, "alkynyl"
includes straight chain alkynyl groups (e.g., ethynyl, propynyl,
butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl),
and branched alkynyl groups. In certain embodiments, a straight
chain or branched alkynyl group has six or fewer carbon atoms in
its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). The term "C.sub.2-C.sub.6"
includes alkynyl groups containing two to six carbon atoms. The
term "C.sub.3-C.sub.6" includes alkynyl groups containing three to
six carbon atoms.
[0292] The term "optionally substituted alkynyl" refers to
unsubstituted alkynyl or alkynyl having designated substituents
replacing one or more hydrogen atoms on one or more hydrocarbon
backbone carbon atoms. Such substituents can include, for example,
alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
amino (including alkylamino, dialkylamino, arylamino, diarylamino
and alkylarylamino), acylamino (including alkylcarbonylamino,
arylcarbonylamino, carbamoyl and ureido), amidino, imino,
sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,
alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0293] Other optionally substituted moieties (such as optionally
substituted cycloalkyl, heterocycloalkyl, aryl, or heteroaryl)
include both the unsubstituted moieties and the moieties having one
or more of the designated substituents.
[0294] "Aryl" includes groups with aromaticity, including
"conjugated," or multicyclic systems with at least one aromatic
ring and do not contain any heteroatom in the ring structure.
Examples include phenyl, benzyl, 1,2,3,4-tetrahydronaphthalenyl,
etc.
[0295] "Heteroaryl" groups are aryl groups, as defined above,
except having from one to four heteroatoms in the ring structure,
and may also be referred to as "aryl heterocycles" or "hetero
aromatic s." As used herein, the term "heteroaryl" is intended to
include a stable 5- or 6-membered monocyclic or 7-, 8-, 9-, 10-,
11- or 12-membered bicyclic aromatic heterocyclic ring which
consists of carbon atoms and one or more heteroatoms, e.g., 1 or
1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, or e.g. 1, 2, 3, 4, 5,
or 6 heteroatoms, independently selected from the group consisting
of nitrogen, oxygen and sulfur. The nitrogen atom may be
substituted or unsubstituted (i.e., N or NR wherein R is H or other
substituents, as defined). The nitrogen and sulfur heteroatoms may
optionally be oxidized (i.e., N.fwdarw.O and S(O).sub.p, where p=1
or 2). It is to be noted that total number of S and O atoms in the
aromatic heterocycle is not more than 1. Examples of heteroaryl
groups include pyrrole, furan, thiophene, thiazole, isothiazole,
imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole,
pyridine, pyrazine, pyridazine, pyrimidine, and the like.
[0296] Furthermore, the terms "aryl" and "heteroaryl" include
multicyclic aryl and heteroaryl groups, e.g., tricyclic, bicyclic,
e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole,
benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,
isoquinoline, naphthrydine, indole, benzofuran, purine, benzofuran,
deazapurine, indolizine.
[0297] In the case of multicyclic aromatic rings, only one of the
rings needs to be aromatic (e.g., 2,3-dihydroindole), although all
of the rings may be aromatic (e.g., quinoline). The second ring can
also be fused or bridged.
[0298] The aryl or heteroaryl aromatic ring can be substituted at
one or more ring positions with such substituents as described
above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxyl,
alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,
aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl,
arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato,
phosphinato, amino (including alkylamino, dialkylamino, arylamino,
diarylamino and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or hetero aromatic moiety. Aryl groups can also be fused
or bridged with alicyclic or heterocyclic rings, which are not
aromatic so as to form a multicyclic system (e.g., tetralin,
methylenedioxyphenyl).
[0299] As used herein, "carbocycle" or "carbocyclic ring" is
intended to include any stable monocyclic, bicyclic or tricyclic
ring having the specified number of carbons, any of which may be
saturated, unsaturated, or aromatic. For example, a
C.sub.3-C.sub.14 carbocycle is intended to include a monocyclic,
bicyclic or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13 or 14 carbon atoms. Examples of carbocycles include, but are not
limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl,
cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl,
cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl,
cyclooctadienyl, fluorenyl, phenyl, naphthyl, indanyl, adamantyl
and tetrahydronaphthyl. Bridged rings are also included in the
definition of carbocycle, including, for example,
[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane
and [2.2.2]bicyclooctane. A bridged ring occurs when one or more
carbon atoms link two non-adjacent carbon atoms. In one embodiment,
bridge rings are one or two carbon atoms. It is noted that a bridge
always converts a monocyclic ring into a tricyclic ring. When a
ring is bridged, the substituents recited for the ring may also be
present on the bridge. Fused (e.g., naphthyl, tetrahydronaphthyl)
and spiro rings are also included.
[0300] As used herein, "heterocycle" includes any ring structure
(saturated or partially unsaturated) which contains at least one
ring heteroatom (e.g., N, O or S). Examples of heterocycles
include, but are not limited to, morpholine, pyrrolidine,
tetrahydrothiophene, piperidine, piperazine and tetrahydrofuran.
Examples of heterocyclic groups include, but are not limited to,
acridinyl, azocinyl, benzimidazolyl, benzofuranyl,
benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl,
carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl,
2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran,
furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl,
1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,
3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,
isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,
methylenedioxyphenyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,
pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,
phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,
piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,
pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,
pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,
pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,
quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,
tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,
tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,
1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,
thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,
thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and
xanthenyl.
[0301] The term "substituted," as used herein, means that any one
or more hydrogen atoms on the designated atom is replaced with a
selection from the indicated groups, provided that the designated
atom's normal valency is not exceeded, and that the substitution
results in a stable compound. When a substituent is oxo or keto
(i.e., .dbd.O), then 2 hydrogen atoms on the atom are replaced.
Keto substituents are not present on aromatic moieties. Ring double
bonds, as used herein, are double bonds that are formed between two
adjacent ring atoms (e.g., C.dbd.C, C.dbd.N or N.dbd.N). "Stable
compound" and "stable structure" are meant to indicate a compound
that is sufficiently robust to survive isolation to a useful degree
of purity from a reaction mixture, and formulation into an
efficacious therapeutic agent.
[0302] When a bond to a substituent is shown to cross a bond
connecting two atoms in a ring, then such substituent may be bonded
to any atom in the ring. When a substituent is listed without
indicating the atom via which such substituent is bonded to the
rest of the compound of a given formula, then such substituent may
be bonded via any atom in such formula.
[0303] Combinations of substituents and/or variables are
permissible, but only if such combinations result in stable
compounds.
[0304] When any variable (e.g., R*) occurs more than one time in
any constituent or formula for a compound, its definition at each
occurrence is independent of its definition at every other
occurrence. Thus, for example, if a group is shown to be
substituted with 0-2R* moieties, then the group may optionally be
substituted with up to two R* moieties and R* at each occurrence is
selected independently from the definition of R. Also, combinations
of substituents and/or variables are permissible, but only if such
combinations result in stable compounds.
[0305] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O''.
[0306] As used herein, "halo" or "halogen" refers to fluoro,
chloro, bromo and iodo. The term "perhalogenated" generally refers
to a moiety wherein all hydrogen atoms are replaced by halogen
atoms. The term "haloalkyl" or "haloalkoxyl" refers to an alkyl or
alkoxyl substituted with one or more halogen atoms.
[0307] The term "carbonyl" includes compounds and moieties which
contain a carbon connected with a double bond to an oxygen atom.
Examples of moieties containing a carbonyl include, but are not
limited to, aldehydes, ketones, carboxylic acids, amides, esters,
anhydrides, etc.
[0308] The term "carboxyl" refers to --COOH or its C.sub.1-C.sub.6
alkyl ester.
[0309] "Acyl" includes moieties that contain the acyl radical
(R--C(O)--) or a carbonyl group. "Substituted acyl" includes acyl
groups where one or more of the hydrogen atoms are replaced by, for
example, alkyl groups, alkynyl groups, halogen, hydroxyl,
alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino
(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and
ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,
thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,
sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,
alkylaryl, or an aromatic or heteroaromatic moiety.
[0310] "Aroyl" includes moieties with an aryl or heteroaromatic
moiety bound to a carbonyl group. Examples of aroyl groups include
phenylcarboxy, naphthyl carboxy, etc.
[0311] "Alkoxyalkyl," "alkylaminoalkyl," and "thioalkoxyalkyl"
include alkyl groups, as described above, wherein oxygen, nitrogen,
or sulfur atoms replace one or more hydrocarbon backbone carbon
atoms.
[0312] The term "alkoxy" or "alkoxyl" includes substituted and
unsubstituted alkyl, alkenyl and alkynyl groups covalently linked
to an oxygen atom. Examples of alkoxy groups or alkoxyl radicals
include, but are not limited to, methoxy, ethoxy, isopropyloxy,
propoxy, butoxy and pentoxy groups. Examples of substituted alkoxy
groups include halogenated alkoxy groups. The alkoxy groups can be
substituted with groups such as alkenyl, alkynyl, halogen,
hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,
alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Examples of halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy and trichloromethoxy.
[0313] The term "ether" or "alkoxy" includes compounds or moieties
which contain an oxygen bonded to two carbon atoms or heteroatoms.
For example, the term includes "alkoxyalkyl," which refers to an
alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen
atom which is covalently bonded to an alkyl group.
[0314] The term "ester" includes compounds or moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
[0315] The term "thioalkyl" includes compounds or moieties which
contain an alkyl group connected with a sulfur atom. The thioalkyl
groups can be substituted with groups such as alkyl, alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, carboxyacid,
alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,
alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl,
alkoxyl, amino (including alkylamino, dialkylamino, arylamino,
diarylamino and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moieties.
[0316] The term "thiocarbonyl" or "thiocarboxy" includes compounds
and moieties which contain a carbon connected with a double bond to
a sulfur atom.
[0317] The term "thioether" includes moieties which contain a
sulfur atom bonded to two carbon atoms or heteroatoms. Examples of
thioethers include, but are not limited to alkthioalkyls,
alkthioalkenyls, and alkthioalkynyls. The term "alkthioalkyls"
include moieties with an alkyl, alkenyl, or alkynyl group bonded to
a sulfur atom which is bonded to an alkyl group. Similarly, the
term "alkthioalkenyls" refers to moieties wherein an alkyl, alkenyl
or alkynyl group is bonded to a sulfur atom which is covalently
bonded to an alkenyl group; and alkthioalkynyls" refers to moieties
wherein an alkyl, alkenyl or alkynyl group is bonded to a sulfur
atom which is covalently bonded to an alkynyl group.
[0318] As used herein, "amine" or "amino" refers to unsubstituted
or substituted --NH.sub.2. "Alkylamino" includes groups of
compounds wherein nitrogen of --NH.sub.2 is bound to at least one
alkyl group. Examples of alkylamino groups include benzylamino,
methylamino, ethylamino, phenethylamino, etc. "Dialkylamino"
includes groups wherein the nitrogen of --NH.sub.2 is bound to at
least two additional alkyl groups. Examples of dialkylamino groups
include, but are not limited to, dimethylamino and diethylamino.
"Arylamino" and "diarylamino" include groups wherein the nitrogen
is bound to at least one or two aryl groups, respectively.
"Aminoaryl" and "aminoaryloxy" refer to aryl and aryloxy
substituted with amino. "Alkylarylamino," "alkylaminoaryl" or
"arylaminoalkyl" refers to an amino group which is bound to at
least one alkyl group and at least one aryl group.
[0319] "Alkaminoalkyl" refers to an alkyl, alkenyl, or alkynyl
group bound to a nitrogen atom which is also bound to an alkyl
group. "Acylamino" includes groups wherein nitrogen is bound to an
acyl group. Examples of acylamino include, but are not limited to,
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido
groups.
[0320] The term "amide" or "aminocarboxy" includes compounds or
moieties that contain a nitrogen atom that is bound to the carbon
of a carbonyl or a thiocarbonyl group. The term includes
"alkaminocarboxy" groups that include alkyl, alkenyl or alkynyl
groups bound to an amino group which is bound to the carbon of a
carbonyl or thiocarbonyl group. It also includes "arylaminocarboxy"
groups that include aryl or heteroaryl moieties bound to an amino
group that is bound to the carbon of a carbonyl or thiocarbonyl
group. The terms "alkylaminocarboxy", "alkenylaminocarboxy",
"alkynylaminocarboxy" and "arylaminocarboxy" include moieties
wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively,
are bound to a nitrogen atom which is in turn bound to the carbon
of a carbonyl group. Amides can be substituted with substituents
such as straight chain alkyl, branched alkyl, cycloalkyl, aryl,
heteroaryl or heterocycle. Substituents on amide groups may be
further substituted.
[0321] Compounds of the present invention that contain nitrogens
can be converted to N-oxides by treatment with an oxidizing agent
(e.g., 3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen
peroxides) to afford other compounds of the present invention.
Thus, all shown and claimed nitrogen-containing compounds are
considered, when allowed by valency and structure, to include both
the compound as shown and its N-oxide derivative (which can be
designated as N.fwdarw.O or N.sup.+--O''). Furthermore, in other
instances, the nitrogens in the compounds of the present invention
can be converted to N-hydroxy or N-alkoxy compounds. For example,
N-hydroxy compounds can be prepared by oxidation of the parent
amine by an oxidizing agent such as m-CPBA. All shown and claimed
nitrogen-containing compounds are also considered, when allowed by
valency and structure, to cover both the compound as shown and its
N-hydroxy (i.e., N--OH) and N-alkoxy (i.e., N--OR, wherein R is
substituted or unsubstituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkenyl, C.sub.1-C.sub.6 alkynyl, 3-14-membered carbocycle or
3-14-membered heterocycle) derivatives.
[0322] In the present specification, the structural formula of the
compound represents a certain isomer for convenience in some cases,
but the present invention includes all isomers, such as geometrical
isomers, optical isomers based on an asymmetrical carbon,
stereoisomers, tautomers, and the like. In addition, a crystal
polymorphism may be present for the compounds represented by the
formula. It is noted that any crystal form, crystal form mixture,
or anhydride or hydrate thereof is included in the scope of the
present invention. Furthermore, so-called metabolite which is
produced by degradation of the present compound in vivo is included
in the scope of the present invention.
[0323] "Isomerism" means compounds that have identical molecular
formulae but differ in the sequence of bonding of their atoms or in
the arrangement of their atoms in space. Isomers that differ in the
arrangement of their atoms in space are termed "stereoisomers."
Stereoisomers that are not mirror images of one another are termed
"diastereoisomers," and stereoisomers that are non-superimposable
mirror images of each other are termed "enantiomers" or sometimes
optical isomers. A mixture containing equal amounts of individual
enantiomeric forms of opposite chirality is termed a "racemic
mixture."
[0324] A carbon atom bonded to four nonidentical substituents is
termed a "chiral center." "Chiral isomer" means a compound with at
least one chiral center. Compounds with more than one chiral center
may exist either as an individual diastereomer or as a mixture of
diastereomers, termed "diastereomeric mixture." When one chiral
center is present, a stereoisomer may be characterized by the
absolute configuration (R or S) of that chiral center. Absolute
configuration refers to the arrangement in space of the
substituents attached to the chiral center. The substituents
attached to the chiral center under consideration are ranked in
accordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn
et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et
al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc.
1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J.
Chem. Educ. 1964, 41, 116).
[0325] "Geometric isomer" means the diastereomers that owe their
existence to hindered rotation about double bonds or a cycloalkyl
linker (e.g., 1,3-cyclobutyl). These configurations are
differentiated in their names by the prefixes cis and trans, or Z
and E, which indicate that the groups are on the same or opposite
side of the double bond in the molecule according to the
Cahn-Ingold-Prelog rules.
[0326] It is to be understood that the compounds of the present
invention may be depicted as different chiral isomers or geometric
isomers. It should also be understood that when compounds have
chiral isomeric or geometric isomeric forms, all isomeric forms are
intended to be included in the scope of the present invention, and
the naming of the compounds does not exclude any isomeric
forms.
[0327] Furthermore, the structures and other compounds discussed in
this invention include all atropic isomers thereof. "Atropic
isomers" are a type of stereoisomer in which the atoms of two
isomers are arranged differently in space. Atropic isomers owe
their existence to a restricted rotation caused by hindrance of
rotation of large groups about a central bond. Such atropic isomers
typically exist as a mixture, however as a result of recent
advances in chromatography techniques, it has been possible to
separate mixtures of two atropic isomers in select cases.
[0328] "Tautomer" is one of two or more structural isomers that
exist in equilibrium and is readily converted from one isomeric
form to another. This conversion results in the formal migration of
a hydrogen atom accompanied by a switch of adjacent conjugated
double bonds. Tautomers exist as a mixture of a tautomeric set in
solution. In solutions where tautomerization is possible, a
chemical equilibrium of the tautomers will be reached. The exact
ratio of the tautomers depends on several factors, including
temperature, solvent and pH. The concept of tautomers that are
interconvertable by tautomerizations is called tautomerism.
[0329] Of the various types of tautomerism that are possible, two
are commonly observed. In keto-enol tautomerism a simultaneous
shift of electrons and a hydrogen atom occurs. Ring-chain
tautomerism arises as a result of the aldehyde group (--CHO) in a
sugar chain molecule reacting with one of the hydroxy groups (--OH)
in the same molecule to give it a cyclic (ring-shaped) form as
exhibited by glucose.
[0330] Common tautomeric pairs are: ketone-enol, amide-nitrile,
lactam-lactim, amide-imidic acid tautomerism in heterocyclic rings
(e.g., in nucleobases such as guanine, thymine and cytosine),
amine-enamine and enamine-enamine. Benzimidazoles also exhibit
tautomerism, when the benzimidazole contains one or more
substituents in the 4, 5, 6 or 7 positions, the possibility of
different isomers arises. For example,
2,5-dimethyl-1H-benzo[d]imidazole can exist in equilibrium with its
isomer 2,6-dimethyl-1H-benzo[d] imidazole via tautomerization.
##STR00172##
[0331] It is to be understood that the compounds of the present
invention may be depicted as different tautomers. It should also be
understood that when compounds have tautomeric forms, all
tautomeric forms are intended to be included in the scope of the
present invention, and the naming of the compounds does not exclude
any tautomer form.
[0332] The term "crystal polymorphs", "polymorphs" or "crystal
forms" means crystal structures in which a compound (or a salt or
solvate thereof) can crystallize in different crystal packing
arrangements, all of which have the same elemental composition.
Different crystal forms usually have different X-ray diffraction
patterns, infrared spectral, melting points, density hardness,
crystal shape, optical and electrical properties, stability and
solubility. Recrystallization solvent, rate of crystallization,
storage temperature, and other factors may cause one crystal form
to dominate. Crystal polymorphs of the compounds can be prepared by
crystallization under different conditions.
[0333] Compounds of the invention may be crystalline,
semi-crystalline, non-crystalline, amorphous, mesomorphous,
etc.
[0334] Compounds described herein include the compounds themselves,
as well as their N-oxides, salts, their solvates, and their
prodrugs, if applicable. A salt, for example, can be formed between
an anion and a positively charged group (e.g., amino) on a
substituted purine or 7-deazapurine compound. Suitable anions
include chloride, bromide, iodide, sulfate, bisulfate, sulfamate,
nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,
glutamate, glucuronate, glutarate, malate, maleate, succinate,
fumarate, tartrate, tosylate, salicylate, lactate,
naphthalenesulfonate, and acetate.
[0335] Likewise, a salt can also be formed between a cation and a
negatively charged group (e.g., carboxylate) on a substituted
purine or 7-deazapurine compound. Suitable cations include sodium
ion, potassium ion, magnesium ion, calcium ion, and an ammonium
cation such as tetramethylammonium ion. The substituted purine or
7-deazapurine compounds also include those salts containing
quaternary nitrogen atoms. Examples of prodrugs include esters and
other pharmaceutically acceptable derivatives, which, upon
administration to a subject, are capable of providing active
substituted purine or 7-deazapurine compounds.
[0336] Additionally, the compounds described herein, for example,
the salts of the compounds, can exist in either hydrated or
unhydrated (the anhydrous) form or as solvates with other solvent
molecules. Nonlimiting examples of hydrates include hemihydrates,
monohydrates, dihydrates, trihydrates, etc. Nonlimiting examples of
solvates include ethanol solvates, acetone solvates, etc.
[0337] "Solvate" means solvent addition forms that contain either
stoichiometric or non stoichiometric amounts of solvent. Some
compounds have a tendency to trap a fixed molar ratio of solvent
molecules in the crystalline solid state, thus forming a solvate.
If the solvent is water the solvate formed is a hydrate; and if the
solvent is alcohol, the solvate formed is an alcoholate. Hydrates
are formed by the combination of one or more molecules of water
with one molecule of the substance in which the water retains its
molecular state as H.sub.2O. A hemihydrate is formed by the
combination of one molecule of water with more than one molecule of
the substance in which the water retains its molecular state as
H.sub.2O.
[0338] As used herein, the term "analog" refers to a chemical
compound that is structurally similar to another but differs
slightly in composition (as in the replacement of one atom by an
atom of a different element or in the presence of a particular
functional group, or the replacement of one functional group by
another functional group). Thus, an analog is a compound that is
similar or comparable in function and appearance, but not in
structure or origin to the reference compound.
[0339] As defined herein, the term "derivative" refers to compounds
that have a common core structure, and are substituted with various
groups as described herein. For example, all of the compounds
represented by Formula (I) are substituted purine compounds or
substituted 7-deazapurine compounds, and have Formula (I) as a
common core.
[0340] The term "bioisostere" refers to a compound resulting from
the exchange of an atom or of a group of atoms with another,
broadly similar, atom or group of atoms. The objective of a
bioisosteric replacement is to create a new compound with similar
biological properties to the parent compound. The bioisosteric
replacement may be physicochemically or topologically based.
Examples of carboxylic acid bioisosteres include, but are not
limited to, acyl sulfonimides, tetrazoles, sulfonates and
phosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96,
3147-3176, 1996.
[0341] All isotopes of atoms occurring in the compounds described
herein are intended to be encompassed. Isotopes include those atoms
having the same atomic number but different mass numbers. By way of
general example and without limitation, isotopes of hydrogen
include tritium and deuterium, and isotopes of carbon include C-13
and C-14.
[0342] The present invention is further illustrated by the
following Examples, which in no way should be construed as further
limiting. The entire contents of all of the references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by reference, in
particular for the teaching that is referenced hereinabove.
EXAMPLES
Example 1
Materials and Methods:
[0343] shRNA Cloning
[0344] shRNAs were designed using the RNAi Codex26. 97-mer oligos
(Table 2) These were amplified with the following primer pair (SEQ
ID NOs: 1 and 2)
TABLE-US-00002 Forward: GATGGCTGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG
Reverse: GTCTAGAGGAATTCCGAGGCAGTAGGC.
[0345] PCR products were gel purified, digested with EcoRI and XHoI
and ligated into the MSCV-PM (Openbiosystems) vector. Clones were
verified by sequencing. shRNA targeting the firefly luciferase was
used as a control27. Nanog shRNA was previously described28.
Production of Viral Supernatants
[0346] 293T cells were plated at a density of 2.5.times.106 cells
per 10-cm dish. The next day, cells were transfected with 2 .mu.g
viral vector, 2 .mu.g Gag-Pol vector and 0.22 .mu.g VSV-G plasmid
using 20 .mu.l Fugene 6 (Roche Applied Science #1181509001) in 400
.mu.l DMEM per plate. Supernatant was collected 48 h and 72 h
post-transfection and filtered through 45 .mu.m pore size filters.
For concentration, viral supernatants were mixed with PEG3350
solution (Sigma P3640, dissolved in PBS, 10% final concentration)
and left overnight at 4 C. The next day, supernatants were
centrifuged at 2500 rpm for 20 minutes, and the pellets were
re-suspended in PBS. Titering was performed on 293 Ts. For shRNA
infections, 500 ul of viral supernatant was used to infect 25,000
cells in the presence of 10 ug/ml protamine sulfate. For
fluorescent labeling of dh1fs, we used lentiviruses PRRL-GFP
(Addgene #12252) and FUdGW-Tomato (Addgene #22771).
Reprogramming Assays
[0347] dH1f cells were first infected with shRNA viruses at high
MOI to ensure all cells received at least one vector (Gauged by
puromycin resistance of parallel infected wells). 25,000
shRNA-infected dH1f cells were then plated per well in 12-well
plates and infected overnight with either retroviral (MOI 2.5)7 or
lentiviral (Addgene #21162, 21164; 100-200 .mu.l supernatant)29
reprogramming factors. For 2-factor reprogramming, Oct4 and Sox2
viruses were used at an MOI of 5. 6 days later, cells were
trypsinized and re-plated 1:4 to 1:6 onto 6-well plates. Medium was
changed to hES medium daily until Day 21 when plates were fixed.
Small molecule inhibitor of Dot1L, EPZ004777 (a gift from Epizyme,
Inc., Cambridge, Mass.) was dissolved in DMSO as a 10 mM stock and
was added at the indicated concentrations. For Dot1L rescue
experiments, an MSCV-based retroviral vector encoding human Dot1L
with or without mutations in the SAM binding site (gifts of Y.
Zhang) were mutagenized at the shRNA target site using QuikChange
II XL Site-Directed Mutagenesis Kit (Agilent Technologies). In
certain experiments, Nanog and Lin28 expression was achieved using
lentivirus (Addgene #21163). IMR-90 and MRC5 human diploid
fibroblasts were purchased from ATCC and 50000 cells were used in
reprogramming experiments. Statistical analysis was performed using
a Student's t-test.
Microarray Analysis
[0348] Total RNA was extracted from three independent culture
plates for each conditions with an RNeasy Mini kit (Qiagen).
Synthesis of cRNA from total RNA and hybridization/scanning of
microarrays were performed with Affymetrix GeneChip products
(HGU133A) as described in the GeneChip manual. Normalization of the
raw gene expression data, quality control checks, and subsequent
analyses were done with the open-source R-project statistical
software (R Development Core Team, 2007)(http://www.r-project.org/)
together with Bioconductor packages. Raw data files (.CEL) were
converted into probe set values by RMA normalization. Genes were
selected at a threshold of Log Ratio 0.4. The microarray data have
been deposited in National Center for Biotechnology Information
Gene Expression Omnibus (GEO) and are accessible through GEO Series
accession number GSE29253.
SYBR-Green Real-Time RT-PCR
[0349] Total RNA was extracted using RNeasy Mini kit coupled with
RNase-free DNase set (Qiagen) and reverse transcribed with
Hexanucleotide Mix (Roche). The resulting cDNAs were used for PCR
using SYBR-Green Master PCR mix (Applied Biosystem) in triplicates.
All quantitations were normalized to an endogenous Beta-Actin
control. The relative quantitation value for each target gene
compared to the calibrator for that target is expressed as
2-(Ct-Cc) (Ct and Cc are the mean threshold cycle differences after
normalizing to Beta-Actin). List of primers can be found in Table
3.
Immunostaining
[0350] Immunostaining of reprogramming plates were performed as
described.sup.8. Briefly, cells were fixed with 4% p-formaldehyde
and stained with biotin-anti-Tra-1-60 (eBioscience, #13-8863-82,
1:250) and streptavidin horseradish peroxidase (Biolegend, #405210,
1:500) diluted in PBS (3%), FCS (0.3%) Triton X-100. Staining was
developed with the Vector labs DAB kit (#SK-4100), and iPSC
colonies quantified with ImageJ software. For the characterization
of shDot1l-iPS cells, single colonies were put onto MEF coated
96-well plates. The plates were fixed for 20 min with 4%
p-formaldehyde/PBS (+/+), washed several times with PBS (+/+) and
incubated overnight at 4.degree. C. with primary antibody and
Hoechst diluted in 3% donkey serum/3% BSA Fraction VII/0.01% Triton
X-100/PBS (+/+); Hoechst, Invitrogen #H3570 (1:20,000),
Tra-1-81/A488 (BD #560174), SSEA-4/A647 (BD #560219), Tra-1-60/A647
(BD #560122), Nanog, rabbit polyclonal (Abcam #ab21624), Oct4,
rabbit polyclonal (Abcam #ab19857). For Nanog and Oct4, donkey
anti-rabbit IgG/A555 (Molecular Probes #A31572) secondary antibody
was used. After several washes with PBS (+/+), images were acquired
using a BD Pathway 435 imager equipped with a x 10 objective.
Teratoma Formation Assay
[0351] iPSCs grown on MEFs were harvested with Collagenease IV (1
mg/ml in DMEM/F12). Cell clumps from one 6-well plate were
resuspended in 50 .mu.l DMEM/F12, 100 .mu.l collagen I
(Invitrogen-#A1064401) and 150 .mu.l hESC-qualified matrigel (BD
Biosciences-#354277). Cell clumps were then injected into the hind
limb femoral muscles (100 .mu.l suspension per leg) of Rag2
.gamma./c mice. After 6-8 weeks, teratomas were harvested and fixed
in Bouin's solution overnight. Samples were then embedded in
paraffin, and sections were stained with hematoxylin/eosin (Rodent
Histopathology Core, Harvard Medical School, Boston, Mass.,
USA).
Characterization of iPS Cells
[0352] Embryoid body differentiation was performed as described
(Loewer). To check for the presence of the reprogramming
transgenes, genomic DNA was isolated using DNeasy Blood &
Tissue Kit (Qiagen) and PCR was performed with specific primers to
the endogenous or the viral trangenes.sup.3.
ChIP Assays
[0353] ChIP-seq was performed as described with slight
modifications.sup.25. 300 000 cells were fixed at room temperature
in PBS 1% formalin (v/v) for 10 minutes with gentle agitations.
Fixation was stopped by the addition of glycine (125 mM final
concentration) and agitation for 5 min at room temperature. Fixed
cells were washed twice in ice-cold PBS, resuspended in 100 ul of
SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1).
Chromatin was sheared by sonication to about 100-500 bp fragments
using bioruptor (diagenode, Denville, N.J.) and diluted tenfold
with dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2 mM EDTA,
16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl). Antibodies against specific
histone modifications was added to sonicated chromatin solution and
incubated at 4 degree overnight with gentle agitation. The
antibodies used were anti-H3K27me3 (Millipore 07-449) and
anti-H3K79me2 (abcam 3594). Immune complexes were collected by
incubation with 20 ul of Protein A/G agarose beads (Millipore) for
an hour at 4 degree with gentle agitation. Precipitates were washed
sequentially with ice cold low salt wash (0.1% SDS, 1%
Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH8.1, 150 mM NaCl), high
salt wash (0.1% SDS, 1% Triton-X-100, 2 mM EDTA, 20 mM Tris-HCl, pH
8.1, 500 mM NaCl), LiCl wash (0.25M LiCl, 1% IGEPAL CA-630, 1%
deoxycholic acid, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and TE wash (1
mM EDTA, 10 mM Tris-HCl, pH 8.1) for 5 mins each at 4 degree with
gentle agitation. Samples were centrifuged briefly in between
washes to collect the beads. Immunoprecipitated DNA was eluted by
incubating beads with 150 ul elution buffer (l % SDS, 0.1 M NaHCO3)
with gentle agitation for 15 mins at room temperature. Elution was
repeated once and eluates were combined, sodium chloride (final
concentration of 0.2M) were added to the eluate and eluates were
incubated at 65 degree overnight to reverse crosslinking. DNA was
purified using PCR purification spin column (Qiagen). For ChIP
sequencing, ChIP DNA libraries were made following Illumina
ChIP-seq library preparation kit and subjected to Solexa sequencing
(Illumina) at Center for Cancer Computational Biology, Dana Faber
Cancer Institute.
TABLE-US-00003 TABLE 2 shRNA Sequences evaluated Hairpin
TGCTGTTGACAGTGAGCGAGCGATTGCTCCAGGAATTTAATAGTGAAGCC
ACAGATGTATTAAATTCCTGGAGCAATCGCCTGCCTACTGCCTCGGA (SEQ ID NO: 3)
TGCTGTTGACAGTGAGCGCCGTGCCCATTCCCTGTGTCAATAGTGAAGCC
ACAGATGTATTGACACAGGGAATGGGCACGTTGCCTACTGCCTCGGA (SEQ ID NO: 4)
TGCTGTTGACAGTGAGCGAGGTGATGACTTCAGTCTCTACTAGTGAAGCC
ACAGATGTAGTAGAGACTGAAGTCATCACCCTGCCTACTGCCTCGGA (SEQ ID NO: 5)
TGCTGTTGACAGTGAGCGCGGATGGAGAGGTGTACTGCATTAGTGAAGCC
ACAGATGTAATGCAGTACACCTCTCCATCCTTGCCTACTGCCTCGGA (SEQ ID NO: 6)
TGCTGTTGACAGTGAGCGACGATGCCAGCAGTCATGCAAATAGTGAAGCC
ACAGATGTATTTGCATGACTGCTGGCATCGCTGCCTACTGCCTCGGA (SEQ ID NO: 7)
TGCTGTTGACAGTGAGCGAGCAGCAACGGATACATCTTAATAGTGAAGCC
ACAGATGTATTAAGATGTATCCGTTGCTGCCTGCCTACTGCCTCGGA (SEQ ID NO: 8)
TGCTGTTGACAGTGAGCGACGATGCCAGCAGTCATGCAAATAGTGAAGCC
ACAGATGTATTTGCATGACTGCTGGCATCGCTGCCTACTGCCTCGGA (SEQ ID NO: 9)
TGCTGTTGACAGTGAGCGAGGGATTCAGATGTCACCTTAATAGTGAAGCC
ACAGATGTATTAAGGTGACATCTGAATCCCGTGCCTACTGCCTCGGA (SEQ ID NO: 10)
TGCTGTTGACAGTGAGCGCCGAGTGTTATATTTGTGAATATAGTGAAGCC
ACAGATGTATATICACAAATATAACACTCGTTGCCTACTGCCTCGGA (SEQ ID NO: 11)
TGCTGTTGACAGTGAGCGCGCACCTCTGAACTTCAGAATATAGTGAAGCC
ACAGATGTATATTCTGAAGTTCAGAGGTGCATGCCTACTGCCTCGGA (SEQ ID NO: 12)
TGCTGTTGACAGTGAGCGAGGACGGGAGCTCCACTGTGAATAGTGAAGCC
ACAGATGTATTCACAGTGGAGCTCCCGTCCGTGCCTACTGCCTCGGA (SEQ ID NO: 13)
TGCTGTTGACAGTGAGCGCGGAGCTCACCTTTGATTACAATAGTGAAGCC
ACAGATGTATTGTAATCAAAGGTGAGCTCCTTGCCTACTGCCTCGGA (SEQ ID NO: 14)
TGCTGTTGACAGTGAGCGACCTCGGTATCTCTAAGAGGAATAGTGAAGCC
ACAGATGTATTCCTCTTAGAGATACCGAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 15)
TGCTGTTGACAGTGAGCGACGGGCCTTCGTGTACATCAATTAGTGAAGCC
ACAGATGTAATTGATGTACACGAAGGCCCGCTGCCTACTGCCTCGGA (SEQ ID NO: 16)
TGCTGTTGACAGTGAGCGCGCCCGTTACTGCTTCAGCAATTAGTGAAGCC
ACAGATGTAATTGCTGAAGCAGTAACGGGCATGCCTACTGCCTCGGA (SEQ ID NO: 17)
TGCTGTTGACAGTGAGCGCGCTTAGTATATGTGTACTTAATAGTGAAGCC
ACAGATGTATTAAGTACACATATACTAAGCTTGCCTACTGCCTCGGA (SEQ ID NO: 18)
TGCTGTTGACAGTGAGCGACCAAATCTTCAGGTGTTCAATTAGTGAAGCC
ACAGATGTAATTGAACACCTGAAGATTTGGGTGCCTACTGCCTCGGA (SEQ ID NO: 19)
TGCTGTTGACAGTGAGCGCCCTGATAGTCAGCATGCGAATTAGTGAAGCC
ACAGATGTAATTCGCATGCTGACTATCAGGTTGCCTACTGCCTCGGA (SEQ ID NO: 20)
TGCTGTTGACAGTGAGCGCGGGCTTTCATGTTATCTATAATAGTGAAGCC
ACAGATGTATTATAGATAACATGAAAGCCCATGCCTACTGCCTCGGA (SEQ ID NO: 21)
TGCTGTTGACAGTGAGCGACCTGAAGAGTCCAATGATGATTAGTGAAGCC
ACAGATGTAATCATCATTGGACTCTTCAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 22)
TGCTGTTGACAGTGAGCGCAGGATTCTGGCCAAACAGAAATAGTGAAGCC
ACAGATGTATTTCTGTTTGGCCAGAATCCTTTGCCTACTGCCTCGGA (SEQ ID NO: 23)
TGCTGTTGACAGTGAGCGACGGAGGGCCAAGCACTATAAATAGTGAAGCC
ACAGATGTATTTATAGTGCTTGGCCCTCCGGTGCCTACTGCCTCGGA (SEQ ID NO: 24)
TGCTGTTGACAGTGAGCGAACCGGTTAAGAGATTCTTATTTAGTGAAGCC
ACAGATGTAAATAAGAATCTCTTAACCGGTCTGCCTACTGCCTCGGA (SEQ ID NO: 25)
TGCTGTTGACAGTGAGCGCGGCATTATGCTTGTTGTACAATAGTGAAGCC
ACAGATGTATTGTACAACAAGCATAATGCCATGCCTACTGCCTCGGA (SEQ ID NO: 26)
TGCTGTTGACAGTGAGCGACCATTGTAAGTGTTGTTTCTATAGTGAAGCC
ACAGATGTATAGAAACAACACTTACAATGGGTGCCTACTGCCTCGGA (SEQ ID NO: 27)
TGCTGTTGACAGTGAGCGAGGAAAGAATATGCATAGAATATAGTGAAGCC
ACAGATGTATATTCTATGCATATTCTTTCCGTGCCTACTGCCTCGGA (SEQ ID NO: 28)
TGCTGTTGACAGTGAGCGCCGGAACTCAACCATTAAGCAATAGTGAAGCC
ACAGATGTATTGCTTAATGGTTGAGTTCCGTTGCCTACTGCCTCGGA (SEQ ID NO: 29)
TGCTGTTGACAGTGAGCGCGGGACTGCAATTATTCAGTATTAGTGAAGCC
ACAGATGTAATACTGAATAATTGCAGTCCCTTGCCTACTGCCTCGGA (SEQ ID NO: 30)
TGCTGTTGACAGTGAGCGACCAGTGGCCAGTTCACTGTATTAGTGAAGCC
ACAGATGTAATACAGTGAACTGGCCACTGGCTGCCTACTGCCTCGGA (SEQ ID NO: 31)
TGCTGTTGACAGTGAGCGAGCAGTTACATGCATACTTCAATAGTGAAGCC
ACAGATGTATTGAAGTATGCATGTAACTGCCTGCCTACTGCCTCGGA (SEQ ID NO: 32)
TGCTGTTGACAGTGAGCGCGCTCTGTAATCTCGTTTCAAATAGTGAAGCC
ACAGATGTATTTGAAACGAGATTACAGAGCATGCCTACTGCCTCGGA (SEQ ID NO: 33)
TGCTGTTGACAGTGAGCGCCCTCCTGATTATTCAGAATATTAGTGAAGCC
ACAGATGTAATATTCTGAATAATCAGGAGGTTGCCTACTGCCTCGGA (SEQ ID NO: 34)
TGCTGTTGACAGTGAGCGACGAAGAGCTCTTCTTTGATTATAGTGAAGCC
ACAGATGTATAATCAAAGAAGAGCTCTTCGCTGCCTACTGCCTCGGA (SEQ ID NO: 35)
TGCTGTTGACAGTGAGCGCGCCAGTAACAAGAAAGAGAAATAGTGAAGCC
ACAGATGTATTTCTCTTTCTTGTTACTGGCATGCCTACTGCCTCGGA (SEQ ID NO: 36)
TGCTGTTGACAGTGAGCGACCTGCATCATGACTCAGAATTTAGTGAAGCC
ACAGATGTAAATTCTGAGTCATGATGCAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 37)
TGCTGTTGACAGTGAGCGCCAACATTATGGGCATCGAGAATAGTGAAGCC
ACAGATGTATTCTCGATGCCCATAATGTTGTTGCCTACTGCCTCGGA (SEQ ID NO: 38)
TGCTGTTGACAGTGAGCGACGAGCTACAAAGCATGGGAAATAGTGAAGCC
ACAGATGTATTTCCCATGCTTTGTAGCTCGGTGCCTACTGCCTCGGA (SEQ ID NO: 39)
TGCTGTTGACAGTGAGCGACGTCCGCAGGAACTTAACTTATAGTGAAGCC
ACAGATGTATAAGTTAAGTTCCTGCGGACGCTGCCTACTGCCTCGGA (SEQ ID NO: 40)
TGCTGTTGACAGTGAGCGCCCTGAGGATAACTCAATATAATAGTGAAGCC
ACAGATGTATTATATTGAGTTATCCTCAGGTMCCTACTGCCTCGGA (SEQ ID NO: 41)
TGCTGTTGACAGTGAGCGCCCGGGAACAGAGAATGTTTAATAGTGAAGCC
ACAGATGTATTAAACATTCTCTGTTCCCGGTTGCCTACTGCCTCGGA (SEQ ID NO: 42)
TGCTGTTGACAGTGAGCGCGGTCTCAGGCGCCAGTGGAAATAGTGAAGCC
ACAGATGTATTTCCACTGGCGCCTGAGACCATGCCTACTGCCTCGGA (SEQ ID NO: 43)
TGCTGTTGACAGTGAGCGCGCCTAGTAAATTACAGAAGAATAGTGAAGCC
ACAGATGTATTCTTCTGTAATTTACTAGGCATGCCTACTGCCTCGGA (SEQ ID NO: 44)
TGCTGTTGACAGTGAGCGCGCTTCTAGGCAGAGTTGCTTATAGTGAAGCC
ACAGATGTATAAGCAACTCTGCCTAGAAGCTTGCCTACTGCCTCGGA (SEQ ID NO: 45)
TGCTGTTGACAGTGAGCGACGCATATATTTGCAGTATGAATAGTGAAGCC
ACAGATGTATTCATACTGCAAATATATGCGCTGCCTACTGCCTCGGA (SEQ ID NO: 46)
TGCTGTTGACAGTGAGCGACCGTCCCGTGGAGTCGCTAAATAGTGAAGCC
ACAGATGTATTTAGCGACTCCACGGGACGGGTGCCTACTGCCTCGGA (SEQ ID NO: 47)
TGCTGTTGACAGTGAGCGCGCCCTCCCTGTCCTTTCCAGATAGTGAAGCC
ACAGATGTATCTGGAAAGGACAGGGAGGGCTTGCCTACTGCCTCGGA (SEQ ID NO: 48)
TGCTGTTGACAGTGAGCGCCGCCAGCCTTCGCTTCTGAAATAGTGAAGCC
ACAGATGTATTTCAGAAGCGAAGGCTGGCGTTGCCTACTGCCTCGGA (SEQ ID NO: 49)
TGCTGTTGACAGTGAGCGCGAGCTTCATGGGATTGGTAAATAGTGAAGCC
ACAGATGTATTTACCAATCCCATGAAGCTCATGCCTACTGCCTCGGA (SEQ ID NO: 50)
TGCTGTTGACAGTGAGCGAACCTTTCCAGCCATAGAGATTTAGTGAAGCC
ACAGATGTAAATCTCTATGGCTGGAAAGGTGTGCCTACTGCCTCGGA (SEQ ID NO: 51)
TGCTGTrGACAGTGAGCGCGCTTTCAAGCTCATCTGTTATTAGTGAAGCC
ACAGATGTAATAACAGATGAGCTTGAAAGCTTGCCTACTGCCTCGGA (SEQ ID NO: 52)
TGCTGTTGACAGTGAGCGAACAGTTGGATTCTTTAGAGAATAGTGAAGCC
ACAGATGTATTCTCTAAAGAATCCAACTGTCTGCCTACTGCCTCGGA (SEQ ID NO: 53)
TGCTGTTGACAGTGAGCGACGAGAGAGTTAGCTGACTTTATAGTGAAGCC
ACAGATGTATAAAGTCAGCTAACTCTCTCGGTGCCTACTGCCTCGGA (SEQ ID NO: 54)
TGCTGTTGACAGTGAGCGACCTGATTATATCCAGTAACACTAGTGAAGCC
ACAGATGTAGTGTTACTGGATATAATCAGGGTGCCTACTGCCTCGGA (SEQ ID NO: 55)
TGCTGTTGACAGTGAGCGCGCCCAAGGTCAAGGAGATTATTAGTGAAGCC
ACAGATGTAATAATCTCCTTGACCTTGGGCTTGCCTACTGCCTCGGA (SEQ ID NO: 56)
TGCTGTTGACAGTGAGCGCGGCATCCACTGTGAATGATAATAGTGAAGCC
ACAGATGTATTATCATTCACAGTGGATGCCATGCCTACTGCCTCGGA (SEQ ID NO: 57)
TGCTGTTGACAGTGAGCGCGCTGTCTCTCTTTGATGGAATTAGTGAAGCC
ACAGATGTAATTCCATCAAAGAGAGACAGCATGCCTACTGCCTCGGA (SEQ ID NO: 58)
TGCTGTTGACAGTGAGCGCGCCTGCAAGGACATGGTTAAATAGTGAAGCC
ACAGATGTATTTAACCATGTCCTTGCAGGCTTGCCTACTGCCTCGGA (SEQ ID NO: 59)
TGCTGTTGACAGTGAGCGACGCACCTACTCCAAGTTCAAATAGTGAAGCC
ACAGATGTATTTGAACTTGGAGTAGGTGCGCTGCCTACTGCCTCGGA (SEQ ID NO: 60)
TGCTGTTGACAGTGAGCGCCGAGTCTGGCTTTGAGAGTTATAGTGAAGCC
ACAGATGTATAACTCTCAAAGCCAGACTCGTTGCCTACTGCCTCGGA (SEQ ID NO: 61)
TGCTGTTGACAGTGAGCGAGCCATGGAAATGCTATCAATGTAGTGAAGCC
ACAGATGTACATTGATAGCATTTCCATGGCCTGCCTACTGCCTCGGA (SEQ ID NO: 62)
TGCTGTTGACAGTGAGCGCAGATGGAAGATGATATAGATATAGTGAAGCC
ACAGATGTATATCTATATCATCTTCCATCTTTGCCTACTGCCTCGGA (SEQ ID NO: 63)
TGCTGTTGACAGTGAGCGCCCAAATCTTCTCCTGTCAGTATAGTGAAGCC
ACAGATGTATACTGACAGGAGAAGATTTGGATGCCTACTGCCTCGGA (SEQ ID NO: 64)
TGCTGTTGACAGTGAGCGAAGAGATTATTTCTCAAGATGATAGTGAAGCC
ACAGATGTATCATCTTGAGAAATAATCTCTCTGCCTACTGCCTCGGA (SEQ ID NO: 65)
TGCTGTTGACAGTGAGCGCAGAGGGAAAGTGTATGATAAATAGTGAAGCC
ACAGATGTATTTATCATACACTTTCCCTCTTTGCCTACTGCCTCGGA (SEQ ID NO: 66)
TGCTGTTGACAGTGAGCGCGGAAAGAACGGAAATCTTAAATAGTGAAGCC
ACAGATGTATTTAAGATTTCCGTTCTTTCCATGCCTACTGCCTCGGA (SEQ ID NO: 67)
TGCTGTTGACAGTGAGCGCGCAGTTATGCTCTIAATGCTTTAGTGAAGCC
ACAGATGTAAAGCATTAAGAGCATAACTGCTTGCCTACTGCCTCGGA (SEQ ID NO: 68)
TGCTGTTGACAGTGAGCGCGCATGCATGACTTTAATCTTATAGTGAAGCC
ACAGATGTATAAGATTAAAGTCATGCATGCTTGCCTACTGCCTCGGA (SEQ ID NO: 69)
TGCTGTTGACAGTGAGCGAAACATGTGTAAGCTGCGGCCCTAGTGAAGCC
ACAGATGTAGGGCCGCAGCTTACACATGTTCTGCCTACTGCCTCGGA (SEQ ID NO: 70)
TGCTGTTGACAGTGAGCGAAAGGATGTGGTCCGAGTGTGGTAGTGAAGCC
ACAGATGTACCACACTCGGACCACATCCTTCTGCCTACTGCCTCGGA (SEQ ID NO: 71)
TABLE-US-00004 TABLE 3 qRT PCR primers used qRT-PCR SEQ SEQ primers
Forward ID ID Reverse ActB TGAAGTGTGACGTGGACATC 72 73
GGAGGAGCAATGATCTTGAT DNMT1 GAATCTCTTGCACGAATTTCTGC 74 75
CATGAGCACCGTTCTCCAAGG DNMT3A CCGATGCTGGGGACAAGAAT 76 77
CCCGTCATCCACCAAGACAC Eed GCGGAGGAATATGTCCGAGAG 78 79
AGAGGTCTGGATTGCTGTTCT ezh2 ATGGGCCAGACTGGGAAGAA 80 81
TGGAAAATCCAAGTCACTGGTC suz12 AGTTGTCCAATAAGGCAAGTTCC 82 83
ACGAGTCACTCTAAATAGCAACG hG9a CATTTCCGCATGAGTGATGATGT 84 85
CAGGCCACCTCCTGAGTTC MBD1 TTACCCCAGGTGAAGCAAGAG 86 87
CCAATACGGGAGAAGTCAGGAC MBD2 CCCACAACGAATGAATGAACAGC 88 89
TGAAGACCTTTGGGTAGTTCCA MBD3 CTGGGAGAGGGAAGAAGTGC 90 91
CGGAAGTCGAAGGTGCTCAG MeCP2 AGCAGAGACATCAGAAGGGTC 92 93
CGGCCAGATTTCCTTTGCTT Dot1L GCTGCCGGTCTACGATAAACA 94 95
AGCTTGAGATCCGGGATTTCT Suv39h1 ATATCCAGACTCAGAGAGCACC 96 97
CAGCTCCCTTTCTAAGTCCTTG Setdb1 TGGATGACAAAAGATGTGAGTGG 98 99
CCATATTTGGACGTGTCCTGAG smyd2 GCCCTACAGTAAGCACTATCCT 100 101
AGTCTCCCTAGCTTCAACCAC bmi1 CCACCTGATGTGTGTGCTTTG 102 103
TTCAGTAGTGGTCTGGTCTTGT ring1 TCAGAACTCATGTGCCCTATCT 104 105
GCAGGTAGGACACTCCTTGT yy1 AGAAGAGCGGCAAGAAGAGTT 106 107
CAACCACTGTCTCATGGTCAATA ezh1 TCAATGAAGCCTGTGAGTGGA 108 109
CAATGCAACTGTGTTCAGTGAC Nr2f1 CAGGCCAGTACGCACTCAC 110 111
TGTTCTCGATGCCCATAATGTTG MBD4 CCCCACCGTCACCTCTAGT 112 113
GTAGCACCAAACTGAGCAGAA suv39h2 GGCTAAACAAAGGATAGCTCTGC 114 115
TGAAAGAAGCAATCTGTGCATGA ehmt1 CAACGCCGTAGACAGCGAG 116 117
CTCCCCGTCCTTATTGTCGAG RunX1 CCCTAGGGGATGTTCCAGAT 118 119
TGAAGCTTTTCCCTCTTCCA AFP AGCTTGGTGGTGGATGAAAC 120 121
CCCTCTTCAGCAAAGCAGAC GATA4 CTAGACCGTGGGTTTTGCAT 122 123
TGGGTTAAGTGCCCCTGTAG Brachury ACCCAGTTCATAGCGGTGAC 124 125
CAATTGTCATGGGATTGCAG Ncam ATGGAAACTCTATTAAAGTGAACCTG 126 127
TAGACCTCATACTCAGCATTCCAGT
Example 2: Screening for Inhibitors and Enhancers of
Reprogramming
[0354] To examine the influence of modifiers on somatic cell
reprogramming, a loss-of-function approach was employed to
interrogate the role of 22 select genes in DNA and histone
methylation pathways. FIG. 1 shows a non-limiting outline of a
screen for inhibitors and enhancers of reprogramming. FIG. 1A
illustrates a protocol for screening shRNA pools in iPS cell
generation.
[0355] In one example, a pool of 3 hairpins was tested for each of
22 target genes and knockdown efficiencies were observed of >60%
for 21 out of 22 targets by qRT-PCR (FIG. 6). FIG. 6 shows the
measurement of knockdown efficiency of the indicated shRNA pools in
dH1f cells measured by quantitative reverse transcription PCR
(qRT-PCR). Expression values for each gene were normalized to those
measured in control shRNA fibroblasts. Human fibroblasts were used
as a system in which to study reprogramming. Human fibroblasts were
differentiated from H1 human embryonic stem cells (dH1fs), which
have a higher reprogramming efficiency than primary adult dermal
fibroblasts, thus yielding a reproducible baseline.sup.7,8. dH1fs
were infected with shRNA pools (at high multiplicity of infection
to ensure all cells received an shRNA vector) followed by
super-infection with reprogramming vectors expressing Oct4, Sox2,
Klf4 and c-Myc (OSKM), and identified the resulting iPSCs by
Tra-1-60 staining (FIG. 1A).
[0356] Eight shRNA pools negatively affected reprogramming (FIG.
1B). FIG. 1B shows the number of Tra-1-60+ colonies, as quantified
by ImageJ, 21 days after OSKM transduction of 25,000 dH1f cells
previously infected with pools of shRNAs against the indicated
genes (3 vectors per gene). Corresponding images of representative
Tra-1-60-stained reprogramming wells are shown in the lower panel.
The dotted lines indicate 3 standard deviations from the mean
number of colonies detected in control shRNA wells. Among the
target genes were Pou5F1 (Oct4, included as a control), and Ehmt1
and SetDB1, two H3K9 methyltransferases whose histone mark was
associated with transcriptional repression. The remaining five
shRNA pools all targeted components of polycomb repressive
complexes (PRC), which are major mediators of gene silencing and
heterochromatin formation. Inhibition of PRC1 (Bmi1, Ring1) and
PRC2 components (Ezh2, Eed, Suz12) did not have a significant
effect on fibroblast proliferation but resulted in significantly
fewer iPSC colonies than the control shRNA (FIG. 1C, FIG. 7). FIG.
1C shows the validation of primary screen hits that decrease
reprogramming efficiency. Quantification of Tra-1-60+ iPSC colonies
expressed as fold-change relative to control shRNA. Data correspond
to the average and s.e.m. *P<0.05, **P<0.01 compared to
control shRNA-expressing fibroblasts. Representative
Tra-1-60-stained reprogramming wells are shown in the lower panel.
FIG. 7 shows cell proliferation upon PRC1/2 knockdown. Depicted are
relative cell growth rates of fibroblasts infected with the
indicated shRNA vectors targeting PRC1 and PRC2 components.
Relative control shRNA infected fibroblasts 5 days after shRNA
transduction (n=3; error bars, .+-.s.e.m).
[0357] This is of particular significance given the recent finding
that PRC2 component Ezh2 is necessary for fusion-based
reprogramming.sup.9. Thus, interference with chromatin-modifying
enzymes that mediate repressive chromatin domains reduces
reprogramming and is consistent with the importance of gene
silencing of the somatic cell program during generation of
iPSC.
Example 3: Suppression of Dot1L Expression Enhances Reprogramming
Efficiency and Substitutes for Klf4 and Myc
[0358] In contrast to genes whose functions appear to be required
for reprogramming, inhibition of three genes enhanced
reprogramming:YY1, Suv39H1, and Dot1L (FIG. 1D). FIG. 1D shows the
validation of primary screen hits that increase reprogramming
efficiency. Quantification of Tra-1-60+ iPSC colonies expressed as
fold-change relative to control shRNA. Data correspond to the
average and s.e.m. *P<0.05, **P<0.01 compared to control
shRNA-expressing fibroblasts. Representative Tra-1-60-stained
reprogramming wells are shown in the lower panel. YY1 is a
transcription factor that activates or represses transcription in a
context-dependent manner.sup.10,11, whereas Suv39H1 is a histone
H3K9 methyltransferase implicated in heterochromatin
formation.sup.12. Enzymes that modify H3K9 were associated with
both inhibition and enhancement of reprogramming in this study,
which suggested that unraveling the precise mechanisms for their
effects might be challenging. The studies focused on Dot1L, a
histone H3 Lysine 79 methyltransferase whose role in reprogramming
has not been previously studied.sup.13. Three Dot1L-targeting
hairpin vectors were evaluated independently for knockdown
efficiency and utilized the two shRNAs that resulted in the most
significant downregulation of Dot1L and concomitant decrease in
global H3K79 levels (FIG. 8). FIG. 8 shows Dot1L mRNA and total
H3K79me2 levels under knock-down conditions. FIG. 8A shows the
knock-down efficiency of individual hairpins targeting Dot1L as
measured by qRT-PCR. FIG. 8B shows the total H3K79me2 levels in
control and shDot1L infected fibroblasts assessed by western
blotting 6 days after shRNA transduction.
[0359] Following infection with OSKM, fibroblasts expressing Dot1L
shRNA formed significantly more iPSC colonies than control
fibroblasts, and the degree of enhancement in reprogramming
correlated with the degree of Dot1L knockdown (FIG. 2A, FIG. 7A).
In addition, the enhanced reprogramming phenotype elicited by Dot1L
knockdown could be reversed by overexpressing an shRNA-resistant
wildtype Dot1L, but not by a catalytically-inactive Dot1L,
indicating that inhibition of catalytic activity of Dot1L is key to
reprogramming.sup.14 (FIG. 2A). FIG. 2 shows that Dot1L inhibition
enhances reprogramming efficiency and substitutes for Klf4 and Myc.
FIG. 2A shows the fold change in the reprogramming efficiency of
dH1f cells infected with 2 independent Dot1L shRNAs or co-infected
with shRNA-2 and a vector expressing an shRNA-resistant wild-type
or catalytically dead mutant Dot1L. Data correspond to the average
and s.e.m.; n-independent experiments. *P<0.01 control
shRNA-expressing fibroblasts.
[0360] To further document the competitive advantage of Dot1L
shRNA-expressing fibroblasts (shDot1L) during reprogramming,
experiments were performed in which fibroblasts infected with Dot1L
shRNA or a control vector were differentially labeled with either
tdTomato or GFP and co-cultured prior to superinfection with OSKM
(FIG. 9).
[0361] FIG. 9 shows same-well reprogramming of admixed control and
Dot1L inhibited cell populations. Control and shDot1L dH1f cells
were infected with either a GFP- or tdTomato-expressing lentivirus,
and mixed in a 1:1 ratio prior to OSKM infection. The middle panel
shows a quantification of morphologically discernible iPS-like
colonies that were scored on the basis of the fluorescent protein
expression. Data correspond to the mean ratio of GFP+ colonies to
Tomato+ colonies in each co-mixture and s.d. (n=2). Lower panels
show representative images of either GFP or tdTomato expressing
iPSC colonies (arrowheads) derived from the indicated co-mixed cell
populations.
[0362] The emerging iPSC colonies were then scored for tdTomato or
GFP fluorescence, which indicated their cell of origin. Co-mixture
of tdTomato- and GFP-labeled control cells resulted in a 1:1 ratio
of red and green iPSC colonies indicating that labeling per se did
not affect relative reprogramming efficiency. In contrast, wells
containing co-mixtures of tdTomato-shDot1L and GFP-shCntrl cells
generated 3-fold more red colonies than green colonies, indicating
that Dot1L-inhibited cells reprogram more efficiently under
identical conditions (FIG. 9). The experiments were repeated with
additional strains of human fibroblasts, and a 3-fold and 6-fold
increases in reprogramming efficiency for IMR-90 and MRC-5 cells
was observed, respectively, indicating that findings with dH1fs
were broadly applicable to other human fibroblast strains (FIG.
2B). FIG. 2B shows the number of Tra-1-60+ colonies derived from
50,000 control and Dot1L shRNA-expressing IMR-90 and MRC5 human
diploid fibroblasts. Data correspond to the average and s.d.
*P<0.05 compared to control shRNA-expressing fibroblasts (n=2).
Lower panels show representative Tra-1-60-stained wells from the
indicated conditions. iPS cells generated through Dot1L inhibition
exhibited a morphology characteristic of embryonic stem cells and
stained positively for SSEA4, SSEA3, Tra-1-81, Oct4 and Nanog (FIG.
2C). FIG. 2C shows immunohistochemistry expression analysis of
pluripotency markers, SSEA4, SSEA3, Oct4, Nanog and Tra-1-81 in
expanded shDot1L-iPS colonies derived from 4-factor (OSKM) and
2-factor (OS) reprogramming.
[0363] Additionally, these cells were able to differentiate into
all three embryonic germ layers in vitro and in teratomas (FIG. 2D
and FIG. 10A), indicating that iPSCs generated following Dot1L
inhibition display all of the hallmarks of pluripotency. FIG. 2D
shows hematoxylin and eosin staining of representative OS-, OSM-,
OSKM-shDot1L-iPS teratomas exhibiting ectoderm (neural rosettes),
mesoderm (cartilage), and endoderm (gut-like endothelium)
differentiation. FIG. 10 shows a characterization of shDot1L iPS
cells. FIG. 10A shows the downregulation of endogenous OCT4 mRNA,
as well as the upregulation of differentiation markers GATA4 and
AFP (endoderm), RUNX1 and Brachury (mesoderm), and NCAM (ectoderm)
in day 8 EBs derived from shCntrl-OSKM, shDot1L-OSKM, and
shDot1L-OS iPS cells as judged by qRT-pCR. Expression values are
represented relative to undifferentiated controls.
[0364] To assess whether Dot1L inhibition could replace any of the
4 exogenous reprogramming factors, shDot1L and shCntrl fibroblasts
were infected with 3 factors, omitting one factor at a time. In the
absence of Oct4 or Sox2 no iPSC colonies emerged in both types of
fibroblasts. When either Klf4 or Myc was omitted, shDot1L
fibroblasts were able to give rise to robust numbers of Tra-1-60
positive colonies while control cells yielded very few colonies as
reported previously.sup.3. It was examined whether Dot1L
suppression would suffice to reprogram fibroblasts in the absence
of both Klf4 and c-Myc. Indeed, shDot1L fibroblasts infected with
only Oct4 and Sox2 gave rise to Tra-1-60-positive colonies, whereas
control fibroblasts did not (FIG. 2E). FIG. 2E shows Tra-1-60
staining of whole plates of shCntrl and shDot1L fibroblasts 21 days
after reprogramming in the absence of each factor or both Klf4 and
c-Myc. Two-factor iPSCs derived by Dot1L inhibition exhibited a
typical ES cell morphology and had silenced GFP expression from the
exogenous reprogramming vectors (FIG. 10B). FIG. 10B shows the
morphology changes and retroviral silencing in colonies emerging in
4-factor (OSKM) and 2-factor (OS) reprogramming of control and
Dot1L-inhibited cells. Green fluorescence indicates persistent GFP
expression derived from the pMIG reprogramming vectors. Note that
after 2-factor (OS) transduction, colonies arising from only
shDot1L or iDot1L-treated fibroblasts silence the transgenes as
indicated by the absence of GFP expression, whereas shCntrl
fibroblasts yield transformed cell clusters that retain factor
expression. PCR on genomic DNA isolated from expanded colonies
indicated the presence of Oct4 and Sox2, but not the Klf4 and c-Myc
transgenes (FIG. 10C). FIG. 10C shows PCR-based detection of
transgenes in the genomic DNA of 2- and 3-factor iPSC lines derived
from shDot1L cells with primers designed to amplify either
endogenous loci or the virally-encoded transgenes. H9ES and dH1f
cells served as negative controls for the transgenes and vector
plasmids and OKSM-derived iPSC lines were used as positive
controls.
[0365] The OS-shDot1L-iPS cells had all of the hallmarks of
pluripotency as gauged by endogenous pluripotency factor expression
and the ability to form all three embryonic germ layers in vitro
and in teratomas (FIG. 2C, 2D and FIG. 10A). These findings
indicate that Dot1L inhibition can reprogram cells to pluripotency
in the presence of only Oct4 and Sox2.
[0366] It was examined whether the cellular mechanisms by which
Dot1L inhibition promotes reprogramming. It was observed that in
established human iPS clones derived from shDot1L fibroblasts,
Dot1L inhibition was no longer evident, reflecting the known
silencing of retroviruses that occurs during reprogramming (FIG.
11A).
FIG. 11 shows Dot1L knockdown dynamics during reprogramming. FIG.
11A shows Dot1L mRNA levels in iPSCs derived from shCntrl and
shDot1L iPSCs relative to levels in the starting control dh1F cells
as measured by qRT-PCR. FIG. 11B shows Dot1L mRNA levels in shDot1L
cells measured by qRT-PCR every 3 days after OSKM expression
normalized to levels observed in control cells prior to OSKM
expression. FIG. 11C shows Dot1L mRNA levels in control cells
measured by qRT-PCR every 3 days after OSKM expression normalized
to levels observed in control cells prior to OSKM expression.
[0367] Examination of Dot1L knockdown levels during the course of
reprogramming revealed that silencing occurred by day 15 after OSKM
transduction (FIGS. 11B, C). Since Dot1L inhibition seemed to work
in the initial phases of reprogramming, it was examined if the
proliferation rates of shDot1L and shCntrl cells before and after
infection with the OSKM reprogramming factors and found them to be
comparable (FIGS. 12A and 12B). FIG. 12 shows the growth dynamics
of shDot1L cells pre- and post-OSKM transduction. FIG. 12A shows
the cumulative population doubling rates of shCntrl and shDot1L
cells over a period of 14 days prior to reprogramming (n=3; error
bars, .+-.s.e.m). FIG. 12B shows the relative cell growth rates of
shCntrl and shDot1L cells prior to and 6 days after OSKM
transduction (n-3; error bars, .+-.s.e.m).
[0368] There was also no difference in the number of senescent
cells, as gauged by senescence-associated .beta.-gal staining
following OSKM infection (FIG. 12C, data not shown). In addition,
the level of retroviral reprogramming factor expression was similar
between control and Dot1L-inhibited cells (FIG. 12C). FIG. 12C
shows qRT-PCR quantification of viral transcript levels using
transgene-specific primers in control or iDot1L treated dH1f cells
3 days after infection with OSM or OS expressing retroviruses.
[0369] Previous studies indicated that Dot1L null cells have
increased apoptosis and accumulation of cells in G2 phase.sup.13.
However, a significant increase in apoptosis or change in the cell
cycle profile of Dot1L knock-down fibroblasts was not observed
(FIG. 13). FIG. 13 shows apoptosis and a cell cycle profile of
Dot1L-inhibited cells. FIG. 13A shows the percentage of apoptotic
cells in the indicated cell populations 5 days after treatment
(iDot1L-10 um) or shRNA infection measured by PI/Annexin staining
(n=3; error bars, .+-.s.d.). FIG. 13B shows a cell cycle profile of
untreated or iDot1L (10 uM for 5 days) treated fibroblasts as
measured by PI staining.
[0370] It was also examined whether Dot1L knockdown, in addition to
increasing reprogramming efficiency, accelerated the kinetics of
reprogramming. FIG. 14 shows the kinetics of iPSC colony formation
upon Dot1L knockdown. FIG. 14A shows the number of Tra-1-60+ iPSC
colonies generated by shCntrl and shDot1L dH1f cells on Day 14 and
Day 21 of reprogramming (n=3; error bars, .+-.s.e.m). FIG. 14B
shows the number of Tra-1-60+ cell clusters per field as judged by
live-staining and imaging on Day 10 after OSKM transduction (n=12
fields, .+-.s.e.m). Immunofluorescence analysis during
reprogramming revealed significantly greater numbers of
Tra-1-60-positive cell clusters on day 10 (FIG. 14A) and
recognizable Tra-1-60-positive colonies on days 14 and 21 in
shDot1L cultures (FIG. 14B), indicating that the emergence of iPSC
colonies is accelerated upon Dot1L inhibition. FIG. 12C shows
qRT-PCR quantification of viral transcript levels using
transgene-specific primers in control or iDot1L treated dH1f cells
3 days after infection with OSM or OS expressing retroviruses.
[0371] When the reprogramming experiments were extended by 10 more
days (at which time the already-formed iPSC colonies begin to
differentiate), shDot1L cells still yielded more iPSC colonies than
controls (FIG. 14C). FIG. 14C shows a modified schema for testing
the reprogramming efficiency of 10,000 OSKM infected dH1f cells
without replating onto MEFs and a longer incubation period of 30
days. The graph on the right shows the number of Tra-1-60+ iPSC
colonies generated through this modified protocol (n=2; error bars,
.+-.s.e.m) When the reprogramming experiments were extended by 10
more days (at which time the already-formed iPSC colonies begin to
differentiate), shDot1L cells still yielded more iPSC colonies than
controls (FIG. 14C). Taken together, Dot1L inhibition both
accelerates the emergence of iPSCs and increases the total yield of
the reprogramming process.
Example 4: Small Molecule Inhibitor of Dot1L Enhances Reprogramming
and Replaces Klf4 and Myc
[0372] In addition to the shRNA mediated knockdown of Dot1L, a
small molecule inhibitor of Dot1L catalytic activity was used to
further validate the findings.sup.15. This inhibitor (EPZ004777,
referred to as iDot1L and shown below) abrogated H3K79 methylation
robustly at 1 uM to 10 uM concentration range and led to 3-4 fold
enhancement of reprogramming of human fibroblasts (FIG. 3A, FIG.
15).
##STR00173##
FIG. 3 shows that the small molecule inhibitor of Dot1L increases
efficiency and substitutes for Klf4 and Myc. FIG. 3A shows the fold
change in the reprogramming efficiency of dH1f cells treated with
iDot1L at the indicated concentrations for 21 days. Data correspond
to the average and s.d.; n=3. *P<0.001 compared untreated
fibroblasts. FIG. 15 shows that the small molecule inhibitor of
Dot1L increases reprogramming efficiency. FIG. 15A shows total
H3K79me2 levels in fibroblasts treated with EPZ004777 (iDot1L) at
the indicated concentrations for 5 days as assessed by western
blotting. FIG. 15B shows representative images of control or iDot1L
treated reprogramming plates stained with Tra-1-60 on day 21.
[0373] Knockdown of Dot1L protein in combination with inhibitor
treatment did not result in a further increase of reprogramming
efficiency thereby reinforcing the previous observation that
inhibition of catalytic activity of Dot1L is key to reprogramming
(FIG. 3B). FIG. 3B shows the number of Tra-1-60+ colonies 21 days
after OSKM transduction of 25,000 control fibroblasts, iDot1L
treated fibroblasts (10 uM) and iDot1L treated fibroblasts
expressing the Dot1L shRNA. Data correspond to average and s.d
(n=2). Representative Tra-1-60-stained reprogramming wells are in
the lower panel. Similar to shRNA mediated suppression of Dot1L,
chemical inhibition of Dot1L allowed reprogramming in the absence
of either Klf4 or Myc and was able to replace both of these
exogenous factors during reprogramming (FIG. 3C). FIG. 3C shows
Tra-1-60 staining of whole plates of untreated or iDot1L(10 uM)
fibroblasts 21 days after reprogramming in the absence of Klf4,
c-Myc or both. Thus, it was possible to generate two-factor iPSCs
robustly and reproducibly either by shRNA mediated suppression of
Dot1L or chemical inhibition of its methyl transferase
activity.
[0374] It was assessed whether these observations can be extended
to the murine system. It was observed that, similar to human
fibroblasts, iDot1L treatment led to 3-fold enhancement of
reprogramming of Oct4-GFP MEFs (FIG. 3D). FIG. 3D shows the number
of AP+ colonies derived from OSKM transduced untreated or iDot1L
treated (10 um) Oct4-GFP Mefs. Data correspond to the average and
s.d.; n=4. *P<0.001 compared untreated MEFs. Representative
AP-stained wells and GFP-positive iPS colonies derived from each
condition are shown in the lower panel.
[0375] The colonies resulting from Dot1L inhibition were
GFP-positive indicating the activation of the endogenous Oct4 gene.
Furthermore, reprogramming of tail-tip fibroblasts (TTFs) derived
from a conditional knockout Dot1L mouse strain yielded
significantly more iPS colonies upon Cre mediated excision of Dot1L
(FIG. 16A). FIG. 16 shows the reprogramming of Dot1L conditional
knockout tail-tip fibroblasts TTFs. FIG. 16A shows an example of
fold change in Alkaline Phosphatase positive (AP+) colonies upon
OSKM transduction of TTFs derived from Dot1L fl/fl mice. TTFs were
first infected with a MSCV-CRE-ER vector. Vehicle(Ethanol) or 4-OHT
was added to cultures at the same time as OSKM infection. Data
correspond to average and s.e.m. (n-6). The complete excision of
both floxed Dot1L alleles in iPSC clones derived from homozygous
TTFs was confirmed by genomic PCR (FIG. 16B). FIG. 16B shows
genomic PCR to detect wildtype, floxed or deleted Dot1L alleles in
iPS colonies derived from reprogramming of the indicated starting
Cre-expressing TTFs in the presence or absence of 4OHT. Dot1L
inhibition also increased reprogramming efficiency of inducible
iPS-derived "secondary" MEFs (FIG. 16C). FIG. 16C shows the number
of AP+ colonies upon doxycycline addition to iPS-derived secondary
MEFs in the presence of iDot1L (10 um). Data correspond to average
and s.d (n=2).
[0376] Taken together these results demonstrate that Dot1L
inhibition enhances reprogramming of mouse cells as well.
[0377] To further dissect out the crucial time window for Dot1L
inhibition, human fibroblasts undergoing reprogramming were treated
with iDot1L at 1 week intervals. It was observed that Dot1L
inhibition either in the first or the second week was sufficient to
enhance reprogramming whereas pretreatment for 5 days prior to OSKM
transduction had no effect (FIG. 3E). FIG. 3E shows the number of
Tra-1-60+ colonies 21 days after OSKM transduction of 25,000
untreated fibroblasts, and fibroblasts treated with iDot1L(10 uM)
for the indicated time periods during reprogramming. Data
correspond to average and s.d (n=3). Representative
Tra-1-60-stained reprogramming wells are shown in the upper panel.
These. findings indicate that Dot1L inhibition at early to middle
stages in the reprogramming process facilitates the acquisition of
pluripotency.
Example 5: Dot1L Inhibition During Reprogramming Induces Nanog and
Lin28 Expression
[0378] Since the effects of Dot1L inhibition were evident early in
the reprogramming process, it was investigate if gene expression
changes in Dot1L-inhibited cells soon after transduction with
reprogramming factors could reveal insights into the molecular
mechanisms involved. A global gene-expression analyses on control
and shDot1L fibroblasts prior to and 6 days after OSKM transduction
was performed along with cells that were treated with iDot1L.
Although thousands of genes were induced or repressed upon OSKM
expression, relatively few genes were differentially expressed in
shDot1L cells on Day 6 of reprogramming (22 up, 23 down;). At this
time point, inhibitor treated cells exhibited broader gene
expression changes (405 up and 175 down), presumably due to more
complete inhibition of K79me2 levels.
[0379] To understand the mechanism by which Dot1L inhibition
substitutes for Klf4, gene expression analyses on control and
shDot1L cells were performed upon 3-factor infection with OSM.
While 94 genes were differentially upregulated in shDot1L cells in
the absence of Klf4, the intersection of this set of genes with the
set differentially upregulated in 4-factor reprogramming of shDot1L
and inhibitor treated cells yielded only 5 common genes (FIG. 4A,
4B). These five genes were Lefty1, Lin28A, Lum, Upp1 and Nanog.
Nanog and Lin28 were upregulated in all three instances of Dot1L
inhibition. These two genes are part of the core pluripotency
network of human ES cells.sup.16,17,18 and have previously been
shown to be sufficient for reprogramming human fibroblasts into
iPSC when used in combination with Oct4 and Sox2.sup.4.
[0380] FIG. 4 shows that Nanog and Lin28 are important for the
enhancement of reprogramming by Dot1L inhibition. FIG. 4A shows the
overlap of differentially upregulated genes in shDot1L cells 6 Days
post-OSKM and OSM transduction with the genes upregulated in OSKM
transduced iDotL-treated cells. FIG. 4B shows heat maps showing
differential expression levels of commonly upregulated genes in
OSKM transduced Dot1L-inhibited cells.
[0381] The possibility was explored that Nanog and Lin28
upregulation was responsible for the enhanced reprogramming
observed following Dot1L inhibition and validated their
upregulation in shDot1L fibroblasts compared to control fibroblasts
6 days after transduction with reprogramming factors (FIG. 4C).
FIG. 4C shows the expression levels of Nanog, Lin28, Rex1, and
Dnmt3b in shDot1L cells 6 days post-OSM or -OS transduction
relative to shCntrl cells as measured by qRT-PCR. Interestingly at
this early time-point, no upregulation of Rex1 and Dnmt3b was
observed, two other well-characterized pluripotency genes,
suggesting that Dot1L inhibition does not broadly upregulate the
pluripotency network. Suppression of either Nanog or Lin28
expression using lentiviral shRNAs abrogated the 2-factor
reprogramming of shDot1L fibroblasts, indicating the essential
roles of Nanog and Lin28 in this process (FIG. 4D, FIG. 17B). FIG.
4D shows the number of Tra-1-60+ iPSC colonies upon knockdown of
Nanog or Lin28 in 2-factor reprogramming of shDot1L cells (n=2;
error bars, +s.e.m). FIG. 17 shows an example of the knockdown
efficiency of Nanog and Lin28 during 2-factor reprogramming. FIG.
17A shows Lin28 and Nanog mRNA levels in shCntrl, shDot1L or
iDot1L-treated cells prior to reprogramming. Expression values are
represented relative to HIES cells. FIG. 17B shows Lin28 and Nanog
mRNA levels in shDot1L cells expressing Lin28 shRNA or Nanog shRNA
6-days after OS transduction. Expression values are represented
relative to shDot1L cells prior to OS infection.
[0382] It was hypothesized that if Nanog and Lin28 upregulation is
the major mechanism by which Dot1L inhibition enhances
reprogramming, then inclusion of Nanog and Lin28 in the OSKM
reprogramming cocktail would not confer any additional enhancement
to shDot1L cells. In fact, no significant difference in the number
of colonies generated between 4-factor (OSKM) and 6-factor (OSKMNL)
reprogramming of shDot1L cells was observed, although the colonies
were larger in the latter conditions (FIG. 4E, FIG. 17C). FIG. 4E
shows the fold-change in Tra-1-60+ iPSC colonies in 4-factor (OSKM)
and 6-factor (OSKM+Nanog+Lin28) reprogramming of shCntrl and
shDot1L fibroblasts relative to control 4-factor reprogramming of
shCntrl cells. Plates were stained on Day 20 of reprogramming.
Corresponding images of representative Tra-1-60 stained
reprogramming wells are shown above (n=2; error bars, .+-.s.e.m).
FIG. 17C shows the average Tra-1-60 positive colony size in
4-factor (OSKM) and 6-factor (OSKM+Nanog+Lin28) reprogramming of
shCntrl and shDot1L fibroblasts relative to control 4-factor
reprogramming of shCntrl cells as shown in FIG. 4C. In control
fibroblasts, Nanog and Lin28 expression did enhance 4-factor
reprogramming by 2.4 fold, and thus significantly phenocopied Dot1L
inhibition (FIG. 4E). Taken together, these data indicate that
Dot1L inhibition requires the action of both Nanog and Lin28 to
substitute for Klf4 and c-Myc and enhance reprogramming.
Example 6: Genome-Wide Analysis of H3K79Me2 Marks During
Reprogramming
[0383] To gain insight into the genome-wide chromatin changes that
are facilitated by Dot1L inhibition during reprogramming, ChIP-seq
for H3K79me2 and H3K27me3 in human ES cells as well as fibroblasts
undergoing reprogramming with or without the iDot1L treatment were
performed (FIG. 18). FIG. 18 shows a Chip-seq experimental design.
FIG. 18A shows dH1f fibroblasts that were pre-treated with iDot1L
for 5 days at 10 uM (Day 0) and then infected with OSKM. 6 days
later, cells were harvested for the ChIP (Day 6). FIG. 18B shows
the number of raw and aligned reads from Illumina sequencing for
each ChIP sample. In both ES cells and fibroblasts, the presence
H3K27me3 and H3K79me2 was mutually exclusive (FIG. 19). FIG. 19
shows the relationship between H3K79me2 and H3K27me3. FIG. 19A
shows a genome-wide representation of the relation between H3K79me2
and H3K27me3 in ES cells. FIG. 19B shows a genome-wide
representation of the relation between H3K79me2 and H3K27me3 in
Fibroblasts. Genes marked by H3K79me2 specifically in ES cells were
pluripotency factors, their downstream targets and genes involved
in epithelial cell adhesion such as CDH1 (165 genes, FIG. 20). In
contrast, genes marked by H3K79me2 specifically in fibroblasts were
significantly enriched in gene sets associated with epithelial to
mesenchymal transitions (EMTs) (119 genes, FIG. 20). FIG. 20 shows
genes marked with K79me2 specifically in fibroblasts, in ESCs and
in both cell types. Genes that have 10-fold or more H3K79me2 in
fibroblasts than in ES cells were designated as
Fibroblasts-specific K79me2 marked genes (upper left dotted line).
Genes that have 10-fold or more H3K79me2 in ES cells than in
fibroblasts were designated as ES-specific K79me2 marked genes
(lower right dotted line). Top 5 gene sets that overlap with these
set of genes are indicated in the boxes. Interestingly, it was
observed that 6 Days after OSKM expression, 143 genes had lost
H3K79me2 2-fold or more (FIG. 5A). FIG. 5 shows genome-wide
analysis of H3K79me2 marks during reprogramming. FIG. 5A shows
genome-wide representation of H3K79me2 marked genes in fibroblasts
and in fibroblasts 6 days into reprogramming. Each dot represents a
gene and the shade indicates the H3K79me2 enrichment of that gene
in ES cells. The dotted line indicates the set of genes that lose
K79me2 2-fold or more upon OSKM transduction. The top 5 gene sets
that overlap with these genes are indicated in the box below. Gene
set overlap analysis indicated that these 143 genes were also
highly significantly represented in gene sets associated with EMT
phenotypes. Only a few of these genes had already decreased in
expression at Day 6 (9 out of 143), but a majority of them would
lose this mark in the pluripotent state (115 out of 143 devoid of
H3K79me2 in ES cells). This finding lead to the question whether
Dot1L inhibition promotes the removal of K79me2 from such
fibroblast specific, EMT-associated genes. To explore this notion,
ChIP-seq for K79me2 on OSKM expressing fibroblasts treated with
iDot1L was performed. It was observed that upon inhibitor
treatment, K79me2 levels were reduced on almost all genes with the
exception of a subset that comprised mostly of housekeeping genes.
This subset of genes also had high levels of K79me2 in ES cells
indicating that these active loci turnover H3K79me2 slowly (FIG.
5B). FIG. 5B shows genome-wide representation of H3K79me2 marked
genes in fibroblasts and in fibroblasts treated with iDot1L 6 days
into reprogramming. Each dot represents a gene and the shade
indicates the H3K79me2 enrichment of that gene in ES cells. Note
that genes that have high K79me2 in ES cells retain this mark
despite Dot1L inhibition. The dotted line indicates the set of
genes that lose K79me2 10-fold or more in iDot1L-treated
fibroblasts. The top 5 gene sets that overlap with these genes are
indicated in the box below. Strikingly, the genes that lost
proportionally the most K79me2 in inhibitor-treated fibroblasts
during reprogramming compared to the initial fibroblasts were again
highly significantly represented in gene sets associated with EMTs
(FIG. 5B). In fact, master regulators of mesenchymal states such as
Zeb1/2, Snai1/2, Grem1 and TGFB2, were among these genes (FIG.
5C).sup.19. FIG. 5C shows H3K79me2 ChIP-sequencing tracks for
select EMT-associated genes in fibroblasts and H3K27me3 in ES
cells.
[0384] These observations support the notion that Dot1L inhibition
collaborates with OSKM in facilitating the loss of K79me2 from
fibroblast specific regulators.
Methods Summary
[0385] shRNAs were designed using the RNAi Codex.sup.26. 97-mer
oligonucleotides (Table 2) were PCR amplified and cloned into
MSCV-PM.sup.27 vector. Control shRNA targeting the firefly
luciferase.sup.27 and the Nanog shRNA were previously
described.sup.28. Reprogramming assays were carried out with either
retroviral.sup.7 or lentiviral.sup.29 reprogramming factors. dH1f
cells were previously described.sup.3. IMR-90 and MRC5 human
diploid fibroblasts were purchased from ATCC. Immunostaining of
reprogramming plates were performed as described.sup.8. For gene
expression analyses, total RNA was extracted from three independent
culture plates for each condition and transcriptional profiling was
performed using Affymetrix U133A microarrays. Primers used for
quantitative real-time PCR can be found in Table 3. ChIP-seq was
performed as described with slight modifications.sup.25.
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[0415] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
examples provided, since the examples are intended as a single
illustration of one aspect of the invention and other functionally
equivalent embodiments are within the scope of the invention.
Various modifications of the invention in addition to those shown
and described herein will become apparent to those skilled in the
art from the foregoing description and fall within the scope of the
appended claims. The advantages and objects of the invention are
not necessarily encompassed by each embodiment of the
invention.
[0416] The contents of all references, patents and published patent
applications cited throughout this application are incorporated
herein by reference in their entirety, particularly for the use or
subject matter referenced herein.
Sequence CWU 1
1
127141DNAArtificial SequenceSynthetic Oligonucleotide 1gatggctgct
cgagaaggta tattgctgtt gacagtgagc g 41227DNAArtificial
SequenceSynthetic Oligonucleotide 2gtctagagga attccgaggc agtaggc
27397DNAArtificial SequenceSynthetic Oligonucleotide 3tgctgttgac
agtgagcgag cgattgctcc aggaatttaa tagtgaagcc acagatgtat 60taaattcctg
gagcaatcgc ctgcctactg cctcgga 97497DNAArtificial SequenceSynthetic
Oligonucleotide 4tgctgttgac agtgagcgcc gtgcccattc cctgtgtcaa
tagtgaagcc acagatgtat 60tgacacaggg aatgggcacg ttgcctactg cctcgga
97597DNAArtificial SequenceSynthetic Oligonucleotide 5tgctgttgac
agtgagcgag gtgatgactt cagtctctac tagtgaagcc acagatgtag 60tagagactga
agtcatcacc ctgcctactg cctcgga 97697DNAArtificial SequenceSynthetic
Oligonucleotide 6tgctgttgac agtgagcgcg gatggagagg tgtactgcat
tagtgaagcc acagatgtaa 60tgcagtacac ctctccatcc ttgcctactg cctcgga
97797DNAArtificial SequenceSynthetic Oligonucleotide 7tgctgttgac
agtgagcgac gatgccagca gtcatgcaaa tagtgaagcc acagatgtat 60ttgcatgact
gctggcatcg ctgcctactg cctcgga 97897DNAArtificial SequenceSynthetic
Oligonucleotide 8tgctgttgac agtgagcgag cagcaacgga tacatcttaa
tagtgaagcc acagatgtat 60taagatgtat ccgttgctgc ctgcctactg cctcgga
97997DNAArtificial SequenceSynthetic Oligonucleotide 9tgctgttgac
agtgagcgac gatgccagca gtcatgcaaa tagtgaagcc acagatgtat 60ttgcatgact
gctggcatcg ctgcctactg cctcgga 971097DNAArtificial SequenceSynthetic
Oligonucleotide 10tgctgttgac agtgagcgag ggattcagat gtcaccttaa
tagtgaagcc acagatgtat 60taaggtgaca tctgaatccc gtgcctactg cctcgga
971197DNAArtificial SequenceSynthetic Oligonucleotide 11tgctgttgac
agtgagcgcc gagtgttata tttgtgaata tagtgaagcc acagatgtat 60attcacaaat
ataacactcg ttgcctactg cctcgga 971297DNAArtificial SequenceSynthetic
Oligonucleotide 12tgctgttgac agtgagcgcg cacctctgaa cttcagaata
tagtgaagcc acagatgtat 60attctgaagt tcagaggtgc atgcctactg cctcgga
971397DNAArtificial SequenceSynthetic Oligonucleotide 13tgctgttgac
agtgagcgag gacgggagct ccactgtgaa tagtgaagcc acagatgtat 60tcacagtgga
gctcccgtcc gtgcctactg cctcgga 971497DNAArtificial SequenceSynthetic
Oligonucleotide 14tgctgttgac agtgagcgcg gagctcacct ttgattacaa
tagtgaagcc acagatgtat 60tgtaatcaaa ggtgagctcc ttgcctactg cctcgga
971597DNAArtificial SequenceSynthetic Oligonucleotide 15tgctgttgac
agtgagcgac ctcggtatct ctaagaggaa tagtgaagcc acagatgtat 60tcctcttaga
gataccgagg gtgcctactg cctcgga 971697DNAArtificial SequenceSynthetic
Oligonucleotide 16tgctgttgac agtgagcgac gggccttcgt gtacatcaat
tagtgaagcc acagatgtaa 60ttgatgtaca cgaaggcccg ctgcctactg cctcgga
971797DNAArtificial SequenceSynthetic Oligonucleotide 17tgctgttgac
agtgagcgcg cccgttactg cttcagcaat tagtgaagcc acagatgtaa 60ttgctgaagc
agtaacgggc atgcctactg cctcgga 971897DNAArtificial SequenceSynthetic
Oligonucleotide 18tgctgttgac agtgagcgcg cttagtatat gtgtacttaa
tagtgaagcc acagatgtat 60taagtacaca tatactaagc ttgcctactg cctcgga
971997DNAArtificial SequenceSynthetic Oligonucleotide 19tgctgttgac
agtgagcgac caaatcttca ggtgttcaat tagtgaagcc acagatgtaa 60ttgaacacct
gaagatttgg gtgcctactg cctcgga 972097DNAArtificial SequenceSynthetic
Oligonucleotide 20tgctgttgac agtgagcgcc ctgatagtca gcatgcgaat
tagtgaagcc acagatgtaa 60ttcgcatgct gactatcagg ttgcctactg cctcgga
972197DNAArtificial SequenceSynthetic Oligonucleotide 21tgctgttgac
agtgagcgcg ggctttcatg ttatctataa tagtgaagcc acagatgtat 60tatagataac
atgaaagccc atgcctactg cctcgga 972297DNAArtificial SequenceSynthetic
Oligonucleotide 22tgctgttgac agtgagcgac ctgaagagtc caatgatgat
tagtgaagcc acagatgtaa 60tcatcattgg actcttcagg gtgcctactg cctcgga
972397DNAArtificial SequenceSynthetic Oligonucleotide 23tgctgttgac
agtgagcgca ggattctggc caaacagaaa tagtgaagcc acagatgtat 60ttctgtttgg
ccagaatcct ttgcctactg cctcgga 972497DNAArtificial SequenceSynthetic
Oligonucleotide 24tgctgttgac agtgagcgac ggagggccaa gcactataaa
tagtgaagcc acagatgtat 60ttatagtgct tggccctccg gtgcctactg cctcgga
972597DNAArtificial SequenceSynthetic Oligonucleotide 25tgctgttgac
agtgagcgaa ccggttaaga gattcttatt tagtgaagcc acagatgtaa 60ataagaatct
cttaaccggt ctgcctactg cctcgga 972697DNAArtificial SequenceSynthetic
Oligonucleotide 26tgctgttgac agtgagcgcg gcattatgct tgttgtacaa
tagtgaagcc acagatgtat 60tgtacaacaa gcataatgcc atgcctactg cctcgga
972797DNAArtificial SequenceSynthetic Oligonucleotide 27tgctgttgac
agtgagcgac cattgtaagt gttgtttcta tagtgaagcc acagatgtat 60agaaacaaca
cttacaatgg gtgcctactg cctcgga 972897DNAArtificial SequenceSynthetic
Oligonucleotide 28tgctgttgac agtgagcgag gaaagaatat gcatagaata
tagtgaagcc acagatgtat 60attctatgca tattctttcc gtgcctactg cctcgga
972997DNAArtificial SequenceSynthetic Oligonucleotide 29tgctgttgac
agtgagcgcc ggaactcaac cattaagcaa tagtgaagcc acagatgtat 60tgcttaatgg
ttgagttccg ttgcctactg cctcgga 973097DNAArtificial SequenceSynthetic
Oligonucleotide 30tgctgttgac agtgagcgcg ggactgcaat tattcagtat
tagtgaagcc acagatgtaa 60tactgaataa ttgcagtccc ttgcctactg cctcgga
973197DNAArtificial SequenceSynthetic Oligonucleotide 31tgctgttgac
agtgagcgac cagtggccag ttcactgtat tagtgaagcc acagatgtaa 60tacagtgaac
tggccactgg ctgcctactg cctcgga 973297DNAArtificial SequenceSynthetic
Oligonucleotide 32tgctgttgac agtgagcgag cagttacatg catacttcaa
tagtgaagcc acagatgtat 60tgaagtatgc atgtaactgc ctgcctactg cctcgga
973397DNAArtificial SequenceSynthetic Oligonucleotide 33tgctgttgac
agtgagcgcg ctctgtaatc tcgtttcaaa tagtgaagcc acagatgtat 60ttgaaacgag
attacagagc atgcctactg cctcgga 973497DNAArtificial SequenceSynthetic
Oligonucleotide 34tgctgttgac agtgagcgcc ctcctgatta ttcagaatat
tagtgaagcc acagatgtaa 60tattctgaat aatcaggagg ttgcctactg cctcgga
973597DNAArtificial SequenceSynthetic Oligonucleotide 35tgctgttgac
agtgagcgac gaagagctct tctttgatta tagtgaagcc acagatgtat 60aatcaaagaa
gagctcttcg ctgcctactg cctcgga 973697DNAArtificial SequenceSynthetic
Oligonucleotide 36tgctgttgac agtgagcgcg ccagtaacaa gaaagagaaa
tagtgaagcc acagatgtat 60ttctctttct tgttactggc atgcctactg cctcgga
973797DNAArtificial SequenceSynthetic Oligonucleotide 37tgctgttgac
agtgagcgac ctgcatcatg actcagaatt tagtgaagcc acagatgtaa 60attctgagtc
atgatgcagg gtgcctactg cctcgga 973897DNAArtificial SequenceSynthetic
Oligonucleotide 38tgctgttgac agtgagcgcc aacattatgg gcatcgagaa
tagtgaagcc acagatgtat 60tctcgatgcc cataatgttg ttgcctactg cctcgga
973997DNAArtificial SequenceSynthetic Oligonucleotide 39tgctgttgac
agtgagcgac gagctacaaa gcatgggaaa tagtgaagcc acagatgtat 60ttcccatgct
ttgtagctcg gtgcctactg cctcgga 974097DNAArtificial SequenceSynthetic
Oligonucleotide 40tgctgttgac agtgagcgac gtccgcagga acttaactta
tagtgaagcc acagatgtat 60aagttaagtt cctgcggacg ctgcctactg cctcgga
974197DNAArtificial SequenceSynthetic Oligonucleotide 41tgctgttgac
agtgagcgcc ctgaggataa ctcaatataa tagtgaagcc acagatgtat 60tatattgagt
tatcctcagg ttgcctactg cctcgga 974297DNAArtificial SequenceSynthetic
Oligonucleotide 42tgctgttgac agtgagcgcc cgggaacaga gaatgtttaa
tagtgaagcc acagatgtat 60taaacattct ctgttcccgg ttgcctactg cctcgga
974397DNAArtificial SequenceSynthetic Oligonucleotide 43tgctgttgac
agtgagcgcg gtctcaggcg ccagtggaaa tagtgaagcc acagatgtat 60ttccactggc
gcctgagacc atgcctactg cctcgga 974497DNAArtificial SequenceSynthetic
Oligonucleotide 44tgctgttgac agtgagcgcg cctagtaaat tacagaagaa
tagtgaagcc acagatgtat 60tcttctgtaa tttactaggc atgcctactg cctcgga
974597DNAArtificial SequenceSynthetic Oligonucleotide 45tgctgttgac
agtgagcgcg cttctaggca gagttgctta tagtgaagcc acagatgtat 60aagcaactct
gcctagaagc ttgcctactg cctcgga 974697DNAArtificial SequenceSynthetic
Oligonucleotide 46tgctgttgac agtgagcgac gcatatattt gcagtatgaa
tagtgaagcc acagatgtat 60tcatactgca aatatatgcg ctgcctactg cctcgga
974797DNAArtificial SequenceSynthetic Oligonucleotide 47tgctgttgac
agtgagcgac cgtcccgtgg agtcgctaaa tagtgaagcc acagatgtat 60ttagcgactc
cacgggacgg gtgcctactg cctcgga 974897DNAArtificial SequenceSynthetic
Oligonucleotide 48tgctgttgac agtgagcgcg ccctccctgt cctttccaga
tagtgaagcc acagatgtat 60ctggaaagga cagggagggc ttgcctactg cctcgga
974997DNAArtificial SequenceSynthetic Oligonucleotide 49tgctgttgac
agtgagcgcc gccagccttc gcttctgaaa tagtgaagcc acagatgtat 60ttcagaagcg
aaggctggcg ttgcctactg cctcgga 975097DNAArtificial SequenceSynthetic
Oligonucleotide 50tgctgttgac agtgagcgcg agcttcatgg gattggtaaa
tagtgaagcc acagatgtat 60ttaccaatcc catgaagctc atgcctactg cctcgga
975197DNAArtificial SequenceSynthetic Oligonucleotide 51tgctgttgac
agtgagcgaa cctttccagc catagagatt tagtgaagcc acagatgtaa 60atctctatgg
ctggaaaggt gtgcctactg cctcgga 975297DNAArtificial SequenceSynthetic
Oligonucleotide 52tgctgttgac agtgagcgcg ctttcaagct catctgttat
tagtgaagcc acagatgtaa 60taacagatga gcttgaaagc ttgcctactg cctcgga
975397DNAArtificial SequenceSynthetic Oligonucleotide 53tgctgttgac
agtgagcgaa cagttggatt ctttagagaa tagtgaagcc acagatgtat 60tctctaaaga
atccaactgt ctgcctactg cctcgga 975497DNAArtificial SequenceSynthetic
Oligonucleotide 54tgctgttgac agtgagcgac gagagagtta gctgacttta
tagtgaagcc acagatgtat 60aaagtcagct aactctctcg gtgcctactg cctcgga
975597DNAArtificial SequenceSynthetic Oligonucleotide 55tgctgttgac
agtgagcgac ctgattatat ccagtaacac tagtgaagcc acagatgtag 60tgttactgga
tataatcagg gtgcctactg cctcgga 975697DNAArtificial SequenceSynthetic
Oligonucleotide 56tgctgttgac agtgagcgcg cccaaggtca aggagattat
tagtgaagcc acagatgtaa 60taatctcctt gaccttgggc ttgcctactg cctcgga
975797DNAArtificial SequenceSynthetic Oligonucleotide 57tgctgttgac
agtgagcgcg gcatccactg tgaatgataa tagtgaagcc acagatgtat 60tatcattcac
agtggatgcc atgcctactg cctcgga 975897DNAArtificial SequenceSynthetic
Oligonucleotide 58tgctgttgac agtgagcgcg ctgtctctct ttgatggaat
tagtgaagcc acagatgtaa 60ttccatcaaa gagagacagc atgcctactg cctcgga
975997DNAArtificial SequenceSynthetic Oligonucleotide 59tgctgttgac
agtgagcgcg cctgcaagga catggttaaa tagtgaagcc acagatgtat 60ttaaccatgt
ccttgcaggc ttgcctactg cctcgga 976097DNAArtificial SequenceSynthetic
Oligonucleotide 60tgctgttgac agtgagcgac gcacctactc caagttcaaa
tagtgaagcc acagatgtat 60ttgaacttgg agtaggtgcg ctgcctactg cctcgga
976197DNAArtificial SequenceSynthetic Oligonucleotide 61tgctgttgac
agtgagcgcc gagtctggct ttgagagtta tagtgaagcc acagatgtat 60aactctcaaa
gccagactcg ttgcctactg cctcgga 976297DNAArtificial SequenceSynthetic
Oligonucleotide 62tgctgttgac agtgagcgag ccatggaaat gctatcaatg
tagtgaagcc acagatgtac 60attgatagca tttccatggc ctgcctactg cctcgga
976397DNAArtificial SequenceSynthetic Oligonucleotide 63tgctgttgac
agtgagcgca gatggaagat gatatagata tagtgaagcc acagatgtat 60atctatatca
tcttccatct ttgcctactg cctcgga 976497DNAArtificial SequenceSynthetic
Oligonucleotide 64tgctgttgac agtgagcgcc caaatcttct cctgtcagta
tagtgaagcc acagatgtat 60actgacagga gaagatttgg atgcctactg cctcgga
976597DNAArtificial SequenceSynthetic Oligonucleotide 65tgctgttgac
agtgagcgaa gagattattt ctcaagatga tagtgaagcc acagatgtat 60catcttgaga
aataatctct ctgcctactg cctcgga 976697DNAArtificial SequenceSynthetic
Oligonucleotide 66tgctgttgac agtgagcgca gagggaaagt gtatgataaa
tagtgaagcc acagatgtat 60ttatcataca ctttccctct ttgcctactg cctcgga
976797DNAArtificial SequenceSynthetic Oligonucleotide 67tgctgttgac
agtgagcgcg gaaagaacgg aaatcttaaa tagtgaagcc acagatgtat 60ttaagatttc
cgttctttcc atgcctactg cctcgga 976897DNAArtificial SequenceSynthetic
Oligonucleotide 68tgctgttgac agtgagcgcg cagttatgct cttaatgctt
tagtgaagcc acagatgtaa 60agcattaaga gcataactgc ttgcctactg cctcgga
976997DNAArtificial SequenceSynthetic Oligonucleotide 69tgctgttgac
agtgagcgcg catgcatgac tttaatctta tagtgaagcc acagatgtat 60aagattaaag
tcatgcatgc ttgcctactg cctcgga 977097DNAArtificial SequenceSynthetic
Oligonucleotide 70tgctgttgac agtgagcgaa acatgtgtaa gctgcggccc
tagtgaagcc acagatgtag 60ggccgcagct tacacatgtt ctgcctactg cctcgga
977197DNAArtificial SequenceSynthetic Oligonucleotide 71tgctgttgac
agtgagcgaa aggatgtggt ccgagtgtgg tagtgaagcc acagatgtac 60cacactcgga
ccacatcctt ctgcctactg cctcgga 977220DNAArtificial SequenceSynthetic
Oligonucleotide 72tgaagtgtga cgtggacatc 207320DNAArtificial
SequenceSynthetic Oligonucleotide 73ggaggagcaa tgatcttgat
207423DNAArtificial SequenceSynthetic Oligonucleotide 74gaatctcttg
cacgaatttc tgc 237521DNAArtificial SequenceSynthetic
Oligonucleotide 75catgagcacc gttctccaag g 217620DNAArtificial
SequenceSynthetic Oligonucleotide 76ccgatgctgg ggacaagaat
207720DNAArtificial SequenceSynthetic Oligonucleotide 77cccgtcatcc
accaagacac 207821DNAArtificial SequenceSynthetic Oligonucleotide
78gcggaggaat atgtccgaga g 217921DNAArtificial SequenceSynthetic
Oligonucleotide 79agaggtctgg attgctgttc t 218020DNAArtificial
SequenceSynthetic Oligonucleotide 80atgggccaga ctgggaagaa
208122DNAArtificial SequenceSynthetic Oligonucleotide 81tggaaaatcc
aagtcactgg tc 228223DNAArtificial SequenceSynthetic Oligonucleotide
82agttgtccaa taaggcaagt tcc 238323DNAArtificial SequenceSynthetic
Oligonucleotide 83acgagtcact ctaaatagca acg 238423DNAArtificial
SequenceSynthetic Oligonucleotide 84catttccgca tgagtgatga tgt
238519DNAArtificial SequenceSynthetic Oligonucleotide 85caggccacct
cctgagttc 198621DNAArtificial SequenceSynthetic Oligonucleotide
86ttaccccagg tgaagcaaga g 218722DNAArtificial SequenceSynthetic
Oligonucleotide 87ccaatacggg agaagtcagg ac 228823DNAArtificial
SequenceSynthetic Oligonucleotide 88cccacaacga atgaatgaac agc
238922DNAArtificial SequenceSynthetic Oligonucleotide 89tgaagacctt
tgggtagttc ca 229020DNAArtificial SequenceSynthetic Oligonucleotide
90ctgggagagg gaagaagtgc 209120DNAArtificial SequenceSynthetic
Oligonucleotide 91cggaagtcga aggtgctcag
209221DNAArtificial SequenceSynthetic Oligonucleotide 92agcagagaca
tcagaagggt c 219320DNAArtificial SequenceSynthetic Oligonucleotide
93cggccagatt tcctttgctt 209421DNAArtificial SequenceSynthetic
Oligonucleotide 94gctgccggtc tacgataaac a 219521DNAArtificial
SequenceSynthetic Oligonucleotide 95agcttgagat ccgggatttc t
219622DNAArtificial SequenceSynthetic Oligonucleotide 96atatccagac
tcagagagca cc 229722DNAArtificial SequenceSynthetic Oligonucleotide
97cagctccctt tctaagtcct tg 229823DNAArtificial SequenceSynthetic
Oligonucleotide 98tggatgacaa aagatgtgag tgg 239922DNAArtificial
SequenceSynthetic Oligonucleotide 99ccatatttgg acgtgtcctg ag
2210022DNAArtificial SequenceSynthetic Oligonucleotide
100gccctacagt aagcactatc ct 2210121DNAArtificial SequenceSynthetic
Oligonucleotide 101agtctcccta gcttcaacca c 2110221DNAArtificial
SequenceSynthetic Oligonucleotide 102ccacctgatg tgtgtgcttt g
2110322DNAArtificial SequenceSynthetic Oligonucleotide
103ttcagtagtg gtctggtctt gt 2210422DNAArtificial SequenceSynthetic
Oligonucleotide 104tcagaactca tgtgccctat ct 2210520DNAArtificial
SequenceSynthetic Oligonucleotide 105gcaggtagga cactccttgt
2010621DNAArtificial SequenceSynthetic Oligonucleotide
106agaagagcgg caagaagagt t 2110723DNAArtificial SequenceSynthetic
Oligonucleotide 107caaccactgt ctcatggtca ata 2310821DNAArtificial
SequenceSynthetic Oligonucleotide 108tcaatgaagc ctgtgagtgg a
2110922DNAArtificial SequenceSynthetic Oligonucleotide
109caatgcaact gtgttcagtg ac 2211019DNAArtificial SequenceSynthetic
Oligonucleotide 110caggccagta cgcactcac 1911123DNAArtificial
SequenceSynthetic Oligonucleotide 111tgttctcgat gcccataatg ttg
2311219DNAArtificial SequenceSynthetic Oligonucleotide
112ccccaccgtc acctctagt 1911321DNAArtificial SequenceSynthetic
Oligonucleotide 113gtagcaccaa actgagcaga a 2111423DNAArtificial
SequenceSynthetic Oligonucleotide 114ggctaaacaa aggatagctc tgc
2311523DNAArtificial SequenceSynthetic Oligonucleotide
115tgaaagaagc aatctgtgca tga 2311619DNAArtificial SequenceSynthetic
Oligonucleotide 116caacgccgta gacagcgag 1911721DNAArtificial
SequenceSynthetic Oligonucleotide 117ctccccgtcc ttattgtcga g
2111820DNAArtificial SequenceSynthetic Oligonucleotide
118ccctagggga tgttccagat 2011920DNAArtificial SequenceSynthetic
Oligonucleotide 119tgaagctttt ccctcttcca 2012020DNAArtificial
SequenceSynthetic Oligonucleotide 120agcttggtgg tggatgaaac
2012120DNAArtificial SequenceSynthetic Oligonucleotide
121ccctcttcag caaagcagac 2012220DNAArtificial SequenceSynthetic
Oligonucleotide 122ctagaccgtg ggttttgcat 2012320DNAArtificial
SequenceSynthetic Oligonucleotide 123tgggttaagt gcccctgtag
2012420DNAArtificial SequenceSynthetic Oligonucleotide
124acccagttca tagcggtgac 2012520DNAArtificial SequenceSynthetic
Oligonucleotide 125caattgtcat gggattgcag 2012626DNAArtificial
SequenceSynthetic Oligonucleotide 126atggaaactc tattaaagtg aacctg
2612725DNAArtificial SequenceSynthetic Oligonucleotide
127tagacctcat actcagcatt ccagt 25
* * * * *
References