U.S. patent application number 11/256381 was filed with the patent office on 2006-03-16 for mll translocations specify a distinct gene expression profile, distinguishing a unique leukemia.
Invention is credited to Scott A. Armstrong, Todd R. Golub, Stanley J. Korsmeyer.
Application Number | 20060057630 11/256381 |
Document ID | / |
Family ID | 23183813 |
Filed Date | 2006-03-16 |
United States Patent
Application |
20060057630 |
Kind Code |
A1 |
Golub; Todd R. ; et
al. |
March 16, 2006 |
MLL translocations specify a distinct gene expression profile,
distinguishing a unique leukemia
Abstract
The present invention relates to the diagnosis of mixed lineage
leukemia (MLL), acute lymphoblastic leukemia (ALL), and acute
myelogenous leukemia (AML) according to the gene expression profile
of a sample from an individual, as well as to methods of therapy
and screening that utilize the genes identified herein as
targets.
Inventors: |
Golub; Todd R.; (Newton,
MA) ; Armstrong; Scott A.; (Wayland, MA) ;
Korsmeyer; Stanley J.; (Weston, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
23183813 |
Appl. No.: |
11/256381 |
Filed: |
October 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10198064 |
Jul 17, 2002 |
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11256381 |
Oct 20, 2005 |
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60306103 |
Jul 17, 2001 |
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Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
G01N 2800/52 20130101;
G01N 2333/475 20130101; C12Q 2600/112 20130101; C12Q 2600/158
20130101; C12Q 2600/136 20130101; C12Q 2600/106 20130101; C12Q
1/6886 20130101; G01N 33/57426 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a grant
P01CA68484 from the National Institutes of Health. The Government
has certain rights in the invention.
Claims
1. A method of identifying a candidate compound for use in treating
mixed lineage leukemia comprising: a) contacting a cell sample with
a candidate compound, wherein the cell is selected from the group
consisting of mononuclear blood cells and bone marrow cells; and b)
detecting an alteration of a gene expression profile of a gene
expression product from at least one informative gene from the cell
sample, wherein a candidate compound that increases the gene
expression profile of at least one informative gene which is
decreased in mixed lineage leukemia or decreases the gene
expression profile of at least one informative gene which is
increased in mixed lineage leukemia, is a candidate compound for
use in treating mixed lineage leukemia.
2. The method of claim 1, wherein the candidate compound decreases
the gene expression profile of at least one informative gene which
is increased in mixed lineage leukemia wherein said gene selected
from the group consisting of FLT3, MEIS1, and HoxA9.
3. A method of identifying a compound for use in treating mixed
lineage leukemia, comprising: a) determining a gene expression
profile of a gene expression product from at least one informative
gene from one or more cells selected from the group consisting of
mononuclear blood cells and bone marrow cells of an individual with
mixed lineage leukemia; b) administering a candidate compound to
the individual; c) determining a gene expression profile of the
gene expression product(s) of step a) from one or more cells
selected from the group consisting of mononuclear blood cells and
bone marrow cells from the individual of step b); and d) comparing
the gene expression profiles of step a) to the gene expression
profile of step c), wherein if the gene expression profile from the
individual after administration of the agent is correlated with
effective treatment of mixed lineage leukemia, then the candidate
compound is a compound for use in treating mixed lineage
leukemia.
4. The method of claim 3, wherein the candidate compound decreases
the gene expression profile of at least one informative gene which
is increased in mixed lineage leukemia wherein said gene selected
from the group consisting of FLT3, MEIS1, and HoxA9.
5. A method of identifying a candidate compound for use in treating
acute lymphoblastic leukemia, comprising: a) contacting a cell
sample with a candidate compound, wherein the cell is selected from
the group consisting of mononuclear blood cells and bone marrow
cells; and b) detecting an alteration of a gene expression profile
of a gene expression product from at least one informative gene
from the cell sample, wherein a candidate compound that increases
the gene expression profile of at least one informative gene which
is decreased in acute lymphoblastic leukemia or decreases the gene
expression profile of at least one informative gene which is
increased in acute lymphoblastic leukemia, is a candidate compound
for use in treating mixed lineage leukemia acute lymphoblastic
leukemia.
6. A method of identifying a compound for use in treating acute
lymphoblastic leukemia, comprising: a) determining a gene
expression profile of a gene expression product from at least one
informative gene from one or more cells selected from the group
consisting of mononuclear blood cells and bone marrow cells of an
individual with acute lymphoblastic leukemia; b) administering a
candidate compound to the individual; c) determining a gene
expression profile of the gene expression product(s) of step a)
from one or more cells selected from the group consisting of
mononuclear blood cells and bone marrow cells from the individual
of step b); and d) comparing the gene expression profiles of step
a) to the gene expression profile of step c), wherein if the gene
expression profile from the individual after administration of the
agent is correlated with effective treatment of acute lymphoblastic
leukemia, then the candidate compound is a compound for use in
treating acute lymphoblastic leukemia.
7. A method of identifying a candidate compound for use in treating
acute myelogenous leukemia comprising: a) contacting a cell sample
with a candidate compound, wherein the cell is selected from the
group consisting of mononuclear blood cells and bone marrow cells;
and b) detecting an alteration of a gene expression profile of a
gene expression product from at least one informative gene from the
cell sample, wherein a candidate compound that increases the gene
expression profile of at least one informative gene which is
decreased in acute myelogenous leukemia or decreases the gene
expression profile of at least one informative gene which is
increased in acute myelogenous leukemia, is a candidate compound
for use in treating acute myelogenous leukemia.
8. A method of identifying a compound for use in treating acute
myelogenous leukemia, comprising: a) determining a gene expression
profile of a gene expression product from at least one informative
gene from one or more cells selected from the group consisting of
mononuclear blood cells and bone marrow cells of an individual with
acute myelogenous leukemia; b) administering a candidate compound
to the individual; c) determining a gene expression profile of the
gene expression product(s) of step a) from one or more cells
selected from the group consisting of mononuclear blood cells and
bone marrow cells from the individual of step b); and d) comparing
the gene expression profiles of step a) to the gene expression
profile of step c), wherein if the gene expression profile from the
individual after administration of the agent is correlated with
effective treatment of acute myelogenous leukemia, then the
candidate compound is a compound for use in treating acute
myelogenous leukemia.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Application No.
10/198,064, filed Jul. 17, 2002, which claims the benefit of U.S.
Provisional Application No. 60/306,103 filed on Jul. 17, 2001. The
entire teachings of the above applications are incorporated herein
by reference.
BACKGROUND OF THE INVENTION
[0003] A subset of human acute leukemias with a decidedly
unfavorable prognosis possess a chromosomal translocation involving
the Mixed Lineage Leukemia (MLL, HRX, AU-1) gene on chromosome
segment 11q23. The leukemic cells, which typically have a
lymphoblastic morphology, have been classified as Acute
Lymphoblastic Leukemia (ALL). However, unlike the majority of
childhood ALL, the presence of the MLL translocations often results
in an early relapse after chemotherapy. As MLL translocations are
typically found in leukemias of infants and chemotherapy-induced
leukemia, it has remained uncertain whether host related factors or
tumor-intrinsic biological differences are responsible for the poor
survival in patients with the translocations. Lymphoblastic
leukemias with either rearranged or germline MLL are similar with
respect to most morphological and histochemical characteristics.
Inmunophenotypic differences associated with lymphoblasts bearing
an MLL translocation include the lack of the early lymphocyte
antigen CD10, expression of the proteoglycan NG2, and the
propensity to co-express the myeloid antigens CD15 and CD65. This
prompted the corresponding disease to be called Mixed Lineage
Leukemia and suggested models, largely unresolved, in which the
leukemia reflects disordered cell fate decisions or the
transformation of a more multi-potential progenitor.
[0004] Generally, therapeutic treatment is more successful when
tailored to the specific type of leukemia. Thus, a need exists for
accurate and efficient methods for diagnosis of leukemia and
identification of subclasses of leukemias.
SUMMARY OF THE INVENTION
[0005] As described herein, MLL is significantly different from ALL
and AML, as assessed by gene expression profiling. The expression
profiles reported here reveal that lymphoblastic leukemias bearing
MLL translocations display a remarkably uniform and highly distinct
pattern that clearly distinguishes them from conventional ALL or
AML and warrants designation as a distinct disease, MLL.
[0006] In one embodiment, the invention relates to a method of
diagnosing mixed lineage leukemia, acute lymphoblastic leukemia or
acute myelogenous leukemia, comprising determining a gene
expression profile of a gene expression product from at least one
informative gene from one or more cells, wherein the cells are
selected from the group consisting of mononuclear blood cells and
bone marrow cells, and wherein the gene expression profile is
correlated with mixed lineage leukemia, acute lymphoblastic
leukemia or acute myelogenous leukemia. In one embodiment, the gene
expression product is RNA. In a preferred embodiment, the gene
expression profile is determined utilizing specific hybridization
probes. In a particularly preferred embodiment, the gene expression
profile is determined utilizing oligonucleotide microarrays. In a
preferred embodiment, the gene expression profile is determined
utilizing antibodies. In particular embodiments, the informative
gene(s) is selected from the group consisting of the genes in FIGS.
1A, 1B, 2A-2F, 3A-3D, and 5, and Tables 1 and 2.
[0007] The invention further relates to a method of diagnosing
mixed lineage leukemia, acute lymphoblastic leukemia or acute
myelogenous leukemia, comprising determining a gene expression
profile of mRNA from at least one informative gene, wherein the
mRNA is isolated from one or more cells of an individual selected
from the group consisting of mononuclear blood cells and bone
marrow cells; and comparing the obtained gene expression profile to
a gene expression profile of a control sample selected from the
group consisting of a mixed lineage leukemia sample, an acute
lymphoblastic leukemia sample and an acute myelogenous leukemia
sample, wherein the gene expression profile of the cell from the
individual is indicative of mixed lineage leukemia, acute
lymphoblastic leukemia or acute myelogenous leukemia.
[0008] The invention also relates to a method of diagnosing mixed
lineage leukemia, comprising determining a gene expression profile
of a gene expression product from at least one informative gene
from one or more cells selected from the group consisting of
mononuclear cells and bone marrow cells, wherein the gene
expression profile is correlated with mixed lineage leukemia.
[0009] The invention further relates to a method of identifying a
compound for use in treating mixed lineage leukemia, acute
lymphoblastic leukemia, or acute myelogenous leukemia, comprising
determining a gene expression profile of a gene expression product
from at least one informative gene from one or more cells selected
from the group consisting of mononuclear blood cells and bone
marrow cells of an individual with mixed lineage leukemia, acute
lymphoblastic leukemia, or acute myelogenous leukemia;
administering a test agent to the individual; determining a gene
expression profile of a gene expression product from at least one
informative gene from one or more cells selected from the group
consisting of mononuclear blood cells and bone marrow cells from
the individual; and comparing the two gene expression profiles,
wherein if the gene expression profile from the individual after
administration of the agent is correlated with effective treatment
of mixed lineage leukemia, acute lymphoblastic leukemia, or acute
myelogenous leukemia, the test agent is a therapeutic agent. In one
embodiment, the disease is mixed lineage leukemia, and a decrease
in the expression of the informative gene selected from the group
consisting of FLT3, MEIS1, and HoxA9, is indicative of effective
treatment of mixed lineage leukemia. In another embodiment, the
gene expression profiles compared prior to and after administration
of the test agent consist of one or more of the same informative
genes.
[0010] The invention also relates to a method for evaluating drug
candidates for their effectiveness in treating mixed lineage
leukemia, acute lymphoblastic leukemia, or acute myelogenous
leukemia, comprising contacting a cell sample or lysate thereof
with a candidate compound, wherein the cell is selected from the
group consisting of mononuclear blood cells and bone marrow cells;
and detecting an alteration of a gene expression profile of a gene
expression product from at least one informative gene from the cell
sample or lysate thereof, wherein a compound that increases the
gene expression profile of at least one informative gene which is
decreased in mixed lineage leukemia, acute lymphoblastic leukemia,
or acute myelogenous leukemia is a compound for use in treating
mixed lineage leukemia, acute lymphoblastic leukemia, or acute
myelogenous leukemia.
[0011] The invention further relates to a method of identifying a
compound for use in treating mixed lineage leukemia, acute
lymphoblastic leukemia, or acute myelogenous leukemia, comprising
contacting a cell sample or lysate thereof with a candidate
compound, wherein the cell is selected from the group consisting of
mononuclear blood cells and bone marrow cells; and detecting an
alteration of a gene expression profile of a gene expression
product from at least one informative gene from the cell sample or
lysate thereof, wherein a compound that decreases the gene
expression profile of at least one informative gene which is
increased in mixed lineage leukemia, acute lymphoblastic leukemia,
or acute myelogenous leukemia is a compound for use in treating
mixed lineage leukemia, acute lymphoblastic leukemia, or acute
myelogenous leukemia.
[0012] The invention further relates to a method of identifying a
compound for use in treating mixed lineage leukemia, acute
lymphoblastic leukemia, or acute myelogenous leukemia, comprising
contacting a cell sample or lysate thereof with a candidate
compound, wherein the cell is selected from the group consisting of
mononuclear blood cells and bone marrow cells; and detecting an
alteration of a gene expression profile of a gene expression
product from at least one informative gene from the cell sample or
lysate thereof, wherein a compound that increases the gene
expression profile of at least one informative gene which is
decreased in mixed lineage leukemia, acute lymphoblastic leukemia,
or acute myelogenous leukemia is a compound for use in treating
acute lymphoblastic leukemia. In a preferred embodiment, the
disease is mixed lineage leukemia, and the informative gene is
selected from the group consisting of FLT3, MEIS1, and HoxA9.
[0013] In another aspect, the invention relates to a method of
identifying a compound that modulates (increases or decreases) the
biological activity of an informative gene.
[0014] In still another aspect, the invention features a method of
identifying a compound that decreases the biological activity of an
informative gene expression product having increased expression in
MLL, AML, or ALL. The method comprises contacting the informative
gene expression product with a candidate compound under conditions
suitable for activity of the informative gene expression product;
and assessing the biological activity level of the informative gene
expression product. A candidate compound that decreases the
biological activity level of the informative gene expression
product relative to a control is a compound that decreases the
biological activity of the informative gene expression product
having increased expression in MLL, AML, or ALL. In one embodiment,
the method is carried out in a cell or animal. In another
embodiment, the method is carried out in a cell-free system. In
still another embodiment the informative gene expression product is
selected from the gene expression products encoded by the genes in
FIGS. 1A, 1B, 2A-2F, 3A-3D, and 5, and Tables 1 and 2.
[0015] In another aspect, the invention features a method of
identifying a compound that increases the biological activity of an
informative gene expression product having decreased expression in
MLL, AML, or ALL. The method comprises contacting the informative
gene expression product with a candidate compound under conditions
suitable for biological activity of the informative gene expression
product; and assessing the biological activity level of the
informative gene expression product. A candidate compound that
increases the biological activity level of the informative gene
expression product relative to a control is a compound that
increases the biological activity of the informative gene
expression product having decreased expression in MLL, AML, or ALL.
In one embodiment, the method is carried out in a cell or animal.
In another embodiment, the method is carried out in a cell-free
system. In still another embodiment the informative gene expression
product is selected from the gene expression products encoded by
the genes in FIGS. 1A, 1B, 2A-2F, 3A-3D, and 5, and Tables 1 and
2.
[0016] In other embodiments, screens can be carried out for
compounds that further increase the expression of a gene or the
biological activity of a gene expression product already
overexpressed in MLL, ALL, or AML, or that further decrease the
expression of a gene or the biological activity of a gene
expression product already underexpressed in MLL, ALL, or AML.
These compounds can be identified according the screening methods
described herein. These compounds should be avoided during
treatment regimens for MLL, ALL, or AML.
[0017] In still another aspect, the invention features a method of
identifying a polypeptide that interacts with an informative gene
expression product having increased or decreased expression in MLL,
AML or ALL in a yeast two-hybrid system. The method comprises
providing a first nucleic acid vector comprising a nucleic acid
molecule encoding a DNA binding domain and a polypeptide encoded by
the informative gene that is increased or decreased in MLL, AML, or
ALL; providing a second nucleic acid vector comprising a nucleic
acid encoding a transcription activation domain and a nucleic acid
encoding a test polypeptide; contacting the first nucleic acid
vector with the second nucleic acid vector in a yeast two-hybrid
system; and assessing transcriptional activation in the yeast
two-hybrid system. An increase in transcriptional activation
relative to a control indicates that the test polypeptide is a
polypeptide that interacts with the informative gene expression
product having increased or decreased expression in MLL, AML or
ALL.
[0018] The invention also relates to compounds identified according
to the above-described screening methods. Such compounds can be
used to treat mixed lineage leukemia, acute lymphoblastic leukemia,
or acute myelogenous leukemia, as appropriate.
[0019] The invention further relates to a method for evaluating a
drug candidate for effectiveness in treating mixed lineage
leukemia, acute lymphoblastic leukemia, or acute myelogenous
leukemia, comprising determining a gene expression profile of a
gene expression product from at least one informative gene, wherein
the gene expression product is isolated from cells derived from a
blood or bone marrow sample from an individual to whom the drug
candidate has been administered, wherein the gene expression
profile is indicative of the effectiveness of the drug candidate in
treating mixed lineage leukemia, acute lymphoblastic leukemia, or
acute myelogenous leukemia.
[0020] The invention also relates to a method for monitoring the
efficacy of a mixed lineage leukemia, acute lymphoblastic leukemia,
or acute myelogenous leukemia treatment, comprising determining the
gene expression profile a gene expression product from at least one
informative gene in a cell from blood samples derived from an
individual being treated, wherein the samples are obtained at
various time points; and comparing the treatment outcome of the
samples at various times during treatment, wherein the efficacy of
mixed lineage leukemia, acute lymphoblastic leukemia, or acute
myelogenous leukemia treatment is determined. In one embodiment the
gene expression profiles obtained over time is compared to gene
expression profiles from individuals who do not have MLL, ALL, or
AML (normal individuals). In another embodiment, the gene
expression profiles determined at various time points include one
or more of the same informative genes.
[0021] The invention also encompasses a method of predicting the
efficacy of treating mixed lineage leukemia, acute lymphoblastic
leukemia, or acute myelogenous leukemia, comprising determining a
gene expression profile of a gene expression product from at least
one informative gene, the gene expression product isolated from one
or more cells selected from the group consisting of mononuclear
cells and bone marrow cells of an individual with mixed lineage
leukemia, acute lymphoblastic leukemia, or acute myelogenous
leukemia, wherein the gene expression profile is correlated with a
treatment outcome. In one embodiment the gene expression profiles
obtained is compared to gene expression profiles from individuals
who do not have MLL, ALL, or AML (normal individuals)
[0022] The invention also relates to a method of treating mixed
lineage leukemia, comprising administering to an individual in need
thereof a therapeutic amount of an agent that inhibits the activity
of a gene product that is increased in mixed lineage leukemia. In a
preferred embodiment, gene product is encoded by an informative
gene selected from the group consisting of FLT3, MEIS1, and
HoxA9.
[0023] The invention further relates to a method of treating mixed
lineage leukemia, comprising administering to an individual in need
thereof a therapeutic amount of an agent which enhances the
activity of a gene product which is decreased in mixed lineage
leukemia.
[0024] In any of the above methods, the gene expression product may
be RNA and the gene expression profile can be determined utilizing
specific hybridization probes. In a particularly preferred
embodiment, the gene expression profile is determined utilizing
oligonucleotide microarrays. In another preferred embodiment, the
gene expression profile is determined utilizing antibodies. In
particular embodiments, the informative gene(s) is selected from
the group consisting of the genes in FIGS. 1A, 1B, 2A-2F, 3A-3D,
and 5, and Tables 1 and 2.
[0025] The invention also relates to an oligonucleotide microarray
having immobilized thereon a plurality of oligonucleotide probes
specific for one or more informative genes for diagnosing mixed
lineage leukemia, acute lymphoblastic leukemia, or acute
myelogenous leukemia wherein the informative genes are selected
from the group consisting of the genes in FIGS. 1A, 1B, 2A-2F,
3A-3D, and 5, and Tables 1 and 2.
[0026] It is well known that proper diagnosis of disease is
essential for successful treatment of individuals. The present
invention will significantly improve the diagnosis of MLL, ALL, and
ALL, and thereby improve the treatment of leukemic individuals.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0028] FIG. 1A illustrates genes that distinguish ALL from MLL. The
100 genes most highly correlated with the class distinction are
shown. Each column represents a leukemia sample and each row
represents an individual gene. Expression levels are normalized for
each gene where the mean is 0. Expression levels greater than the
mean are shown in red, whereas levels less than the mean are shown
in blue. Increasing distance from the mean is represented by
increasing color intensity.
[0029] FIG. 1B illustrates genes that distinguish ALL (left-most 20
columns) from MLL (right-most 17 columns). The 100 genes most
highly correlated with the class distinction are shown. Each column
represents a leukemia sample and each row represents an individual
gene. Expression levels are normalized for each gene where the mean
is 0. Expression levels greater than the mean are shown in red,
whereas levels less than the mean are shown in blue. Increasing
distance from the mean is represented by increasing color
intensity.
[0030] FIG. 2A illustrates selected early lymphocyte gene
expression in ALL and MLL. Relative levels of expression of CD10 in
ALL and MLL samples are shown. Each bar represents an individual
leukemia sample. The expression values are raw data obtained from
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays based
on the scaling described in the Examples.
[0031] FIG. 2B illustrates selected early lymphocyte gene
expression in ALL and MLL. Relative levels of expression of CD19 in
ALL and MLL samples are shown. Each bar represents an individual
leukemia sample. The expression values are raw data obtained from
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays based
on the scaling described in the Examples.
[0032] FIG. 2C illustrates selected early lymphocyte gene
expression in ALL and MLL. Relative levels of expression of IgB in
ALL and MLL samples are shown. Each bar represents an individual
leukemia sample. The expression values are raw data obtained from
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays based
on the scaling described in the Examples.
[0033] FIG. 2D illustrates selected early lymphocyte gene
expression in ALL and MLL. Relative levels of expression of CD24 in
ALL and MLL samples are shown. Each bar represents an individual
leukemia sample. The expression values are raw data obtained from
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays based
on the scaling described in the Examples.
[0034] FIG. 2E illustrates selected early lymphocyte gene
expression in ALL and MLL. Relative levels of expression of CD43 in
ALL and MLL samples are shown. Each bar represents an individual
leukemia sample. The expression values are raw data obtained from
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays based
on the scaling described in the Examples.
[0035] FIG. 2F illustrates selected early lymphocyte gene
expression in ALL and MLL. Relative levels of expression of CD44 in
ALL and MLL samples are shown. Each bar represents an individual
leukemia sample. The expression values are raw data obtained from
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays based
on the scaling described in the Examples.
[0036] FIG. 3A illustrates selected HOXA9 gene expression in ALL
and MLL. Relative levels of expression of HOXA9 in ALL and MLL
samples are shown. The expression values are obtained using
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays as
described in the Examples.
[0037] FIG. 3B illustrates selected HOXA5 gene expression in ALL
and MLL. Relative levels of expression of HOXA5 in ALL and MLL
samples are shown. The expression values are obtained using
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays as
described in the Examples.
[0038] FIG. 3C illustrates selected HOXA4 gene expression in ALL
and MLL. Relative levels of expression of HOXA4 in ALL and MLL
samples are shown. The expression values are obtained using
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays as
described in the Examples.
[0039] FIG. 3D illustrates selected HOXA7 gene expression in ALL
and MLL. Relative levels of expression of HOXA7 in ALL and MLL
samples are shown. The expression values are obtained using
Affymetrix GENECHIP.RTM. analysis after scaling of the arrays as
described in the Examples.
[0040] FIG. 4A illustrates the comparison of gene expression
between ALL, MLL and AML, and shows the principal component
analysis (PCA) plot of ALL (red), MLL (blue), and AML (yellow)
performed using 8700 genes that passed filtering.
[0041] FIG. 4B illustrates the comparison of gene expression
between ALL, MLL and AML, and shows the PCA plot comparing ALL
(red), MLL (blue), and AML (yellow) using the 500 genes that best
distinguished ALL from AML.
[0042] FIG. 5 illustrates genes specifically expressed in MLL, ALL
or AML. The top 15 genes, and their corresponding GenBank Accession
Numbers, that are most highly correlated with one type of leukemia
versus the other two are shown. Each column represents a leukemia
sample and each row a gene. The relative levels of expression are
shown in red (relatively high) and blue (relatively low) as
described in FIGS. 1A and 1B1.
[0043] FIG. 6 illustrates the classification of ALL, MLL and AML
based on gene expression profile through a plot showing the error
rate in class prediction using a cross-validation approach. One
sample was withheld, and the class membership of this sample
predicted based on gene expression levels. The genes used are the
top 1-250 genes that are best correlated with the ALL/MLL/AML
three-class distinction.
DETAILED DESCRIPTION OF THE INVENTION
[0044] Early and accurate diagnosis of disease is of paramount
importance in rendering effective treatment. The present invention
relates to the diagnosis of mixed lineage leukemia (MLL), acute
lymphoblastic leukemia (ALL), and acute myelogenous leukemia (AML)
according to the gene expression profile of a sample from an
individual, as well as to methods of therapy and screening that
utilize the genes identified herein as targets.
[0045] In one embodiment, the present invention is directed to a
method of diagnosing mixed lineage leukemia, acute lymphoblastic
leukemia and acute myelogenous leukemia, comprising isolating a
gene expression product from at least one informative gene from one
or more cells of an individual selected from the group consisting
of mononuclear blood cells and bone marrow cells; and determining a
gene expression profile of at least one informative gene, wherein
the gene expression profile is correlated with mixed lineage
leukemia, acute lymphoblastic leukemia and acute myelogenous
leukemia.
[0046] In another embodiment, the present invention is directed
toward a method of diagnosing mixed lineage leukemia, acute
lymphoblastic leukemia and acute myelogenous leukemia, comprising
isolating mRNA from one or more cells of an individual, wherein the
cells are selected from the group consisting of mononuclear blood
cells and bone marrow cells, determining a gene expression profile
of at least one informative gene, and comparing the gene expression
profile with a gene expression profile of a control sample selected
from the group consisting of mixed lineage leukemia sample, acute
lymphoblastic leukemia sample and acute myelogenous leukemia
sample, wherein the gene expression profile obtained from the cells
of the individual is indicative of mixed lineage leukemia, acute
lymphoblastic leukemia or acute myelogenous leukemia.
[0047] In one example of the above method, if the gene expression
product obtained from the sample is similar to the gene expression
product of MLL, then the individual is diagnosed as having MLL; and
if the gene expression product obtained from the sample is similar
to the gene expression product of ALL, then the individual is
diagnosed as having ALL; and if the gene expression product
obtained from the sample is similar to the gene expression product
of AML, then the individual is diagnosed as having AML. Using
similar methods, the diagnosis of certain types of leukemias (MLL,
ALL, or AML) can also be ruled out.
[0048] "Gene expression profile" as used herein is defined as the
level or amount of gene expression of particular genes as assessed
by methods described herein. The gene expression profile can
comprise data for one or more genes and can be measured at a single
time point or over a period of time.
[0049] As used herein, "gene expression products" are proteins,
polypeptides, or nucleic acid molecules (e.g., mRNA, tRNA, rRNA, or
cRNA) that result from transcription or translation of genes. The
present invention can be effectively used to analyze proteins,
peptides or nucleic acid molecules that are the result of
transcription or translation. The nucleic acid molecule levels
measured can be derived directly from the gene or, alternatively,
from a corresponding regulatory gene or regulatory sequence
element. All forms of gene expression products can be measured.
Additionally, variants of genes and gene expression products
including, for example, spliced variants and polymorphic alleles,
can be measured. Similarly, gene expression can be measured by
assessing the level of protein or derivative thereof translated
from mRNA. The sample to be assessed can be any sample that
contains a gene expression product. Suitable sources of gene
expression products, e.g., samples, can include intact cells, lysed
cells, cellular material for determining gene expression, or
material containing gene expression products. Examples of such
samples are brain, blood, bone marrow, plasma, lymph, urine,
tissue, mucus, sputum, saliva or other cell samples. Methods of
obtaining such samples are known in the art. In a preferred
embodiment, mononuclear bloods cells are used. In another preferred
embodiment, bone marrow tissue is used.
[0050] In one embodiment, the gene expression product is a protein
or polypeptide. In this embodiment the determination of the gene
expression profile can be made using techniques for protein
detection and quantitation known in the art. For example,
antibodies specific for the protein or polypeptide can be obtained
using methods which are routine in the art, and the specific
binding of such antibodies to protein or polypeptide gene
expression products can be detected and measured.
[0051] The present invention also provides methods for classifying
the sample. A sample can be classified in many ways including but
not limited to leukemia subclass (e.g., ALL, AML, or MLL), response
to a particular treatment, referred to herein as treatment outcome,
or treatment efficacy. Informative genes include, but are not
limited to, those shown in FIGS. 1A, 1B, 2A-2F, 3A-3D, and 5, and
Tables 1 and 2. Using the methods described herein, expression of
numerous genes can be measured simultaneously. The assessment of
numerous genes provides for a more accurate evaluation of the
sample because there are more genes that can assist in classifying
the sample.
[0052] In a preferred embodiment, the gene expression product is
mRNA and the gene expression levels are obtained, e.g., by
contacting the sample with a suitable microarray, and determining
the extent of hybridization of the nucleic acid in the sample to
the probes on the microarray.
[0053] The gene expression value measured or assessed is the
numeric value obtained from an apparatus that can measure gene
expression levels. Gene expression levels refer to the amount of
expression of the gene expression product, as described herein. The
values are raw values from the apparatus, or values that are
optionally rescaled, filtered and/or normalized. Such data is
obtained, for example, from a GeneChip.RTM. probe array or
Microarray (Affymetrix, Inc.) (U.S. Pat. Nos. 5,631,734, 5,874,219,
5,861,242, 5,858,659, 5,856,174, 5,843,655, 5,837,832, 5,834,758,
5,770,722, 5,770,456, 5,733,729, 5,556,752, all of which are
incorporated herein by reference in their entirety), and the
expression levels are calculated with software (e.g., Affymetrix
GENECHIP.RTM. software). Nucleic acids (e.g., mRNA) from a sample
which has been subjected to particular stringency conditions
hybridize to the probes on the chip. The nucleic acid to be
analyzed (e.g., the target) is isolated, amplified and labeled with
a detectable label, (e.g., .sup.32P or fluorescent label) prior to
hybridization to the arrays. Once hybridization occurs, the arrays
are inserted into a scanner which can detect patterns of
hybridization. The hybridization data are collected as light
emitted from the labeled groups which is now bound to the probe
array. The probes that perfectly match the target produce a
stronger signal than those that have mismatches. Since the sequence
and position of each probe on the array are known, by
complementarity, the identity of the target nucleic acid applied to
the probe is determined. Quantitation of gene profiles from the
hybridization of labeled mRNA /DNA microarray can be performed by
scanning the microarrays to measure the amount of hybridization at
each position on the microarray with an Affymetrix scanner
(Affymetrix, Santa Clara, Calif.). For each stimulus a time series
of mRNA levels (C={C1,C2,C3, . . . Cn}) and a corresponding time
series of mRNA levels (M={M1,M2,M3, . . . Mn}) in control medium in
the same experiment as the stimulus is obtained. Quantitative data
is then analyzed. Ci and Mi are defined as relative steady-state
mRNA levels, where i refers to the ith timepoint and n to the total
number of timepoints of the entire timecourse. .mu.M and .sigma.M
are defined as the mean and standard deviation of the control time
course, respectively. Microarrays are only one method of obtaining
gene expression values. Other methods for obtaining gene expression
values known in the art or developed in the future can be used with
the present invention.
[0054] Once the gene expression values are prepared, the sample can
be classified. Genes that are particularly relevant for
classification have been identified as a result of work described
herein and are shown in FIGS. 1A, 1B, 2A-2F, 3A-3D, and 5, and
Tables 1 and 2. The genes that are relevant for classification are
referred to herein as "informative genes." Not all informative
genes for a particular class distinction must be assessed in order
to classify a sample. For example, a subset of the informative
genes which demonstrate a high correlation with a class distinction
can be used. This subset can be, for example, one or more genes,
for example 2, 3, or 4 genes, 5 or more genes, for example 6, 7, 8,
or 9 genes, 10 or more genes, 25 or more genes, 45 or more genes,
or 50 or more genes. Typically the accuracy of the classification
will increase with the number of informative genes assessed.
[0055] The correlation between gene expression profiles and class
distinction can be determined using a variety of methods. Methods
of defining classes and classifying samples are described, for
example, in U.S. patent application Ser. No. 09/544,627, filed Apr.
6, 2000 by Golub et al., the teachings of which are incorporated
herein by reference in their entirety. The information provided by
the present invention, alone or in conjunction with other test
results, aids in sample classification and diagnosis of
disease.
[0056] The present invention also provides methods for monitoring
the effect of a treatment regimen in an individual by monitoring
the gene expression profile for one or more informative genes.
Treatment efficacy classification can be made by comparing the gene
expression profile of a sample at several time points during
treatment with respect to one or more informative genes. A
treatment can be considered efficacious if the gene expression
profile with regard to one or more informative genes tends toward a
normal gene expression profile. That is, for example, treatment can
be considered efficacious if a gene having increased expression in
a disorder (e.g., MLL) shows reduced expression (i.e., expression
tending toward normal expression) as a result of treatment. For
example, in one method, a baseline gene expression profile for the
individual can be determined, and repeated gene expression profiles
can be determined at time points during treatment. A shift in gene
expression profile from a profile correlated with poor treatment
outcome to profile correlated with improved treatment outcome is
evidence of an effective therapeutic regimen, while a repeated
profile correlated with poor treatment outcome is evidence of an
ineffective therapeutic regimen. For example, HOXA9 and MEIS1
upregulation has been correlated with a poor prognosis. An
effective therapeutic regimen might be expected to reduce the level
of HOXA9 and MEIS1 expression. Similarly, as described herein,
expression of FLT3 is correlated with MLL. Thus, a reduction in the
baseline level of FLT3 or its kinase activity can be indicative of
an effective therapeutic. FIGS. 1A, 1B, 2A-2F, 3A-3D, and 5, and
Tables 1 and 2 provide additional gene products which can be useful
in evaluating the efficacy of treatment.
[0057] The present invention also provides information regarding
the genes that are important in MLL treatment response, thereby
providing additional targets for diagnosis and therapy. It is also
clear that the present invention can be used to generate databases
comprising informative genes which will have many applications in
medicine, research and industry.
[0058] Also encompassed in the present invention is the use of gene
expression profiles to screen for therapeutic agents. In one
embodiment, the present invention is directed to a method of
screening for a therapeutic agent for an individual with mixed
lineage leukemia, comprising isolating a gene expression product
from at least one informative gene from one or more cells of the
individual with mixed lineage leukemia; identifying a therapeutic
agent by determining a gene expression profile of at least one
informative gene before and after administration of the agent,
wherein if the gene expression profile from the individual after
administration of the agent is correlated with effective treatment
of mixed lineage leukemia the agent is identified as a therapeutic
agent. In another embodiment, the cells are selected from the group
consisting of mononuclear blood cells and bone marrow cells.
Alternatively, the above method can utilize a cell line derived
from an individual with mixed lineage leukemia.
[0059] The invention also provides methods (also referred to herein
as "screening assays") for identifying agents or compounds (e.g.,
fusion proteins, polypeptides, peptidomimetics, prodrugs,
receptors, binding agents, antibodies, small molecules or other
drugs, or ribozymes) that alter or modulate (e.g., increase or
decrease) the activity of the gene expression products of the
informative genes (e.g., polypeptides encoded by the informative
genes) as described herein, or that otherwise interact with the
informative genes and/or polypeptides described herein. Such
compounds can be compounds or agents that bind to informative gene
expression products described herein (e.g., the polypeptides
encoded by the informative genes in FIGS. 1A, 1B, 2A-2F, 3A-3D, and
5, and Tables 1 and 2), and that have a stimulatory or inhibitory
effect on, for example, activity of the polypeptide encoded by an
informative gene described herein; or that change (e.g., enhance or
inhibit) the ability of a polypeptide encoded by an informative
gene to interact with compounds or agents that bind such an
informative gene polypeptide; or the alter post-translational
processing of such a polypeptide (e.g., agents that alter
proteolytic processing to direct the polypeptide from where it is
normally synthesized to another location in the cell, such as the
cell surface or the nucleus; or agents that alter proteolytic
processing such that more polypeptide is released from the cell,
etc.). In one example, the binding agent is an MLL binding agent.
As used herein, by an MLL binding agent" is meant an agent as
described herein that binds to a polypeptide encoded by an
informative gene of the present invention and modulates the
occurrence, severity, or progression of mixed lineage leukemia. The
modulation can be an increase or a decrease in the occurrence,
severity, or progression of prostate cancer. In addition, an MLL
binding agent includes an agent that binds to a polypeptide that is
upstream (earlier) or downstream (later) of the cell signaling
events mediated by a polypeptide encoded by an informative gene of
the present invention, and thereby modulates the overall activity
of the signaling pathway; in turn, the mixed lineage leukemia
disease state of is modulated.
[0060] The candidate compound can cause an alteration in the
activity of a polypeptide encoded by an informative gene of the
present invention. For example, the activity of the polypeptide can
be altered (increased or decreased) by at least 1.5-fold to 2-fold,
at least 3-fold, or, at least 5-fold, relative to the control.
Alternatively, the polypeptide activity can be altered, for
example, by at least 10%, at least 20%, 40%, 50%, or 75%, or by at
least 90%, relative to the control.
[0061] In one embodiment, the invention provides assays for
screening candidate compounds or test agents to identify compounds
that bind to or modulate the activity of a polypeptide encoded by
an informative gene described herein (or biologically active
portion(s) thereof), as well as agents identifiable by the assays.
As used herein, a "candidate compound" or "test agent" is a
chemical molecule, be it naturally-occurring or
artificially-derived, and includes, for example, peptides,
proteins, synthesized molecules, for example, synthetic organic
molecules, naturally-occurring molecule, for example, naturally
occurring organic molecules, nucleic acid molecules, and components
thereof.
[0062] In general, candidate compounds for use in the present
invention may be identified from large libraries of natural
products or synthetic (or semi-synthetic) extracts or chemical
libraries according to methods known in the art. Those skilled in
the field of drug discovery and development will understand that
the precise source of test extracts or compounds is not critical to
the screening procedure(s) of the invention. Accordingly, virtually
any number of chemical extracts or compounds can be screened using
the exemplary methods described herein. Examples of such extracts
or compounds include, but are not limited to, plant-, fungal-,
prokaryotic- or animal-based extracts, fermentation broths, and
synthetic compounds, as well as modification of existing compounds.
Numerous methods are also available for generating random or
directed synthesis (e.g., semi-synthesis or total synthesis) of any
number of chemical compounds, including, but not limited to,
saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
Synthetic compound libraries are commercially available, e.g., from
Brandon Associates (Merrimack, N.H.) and Aldrich Chemical
(Milwaukee, Wis.). Alternatively, libraries of natural compounds in
the form of bacterial, fungal, plant, and animal extracts are
commercially available from a number of sources, including Biotics
(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics
Institute (Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge,
Mass.). In addition, natural and synthetically produced libraries
are generated, if desired, according to methods known in the art,
e.g., by standard extraction and fractionation methods. For
example, candidate compounds can be obtained using any of the
numerous approaches in combinatorial library methods known in the
art, including: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
limited to polypeptide libraries, while the other four approaches
are applicable to polypeptide, non-peptide oligomer or small
molecule libraries of compounds (Lam, Anticancer Drug Des., 12: 145
(1997)). Furthermore, if desired, any library or compound is
readily modified using standard chemical, physical, or biochemical
methods.
[0063] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their activities should be employed whenever possible.
[0064] When a crude extract is found to modulate (i.e., stimulate
or inhibit) the expression and/or activity of the informative genes
and/or their encoded polypeptides, further fractionation of the
positive lead extract is necessary to isolate chemical constituents
responsible for the observed effect. Thus, the goal of the
extraction, fractionation, and purification process is the careful
characterization and identification of a chemical entity within the
crude extract having an activity that stimulates or inhibits
nucleic acid expression, polypeptide expression, or polypeptide
biological activity. The same assays described herein for the
detection of activities in mixtures of compounds can be used to
purify the active component and to test derivatives thereof.
Methods of fractionation and purification of such heterogenous
extracts are known in the art. If desired, compounds shown to be
useful agents for treatment are chemically modified according to
methods known in the art. Compounds identified as being of
therapeutic value may be subsequently analyzed using animal models
for diseases in which it is desirable to alter the activity or
expression of the nucleic acids or polypeptides of the present
invention.
[0065] In one embodiment, to identify candidate compounds that
alter the biological activity of a polypeptide encoded by an
informative gene as described herein, a cell, tissue, cell lysate,
tissue lysate, or solution containing or expressing a polypeptide
encoded by the informative gene (e.g., a polypeptide encoded by a
gene in any of FIGS. 1A, 1B, 2A-2F, 3A-3D, and 5, and Tables 1 and
2), or a fragment of derivative thereof, can be contacted with a
candidate compound to be tested under conditions suitable for
biological activity of the polypeptide. Alternatively, the
polypeptide can be contacted directly with the candidate compound
to be tested. The level (amount) of polypeptide biological activity
is assessed/measured, either directly or indirectly, and is
compared with the level of biological activity in a control (i.e.,
the level of activity of the polypeptide or active fragment or
derivative thereof in the absence of the candidate compound to be
tested, or in the presence of the candidate compound vehicle only).
If the level of the biological activity in the presence of the
candidate compound differs, by an amount that is statistically
significant, from the level of the biological activity in the
absence of the candidate compound, or in the presence of the
candidate compound vehicle only, then the candidate compound is a
compound that alters the biological activity of the polypeptide
encoded by an informative gene of the invention. For example, an
increase in the level of polypeptide biological activity relative
to a control, indicates that the candidate compound is a compound
that enhances (is an agonist of) the polypeptide biological
activity. Similarly, a decrease in the polypeptide biological
activity relative to a control, indicates that the candidate
compound is a compound that inhibits (is an antagonist of) the
polypeptide biological activity.
[0066] In another embodiment, the level of biological activity of a
polypeptide encoded by an informative gene, or a derivative or
fragment thereof in the presence of the candidate compound to be
tested, is compared with a control level that has previously been
established. A level of polypeptide biological activity in the
presence of the candidate compound that differs from (i.e.,
increases or decreases) the control level by an amount that is
statistically significant indicates that the compound alters the
biological activity of the polypeptide.
[0067] The present invention also relates to an assay for
identifying compounds (e.g., antisense nucleic acids, fusion
proteins, polypeptides, peptidomimetics, prodrugs, receptors,
binding agents, antibodies, small molecules or other drugs, or
ribozymes) that alter (e.g., increase or decrease) expression
(e.g., transcription or translation) of an informative gene or that
otherwise interact with an informative gene described herein, as
well as compounds identifiable by the assays. For example, a
solution containing an informative gene can be contacted with a
candidate compound to be tested. The solution can comprise, for
example, cells containing the informative gene or cell lysate
containing the informative gene; alternatively, the solution can be
another solution that comprises elements necessary for
transcription/translation of the informative gene. Cells not
suspended in solution can also be employed, if desired. The level
and/or pattern of informative gene expression (e.g., the level
and/or pattern of mRNA or protein expressed) is assessed, and is
compared with the level and/or pattern of expression in a control
(i.e., the level and/or pattern of the informative gene expressed
in the absence of the candidate compound, or in the presence of the
candidate compound vehicle only). If the expression level and/or
pattern in the presence of the candidate compound differs by an
amount or in a manner that is statistically significant from the
level and/or pattern in the absence of the candidate compound, or
in the presence of the candidate compound vehicle only, then the
candidate compound is a compound that alters the expression of an
informative gene. Enhancement of informative gene expression
indicates that the candidate compound is an agonist of informative
gene polypeptide activity. Similarly, inhibition of informative
gene expression indicates that the candidate compound is an
antagonist of informative gene polypeptide activity.
[0068] In another embodiment, the level and/or pattern of an
informative gene in the presence of the candidate compound to be
tested, is compared with a control level and/or pattern that has
previously been established. A level and/or pattern informative
gene expression in the presence of the candidate compound that
differs from the control level and/or pattern by an amount or in a
manner that is statistically significant indicates that the
candidate compound alters informative gene expression.
[0069] In another embodiment of the invention, compounds that alter
the expression of an informative gene, or that otherwise interact
with an informative gene described herein, can be identified using
a cell, cell lysate, or solution containing a nucleic acid encoding
the promoter region of the informative gene operably linked to a
reporter gene. As used herein by "promoter" means a minimal
nucleotide sequence sufficient to direct transcription, and by
"operably linked" means that a gene and one or more regulatory
sequences are connected in such a way as to permit gene expression
when the appropriate molecules (e.g., transcriptional activator
proteins) are bound to the regulatory sequences. Examples of
reporter genes and methods for operably linking a reporter gene to
a promoter are known in the art. After contact with a candidate
compound to be tested, the level of expression of the reporter gene
(e.g., the level of mRNA or of protein expressed) is assessed, and
is compared with the level of expression in a control (i.e., the
level of expression of the reporter gene in the absence of the
candidate compound, or in the presence of the candidate compound
vehicle only). If the level of expression in the presence of the
candidate compound differs by an amount or in a manner that is
statistically significant from the level in the absence of the
candidate compound, or in the presence of the candidate compound
vehicle only, then the candidate compound is a compound that alters
the expression of the informative gene, as indicated by its ability
to alter expression of the reporter gene that is operably linked to
the informative gene promoter. Enhancement of the expression of the
reporter gene indicates that the compound is an agonist of the
informative gene polypeptide activity. Similarly, inhibition of the
expression of the reporter gene indicates that the compound is an
antagonist of the informative gene polypeptide activity.
[0070] In another embodiment, the level of expression of the
reporter in the presence of the candidate compound to be tested, is
compared with a control level that has been established previously.
A level in the presence of the candidate compound that differs from
the control level by an amount or in a manner that is statistically
significant indicates that the candidate compound alters
informative gene expression.
[0071] The present invention also features methods of detecting
and/or identifying a compound that alters the interaction between a
polypeptide encoded by an informative gene and a polypeptide (or
other molecule) with which the polypeptide normally interacts with
(e.g., in a cell or under physiological conditions). In one
example, a cell or tissue that expresses or contains a compound
(e.g., a polypeptide or other molecule) that interacts with a
polypeptide encoded by an informative gene (such a molecule is
referred to herein as a "polypeptide substrate") is contacted with
the informative gene polypeptide in the presence of a candidate
compound, and the ability of the candidate compound to alter the
interaction between the polypeptide encoded by the informative gene
and the polypeptide substrate is determined, for example, by
assaying activity of the polypeptide. Alternatively, a cell lysate
or a solution containing the informative gene polypeptide, the
polypeptide substrate, and the candidate compound can be used. A
compound that binds to the informative gene polypeptide or to the
polypeptide substrate can alter the interaction between the
informative gene polypeptide and the polypeptide substrate by
interfering with (inhibiting), or enhancing the ability of the
informative gene polypeptide to bind to, associate with, or
otherwise interact with the polypeptide substrate.
[0072] Determining the ability of the candidate compound to bind to
the informative gene polypeptide or a polypeptide substrate can be
accomplished, for example, by coupling the candidate compound with
a radioisotope or enzymatic label such that binding of the
candidate compound to the informative gene polypeptide or
polypeptide substrate can be determined by directly or indirectly
detecting the candidate compound labeled with .sup.125I, .sup.35S,
.sup.14C, or .sup.3H, and the detecting the radioisotope (e.g., by
direct counting of radioemission or by scintillation counting).
Alternatively, the candidate compound can be enzymatically labeled
with, for example, horseradish peroxidase, alkaline phosphatase, or
luciferase, and the enzymatic label is then detected by
determination of conversion of an appropriate substrate to product.
In another alternative, one of the other components of the
screening assay (e.g., the polypeptide substrate or the informative
gene polypeptide) can be labeled, and alterations in the
interaction between the informative gene polypeptide and the
polypeptide substrate can be detected. In these methods, labeled
unbound components can be removed (e.g., by washing) after the
interaction step in order to accurately detect the effect of the
candidate compound on the interaction between the informative gene
polypeptide and the polypeptide substrate.
[0073] It is also within the scope of this invention to determine
the ability of a candidate compound to interact with the
informative gene polypeptide or polypeptide substrate without the
labeling of any of the interactants. For example, a
microphysiometer can be used to detect the interaction of a
candidate compound with a polypeptide encoded by an informative
gene or a polypeptide substrate without the labeling of either the
candidate compound, the polypeptide encoded by the informative
gene, or the polypeptide substrate (McConnell et al., Science 257:
1906-1912 (1992)). As used herein, a "microphysiometer" (e.g.,
CYTOSENSOR.TM.) is an analytical instrument that measures the rate
at which a cell acidifies its environment using a light-addressable
potentiometric sensor (LAPS). Changes in this acidification rate
can be used as an indicator of the interaction between ligand and
polypeptide.
[0074] In another embodiment of the invention, assays can be used
to identify polypeptides that interact with one or more
polypeptides encoded by an informative gene. For example, a yeast
two-hybrid system such as that described by Fields and Song (Fields
and Song, Nature 340: 245-246 (1989)) can be used to identify
polypeptides that interact with one or more polypeptides encoded by
an informative gene. In such a yeast two-hybrid system, vectors are
constructed based on the flexibility of a transcription factor that
has two functional domains (a DNA binding domain and a
transcription activation domain). If the two domains are separated
but fused to two different proteins that interact with one another,
transcriptional activation can be achieved, and transcription of
specific markers (e.g., nutritional markers such as His and Ade, or
color markers such as lacZ) can be used to identify the presence of
interaction and transcriptional activation. For example, in the
methods of the invention, a first vector is used that includes a
nucleic acid encoding a DNA binding domain and a polypeptide
encoded by an informative gene, or fragment or derivative thereof,
and a second vector is used that includes a nucleic acid encoding a
transcription activation domain and a nucleic acid encoding a
polypeptide that potentially may interact with the informative gene
polypeptide, or fragment or derivative thereof. Incubation of yeast
containing the first vector and the second vector under appropriate
conditions (e.g., mating conditions such as used in the
MATCHMAKER.TM. system from Clontech) allows identification of
colonies that express the markers of the polypeptide(s). These
colonies can be examined to identify the polypeptide(s) that
interact with the polypeptide encoded by the informative gene or a
fragment or derivative thereof. Such polypeptides may be useful as
compounds that alter the activity or expression of an informative
gene polypeptide.
[0075] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize a
polypeptide encoded by an informative gene, or a polypeptide
substrate, or other components of the assay on a solid support, in
order to facilitate separation of complexed from uncomplexed forms
of one or both of the polypeptides, as well as to accommodate
automation of the assay. Binding of a candidate compound to the
polypeptide, or interaction of the polypeptide with a polypeptide
substrate in the presence and absence of a candidate compound, can
be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtitre plates, test
tubes, and micro-centrifuge tubes. In one embodiment, a fusion
protein (e.g., a glutathione-S-transferase fusion protein) can be
provided that adds a domain that allows the informative gene
polypeptide, or the polypeptide substrate to be bound to a matrix
or other solid support.
[0076] This invention further pertains to novel compounds
identified by the above-described screening assays. Accordingly, it
is within the scope of this invention to further use a compound
identified as described herein in an appropriate animal model. For
example, a compound identified as described herein can be used in
an animal model to determine the efficacy, toxicity, or side
effects of treatment with such a compound. Alternatively, a
compound identified as described herein can be used in an animal
model to determine the mechanism of action of such a compound.
Furthermore, this invention pertains to uses of novel compounds
identified by the above-described screening assays for treatments
as described herein. In addition, a compound identified as
described herein can be used to alter activity of a polypeptide
encoded by an informative gene, or to alter expression of the
informative gene, by contacting the polypeptide or the nucleic acid
molecule (or contacting a cell comprising the polypeptide or the
nucleic acid molecule) with the compound identified as described
herein.
[0077] The present invention encompasses a method of treating MLL,
AML or ALL, comprising the administration of an agent which
modulates the expression level or activity of an informative gene
product. A therapeutic agent may increase or decrease the level or
activity of the gene product. For example, an inhibitor of the
kinase FLT3 should be useful in treating MLL. Other suitable
therapeutic targets for drug development include genes described
herein in FIGS. 1A, 1B, 2A-2F, 3A-3D, and 5, and Tables 1 and
2.
[0078] The present invention further relates to antibodies that
specifically bind a polypeptide, preferably an epitope, of an
informative gene of the present invention (as determined, for
example, by immunoassays, a technique well known in the art for
assaying specific antibody-antigen binding). Antibodies of the
invention include, but are not limited to, polyclonal, monoclonal,
multispecific, human, humanized or chimeric antibodies, single
chain antibodies, Fab fragments F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id)
antibodies (including, for example, anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above.
[0079] The term "antibody," as used herein, refers to
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, and more specifically, molecules that
contain an antigen binding site that specifically binds an antigen.
The immunoglobulin molecules of the invention can be of any type
(for example, IgG, IgE, IgM, IgD, IgA and IgY), and of any class
(for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of
an immunoglobulin molecule.
[0080] In one embodiment, the antibodies are antigen-binding
antibody fragments and include, without limitation, Fab, Fab' and
F(ab').sub.2, Fd, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv) and fragments comprising either a
V.sub.L or V.sub.H domain. Antigen-binding antibody fragments,
including single-chain antibodies, can comprise the variable
region(s) alone or in combination with the entirety or a portion of
one or more of the following: hinge region, CH1, CH2, and CH3
domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable region(s)
with a hinge region, CH1, CH2, and/or CH3 domains.
[0081] The antibodies of the invention may be from any animal
origin including birds and mammals. Preferably, the antibodies are
human, murine, donkey, sheep, rabbit, goat, guinea pig, hamster,
horse, or chicken.
[0082] As used herein, "human" antibodies include antibodies having
the amino acid sequence of a human immunoglobulin and include
antibodies produced by human B cells, or isolated from human sera,
human immunoglobulin libraries or from animals transgenic for one
or more human immunoglobulins and that do not express endogenous
immunoglobulins, as described in U.S. Pat. No. 5,939,598 by
Kucherlapati et al., for example.
[0083] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material.
[0084] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention that they recognize or specifically bind.
The epitope(s) of polypeptide portion(s) may be specified, for
example, by N-terminal and/or C-terminal positions, or by size in
contiguous amino acid residues. Antibodies that specifically bind
any epitope or polypeptide encoded by an informative gene of the
present invention may also be excluded. Therefore, the present
invention includes antibodies that specifically bind a polypeptide
encoded by an informative gene of the present invention, and allows
for the exclusion of the same.
[0085] The term "epitope," as used herein, refers to a portion of a
polypeptide which contacts an antigen-binding site(s) of an
antibody or T cell receptor. Specific binding of an antibody to an
antigen having one or more epitopes excludes non-specific binding
to unrelated antigens, but does not necessarily exclude
cross-reactivity with other antigens with similar epitopes.
[0086] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies of the
present invention may not display any cross-reactivity, such that
they do not bind any other analog, ortholog, or homolog of a
polypeptide of the present invention. Alternatively, antibodies of
the invention can bind polypeptides with at least about 95%, 90%,
85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% identity (as calculated
using methods known in the art) to a polypeptide encoded by an
informative gene of the present invention. Further included in the
present invention are antibodies that bind polypeptides encoded by
informative genes that hybridize to an informative gene of the
present invention under stringent hybridization conditions, as will
be appreciated by one of skill in the art.
[0087] Antibodies of the present invention can also be described or
specified in terms of their binding affinity to a polypeptide of
the invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, 10.sup.-12 M, 5.times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-13 M,
5.times.10.sup.-15 M, and 10.sup.-15 M.
[0088] The invention also provides antibodies that competitively
inhibit binding of an antibody to an epitope of a polypeptide of
the invention, as determined by any method known in the art for
determining competitive binding, for example, using immunoassays.
In particular embodiments, the antibody competitively inhibits
binding to the epitope by at least about 90%, 80%, 70%, 60%, or
50%.
[0089] Antibodies of the present invention can act as agonists or
antagonists of polypeptides encoded by the informative genes of the
present invention. For example, the present invention includes
antibodies which disrupt interactions with the polypeptides encoded
by the informative genes of the invention either partially or
fully. The invention also includes antibodies that do not prevent
binding, but prevent activation or activity of the polypeptide.
Activation or activity (for example, signaling) may be determined
by techniques known in the art. Also included are antibodies that
prevent both binding to and activity of a polypeptide encoded by an
informative gene. Likewise included are neutralizing
antibodies.
[0090] Antibodies of the present invention may be used, for
example, and without limitation, to purify, detect, and target the
polypeptides encoded by the informative genes described herein,
including both in vitro and in vivo diagnostic and therapeutic
methods. For example, the antibodies have use in immunoassays for
qualitatively and quantitatively measuring levels of the
polypeptides in biological samples. See, for example, Harlow et
al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988).
[0091] As discussed in more detail below, the antibodies of the
present invention may be used either alone or in combination with
other compositions. The antibodies may further be recombinantly
fused to a heterologous polypeptide at the N- and/or C-terminus or
chemically conjugated (including covalent and non-covalent
conjugations) to polypeptides or other compositions. For example,
antibodies of the present invention may be recombinantly fused or
conjugated to molecules useful as labels in detection assays, or
effector molecules such as heterologous polypeptides, drugs, or
toxins.
[0092] The antibodies of the invention include derivatives that are
modified, for example, by the covalent attachment of any type of
molecule to the antibody such that covalent attachment does not
prevent the antibody from recognizing its epitope. For example, but
not by way of limitation, the antibody derivatives include
antibodies that have been modified, for example, by glycosylation,
acetylation, pegylation, phosphorylation, amidation, derivatization
by known protecting/blocking groups, proteolytic cleavage, or
linkage to a cellular ligand or other protein. Any of numerous
chemical modifications can be carried out by known techniques,
including, but not limited to, specific chemical cleavage,
acetylation, formylation, and metabolic synthesis of tunicamycin.
Additionally, the derivative can contain one or more non-classic
amino acids.
[0093] The antibodies of the present invention can be generated by
any suitable method known in the art. Polyclonal antibodies to an
antigen-of-interest can be produced by various procedures well
known in the art. For example, a polypeptide of the invention can
be administered to various host animals including, but not limited
to, rabbits, mice, rats, or the like, to induce the production of
sera containing polyclonal antibodies specific for the antigen.
Various adjuvants can be used to increase the immunological
response, depending on the host species, and include, but are not
limited to, Freund's adjuvant (complete and incomplete), mineral
gels such as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
well known in the art.
[0094] Monoclonal antibodies can be prepared using a wide variety
of techniques also known in the art, including hybridoma cell
culture, recombinant, and phage display technologies, or a
combination thereof. For example, monoclonal antibodies can be
produced using hybridoma techniques as is known in the art and
taught, for example, in Harlow et al., Antibodies: A Laboratory
Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). The
term "monoclonal antibody" as used herein is not necessarily
limited to antibodies produced through hybridoma technology, but
also refers to an antibody that is derived from a single clone,
including any eukaryotic, prokaryotic, or phage clone.
[0095] Human antibodies are desirable for therapeutic treatment of
human patients. These antibodies can be made by a variety of
methods known in the art including phage display methods using
antibody libraries derived from human immunoglobulin sequences.
Human antibodies can also be produced using transgenic mice that
are incapable of expressing functional endogenous immunoglobulins,
but which can express human immunoglobulin genes. The transgenic
mice are immunized with a selected antigen, for example, all or a
portion of a polypeptide of the invention. Monoclonal antibodies
directed against the antigen can be obtained from the immunized,
transgenic mice using conventional hybridoma technology. The human
immunoglobulin transgenes harbored by the transgenic mice rearrange
during B cell differentiation, and subsequently undergo class
switching and somatic mutation. Thus, using such a technique, it is
possible to produce therapeutically useful IgG, IgA, IgM and IgE
antibodies. For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, for example, PCT
publications WO 98/24893; WO 96/34096; WO 96/33735; and U.S. Pat.
Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5661,016;
5,545,806; 5,814,318; and 5,939,598.
[0096] In another embodiment, antibodies to the polypeptides
encoded by the informative genes as described herein can, in turn,
be utilized to generate anti-idiotype antibodies that "mimic"
polypeptides of the invention using techniques well known to those
skilled in the art. (See, for example, Greenspan & Bona, FASEB
J. 7(5):537-444 (1989) and Nissinoff, J. Immunol. 147(8):2429-2438
(1991)). For example, antibodies that bind to and competitively
inhibit polypeptide multimerization and/or binding of a polypeptide
to a ligand can be used to generate anti-idiotypes that "mimic" the
polypeptide multimerization and/or binding domain and, as a
consequence, bind to and neutralize polypeptide and/or its ligand.
Such neutralizing anti-idiotypes or Fab fragments of such
anti-idiotypes can be used in therapeutic regimens to neutralize
polypeptide ligand. For example, such anti-idiotypic antibodies can
be used to bind a polypeptide encoded by an informative gene and/or
to bind its ligands, and thereby block its biological activity.
[0097] The antibodies or fragments thereof of the present invention
can be fused to marker sequences, such as a peptide to facilitate
their purification. In one embodiment, the marker amino acid
sequence is a hexa-histidine peptide, and HA tag, or a FLAG tag, as
will be readily appreciated by one of skill in the art.
[0098] The present invention further encompasses antibodies or
fragments thereof conjugated to a diagnostic or therapeutic agent.
The antibodies can be used diagnostically, for example, to monitor
the development or progression of a tumor as part of a clinical
testing procedure to determine the efficacy of a given treatment
regimen. Detection can be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include
enzymes (such as, horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase), prosthetic group
(such as streptavidin/biotin and avidin/biotin), fluorescent
materials (such as umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride or phycoerythrin), luminescent materials (such as
luminol), bioluminescent materials (such as luciferase, luciferin,
and aequorin), radioactive materials (such as, .sup.125I,
.sup.131I, .sup.111In, or .sup.99Tc), and positron emitting metals
using various positron emission tomographies, and nonradioactive
paramagnetic metal ions.
[0099] In an additional embodiment, an antibody or fragment thereof
can be conjugated to a therapeutic moiety such as a cytotoxin, for
example, a cytostatic or cytocidal agent, a therapeutic agent or a
radioactive metal ion. A cytotoxin or cytotoxic agent includes any
agent that id detrimental to cells. Examples include paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof. Therapeutic agents include, but are
not limited to, antimetabolites (e.g., methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil
decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin,
mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)
cisplatin), anthracyclines (for example, daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (for example,
actinomycin, bleomycin, mithramycin, and anthramycin (AMC)), and
anti-mitotic agents (for example, vincristine and vinblastine).
[0100] The conjugates of the invention can be used for modifying a
given biological response, the therapeutic agent or drug moiety is
not to be construed as limited to classical chemical therapeutic
agents. For example, the drug moiety may be a protein or
polypeptide possessing a desired biological activity. Such proteins
may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.- interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, a thrombotic agent or an anti-angiogenic agent, for
example, angiostatin or endostatin; or, biological response
modifiers such as, for example, lymphokines, interleukins,
granulocyte macrophase colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[0101] Antibodies of the invention can also be attached to solid
supports. These are particularly useful for immunoassays or
purification of the target antigen. Such solid supports include,
but are not limited to, glass, cellulose, silicon, polyacrylamide,
nylon, polystyrene, polyvinyl chloride or polypropylene. Techniques
for conjugating such therapeutic moiety to antibodies are well
known in the art, see, for example, Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. eds., pp.
243-56 (Alan R. Liss, Inc. 1985).
[0102] Alternatively, an antibody can be conjugated to a second
antibody to form an antibody heteroconjugate as described by Segal
in U.S. Pat. No. 4,676,980.
[0103] An antibody of the invention, with or without conjugation to
a therapeutic moiety, administered alone or in combination with
cytotoxic factor(s) and/or cytokine(s), can be used as a
therapeutic.
[0104] Antisense antagonists of the present invention are also
included. Antisense technology can be used to control gene
expression through antisense DNA or RNA, or through triple-helix
formation. Antisense techniques are discussed for example, in
Okano, J., Neurochem. 56:560 (1991). The methods are based on
binding of a polynucleotide to a complementary DNA or RNA. In one
embodiment, an antisense sequence is generated internally by the
organism, in another embodiment, the antisense sequence is
separately administered (see, for example, O'Connor, J., Neurochem.
56:560 (1991)).
[0105] In one embodiment, the 5' coding portion of an informative
gene can be used to design an antisense RNA oligonucleotide from
about 10 to 40 base pairs in length. Generally, a DNA
oligonucleotide is designed to be complementary to a region of the
gene involved in transcription thereby preventing transcription and
the production of the receptor. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA
molecule into receptor polypeptide.
[0106] In one embodiment, the antisense nucleic acid of the
invention is produced intracellularly by transcription from an
exogenous sequence. For example, a vector or a portion thereof, is
transcribed, producing an antisense nucleic acid of the invention.
Such a vector contains the sequence encoding the antisense nucleic
acid. The vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired
antisense RNA. Vectors can be constructed by recombinant DNA
technology and can be plasmid, viral, or otherwise, as is known to
one of skill in the art.
[0107] Expression can be controlled by any promoter known in the
art to act in the target cells, such as vertebrate cells, and
preferably human cells. Such promoters can be inducible or
constitutive and include, without limitation, the SV40 early
promoter region (Bernoist and Chambon, Nature 29:304-310 (1981),
the promoter contained in the 3' long terminal repeat of Rous
sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980)), the herpes
thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.
78:1441-1445 (1981)), and the regulatory sequences of the
metallothionein gene (Brinster et al., Nature 296:39-42
(1982)).
[0108] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of an informative gene. Absolute complementarity, although
preferred, is not required. A sequence "complementary to at least a
portion of an RNA," referred to herein, means a sequence having
sufficient complementarity to be able to hybridize with the RNA,
forming a stable duplex. The ability to hybridize will depend on
both the degree of complementarity and the length of the antisense
nucleic acid. Generally, the larger the hybridizing nucleic acid,
the more base mismatches with the RNA it may contain and still form
a stable duplex. One skilled in the art can ascertain a tolerable
degree or mismatch by use of standard procedures to determine the
melting point of the hybridized complex.
[0109] Oligonucleotides that are complementary to the 5' end of the
RNA, for example, the 5' untranslated sequence up to and including
the AUG initiation codon, are generally regarded to work most
efficiently at inhibiting translation. However, sequences
complementary to the 3' untranslated sequences of mRNAs have been
shown to be effective at inhibiting translation of mRNAs as well.
Thus, oligonucleotides complementary to either the 5'- or
3'-non-translated, non-coding regions of a nucleotide sequence can
be used in an antisense approach to inhibit mRNA translation.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA can include the complement of the AUG start codon. Antisense
oligonucleotides complementary to mRNA coding regions can also be
used in accordance with the invention. In one embodiment, the
antisense nucleic acids are at least six nucleotides in length, and
are preferably oligonucleotides ranging from about 6 to about 50
nucleotides in length. In other embodiments, the oligonucleotide is
at least about 10, 17, 25 or 50 nucleotides in length.
[0110] The antisense oligonucleotides of the invention can be DNA
or RNA, or chimeric mixtures, or derivatives or modified versions
thereof, single-stranded or double-stranded. The oligonucleotide
can be modified at the base moiety, sugar moiety, or phosphate
backbone, or example, to improve stability of the molecule,
hybridization, and the like. The oligonucleotide can include other
appended groups such as peptides (for example, to target host cell
receptors in vivo), or agents that facilitate transport across the
cell membrane, or the blood-brain barrier, or intercalating
agents.
[0111] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
a-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0112] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0113] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0114] In yet another embodiment, the antisense oligonucleotide is
an .alpha.-anomeric oligonucleotide. An .alpha.-anomeric
oligonucleotide forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .beta.-units, the
strands run parallel to each other (Gautier et al., Nucl. Acids
Res. 15:6625-6641 (1987)). The oligonucleotide is a
2'-O-methylribonucleotide (Inoue et al., Nucl. Acids Res.
15:6131-6148 (1987)), or a chimeric RNA-DNA analog (Inoue et al.,
FEBS Lett. 215:327-330 (1987)).
[0115] Antisense oligonucleotides of the invention may be
synthesized by standard methods known in the art, for example, by
use of an automated DNA synthesizer.
[0116] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme. Hammerhead ribozymes cleave
mRNAs at locations dictated by flanking regions that form
complementary base pairs with the target mRNA. The target mRNA has
the following sequence of two bases: 540 -UG-3'. The construction
and production of hammerhead ribozymes is well known in the art and
is described more fully in Haseloff and Gerlach (Nature 334:585-591
(1988)). Preferably, the ribozyme is engineered so that the
cleavage recognition site is located near the 5' end of the mRNA in
order to increase efficiency and minimize the intracellular
accumulation of non-functional mRNA transcripts.
[0117] Ribozymes of the invention can be composed of modified
oligonucleotides (for example for improved stability, targeting,
and the like). DNA constructs encoding the ribozyme can be under
the control of a strong constitutive promoter, such as, for
example, pol III or pol II promoter, so that a transfected cell
will produce sufficient quantities of the ribozyme to destroy
endogenous target mRNA and inhibit translation. Since ribozymes,
unlike antisense molecules, are catalytic, a lower intracellular
concentration is generally required for efficiency.
[0118] The present invention also provides pharmaceutical
compositions, including both therapeutic and prophylatic
compositions. Compositions within the scope of this invention
include all compositions wherein the therapeutic abent, antibody,
fragment or derivative, antisense oligonucleotide or ribozyme is
contained in an amount effective to achieve its intended purpose.
While individual needs vary, determination of optimal ranges of
effective amounts of each component is within the skill of the art.
The effective does is a function of a number of factors, including
the specific antibody, the antisense construct, ribozyme or
polypeptide of the invention, the presence of a conjugated
therapeutic agent (see below), the patient and their clinical
status.
[0119] Mode of administration may be by parenteral, subcutaneous,
intravenous, intramuscular, intraperitoneal, transdermal, or buccal
routes. Alternatively, or concurrently, administration may be
orally. The dosage administered will be dependent upon the age,
health, and weight of the recipient, kind of concurrent treatment,
if any, frequency of treatment, and the nature of the effect
desired.
[0120] Such compositions generally comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skimmed milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents.
[0121] These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, and the like. Such compositions will contain a
therapeutically effective amount of the compound, preferably in
purified form, together with a suitable amount of carrier so as to
provide the form for proper administration to the patient. The
formulation should suit the mode of administration.
[0122] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to a human. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0123] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, and the like, and those
formed with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0124] The compositions of the invention can be administered alone
or in combination with other therapeutic agents. Therapeutic agents
that can be administered in combination with the compositions of
the invention, include but are not limited to chemotherapeutic
agents, antibiotics, steroidal and non-steroidal
anti-inflammatories, conventional immunotherapeutic agents,
cytokines and/or growth factors. Combinations may be administered
either concomitantly, for example, as an admixture, separately but
simultaneously or concurrently; or sequentially. This includes
presentations in which the combined agents are administered
together as a therapeutic mixture, and also procedures in which the
combined agents are administered separately but simultaneously, for
example, as through separate intravenous lines into the same
individual. Administration "in combination" further includes the
separate administration of one of the compounds or agents given
first, followed by the second.
[0125] Conventional nonspecific immunosuppressive agents, that may
be administered in combination with the compositions of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs, cyclophosphamide methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive agents.
[0126] In a further embodiment the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, tetracycline,
metronidazole, amoxicillin, beta-lactamases, aminoglycosides,
macrolides, quinolones, fluoroquinolones, cephalosporins,
erythromycin, ciprofloxacin, and streptomycin.
[0127] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that can be
administered with the compositions of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
e-acetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0128] In another embodiment, compositions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethinyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
dietylstilbestrol diphosphate, chlorotrianisene, and testolactone);
nitrogen mustard derivatives (e.g., mephalen, chorambucil,
mechlorethamine (nitrogen mustard) and thiotepa); steroids and
combinations (e.g., bethamethasone sodium phosphate); and others
(e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate,
vinblastine sulfate, and etoposide).
[0129] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and
TNF-alpha.
[0130] In additional embodiments, the compositions of the invention
are administered in combination with other therapeutic or
prophylactic regimens, such as, for example, radiation therapy.
[0131] The present invention is further directed to therapies which
involve administering pharmaceutical compositions of the invention
to an animal, preferably a mammal, and most preferably a human
patient for treating one or more of the described disorders.
Therapeutic compositions of the invention include, for example,
therapeutic agents identified in screening assays, antibodies of
the invention (including fragments, analogs and derivatives thereof
as described herein), antisense oligonucleotides, ribozymes and
nucleic acids encoding same. The compositions of the invention can
be used to treat, inhibit, prognose, diagnose or prevent diseases,
disorders or conditions associated with aberrant expression and/or
activity of a polypeptide of the invention, including, but not
limited to, any one or more of the diseases, disorders, or
conditions such as, for example, MLL, AML, or ALL.
[0132] The treatment and/or prevention of diseases and disorders
associated with aberrant expression and/or activity of a
polypeptide of the invention includes, but is not limited to,
alleviating symptoms associated with those diseases and
disorders.
[0133] The amount of the compound of the invention which will be
effective in the treatment, inhibition and prevention of a disease
or disorder associated with aberrant expression and/or activity of
a polypeptide of the invention can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0134] For antibodies, the dosage administered to a patient is
typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
Preferably, the dosage administered to a patient is between 0.1
mg/kg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than
antibodies from other species due to the immune response to the
foreign polypeptides. Thus, lower dosages of human antibodies and
less frequent administration is often possible. Furthermore, the
dosage and frequency of administration of antibodies of the
invention may be reduced by enhancing uptake and tissue penetration
of the antibodies by modifications such as, for example, lipidation
or addition of cell-specific tags.
[0135] The compounds or pharmaceutical compositions of the
invention can be tested in vitro, and then in vivo for the desired
therapeutic or prophylactic activity, prior to use in humans. For
example, in vitro assays to demonstrate the therapeutic or
prophylactic utility of a compound or pharmaceutical composition
include, the effect of a compound on a cell line or a patient
tissue sample. The effect of the compound or composition on the
cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art including, but not
limited to, rosette formation assays and cell lysis assays. In
accordance with the invention, in vitro assays which can be used to
determine whether administration of a specific compound is
indicated, include in vitro cell culture assays in which a patient
tissue sample is grown in culture, and exposed to or otherwise
administered a compound, and the effect of such compound upon the
tissue sample is observed.
[0136] The invention provides methods of treatment, inhibition and
prophylaxis by administration to a subject of an effective amount
of a compound or pharmaceutical composition of the invention. In
one aspect, the compound is substantially purified such that the
compound is substantially free from substances that limit its
effect or produce undesired side-effects. The subject is preferably
an animal, including but not limited to animals such as cows, pigs,
horses, chickens, cats, dogs, etc., and is preferably a mammal, and
most preferably human.
[0137] Various delivery systems are known and can be used to
administer a composition of the invention, for example,
encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the compound,
receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem.
262:4429-4432 (1987)), construction of a nucleic acid as part of a
retroviral or other vector, and the like as will be known by one of
skill in the art.
[0138] Methods of introduction include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
or compositions may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compounds or compositions of the invention into the
central nervous system by any suitable route, including
intraventricular and intrathecal injection, intraventricular
injection may be facilitated by an intraventricular catheter, for
example, attached to a reservoir, such as an Ommaya reservoir.
Pulmonary administration can also be employed, for example, by use
of an inhaler or nebulizer, and formulation with an aerosolizing
agent.
[0139] In a specific embodiment, it may be desirable to administer
the pharmaceutical compounds or compositions of the invention
locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during
surgery, topical application, for example, in conjunction with a
wound dressing after surgery, by injection, by means of a catheter,
by means of a suppository, or by means of an implant, said implant
being of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention,
care must be taken to use materials to which the protein does not
absorb.
[0140] In another embodiment, the compound or composition can be
delivered in a vesicle, such as a liposome (Langer, Science
249:1527-1533 (1990)).
[0141] In yet another embodiment, the compound or composition can
be delivered in a controlled release system. Furthermore, a
controlled release system can be placed in proximity of the
therapeutic target, thus requiring only a fraction of the systemic
dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra, vol. 2, pp. 115-138 (1984)). In a further
embodiment, a pump may be used. In another embodiment, polymeric
materials can be used.
[0142] In a particular embodiment where the compound of the
invention is a nucleic acid encoding a protein, the nucleic acid
can be administered in vivo to promote expression of its mRNA and
encoded protein, by constructing it as part of an appropriate
nucleic acid expression vector and administering, for example, by
use of a retroviral vector, or by direct injection, or by use of
microparticle bombardment for example, a gene gun, or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl.
Acad Sci. USA 88:1864-1868 (1991)). Alternatively, a nucleic acid
can be introduced intracellularly and incorporated within host cell
DNA for expression, by homologous recombination.
[0143] The present invention also provides kits that can be used in
the above methods. In one embodiment, a kit comprises a
pharmaceutical composition of the invention in one or more
containers.
[0144] In another embodiment, the kit is a diagnostic kit for use
in testing biological samples. The kit can include a control
antibody that does not react with the polypeptide of interest in
addition to a specific antibody or antigen-binding fragment thereof
which binds to the polypeptide (antigen) or the invention being
tested for in the biological sample. Such a kit may include a
substantially isolated polypeptide antigen comprising an epitope
that is specifically immunoreactive with at least one
anti-polypeptide antigen antibody. Further, such a kit can include
a means for detecting the binding of said antibody to the antigen
(for example, the antibody may be conjugated to a fluorescent
compound such as fluorescein or rhodamine which can be detected by
flow cytometry). In a further embodiment, the kit may include a
recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to
a solid support.
[0145] In an alternative embodiment, the detecting means of the
above-described kit includes a solid support to which the
polypeptide antigen is attached. The kit can also include a
non-attached reporter-labeled anti-human antibody. Binding of the
antibody to the polypeptide antigen can be detected by binding of
the reporter-labeled antibody.
[0146] In an additional embodiment, the invention includes a
diagnostic kit for use in screening serum samples containing
antigens of the polypeptide of the invention. The diagnostic kit
includes a substantially isolated antibody specifically
immunoreactive with polypeptide or polynucleotide antigens, and
means for detecting the binding of the polynucleotide or
polypeptide antigen to the antibody. In one embodiment, the
antibody is attached to a solid support. In another embodiment, the
antibody may be a monoclonal antibody. The detecting means of the
kit can include a second, labeled monoclonal antibody.
Alternatively, or in addition, the detecting means can include a
labeled, competing antigen.
[0147] In one diagnostic configuration, the test serum sample is
reacted with a solid phase reagent having a surface-bound antigen
obtained by the methods of the present invention. After binding
with specific antigen antibody to the reagent and removing unbound
serum components by washing, the reagent is reacted with
reporter-labeled anti-human antibody to bind reporter to the
reagent in proportion to the amount of bound anti-antigen antibody
on the solid support. Generally, the reagent is washed again to
remove unbound labeled antibody, and the amount of reporter
associated with the reagent is determined. The reporter can be an
enzyme, for example, which is detected by incubating the solid
phase in the presence of a suitable fluorometric, luminescent or
calorimetric substrate, as is standard in the art.
[0148] The solid surface reagent in the above assay is prepared by
known techniques for attaching protein material to solid support
material. Suitable solid support materials include, for example and
without limitation, polymeric beads, dip sticks, 96-well plate or
filter material.
[0149] The invention will be further described with reference to
the following non-limiting examples. The teaching of all patents,
patent applications and all other publications and websites cited
herein are incorporated by reference in their entirety.
EXEMPLIFICATION
[0150] Invariable, MLL translocations result in the production of a
chimeric protein where the NH.sub.2-terminal portion of MLL is
fused to the COOH-terminal portion of one of >20 fusion partners
(Dimartino and Cleary, Br J Haematol 106:614-626 (1999)). This has
prompted models of leukemogenesis in which the MLL-fusion protein
may confer a gain-of-function or neomorphic properties, or
alternatively represent a dominant negative that interferes with
normal MLL function. Moreover, mice heterozygous for MLL (+/-)
demonstrate developmental aberration (Yu et al., Nature 378:505-508
(1995); Hess et al., Blood 90:1799-1806 (1997)), suggesting the
disruption of one allele by chromosomal translocation might also
manifest as haplo-insufficiency in leukemic cells.
[0151] MLL is a homeotic regulator which shares homology with
Drosophila trithorax (trx) and positively regulates the maintenance
of homeotic (Hox), gene expression during development (Yu et al.,
Nature 378:505-508 (1995)). MLL deficient mice indicate that MLL is
required for proper segment identity in the axioskeletal system,
and also regulates hematopoiesis (Hess et al, Blood 90:1799-1806
(1997)). As MLL normally regulates Hox gene expression, its role in
leukemogenesis may include altered patterns of HOX gene expression.
An expanding body of literature shows that HOX genes are important
for appropriate hematopoietic development (Buske and Humphries, Int
J I Hematol 71:301-308 (2000)). Also, the t(7;11)(p15;p15)
translocation found in human acute myelogenous leukemia (AML)
results in a fusion of HOXA9 to the nucleoporin NUP98 (Nakamura et
al., Nat Genet 12:154-158 (1996) and Borrow et al., Nat Genet
12:159-167 (1996)). thus, HOX genes represent one set of
transcriptional targets that warrants assessment in leukemias with
MLL translocation.
[0152] We hypothesized that MLL translocations maintain a gene
expression program that results in a distinct form of leukemia. It
was reasoned that RNA profiles might help resolve whether leukemias
bearing an MLL translocation represent a truly biphenotypic
leukemia of mixed identity, a conventional B-cell precursor ALL
with expression of limited myeloid genes, or a less committed
hematopoietic progenitor cell. Moreover, comparing gene expression
profiles of lymphoblastic leukemias with and without rearranged MLL
is important because of their dramatically different response to
standard ALL therapy, and because such analysis may identify new
molecular targets for therapeutic approaches. The expression
profiles reported here reveal that lymphoblastic leukemias bearing
MLL translocations display a remarkable uniform and highly distinct
pattern that clearly distinguishes them from conventional ALL or
AML and warrants designation as a distinct disease, MLL.
Methods
[0153] Patient samples. After informed consent was obtained,
leukemia samples were obtained from peripheral blood or bone marrow
from patients or diagnosis or relapse. When the samples were
obtained from peripheral blood, the percentage of blasts was
greater than 60% of the total white blood cells present. Fifteen of
the samples with an MLL translocation and all of the conventional
childhood ALL samples were obtained from patients treated on Dana
Farber Cancer Institute protocols between 1980 and 2001. Three of
the infant leukemia samples with MLL rearrangements were obtained
from patients treated on the Interfant99 protocol, and the two
adult patients with MLL rearrangements were obtained from patients
treated at Princess Margaret Hospital in Toronto. Except for one of
the conventional ALL samples and four of the MLL samples that were
obtained at relapse, ass samples were diagnostic specimens. The AML
samples have been previously described (Golub et al., Science
286:531-537 (1999)), and were from both adults and children. Eight
of the MLL rearranged samples contain t(4;11), one t(9; 11), three
t(11;19), one t(3;11) and one t(1;11). Six of the MLL
rearrangements were detected by either FISH or Southern blot, and
thus the translocation partner is unknown. The mononuclear cells
were purified from red blood cells and neutrophils by
ficoll-hypaque density centrifugation and either frozen in liquid
nitrogen with 10% DMSO in fetal calf serum or put directly into
Trizol (Life Sciences) for RNA purification.
[0154] Assessment for the presence of MLL translocations. All
patient samples were assessed by standard cytogenetics. All
childhood ALL patient samples were screened for the presence of a
TEL-AML1 translocation by RT-PCR as previously described, Loh, M.
L. et al., Blood 92:4792-4797 (1998). Any patient sample where
cytogenetics failed and had no TEL-AML I translocation was further
assessed by fluorescence in situ hybridization (FISH) using a probe
that spans the 11q23 breakpoint or by Southern blot (Silverman et
al., Cancer 80:2285-2295 (1997); and Cuthbert et al, Genes
Chromosomes Cancer 29:180-185 (2000)). AML samples were not
assessed for chromosomal translocations.
[0155] RNA purification, labeling and hybridization. A total of
10-20.times.10.sup.6 cells were used to prepare total RNA using the
Trizol (Life Sciences) purification method. This generally yielded
between 5 and 20 .mu.g of total RNA the quality of which was
analyzed by gel electrophoresis. If the rRNA bands were intact, the
RNA was determined to be of good quality and 5-15 .mu.g was used
for subsequent production of biotinylated cRNA as described
previously (Golub et al., Science 286:531-537 (1999)), and were
from both adults and children. Briefly, first strand cDNA synthesis
was generated using a T7-linked oligo-dT primer, followed by second
strand synthesis. An in vitro transcription reaction was done to
generate the cRNA containing biotinylated UTP and CTP, which was
subsequently chemically fragmented at 95.degree. C. for 35 minutes.
Samples were excluded if less than 15 .mu.g of labeled RNA was
produced. Labeled RNA was then hybridized to Affymetrix (Santa
Clara, Calif.) U95A or U95A V2 oligonucleotide arrays at 45.degree.
C. for 16 hours. Arrays were washed and stained with
streptavidin-phycoerytherin (SAPE, Molecular Probes). The signal
was amplified using a biotinylated anti-streptavidin antibody
(Vector Laboratories, Burlingame, Calif.) at 3 .mu.g/ml. This was
followed by a second staining with SAPE. Normal goat IgG was used
as a blocking agent. The scans were performed on Affymetrix canners
and the expression values calculated using Affymetrix GENECHIP
software. The chip image was then scanned visually for obvious
differences between arrays. If there were obvious abnormalities
present in the image, the sample was re-hybridized. The scans were
then normalized based on a linear scaling method as described in
the supplementary material. The raw expression data was obtained
from Affymetrix's GeneChip s re-scaled to account for different
chip intensities. Briefly, each column (sample) in the dataset was
multiplied by 1/slope of at least squares linear fit of the sample
versus the reference (the first sample in the dataset). This linear
fit is done using only genes that have `Present` calls in both the
sample being re-scaled and the reference. The sample chosen as
reference was a typical one (i.e., one with the number of "P" calls
closer to the average over all samples in the dataset). Samples
were disregarded if the scaling factor was greater than 3 fold.
[0156] A threshold of 100 units was imposed before analysis because
at those low values the data is noisy and not very reproducible. A
ceiling of 16,000 units was also imposed due to saturation effects.
After this preprocessing gene expression values were subjected to a
variation filter which excluded genes showing minimal variation
across the samples being analyzed. The variation filter tests for a
fold-change and absolute variation over samples (comparing max/min
and max-min with predefined values and excluding genes not obeying
both conditions). The max/min filter was 5 and the max-min 500 for
all experiments.
[0157] Data analysis. Identification of genes that are correlated
with particular class distinctions was performed as previously
described (Golub et al., Science 286:531-537 (1999)). The
signal-to-noise statistic
(.mu..sub.0-.mu..sub.1)/(.sigma..sub.0+.sigma..sub.1) was used
where .mu. and .sigma. represent the median and standard deviation
of expression, respectively for each class. One hundred
permutations of the samples were performed to determine if the
correlations were greater than would be expected by chance with a
99% confidence.
[0158] The class predictor was performed using a cross-validation
approach and the K-Nearest Neighbors (K-nn) algorithm as follows.
The k-nearest neighbors (k-NN) algorithm predicts the class of a
new sample by calculating the Euclidean distance of the new sample
to samples in a training set whose location has been identified in
expression space. The predicted class of the new sample is then
determined by identifying the class to which the majority of the
k-nearest neighbors belong. The genes used to determine the
location in expression space of each sample were identified by
determining which genes best correlated with the class distinction
as described above using the signal to noise statistic. For all
experiments k=5. The prediction results shown in FIG. 6 were done
using a cross validation approach where 1 of the 57 samples was
withheld, the genes that best correlated with the ALL/MLL/AML
distinction were determined, and those genes were used to build the
k-nn algorithm. This was done for anywhere from 1-250 genes and the
error rate (number of failures/57) is graphed vs. the number of
genes. For the test set samples, the model was built with the 57
train set samples and then the class membership was determined for
each "test sample" by determining the samples "neighbors" in gene
expression space as described above.
[0159] Principal component analysis was performed using S-plus
statistical software and the default settings. A commonly used
technique for data reduction and visualization is principal
component analysis (PCA). In this type of analysis, the linear
combinations of variables are identified as the principal
components that explain the variability in the dataset. To reduce
the dimensionality of the data, the top 2 or 3 components can be
graphed. In our case, the tope 3 principal were thus used to
project the samples in 3-dimensional space based on the gene
expression profile. We first performed the analysis using the 8700
genes that passed the filtering described above. PCA was then
performed using the top 500 genes that correlated with the AML/ALL
class distinction. ALL, MLL, AML samples were then projected in
that 500 gene space. These analysis were performed using S-plus
statistical software using the default settings and covariance,
followed by a three dimensional scatter plot of the coordinates of
the 3 principal components for each sample. Singular value
decomposition was used to derive the eigen values of the covariance
matrix for the 8700 gene analysis. The S-Plus function used was
"princomp( )". The coordinates of the three principal components
for each sample were then used to project the samples in three
dimensions.
Results
MLL is Distinct From ALL
[0160] To further define the biological characteristics specified
by MLL translocations, gene expression profiles of leukemic cells
from patients diagnosed with acute lymphoblastic leukemia bearing
an MLL translocation were compared with conventional ALL which lack
this translocation. Initially, samples from 20 patients with
childhood ALL (denoted ALL), and 17 patients with MLL translocation
(referred to as MLL) were collected. Patient details are presented
in Table 1 (MLL) and Table 2 (ALL). TABLE-US-00001 TABLE 1 MLL
Patient Data Chromo- Sam- Age at FIG./Column Patient some ple
Diagnosis Specimen (MLL) MLL_1 t(4; 11) BM 1 month Diagnostic
Column 1 MLL_2 So.Blot+# BM 3 months Relapse Column 2 MLL_3 t(4;
11) PB 8 months Diagnostic Column 3 MLL_4 FISH+* PB 2 months
Relapse Column 4 MLL_5 FISH+ BM 2 months Diagnostic Column 5 MLL_6
FISH+ PB 18 months Diagnostic Column 6 MLL_7 t(4; 11) BM 8 months
Relapse Column 7 MLL_8 t(4; 11) PB 5 months Diagnostic Column 8
MLL_9 So.Blot+ PB 7 months Diagnostic Column 9 MLL_10 t(1; 11) PB 1
month Diagnostic Column 10 MLL_11 t(3; 11) PB 1 day Diagnostic
(q13; q23) Column 11 MLL_12 t(11; 19) BM 3 months Relapse Column 12
MLL_13 t(4; 11) PB 1 month Diagnostic Column 13 MLL_14 t(11; 19) BM
3 months Diagnostic Column 14 MLL_15 t(11; 19) PB 7 months
Diagnostic Column 15 MLL_16 t(4; 11) PB >21 years Diagnostic
Column 16 MLL_17 t(4; 11) PB >21 years Diagnostic Column 17
MLL_18 FISH+ PB 10 months Diagnostic Column 18 MLL_19 t(9; 11) PB 4
years Diagnostic Column 19 MLL_20 t(4; 11) PB 6 years Diagnostic
Column 20 PB = peripheral blood BM = bone marrow
[0161] TABLE-US-00002 TABLE 2 ALL Patient Data Age at FIG./Column
Patient Tel/AML1 Chromosomes Sample Diagnosis Specimen (ALL) ALL_1
Pos. 46 xy (+6) BM 6 y Diagnostic Column 1 ALL_2 Pos. Diploid BM 5
y Diagnostic Column 2 ALL_3 Pos. No data BM 4 y Diagnostic Column 3
ALL_4 Pos. Hyperdiploid, PB 12 y Diagnostic del7q Column 4 ALL_5
Pos. Hyperdiploid, BM 12 y Relapse del7q Column 5 ALL_6 Pos. Add
1p36, BM 4 y Diagnostic del 12p Column 6 ALL_7 Pos. Diploid BM 2 y
Diagnostic Column 7 ALL_8 Pos. No Data BM 3 y Diagnostic Column 8
ALL_9 Neg. Diploid BM 9 y Diagnostic Column 9 ALL_10 Neg. Del 3p,
BM 5 y Diagnostic Add 12p, Column Add 17q 10 ALL_11 Neg. del 12p13
BM 20 m Relapse Column 11 ALL_12 Neg. Hyperdiploid BM 13 m
Diagnostic Column 12 ALL_13 Neg. Hyperdiploid PB 4 y Relapse Column
13 ALL_14 Neg. Diploid BM 20 m Diagnostic Column 14 ALL_15 Neg.
Diploid BM 10 y Diagnostic Column 15 ALL_16 Neg. Diploid BM 4 y
Diagnostic Column 16 ALL_17 Neg. Del 9p21 PB 11 y Diagnostic Column
17 ALL_18 Neg. Failed PB 15 y Diagnostic (MLL FISH Column Neg.) 18
ALL_19 Neg. Hyperdiploid BM 23 m Diagnostic Column 19 ALL_20 Neg.
Diploid BM 9 y Diagnostic Column 20 ALL-21 Neg. Failed PB 12 m
Diagnostic (MLL FISH Column Neg.) 21 ALL_22 Neg. t(1; 19) PB 7 y
Diagnostic Column 22 ALL_23 Pos. Diploid PB 3 y Diagnostic Column
23 ALL_24 Pos. No Data BM 8 y Diagnostic Column 24 Pos. = positive
for TelAML1 translocation Neg. = negative for TelAML1 translocation
PB = peripheral blood BM = bone marrow m = months y = years
[0162] First, it was determined if there were genes among the
12,600 tested whose expression pattern correlated with the presence
of an MLL translocation. The genes were sorted by their degree of
correlation with the MLL/ALL distinction (FIGS. 1A and 1B), and
permutation testing was used to assess the statistical significance
of the observed differences in gene expression (Golub et al.,
Science 286:531-537 (1999)). For the 37 samples tested,
approximately 1000 genes proved underexpressed in MLL as compared
to conventional ALL while approximately 200 genes were relatively
highly expressed. FIG. 1A shows the top 50 genes that are
relatively underexpressed in MLL, and FIG. 1B shows the bottom 50
genes that are relatively overexpressed in MLL. Genes, and their
GenBank Accession Numbers, are labeled at the right. The top 200
genes that make the ALL/MLL distinction and their GenBank Accession
Numbers can be found in Table 3 (top 100 genes that are
underexpressed in MLL compared to ALL) and Table 4 (top 100 genes
that are overexpressed in MLL compared to ALL). TABLE-US-00003
TABLE 3 Genes Underexpressed in MLL Compared to ALL GenBank No.
Name GenBank No. Name J03779 CD10 AA808961 cDNA nw16h03.s1 AL050105
DKFZp586H0519 AB018303 KIAA0760 L33930 CD24 X74837 HUMM9 Y12735
Dyrk3 AL022723 Chromosome 6 sequence AB020674 KIAA0867 M74719 E2-2
D26070 ITPR3 M63838 IFI16 M11722 TdT U81607 Gravin M61877
a-spectrin U96113 Nedd-4 like ubiquitin ligase X59350 CD22 D13639
KIAK0002 W25798 cDNA 13f12 X53586 Integrin alpha 6 AL049279
DKFZp564I083 U01062 ITPR3 AF032885 FKHR M21535 ERG11 L46922 FHIT
D88827 Zinc finger protein FPM315 S67427 myosin W26406 cDNA 29b7
AL079277 Unknown cDNA U15642 E2F-5 M96803 b-spectrin D43949
KIAA0082 X83441 DNA Ligase IV J011001 TM7XN1 X15357 ANP-receptor
M63928 CD27 M55284 Protein Kinase C-L AB028961 KIAA1038 AI146846
cDNA qb92h04 X55740 5' Nucleotidase AB023176 KIAA0959 L05186 Focal
adhesion kinase AF002999 TRF2 AF070588 cDNA 24554 D26070 ITPR3
U48705 Tyrosine Kinase DDR Y11312 PI3-Kinase AF070614 cDNA 24732
U48959 MLCK U23850 ITPR3 J05243 a-spectrin AF054180 Hematopoietic
zinc finger protein Y14768 Cosmid TN62 AL049471 DKFZp586N012 U01062
ITPR3 AF084481 WFS1 Z49194 OBF-1 U90547 Human Ro V59423 Smad1
U15085 HLA-DMB U29175 Snf2-b AJ001381 cDNA for an allele of myosin
M81141 HLA-DQ-b AL049933 DKFZp564K1216 D87437 KIAA0250 AI198311
cDNA qi61f11.x1 Y00264 Amyloid A4 precursor AL050060 DKFZp566H073
U59912 Smad1 X06318 PKC-b1 AJ007583 acetylglucosaminyltransferase
U43885 Grb2-associated binder-1 L75847 ZNF-45 M31523 E2A
transcription factor D17530 Dreberin E L10373 clone CCG-B7 D86967
KIAA0212 Y11306 TCF-4 J03600 Lipoxygenase X05323 MIRC OX-2 gene
D42055 KIAA0093 X78932 Zinc finger protein HZF9 AL021154 Chromosome
1 PAC W26633 cDNA 34b1 AI761647 cDNA wg66h09 AF052131 clone 23930
AF054815 VAMP5 AL050260 DKFZp564E1082 N36926 cDNA YY38E04 AL080218
DKFZp586N1323 U96113 Nedd-4-like ubiquitin ligase A1561196 cDNA
tq27a01.x1 AB019527 LDOC1 W26023 cDNA 18c3 M34641 FGF Receptor-1
U68186 Extracellular matrix I L29376 MHC class I mRNA X62744 RING6
M60028 HLA-DQ-b X78926 Zinc Finger HZF3
[0163] TABLE-US-00004 TABLE 4 Genes Overexpressed in MLL Compared
to ALL GenBank No. Name GenBank No. Name AI535946 Lectin HL14
U67516 MAPKKK5 M14087 Lectin HL14 AF072099 Immunoglobulin-like
transcript 3 AI201310 cDNAqf71b11 X96753 chondroitin sulfate
proteoglycan NG2 M80899 AHNAK X52075 CD43 AJ001687 NKG2D U38545
Phospholipase I U66838 Cyclin A1 D00017 Annexin II M59040 CD44
U02687 FLT-3 M95929 Phox1 AA570193 cDNAnf38c11.s1 D25217 KIAA0027
U21551 ECA39 AI597616 cDNAtn15f08 X55988 eosinophil derived
neurotoxin W72186 cDNAzd69b10 D78177 quinolinate phosphoribosyl
transferase U41813 HOXA9 Z48579 disintegrin-metalloprotease L05424
CD44 AF039656 NAP-22 L05424 CD44 Y00638 CD45 AC004080 Chromosome 7
PAC J03910 Metallothionein-IG X05908 Annexin I AF030339 VESPR
U78027 chromosome X BAC M28713 NADH-cytochrome B5 reductase Y00062
CD45 AB023209 KIAA0992 AF098641 CD44 L40377 CAP2 Y000638 CD45
D86181 galactocerebrosidase AF004230 monocyte elastase inhibitor
M83215 AML1 W60864 cDNAzd27g05 X61118 LMO2 X55989 Eosinophil CRP
U01147 ABR M93056 Monocyte MIR-7 M96995 GRB2 AF027208 AC133
AF040704 putative tumor suppressor (101F6) Z83844 Chromosome 22
sequence U57971 calcium ATPase AL050396 DKFZp586K1720 AB028948
KIAA1025 AA978353 cDNAoq40b07 U11791 cyclin H D21261 KIAA0120
AF022991 Rigui L08177 EBV induced EBI2 L11669 Tetracycline-like
transporter L19182 MAC25 U39064 MAPKK6 D28364 Annexin II AF054176
Angiotensin D15057 DAD-1 U87947 HNMP-1 AF020044 C-type lectin
precursor L19872 AH-receptor AI138834 cDNAqe04b02 M36035
benzodiazepine receptor X73882 E-MAP-115 U93305 Chromosome X p11
sequence AF026816 Putative Oncogene AF009615 ADAM10 X17042
Proteoglycan I X52541 EGR1 M20867 glutamate dehydrogenase AF044253
potassium channel beta-2 U60060 FEZ1 M26683 Interferon gamma
inducible mRNA AF025529 LIR-6 M31166 TSG-14 AB007888 KIAA0428
R93527 cDNAyq35f10.r1 M13485 Metallothionein I-B X15998 chondroiton
sulfate proteoglycan M26679 HOX A5 X55990 eosinophil cationic
protein AL050267 DKFZp564A032 U73960 ARF-like protein 4 R92331
cDNA03h03 M13452 Lamin A AL050162 DKFZp586B2022 AI560890 cDNA
tq41d05.x1 AI017574 cDNAou23f10 AB024057 vascular rab-gap M62896
Annexin II M97815 CRABP-II AL050374 DKFZp586c1619 M60614 WIT-1
[0164] As shown in FIGS. 1A and 1B and Tables 1 and 2, MLL shows a
dramatically different gene expression profile from ALL.
MLL Shows Multi-Lineage Gene Expression
[0165] Inspection of the genes differentially expressed between MLL
and ALL was instructive (FIGS. 1A and 1B and Tables 1 and 2). Many
underexpressed genes in MLL have a unction in early B cell
development. These include genes expressed in early B-cells (CD10,
CD24, CD22, TdT) (Hardy and Hayakawa, Annu Rev Immunol., 19:595-621
(2001); LeBien, Blood 96:9-23 (2000)), genes required for
appropriate B-cell development (E2A, E2-2, PI3-Kinase, Octamer
Binding Factor-1, and DNA ligase IV) (Murre, Cold Spring Barb Symp
Quant Bio 164:39-44 (1999); Fruman et al., Science 283:393-397
(1999); Schubart et al., Nat Immunol 2:69-74 (2001); and Frank et
al., Nature 396:173-177 (1998)), and genes found to be correlated
with B-precursor ALL in an AML/ALL comparison (Snj2-.beta.) (Golub
et al., Science 286:531-537 (1999)). The relative underexpression
of the forkhead (FKHR), SMAD1 and TCF-4 transcription factors
suggests they may also be involved in later stages of B-cell
differentiation or leukemogenesis. Relatively overexpressed genes
in MLL include the adhesion molecules HL14, Annexin I, Annexin II,
CD44, and CD43. Multiple genes that are expressed in hematopoietic
lineages other than lymphocytes are also highly expressed in MLL.
These include genes expressed in progenitors (AC133, FLT3, LMO2)
(Yin et al., Blood 90:5002-5012 (1997); Rosnet et al., Blood
82:1110-1119 (1993); and Dong et al., Br J Haematol 93:280-286
(1996)), myeloid specific genes (Cyclin A1, monocyte elastase
inhibitor, macrophage capping protein, eosinophil-CRP), (Yang et
al., Blood 93:2067-2074 (1999); Remold-O'Donnell et al., Proc
NatlAcad Sci USA 89:5635-5639 (1992); and Rosenberg et al., J Exp
Med. 170:163-176 (1989)), and at least one natural killer cell
specific gene (NKG2D) (Ho et al., Proc Natl Acad Sci USA,
95:6320-6325 (1998)) (FIGS. 1A, 1B and Tables 1 and 2).
Overexpression of HOXA9 and Proteoglycan I in MLL is of particular
interest as these genes were previously reported to be highly
expressed in AML (Golub et al., Science 286:531-537 (1999)), and
overexpression of HOXA9 has been associated with a poor prognosis
(Golub et al., Science 286:531-537 (1999)).
MLL is Arrested at an Early Stage of Hematopoietic Development
[0166] Since lymphoblasts with MLL rearrangement express many
myeloid specific genes, a detailed assessment of the expression of
lymphoid genes was performed. Genes known to mark early B-lymphoid
commitment such as Ig.beta. and CD19 are expressed in MLL albeit at
lower levels than in ALL (FIGS. 2C and 2B). CD10 (CALLA) is not
expressed in MLL (FIG. 2A), whereas the IL-7 receptor is expressed
at similar levels in ALL and MLL.
[0167] A number of genes have been shown to vary their expression
level as murine hematopoietic cells transition from stem cell, to
common lymphoid progenitor, to pro-B and then pre-B cells.
Ig.beta., CD24, CD44 and CD43 represent early steps of lymphoid
development (Hardy and Hayakawa, Annu Rev Immunol 19:595-621 (2001)
and Kondo et al., Cell 91:661-672 (1997)). Ig.beta. and CD24
expression increases with maturation while CD44 and CD43 levels
decrease (Kondo et al., Cell 91:661-672 (1997)). the MLL samples
express relatively low levels of CD24 and Ig.beta. but high levels
of both CD44 and CD43 (FIGS. 2F and 2E). In total these data
suggest that MLL represents a maturational arrest at an early
lymphoid progenitor stage of development.
Selected HOX Genes are Highly Expressed in MLL versus ALL
[0168] Multiple members of the class I Hox genes are known to be
regulated by Mll (Yu et al., Nature 378:505-508 (1995)) prompting a
detailed comparison of the patterns of HOX gene expression between
ALL and MLL. Several of the 20 class I HOX genes present on the
microarrays demonstrated significant and consistent differences in
expression. HOXA9 and HOXA5 were not expressed in conventional ALL,
but were expressed, often at high levels, in most MLL samples
(FIGS. 3A-3D). Similarly, HOXA4 was typically expressed in MLL, but
rarely in conventional ALL (FIG. 3C). HOXC6 showed mildly elevated
levels of expression in MLL (Supplemental Information at
http://research.dfci.harvard.edu/korsmeyer/MLL.htm). However, the
HOX patterns displayed selectivity as other genes such as HOXA7
showed no obvious difference in their expression pattern (FIG. 3D).
MEIS1, a cofactor for HOX proteins, which can accelerate HoxA9
dependent leukemia (Nakamura et al., Nat Genet 19:149-1531 (1996)),
was also significantly overexpressed in MLL as previously reported
for the t(4;11) containing subset (Rozovskaia et al., Oncogene
20:874-878 (2001)).
MLL is Distinct From Both AML and ALL
[0169] MLL is characterized by the expression of myeloid specific
genes, which raised the possibility that MLL is more closely
related to acute myelogenous leukemia (AML). To determine if this
is the case, or if MLL is separable as a distinct type of leukemia,
a principal component analysis (PCA) was performed using the gene
expression profiles of MLL, ALL and AML specimens. This clustering
algorithm reduces complex multidimensional data to a few specified
dimensions so that it can be visualized effectively (Venables and
Ripley, Modern Applied Statistics with S-Plus, Springer Verlag, New
York (1994)). First, the analysis was performed in an unsupervised
manner using the 8700 genes that showed some variability in
expression level. As expected, the ALL and AML samples displayed
substantial separation (FIG. 4A). Of note, the MLL samples proved
largely separate from either AML or ALL (FIG. 4A). In order to
determine if this separation could be attributed to a difference in
hematopoietic identity, a similar analysis was performed using the
500 genes whose expression best distinguished the separation of AML
versus ALL. When projected into this 500-gene space using PCA the
MLL samples principally fall between the AML and ALL samples (FIG.
4B).
[0170] Since the above clustering analyses supported three distinct
entities of ALL, AML and MLL, it was queried if selected genes
could be identified which distinguished each type of leukemia from
the other two (FIG. 5). Conventional ALL expressed high levels of
the following genes compared to MLL and AML: CD10, CD24, DYRK, TdT,
FKHR, DNA ligase IV, KIAA0867, CD22, OBF-1, B-spectrin,
DKFZp5641083, Snf-2B, MLCK, VAMP5, and cDNA wg66h09) and these
genes were underexpressed in MLL and ALL. AML samples expressed
high levels of the following genes compared to ALL and MLL:
adipsin, cathepsinD, CD13, M6 antigen, gap junction protein,
a-endosulfine, NF-2 transcription factor DP-2, DRAP1, cDNA 20c1,
phosphodiesterase 3B, cosmid 19p13.2, chromosome 19 clone,
chromosome 22q11 clone, and CRYAA, and these genes were
underexpressed in ALL and MLL. MLL samples expressed high levels of
the following genes compared to ALL and AML: AC 133, LMO2, FLT3,
KIAA0428, NKG2D, ADAM10, KIAA1025, Lectin HL14, cDNA ag36c04,
cyclin A1, ADAM10, putative oncogene, DKFZp588o01, KIAA0920, and
LMO2, and these genes were underexpressed in ALL and AML. The
GenBank Accession Numbers for each of these genes is shown in FIG.
5. Permutation analysis indicated that approximately 200 genes were
significantly overexpressed in MLL as compared to the other two
leukemia categories. In combination, the PCA and gene expression
comparisons (FIGS. 4A, 4B and 5) indicate that MLL is a separable,
distinct disease based on gene expression profile. These data also
show that ALL and AML are separable distinct diseases. The genes
shown in FIG. 5, particularly the genes that are over expressed in
each disease type, can be used in gene expression profile studies
to diagnose MLL, ALL, or AML. These genes, including underexpressed
and overexpressed genes for each disease type, can also be used as
target for identifying and/or detecting compounds that alter
expression and/or activity of these genes or their gene products,
for therapeutic methods, and for monitoring efficacy of treatment,
as described herein.
Gene Expression Profiles Correctly Classify ALL, MLL and AML
[0171] A more stringent assessment of the power of the
aforementioned difference in gene expression profiles would be
their capacity to assign individual samples as MLL, ALL, or AML.
The detection of MLL translocations in leukemia samples is
currently most often performed by either cytogenetic analysis or by
fluorescence in situ hybridization (FISH) which can technically
fail or may be unavailable. Thus, other approaches to correctly
assign individual cases to meaningful subsets of leukemia would be
useful. To test this possibility a three-class predictor was
developed based on a k-nearest neighbors algorithm (Dasarathy (ed),
IEEE Computer Society Press, Los Altos, Calif., December 1991.
ISBN: 0818689307). This algorithm assigns a test sample to a class
by identifying the k nearest samples in the training set and
choosing the most common class among these k nearest neighbors. For
this purpose, distances were defined by a euclidean metric based on
the expression levels of a specified number of genes.
[0172] The accuracy of this method was assessed using a cross
validation approach. When one of the 57 samples is removed, the
genes that most closely correlate with the ALL/MLL/AML class
distinction are identified, and the expression of these genes used
to determine the class of the withheld sample. The model assigned
the withheld sample to the appropriate class with 95% accuracy.
Moreover this accuracy was maintained as we extended from 40 to 250
genes to build the predictor (FIG. 6), as further testimony to the
strong distinction among these leukemia categories.
[0173] To assess if the unique signature of gene profiles in MLL
samples could be attributed to their occurrence in infants, the
above model was tested using 10 independent leukemia samples. The
test set consisted of 3 childhood (>12 months) conventional
ALLs, 2 lymphoblastic leukemias of childhood carrying
cytogenetically verified MLL translocations, 2 infant (<12
months) leukemias in which cytogenetic analysis did not detect an
MLL translocation and 3 AML samples. Utilizing the 100 genes that
best correlated with the three-class distinction, nine of ten
samples were correctly classified as MLL, ALL or AML. The one
apparent error was an infant reported to be negative for an MLL
rearrangement by cytogenetics, yet consistently predicted to have a
rearrangement based on gene expression profile. This prompted
further analysis by FISH, which confirmed that this infant leukemia
did indeed possess an MLL translocation and that the prospective
assignment by expression profiling was correct. Taken together
these data show that the unique gene expression profile
characteristic of MLL cannot be attributed merely to the fact that
most samples are from infants.
Discussion
[0174] Gene expression profiles of lymphoblastic leukemias which
possess an MLL translocation are remarkable consistent and differ
significantly from those of other leukemias. Consequently, it is
appropriate that they be considered a distinct disease entitled MLL
for "Mixed Lineage Leukemia." This is supported by their comparison
to conventional B cell precursor ALL that lacks MLL rearrangement
where .about.1000 genes proved underexpressed and .about.200
overexpressed in the MLL rearranged group. Moreover, evaluation of
the expression profiles using principal component analysis
indicated that MLL was clearly separable from conventional ALL and
also AML. The expression differences are so robust that .about.95%
of leukemic samples were correctly classified as MLL, ALL or AML.
As testimony to the extent of divergence of MLL, it remained
separable from ALL and AML when 250 genes were used to build the
class predictor. This provides strong evidence that a specific
chromosomal translocation results in a distinct type of
lymphoblastic leukemia, rather than a model in which all
translocations merely provide transformation events that
subsequently converge upon a common pathway of leukemogenesis. In
addition, the data indicate that MLL is arrested at an earlier
stage of differentiation and/or has a different cell of origin than
ALL. Select Hox genes are overexpressed in MLL-dependent leukemia,
as compared to normal B-cell progenitors and other ALL. FLT3
expression best distinguishes MLL from ALL.
[0175] Gene expression patterns of MLL provide insight into the
proposed models for its cellular origin. A summary of expression
profiles shows that MLL expresses some lymphocyte specific and
myeloid specific genes, but at lower levels than either
conventional ALL or AML, respectively. Based on murine studies that
have defined gene expression patterns during lymphocyte commitment
(Hardy and Hayakawa, Annu Rev Immunol 19:595-621 (2001); and Kondo
et al., Cell 91:661-672 (1997)), the low-level expression of CD24
and Ig.beta., along with high expression of CD43 and CD44 suggests
that MLL is arrested at an earlier stage of development than
conventional ALL. Furthermore, the expression of genes typically
found in progenitor cells suggests MLL represents an early
hematopoietic progenitor. This is consistent with studies that have
shown multi-lineage gene expression in hematopoietic progenitors
prior to full lineage commitment (Hu et al., Genes Dev 11:774-785
(1997)). Of particular interest is the possibility that MLL may
represent the expansion of a bipotential B-macrophage progenitor
(Montecino-Rodriguez et al., Nat Immunol 2:83-88 (2001); and Cumano
et al., Nature 356:612-615 (1992)). Early B-cells can be induced to
differentiate into myelomonocytic cells under certain conditions
(Nutt et al., Nature 401:556-562 (1999)), and derivation of
macrophages from leukemia cell lines has been well documented
(Borrello and Phipps, Immunol Today 17:471-475 (1996)). An
attractive model would hold that the MLL-fusion protein drives the
"transdifferentiation" of an early lymphocyte progenitor. The
expression of many myeloid and monocyte/macrophage specific genes
is consistent with MLL reflecting a very early B cell progenitor
that has initiated transdifferentiation. The multiple HOX genes
that are selectively expressed in MLL are attractive candidates for
direct targets of the MLL-fusion proteins. Mll gene ablated mice
have indicated that select members of the clustered Hox genes
require MLL for their expression. As overexpression of HoxA9 has
also been shown to induce AML in mouse models (Nakamura et al., Nat
Genet 12:149-1531 (1996)), and its expression is controlled by
levels of Mll (Hanson et al., Proc Natl Acad Sci USA,
96:14372-14377 (1999)); misexpression of HOXA9 may be an important
component of MLL-translocation driven leukemogenesis. The findings
here prompt further studies to determine if MLL-fusion proteins
directly activate HOX genes, and thus lead to defects in
hematopoietic differentiation.
[0176] This is the first demonstration in which whole genome
profiling reveals that a chromosomal translocation can specify a
unique gene expression program. This separates MLL as a distinct
disease, which is of both pathogenic and therapeutic importance.
Lymphoblastic leukemias with MLL translocations are recognized as
having a poor prognosis, as standard ALL therapies have been
relatively ineffective. The unique identity of MLL noted here
provides insight into the poor response. As MLL is a distinct
disease, new therapeutic approaches are needed. Of note, pilot
studies have shown that addition of the drug cytarabine, an
important agent in myeloid leukemia treatment, may improve the
outcome for MLL patients (Ludwig et al., Blood 92:1898-1909 (1998);
Silverman et al, Cancer 80:2285-2295 (1997); and Pieters et al.,
Leukemia 12:1344-1348 (1998)). However, it is the
translocation-specific therapies which have recently proven
attractive for their efficacy and lack of toxicity. Other leukemias
in which a translocation specifies a distinct disease are chronic
myelogenous leukemia (CML) with the BCR-ABL fusion and acute
promyelocytic leukemia (APL) with the PML-RAR.alpha. fusion. The
tailored development of the tyrosine kinase inhibitor STI571 and
its treatment of CML and the use of all trans retinoic acid (ATRA)
in APL has substantially improved the outcome in those diseases
(Tallman et al., N Engl J Med 337:1021-1028 (1997) and Druker et
al., N Engl J Med 344:1031-1037 (2001)). While pharmacologic
approaches to the complex regulatory capacity of MLL may prove
challenging, the distinct gene expression signature defined here
for MLL may provide unanticipated molecular targets. Of special
note, FLT3 is the most differentially expressed gene that
distinguishes MLL from ALL and AML (FIG. 5). Aberrations of FLT3,
especially duplication of its juxtamembrane domain, have been noted
in some cases of AML and may be leukemogenic (Nakao et al.,
Leukemia 10:1911-1918 (1996); Zhao et al., Leukemia 14:374-378
(2000); and Tse et al., Leukemia 14:1766-1776 (2000)). As a
tyrosine kinase receptor, FLT3 represents an attractive target for
rational drug development.
[0177] Additional information regarding the methods used to carry
out the above described studies, patient samples, and the
differentially expressed genes identified though these studies can
be found at
http://research.dfci.harvard.edu/korsmeyer/MLL.htm, and
http://www-genome.wi.mit.edu/MPR, the teachings of which are
incorporated herein by reference in their entirety.
[0178] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *
References