U.S. patent application number 14/921008 was filed with the patent office on 2016-02-11 for methods for the treatment of cancer.
This patent application is currently assigned to THE TRANSLATIONAL GENOMICS RESEARCH INSTITUTE. The applicant listed for this patent is The Translational Genomics Research Institute. Invention is credited to Aditi Bapat, Kimberly J. Bussey, Michael J. Demeure.
Application Number | 20160038498 14/921008 |
Document ID | / |
Family ID | 51654872 |
Filed Date | 2016-02-11 |
United States Patent
Application |
20160038498 |
Kind Code |
A1 |
Bussey; Kimberly J. ; et
al. |
February 11, 2016 |
METHODS FOR THE TREATMENT OF CANCER
Abstract
The present invention is directed to methods for treating cancer
in a subject, such as a cancer of the endocrine system. In some
aspects, the method includes administering to the subject one or
more of a polo-like kinase 1 inhibitor, a mouse double minute 2
inhibitor, and/or a mitotic catastrophe inducing compound. In other
aspects, the method includes measuring an expression level of one
or more markers, including caspase 8 and caspase 9, to assess the
functionality of the caspase cascade in the subject.
Inventors: |
Bussey; Kimberly J.;
(Phoenix, AZ) ; Demeure; Michael J.; (Phoenix,
AZ) ; Bapat; Aditi; (Phoenix, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Translational Genomics Research Institute |
Phoenix |
AZ |
US |
|
|
Assignee: |
THE TRANSLATIONAL GENOMICS RESEARCH
INSTITUTE
Phoenix
AZ
|
Family ID: |
51654872 |
Appl. No.: |
14/921008 |
Filed: |
October 23, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14245188 |
Apr 4, 2014 |
9198910 |
|
|
14921008 |
|
|
|
|
61808462 |
Apr 4, 2013 |
|
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Current U.S.
Class: |
514/249 |
Current CPC
Class: |
A61K 31/47 20130101;
A61K 31/519 20130101; A61K 31/428 20130101; A61K 31/69 20130101;
A61K 31/428 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 31/496 20130101; A61K 31/366 20130101; A61K
31/69 20130101; A61K 31/4184 20130101; A61K 31/47 20130101; A61K
31/4184 20130101; A61K 45/06 20130101; A61K 31/496 20130101; A61K
31/519 20130101; A61K 31/366 20130101; A61K 31/437 20130101; A61K
31/437 20130101 |
International
Class: |
A61K 31/519 20060101
A61K031/519; A61K 31/496 20060101 A61K031/496 |
Claims
1. A method of treating endocrine cancer in a subject, the method
comprising the steps of: administering a therapeutically effective
amount of a polo-like kinase 1 Inhibitor (PLK1 inhibitor) to the
subject; and administering a therapeutically effective amount of a
mouse double minute 2 inhibitor (MDM2 inhibitor) to the
subject.
2. The method of claim 1, wherein the endocrine cancer is a cancer
of an adrenal gland.
3. The method of claim 1, wherein the endocrine cancer is a
malignant cancer of the adrenal gland.
4. The method of claim 3, wherein the malignant cancer of the
adrenal gland is adrenocortical carcinoma.
5. The method of claim 1, wherein the PLK1 inhibitor is selected
from the group consisting of BI-2536, cyclapolin 9, GW 843682X,
TC-S 7005, Wortmannin, NMS-P937, and GSK461364A.
6. The method of claim 5, wherein the PLK1 inhibitor is
BI-2536.
7. The method of claim 1, wherein the MDM2 inhibitor is selected
from the group consisting of nutlin, caylin-1, HU 373, caylin-2,
JNJ 26854165, NSC 66811, and trans-4-Indo, 4'-boranyl-chalcone.
8. The method of claim 7, wherein the nutlin is nutlin-3.
9. The method of claim 1, wherein the MDM2 inhibitor and the PLK1
inhibitor are administered as a single pharmaceutical
composition.
10. The method of claim 1, wherein the MDM2 inhibitor and the PLK1
inhibitor are administered as individual doses.
11. A method of treating a subject with a caspase cascade defect,
the method comprising the steps of: administering a therapeutically
effective amount of a polo-like kinase 1 Inhibitor (PLK1 inhibitor)
to the subject; and administering a therapeutically effective
amount of a mouse double minute 2 inhibitor (MDM2 inhibitor) to the
subject.
12. The method of claim 11, wherein the PLK1 inhibitor is selected
from the group consisting of BI-2536, cyclapolin 9, GW 843682X,
TC-S 7005, Wortmannin, NMS-P937, and GSK461364A.
13. The method of claim 12, wherein the PLK1 inhibitor is
BI-2536.
14. The method of claim 11, wherein the MDM2 inhibitor is selected
from the group consisting of nutlin, caylin-1, HU 373, caylin-2,
JNJ 26854165, NSC 66811, and trans-4-Indo, 4'-boranyl-chalcone.
15. The method of claim 14, wherein the MDM2 inhibitor is
nutlin-3.
16. The method of claim 11, wherein the MDM2 inhibitor and the PLK1
inhibitor are administered as a single pharmaceutical
composition.
17. A method of treating endocrine cancer in a subject, the method
comprising the steps of: identifying a caspase cascade defect in
the subject; and administering a therapeutically effective amount
of mitotic catastrophe inducing composition to the subject, wherein
the mitotic catastrophe inducing composition comprises a PLK1
inhibitor and an MDM2 inhibitor.
18. The method of claim 17, wherein the endocrine cancer is a
cancer of the adrenal gland.
19. The method of claim 17, wherein the identifying a caspase
cascade defect comprises identifying an expression level of at
least one marker.
20. The method of claim 19, wherein the at least one marker is
selected from the group consisting of caspase 8, caspase 9, and
p53.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 14/245,188, filed Apr. 4, 2014, which claims
priority to U.S. Application No. 61/808,462, filed Apr. 4, 2013,
the entire contents and disclosure of these applications are herein
incorporated by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 61 kilobyte
ASCII (text) file named "ACCSeq_List_ST25" created on Apr. 3,
2014.
FIELD OF THE INVENTION
[0003] This application relates to methods for the treatment of one
or more forms of cancer and more particularly, relates to the
assessment of the functionality of the caspase cascade in a subject
and methods of circumventing defects in the functionality.
BACKGROUND OF THE INVENTION
[0004] Cancers with less than 40,000 cases diagnosed per year in
the U.S. are both an unmet medical need and unparalleled
opportunity. Rare cancers have been the basis of paradigm-defining
concepts in cancer because their rarity comes from the limited
combinations of events that result in that particular type of
cancer. For example, the first instance of a translocation driven
oncogenic event, BRC-ABL, was discovered in seven cases of chronic
myelogenous leukemia. Nowell PC & DA Hungerford, A minute
chromosome in human chronic granulocytic leukemia, Science
(142)1497 (1960). The role of tumor suppressors, particularly p53
and RB1, was found through the investigation of tumors from
patients with Li-Fraumeni syndrome and familial retinoblastoma,
respectively. As researchers find necessary and sufficient genetic
changes that drive rare cancers, these can be further studied to
understand and treat more common cancers. If researchers can
identify what core cancer pathways are operational in a rare tumor,
the knowledge about those pathways from more common cancers can be
leveraged to improve diagnosis and treatment.
[0005] Adrenocortical carcinoma (ACC) is an aggressive cancer of
the adrenal cortex. Recent studies show that the outcome for ACC
patients has remained unchanged in the past 25 years, with a 40%
overall 5-year survival rate of patients undergoing surgical
resection. KY Bilimoria et al., Adrenocortical carcinoma in the
United States: treatment utilization and prognostic factors, Cancer
(113) 3130-3136 (2008). ACC is rare with an incidence of
approximately 300 new cases per year in the United States. Id. The
rarity of ACC suggests either a specific genomic alteration, or a
small number of alterations, is necessary and sufficient for tumor
development. Childhood ACC is a feature of the Li-Fraumeni syndrome
and mutation of p53, but inactivating mutations in p53 are an
uncommon feature of adult ACC. R. Libe et al., Somatic TP53
mutations are relatively rare among adrenocortical cancers with the
frequent 17p13 loss of heterozygosity, Clinical Cancer Res. (13)
844-850 (2007). Comparative genomic hybridization studies
demonstrate that no abnormality has been seen in more than 60% of
ACCs examined. Bussey KJ & MJ Demeure, Genomic and expression
profiling of adrenocortical carcinoma: application to diagnosis,
prognosis and treatment, Future Oncol. (5) 641-655 (2009). This
implies that the necessary and sufficient alteration(s) responsible
for the pathogenesis of ACC exist either below the limit of
detection or resolution of previously used technologies and/or
result from structural events that do not consistently alter copy
number. It could also imply that there is genomic heterogeneity
that converges on a small number of critical pathways resulting in
this rare disease.
[0006] ACC patients who present clinically with large, locally
invasive tumors, have involved margins or present with metastatic
disease, fare considerably worse with 5 year survival rates of
10-20%, largely due to the limited effectiveness of chemotherapy.
The only realistic opportunity for cure is a complete surgical
resection. Unfortunately, metastatic spread is already present in
40-70% of patients at the time of diagnosis precluding cure.
Standard chemotherapy in ACC cases remains based on mitotane which
was first approved in 1960. Mitotane, also known as o,p'-DDD, is a
derivative of the pesticide DDT and an adrenolytic. Tacon, L. J. et
al., Current and emerging therapies for advanced adrenocortical
carcinoma. Oncologist (16) 36-48 (2011); Daffara, F. et al.,
Prospective evaluation of mitotane toxicity in adrenocortical
cancer patients treated adjuvantly Endocr. Relat. Cancer (15)
1043-53 (2008); Netto, A. D. et al., Treatment of Adrenocortical
Cancer with O,P'-Ddd. Ann. Intern. Med. (59) 74-78 (1963); and
Bapat, A. A. et al., A fly in the ointment: reassessing mitotane's
role in the treatment of adrenocortical carcinoma. Pharmacogenomics
(13) 1207-1209 (2012). The response rates to mitotane as a single
agent is a relatively poor 23%, but survival for those patients
whose tumors do respond is improved from 14 to 50 months. Tacon, L.
J. et al., Current and emerging therapies for advanced
adrenocortical carcinoma. Oncologist (16) 36-48 (2011); Daffara, F.
et al., Prospective evaluation of mitotane toxicity in
adrenocortical cancer patients treated adjuvantly. Endocr. Relat.
Cancer, (15) 1043-53 (2008); and Netto, A. D. et al., Treatment of
Adrenocortical Cancer with O,P'-Ddd. Ann. Intern. Med. (59) 74-78
(1963). Mitotane is most often used in combination with etoposide,
doxorubicin, and cisplatin (EDP-M) based on the reported 49%
response rate in a phase II trial. Berruti, A. et al., Etoposide,
doxorubicin and cisplatin plus mitotane in the treatment of
advanced adrenocortical carcinoma: a large prospective phase II
trial Endocr. Relat. Cancer. (12) 657-666 (2005). However, further
study in the phase III FIRM-ACT trial demonstrated the response
rate to EDP-M was 23.2% and the median progression-free survival
interval was 5 months. Fassnacht, M. et al., Combination
Chemotherapy in Advanced Adrenocortical Carcinoma. N. Engl. J. Med.
(366) 2189-2197 (2012). For most patients, mitotane is poorly
tolerated due to its severe toxic side effects, including
obliteration of the healthy contralateral adrenal gland. There is
no approved second-line regimen for those whose disease progresses
on these agents.
[0007] The increased incidence of ACC in patients with Li-Fraumeni
syndrome suggests the p53 pathway is involved in ACC progression.
Fassnacht, M. et al., Adrenocortical carcinoma: a clinician's
update Nat. Rev. Endocrinol. (7) 323-335 (2011). In adults,
however, mutation in p53 is seen in less than 25% of cases
suggesting that other elements of the p53 pathway may be perturbed.
Waldmann, J. et al., Clinical impact of TP53 alterations in
adrenocortical carcinomas Langenbecks Arch. Surg. (397) 209-216
(2012); Libe, R. et al., Somatic TP53 mutations are relatively rare
among adrenocortical cancers with the frequent 17p13 loss of
heterozygosity Clin. Cancer Res. (13) 844-850 (2007); and Hamid, T.
and S. S. Kakar, PTTG/securin activates expression of p53 and
modulates its function Mol. Cancer (3) 18 (2004). It has been found
that p53 is a major driver of differential gene expression when
comparing ACC to normal adrenal glands or when comparing low and
high-grade tumors. Demeure, M. J. et al., PTTG1 overexpression in
adrenocortical cancer is associated with poor survival and
represents a potential therapeutic target Surgery (154) 1405-1416
(2013). This dysregulation is accompanied by perturbations in the
G2/M transition of the cell cycle. Demeure, M. J. et al., PTTG1
overexpression in adrenocortical cancer is associated with poor
survival and represents a potential therapeutic target Surgery
(154) 1405-1416 (2013). Moreover, Polo-like kinase 1 (PLK-1)
negatively modulates p53 functioning, promotes Mouse double minute
2 (MDM2) activity through its phosphorylation, and is involved in
the G2/M transition. X. Liu et al., Polo-like Kinase (Plk) 1
depletion induces apoptosis in cancer cells, Proc. Nat. Acad. Sci.
(100) 5789-5794 (2003).
[0008] p53 is a tumor suppressor and transcription factor that
responds to cellular stress by activating the transcription of
numerous genes involved in cell cycle arrest, apoptosis,
senescence, and DNA repair. Unlike normal cells, which have
infrequent cause for p53 activation, tumor cells are under constant
cellular stress from various insults including hypoxia and
pro-apoptotic oncogene activation. Thus, there is a strong
selective advantage for inactivation of the p53 pathway in tumors,
eliminating p53 function may be a prerequisite for tumor survival.
Mouse models have been used to demonstrate that absence of p53
function is a continuous requirement for the maintenance of
established tumors; when p53 function is restored to tumors with
inactivated p53, the tumors regressed.
[0009] p53 is inactivated by mutation and/or loss in 50% of solid
tumors and 10% of liquid tumors. Other key members of the p53
pathway are also genetically or epigenetically altered in cancer.
MDM2, an oncoprotein, inhibits p53 function, and it is activated by
gene amplification at incidence rates that are reported to be as
high as 10%. MDM2, in turn, is inhibited by another tumor
suppressor, p14ARF. Alterations downstream of p53 may be
responsible for at least partially inactivating the p53 pathway in
p53 wild type tumors (p53WT). In support of this concept, some
p53WT tumors appear to exhibit reduced apoptotic capacity, although
their capacity to undergo cell cycle arrest remains intact. MDM2
inhibits p53 activity by three mechanisms: 1) acting as an E3
ubiquitin ligase to promote p53 degradation; 2) binding to and
blocking the p53 transcriptional activation domain; and 3)
exporting p53 from the nucleus to the cytoplasm.
[0010] Apoptosis is an active form of cell death that is involved
in multiple processes of normal cell development as well as in
malignant cell transformations. Mechanism of apoptosis is engaged
in biological events induced by various types of drugs, cytokines,
and growth factors, oxidative stress, radiation, aging, autoimmune
diseases, and immune rejection within organ transplantation. Recent
studies on apoptosis demonstrate that common molecular mechanisms
are employed in various types of apoptosis, induced by hormones,
cytokines, growth factor deprivation, chemotherapeutic agents,
ionizing radiation, immunological disorders, AIDS, cancer and
aging.
[0011] Cascade-like activation of caspase proteases represents a
point in the induction of apoptosis. Two types of apoptosis
signaling mediated by the caspase cascade have been described:
receptor-dependent and receptor-independent. The initial phase of
receptor-depending triggering of apoptosis includes activation of
appropriate death receptors by specific ligands, such as TNF or
FasL, which are presently the most studied inductors of apoptosis.
Upon activation, cell surface death receptors, Fas (CD95) or TNFR1,
are attached to cytosolic adapter proteins (FADD, MORT, RIP,
TRADD), which in turn recruit caspase-8 to activate the
interleukin-1-.beta.-converting enzyme ICE/CED-3 family caspase
cascade, followed by activation of CPP32/caspase-3-subfamily of
cysteine proteases, whose members are localized in the cytoplasm in
the form of latent precursors known as procaspases.
Receptor-independent types of caspase cascade-mediated apoptosis
usually include important cytochrome c-inducible mechanism that
requires the formation of tertiary complex of cytochrome c, dATP,
Apaf-1 and procaspase-9, which lead to the activation of the latter
via autoproteolysis and homodimerization, and subsequent caspase
cascade activation. See D. R. Mcllwain et al. Caspase Functions in
Cell Death and Disease Cold Spring Harbor Perspectives in Biology
(5) a008656 (2013). In general, caspases involved in mediating
apoptosis have been generally divided by function: (i) initiator
caspases (i.e., caspases 8 and 9) and (ii) executioner caspases
(i.e., caspases 3, 6, and 7).
[0012] Restoring cell death functionality as a therapy for cancers
of the endocrine system, including ACC, is a desirable strategy, as
many current therapies rely on use of the apoptosis-inducing
caspase cascade. Apoptosis-inducing mechanisms in patients with
different cancers, however, may not properly function, which may
lead to poor patient responses to one or more therapeutics.
Embodiments of the present invention provide methods that can
overcome at least some of these shortcomings in the
therapeutic-strategy field. The present invention provides methods
of treatment that can be tailored by determining which, if any
caspases, are expressed and/or methods of the present invention can
be applied as a clinical assay to guide treatment decisions. Some
aspects of the invention may also function as a screening model to
identify new therapies with alternative modes of cellular
death.
[0013] The articles, treatises, patents, references, and published
patent applications described above and herein are hereby
incorporated by reference in their entirety for all purposes.
SUMMARY
[0014] Embodiments of the invention provide a method of treating
endocrine cancer in a subject. The method can include administering
to the subject a therapeutically effective amount of a polo-like
kinase 1 (PLK1) inhibitor. The method may also include
administering to the subject a therapeutically effective amount of
mouse double minute 2 (MDM2) inhibitor. For example, the PLK1
inhibitor can be selected from the group consisting of BI-2536,
cyclapolin 9, GW 843682X, TC-S 7005, Wortmannin, NMS-P937, and
GSK461364A. In particular aspects, the PLK1 inhibitor is BI-2536.
In addition, the MDM2 inhibitor can be selected from the group
consisting of a nutlin, caylin-1, HLI 373, caylin-2, JNJ 26854165,
NSC 66811, and trans-4-Iodo, 4'-boranyl-chalcone. In some
particular embodiments, the MDM2 is a nutlin, such as nutlin-3.
[0015] In some aspects, the endocrine cancer being treated can
comprise a cancer of the adrenal gland of the subject. In one
embodiment, the cancer of the adrenal gland can comprise a
malignant cancer. In a preferred embodiment, the malignant adrenal
gland cancer can be adrenocortical carcinoma.
[0016] In some embodiments, the PLK1 and MDM2 inhibitors can be
administered as a single pharmaceutical composition that is
administered to the subject as one or more single doses. In other
aspects, the PLK1 and MDM2 inhibitors are administered as
individual doses that can be administered at the same time,
sequentially, or at any other time interval. In some embodiments,
the method may also include determining if a marker having a
sequence selected from the group consisting of SEQ ID NO. 3 and SEQ
ID NO. 6 comprises a p53 wild type sequence in the subject being
treated.
[0017] Some embodiments of the invention provide a method of
treating endocrine cancer in a subject. The method may include
receiving a sample of a tumor from a subject and forming a mixture.
The mixture may include at least a portion of the sample and a
reagent (e.g., an oligonucleotide or an antibody) that specifically
binds to a first marker having a sequence selected from the group
consisting of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID
NO. 5. The method also includes subjecting the mixture to
conditions that allow detection of binding of the reagent to the
marker and then assessing an expression level of the marker in the
sample based on a level of binding of the reagent to the marker.
The method may also include administering a therapeutically
effective amount of a pharmaceutical composition comprising a
mitotic catastrophe inducing composition to the subject when the
expression level of the first marker in the sample is reduced
compared to a control sample. In some embodiments, the mitotic
catastrophe inducing composition is a mouse double minute 2 (MDM2)
inhibitor. The method may also include determining if a second
marker having a sequence substantially similar a sequence selected
from the group consisting of SEQ ID NO. 3 and SEQ ID NO. 6
comprises a p53 wild type sequence in the subject being
treated.
[0018] In some aspects, the endocrine cancer being treated can
comprise a cancer of the adrenal gland of the subject. In one
embodiment, the cancer of the adrenal gland can comprise a
malignant cancer. In a preferred embodiment, the malignant adrenal
gland cancer can be adrenocortical carcinoma.
[0019] Embodiments of the invention provide a method of treating a
subject with a caspase cascade defect. The method can include
administering to the subject a therapeutically effective amount of
mitotic catastrophe inducing composition to the subject. For
example, the mitotic catastrophe inducing composition can comprise
a PLK1 and/or an MDM2 inhibitor. For example, the PLK1 inhibitor
can be selected from the group consisting of BI-2536, cyclapolin 9,
GW 843682X, TC-S 7005, Wortmannin, NMS-P937, and GSK461364A. In
particular aspects, the PLK1 inhibitor is BI-2536. In addition, the
MDM2 inhibitor can be selected from the group consisting of a
nutlin, caylin-1, HLI 373, caylin-2, JNJ 26854165, NSC 66811, and
trans-4-lodo, 4'-boranyl-chalcone. In some particular embodiments,
the MDM2 is a nutlin, such as nutlin-3. In some aspects, the
caspase cascade defect comprises reduced expression of a first
marker having a sequence selected from the group consisting of SEQ
ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO. 5 in a sample
from the subject compared to a control sample.
[0020] Additional objectives, advantages and novel features will be
set forth in the description which follows or will become apparent
to those skilled in the art upon examination of the drawings and
detailed description which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is an illustration of caspase cascade defects in ACC.
Integrated analysis revealed a universal under-expression of
caspase 9, mutation or deletion of caspase 8, and perturbations of
other parts of the pathway. Green arrows are activating changes.
C=Cleavage; B=Binding; CR=complex formation; Tn=translocation
between cellular compartments. Large red arrows indicate
under-expression. Large red circles signify mutation or deletion
events. The number of tumors affected out the total is indicated in
black. This illustration was prepared using the Pathway Map Creator
in MetaCore.
[0022] FIG. 2 is a series of graphs that illustrate that ACC tumors
and cell lines are characterized by a lack of caspase 9 expression
at the mRNA level. (A) RT-qPCR values of caspases 1-10 compared to
normal adrenal RNA from each of the tumors assessed. (B) RT-qPCR
values of caspases 1-10 in the two commercially available ACC cell
lines, SW-13 and H295R. Data plotted is the mean with the SEM of
three biological replicates with three technical replicates per
biological replicate. ** p<0.01, *** p<0.0001.
[0023] FIG. 3 is a series of graphs that illustrate that the lack
of caspase 9 expression results in a delay of caspase 3/7
activation after doxorubicin exposure in all ACC cell lines, except
SW-13. Bars indicate comparisons that were not significantly
different using a corrected p-value of <0.05 as the cut-off.
[0024] FIG. 4 illustrates that the inhibition of Polo-like kinase 1
(PLK-1) reduced the viability of ACC cell lines. (A) ACC cell
lines, H295R and SW-13 were treated with PLK-1 siRNA and amount of
PLK-1 protein was determined after 72 hours of siRNA treatment. (B)
Quantitation of PLK-1 protein western after siRNA knockdown
expressed relative to .beta.-actin. (C) Knocking down expression of
the PLK-1 protein reduced viability of the H295R & SW-13 cell
lines as compared to the controls. (D and E) Treating the cells
with BI-2536, an inhibitor of PLK-1, reduced the viability of both
the H295R and SW-13 ACC cell lines. All experiments were performed
in at least 3 technical replicates and data are represented as
means with standard error. TF=Transfection agent and
NT=Non-targeting siRNA.
[0025] FIG. 5 illustrates that inhibition of PLK-1 reduces
expression of mutant p53 protein in SW13 cells. As PLK-1 has been
shown to physically interact with p53 to control its functions, the
levels of the wild type p53 protein should not be affected after
inhibition of PLK-1. (A) BI-2536 treatment resulted in a decrease
in the amount of mutant p53 protein in the SW-13 cells, but did not
change expression of the wild type p53 protein in the H295R cells.
(B and C) Quantitation of PLK-1 and p53 protein western blots shown
in panel (A) after BI-2536 treatment expressed relative to
.beta.-actin. (D) Inhibition of PLK-1 with BI-2536 did not reduce
transcript levels of either wild type or mutant p53 as determined
by qRT-PCR. All experiments were performed in at least 3 technical
replicates and data are represented as means with standard
error.
[0026] FIG. 6 illustrates that inhibition of PLK-1 restores p53
functioning. Since PLK-1 is a negative regulator of p53, inhibition
of PLK-1 should restore p53 transactivation and apoptotic
functions. (A) Treatment of H295R and SW-13 cells with BI-2536
resulted in the restoration of wild type p53's transactivation
functions as seen by the increased transcription of its downstream
gene, CDKN1A (p21), as determined by qRT-PCR. (B) BI-2536 treatment
of the SW-13 did not increase the transcription of CDKNIA, as this
cell line possess a mutant p53 gene. (C) Furthermore, inhibition of
PLK-1 restored wild type p53's apoptotic response in H295R cells as
determined by the Caspase 3/7 glo assay. (D) The apoptotic response
was slightly delayed in the SW-13 cells with mutant p53 with
maximum response observed at 48 hours. Doxorubicin was used as a
positive control for apoptosis. All experiments were performed in
at least 3 technical replicates and data are represented as means
with standard error.
[0027] FIG. 7 illustrates that inhibition of MDM2 by nutlin-3
restores wild type p53's apoptotic response. Since PLK-1 also
indirectly controls p53's activity via MDM2, the effect of PLK-1
inhibition on MDM2 functioning was determined. (A) Inhibition of
PLK-1 with BI-2536 did not change the levels of the MDM2 protein.
(B) Quantitation of the MDM2 protein western blot after BI-2536
treatment expressed relative to .beta.-actin. (C) Treating the
cells with nutlin-3, an inhibitor of MDM2, reduced the viability of
both the H295R and SW-13 ACC cell lines. (D) Inhibition of MDM2
restored wild type p53's apoptotic response in H295R cells as
determined by the Caspase 3/7 glo assay. (E) A small increase in
apoptosis was observed in the SW-13 cells with mutant p53.
Doxorubicin was used as a positive control for apoptosis. All
experiments were performed in at least 3 technical replicates and
data are represented as means with standard error.
[0028] FIG. 8 illustrates the synergy of PLK-1 inhibition by
BI-2536 with MDM2 inhibition by nutlin-3. Since PLK-1 plays a role
in controlling p53 directly and indirectly via MDM2, the synergy of
inhibition of PLK-1 and MDM2 inhibition was investigated. (A and B)
Treatment of H295R (A) and SW-13 (B) cells with BI-2536 sensitized
both the cell lines to nutlin-3 treatment. (C and D) Dual
inhibition of PLK-1 and MDM2 resulted in an additive response in
the H295R cells (C), with wild-type p53, whereas no additive
apoptotic response was seen in the SW-13 cells (D) with mutant p53.
Doxorubicin was used as a positive control for apoptosis. All
experiments were performed in at least 3 technical replicates and
data are represented as means with standard error.
[0029] The headings used in the figures should not be interpreted
to limit the scope of the claims.
DETAILED DESCRIPTION
[0030] Some aspects of the present invention can be used in
providing treatments to subjects with cancer that exhibit one or
more cellular defects. Some conventional therapeutic strategies for
use by endocrine cancer patients include one or more pharmaceutical
compositions that rely on cellular processes (e.g., the caspase
cascade) to provide a desired outcome. For example, some
therapeutic strategies rely on the intrinsic and/or extrinsic
caspase cascades to trigger cell death mechanisms, including
apoptosis, necroptosis, necrosis, etc. The therapeutic strategies
that rely on these cellular processes, however, will be ineffective
in subjects with one or more defects in the caspase cascade.
[0031] Subjects with some non-functional or malfunctioning cellular
processes, including those with caspase cascade defects, can be
treated using some aspects of the invention. The treatment methods
may include the administration of one or more pharmaceutical
compositions, alone or in combination, which can circumvent the
caspase cascade defects. For example, the treatment methods provide
a manner in which subjects with diseases associated with caspase
cascade defects receive an efficacious treatment in spite of the
defects of the caspase cascade.
[0032] Prior to treatment, some subjects or physicians treating the
subjects may find it helpful to determine whether any caspase
cascade defects are present within the subject. By determining the
state of caspase cascade defects within the subject, appropriate
therapeutic-strategy decisions can be made by the subject and/or
the physician. Some aspects of the invention provide methods of
assessing the expression of one or more caspases (e.g., caspases 8
and/or 9) and making therapy-related decisions based, at least in
part, on the expression of these caspases. For example, a reduction
in expression of some caspases indicates that a caspase cascade
defect is present within the subject such that a therapy should be
administered that can circumvent the caspase cascade defect.
[0033] Some embodiments of the invention may include providing one
or more therapeutic strategies that can rely on mitotic catastrophe
as a manner of inducing cell death in endocrine cancer. For
example, mitotic catastrophe can be initiated using one or more
compounds or techniques and can rely on one or more caspases that
are properly functioning (e.g., caspase 2). As such, even though
other initiator caspases (e.g., caspase 8 and 9) may be
non-functional, treatment strategies that rely on mitotic
catastrophe can circumvent the non-functional caspases by relying
on caspase 2.
[0034] The present invention is directed to methods for treating
one or more pathologies in a subject, including cancer. For
example, methods according to some embodiments may comprise
providing therapeutically effective amounts of one or more
pharmaceutical compositions, alone or in combination, to the
subject with cancer of the endocrine system. In some aspects, the
method may include administering one or more pharmaceutical
compositions, alone or in combination, that can circumvent one or
more defects in normal cellular processes that may be associated
with the endocrine cancer (e.g., correlated with cancer or
causatively associated cancer). In particular, the administration
of one or more pharmaceutical compositions may initiate one or more
cellular processes that can trigger the death of one or more cells
associated with the cancer. For example, the pharmaceutical
compositions can trigger apoptosis, necroptosis, or any other
extrinsically or intrinsically triggered cellular processes that
result in the cell death or cell senescence.
[0035] Some embodiments of the invention provide methods of
treating endocrine cancer in a subject, which include an assessment
of one or more markers to select a treatment. The method may
include assessing an expression level of the markers to make
determinations regarding the operability/functionality of one or
more cell processes. For example, prior to the administration of
one or more pharmaceutical compositions, an assessment of the
expression level of one or more initiator caspases (i.e., caspases
8 and 9) can be performed to determine whether circumvention of the
initiator caspase(s) is a preferred manner of treating the subject.
For example, in the event that the expression of one or more of the
initiator caspases is reduced, other therapeutic strategies (i.e.,
induction of mitotic catastrophe) can be employed using other
initiator caspases. The method may also include assessing an
allelic state of the subject's p53 gene and/or protein to determine
whether the subject possess a wild type or mutated form of p53.
[0036] Some embodiments of the invention provide methods of
treating caspase cascade defects. The method may include initially
determining whether the subject is experiencing a caspase cascade
defect, which may include non-functioning or malfunctioning aspects
of the apoptosis-inducing caspase cascade. For example, caspase
cascade defects may include an inability of one or more initiator
caspases to activate one or more executioner caspases. The method
may also include administering therapeutically effective amounts of
one or more pharmaceutical compositions, alone or in combination,
to the subject with the caspase cascade defect. In some aspects,
the method may include administering one or more pharmaceutical
compositions, alone or in combination, that can circumvent the one
or more caspase cascade defects. In particular, the administration
of one or more pharmaceutical compositions may generally correct
and/or circumvent one or more of the caspase cascade defects. For
example, the pharmaceutical compositions can trigger apoptosis,
necroptosis, mitotic catastrophe, or any other extrinsically or
intrinsically triggered cellular processes that result in the cell
death or cell senescence.
[0037] Generally, some embodiments of the present invention can be
used to identify a marker and/or used to assess a level of
expression of the marker. Some embodiments of the invention may
also be directed to assessing a status of the marker. For example,
embodiments of the invention may include determining whether a
marker has a sequence (i.e., a DNA sequence, an RNA sequence,
and/or a protein sequence) that is generally recognized as a wild
type sequence or whether the sequence has one or more mutations
therein.
[0038] A marker may be any molecular structure produced by a cell,
expressed inside the cell, accessible on the cell surface, or
secreted by the cell. A marker may be any protein, carbohydrate,
fatty acid, nucleic acid, catalytic site, or any combination of
these such as an enzyme, glycoprotein, cell membrane, virus, a
particular cell, or other uni- or multimolecular structure. A
marker may be represented by a sequence of a nucleic acid or any
other molecules derived from the nucleic acid. Examples of such
nucleic acids include miRNA, tRNA, siRNA, mRNA, cDNA, genomic DNA
sequences, or complementary sequences thereof. Alternatively, a
marker may be represented by a protein sequence. The concept of a
marker is not limited to the exact nucleic acid sequence or protein
sequence or products thereof, rather it encompasses all molecules
that may be detected by a method of assessing the marker. Without
being limited by the theory, the detection of the marker may
encompass the detection and/or determination of a change in copy
number (e.g., copy number of a gene or other forms of nucleic acid)
or in the detection of one or more translocations.
[0039] Therefore, examples of molecules encompassed by a marker
represented by a particular sequence further include alleles of the
gene used as a marker. An allele or an allelic status includes any
form of a particular nucleic acid that may be recognized as a form
of the particular nucleic acid on account of its location,
sequence, or any other characteristic that may identify it as being
a form of the particular gene. Alleles include but need not be
limited to forms of a gene that include point mutations, silent
mutations, deletions, frameshift mutations, single nucleotide
polymorphisms (SNPs), inversions, translocations, heterochromatic
insertions, and differentially methylated sequences relative to a
reference gene, whether alone or in combination. An allele of a
gene may or may not produce a functional protein; may produce a
protein with altered function, localization, stability,
dimerization, or protein-protein interaction; may have
overexpression, underexpression or no expression; may have altered
temporal or spatial expression specificity; or may have altered
copy number (e.g., greater or less numbers of copies of the
allele). An allele may also be called a mutation or a mutant. An
allele may be compared to another allele that may be termed a wild
type form of an allele. In some cases, the wild type allele is more
common than the mutant.
[0040] "Apoptosis" is defined as a conserved, genetically
programmed form of cellular suicide characterized by distinct
morphological changes such as cytoskeletal disruption, cell
shrinkage, membrane blebbing, nuclear condensation, fragmentation
of DNA, and loss of mitochondrial function.
[0041] "Caspase" refers to a cysteine protease of the
interleukin-1.beta./CED-3 family. "The caspase cascade" is a
sequential activation of at least two caspases, or the activation
of caspase activity that behaves as if it involves the sequential
activation of at least two caspases.
[0042] "Initiator caspase(s)" refers to caspase 2, caspase 8, and
caspase 9, which function as the first caspases activated in the
extrinsic apoptotic pathway and the intrinsic apoptotic pathways.
Initiator caspases generally exist as inactive pro-caspases that
are activated by dimerization rather than cleavage, unlike other
caspases. Initiator caspase dimerization results from upstream
signaling events and facilitates autocatalytic cleavage of
initiator caspases, which results in stabilization of the
dimer.
[0043] "Executioner caspase(s)" refers to caspase 3, caspase 6, and
caspase 7, which function as effector caspases after activation
(i.e., cleavage) by the initiator caspases. Once activated, one
executioner caspase can cleave the remaining executioner caspases
to irreversibly trigger the caspase cascade and apoptosis.
[0044] "Defects in the caspase cascade" or "caspase cascade
defects" refer to an inability of one or more of the initiator
caspases to activate the executioner caspases. The caspase cascade
defects may occur due to reduced or non-detectable expression of
the initiator caspases. The defects may arise because of one or
more mutations in the nucleotide (e.g., DNA or RNA) sequence or the
amino acid sequence of the initiator caspase(s) protein. Moreover,
the defects may also arise due to post-translational changes in the
initiator caspase(s) protein. Caspase cascade defects can result in
one or more pathological states, including cancer and other
proliferative disorders. Caspase cascade defects may be assessed
using any of the techniques described below. In some embodiments,
two of the initiator caspases may be non-functional (i.e., through
mutation and/or reduced expression) such that only one other
initiator caspase can function to activate the caspase cascade. For
example, in the event that one or more of the initiator caspases
are non-functional, activation of one of the functional initiator
caspases can be achieved to provide activation of the caspase
cascade.
[0045] By way of example only, some embodiments may provide
assessing an expression level of a marker, such as caspase 8 or
caspase 9. The invention includes determining the expression of
these markers using one or more molecular techniques described
herein or known to those having skill in the art. Moreover, the
invention may provide methods for determining whether the cells of
a subject contain at least one mutation in the p53 gene or protein.
The invention may further include testing for at least one mutation
in the p53 gene or protein using tests that are well known to those
of ordinary skill in the art. For example, see Flaman, J.-M. et
al., Proc. Natl. Acad. Sci. USA 92: 3963-3967 (1995). In one
embodiment, the mutation(s) are detected by direct sequencing of
the gene or the protein. In another embodiment, the mutation(s) are
detected by PCR.
[0046] In a particular embodiment of the invention that relates to
the treatment of endocrine cancers, the cancer can be identified as
p53 wild type (p53WT). In another aspect, the present invention
provides a diagnostic for determining which patients should be
administered a treatment according to some embodiments of the
invention. For example, a sample from a patient/subject may be
taken and analyzed to determine the allelic status of the sample
(e.g., cancer cells) with respect to p53. In one aspect, a patient
having a cancer that is p53WT will be selected for treatment. In
another aspect, a patient having a cancer that has p53WT protein is
selected over a patient that does not have these characteristics.
The taking of cancer cells for analyses is well known to those
skilled in the art. The term "p53WT" or "p53 wild type" means a
protein encoded by a nucleic acid sequence that is substantially
similar to the sequence of SEQ ID NO. 3 or a protein comprising an
amino acid sequence that is substantially similar to the amino acid
sequence of SEQ ID NO. 6.
[0047] The term "mitosis" refers to the generally accepted theory
of eukaryotic cellular growth. Mitosis is the process of the cell
cycle by which a cell duplicates into two generally genetically
identical daughter cells. In general, during mitosis, chromosomes
in the nucleus are replicated and separated into two sets of
chromosomes, each having its own nucleus. Successful mitosis is
generally followed by cytokinesis, which divides cellular
constituents into two daughter cells.
[0048] The term "mitotic catastrophe" is method of cell death that
refers to a mechanism to prevent genomic instability in response to
DNA damage or perturbations to cellular components during mitosis.
The perturbations could include damage to the chromosomes or the
cellular components that function to replicate and/or segregate the
chromosomes during mitosis. In addition, the accumulation of
unrepaired DNA damage (e.g., mutations, amplifications, deletions,
SNPs, etc.) may also trigger mitotic catastrophe. See R. Mu et al.,
Depletion of pre-mRNA splicing factor Cdc5L inhibits mitotic
progression and triggers mitotic catastrophe, Cell Death and
Disease (5) e11151 (2014). In particular, mitotic catastrophe can
be characterized by the occurrence of aberrant mitosis or the
missegregation of the chromosomes followed by cell division.
Mitotic catastrophe can function as an innate oncosuprresive
mechanism or a mechanism that is triggered by one or more mitotic
catastrophe-inducing agents. For example, mitotic catastrophe is
one of the modes of cell death following treatment with ionizing
radiation and can occur in response to other pharmaceutical
compositions. See D. Morse et al., Docetaxel induces cell death
through mitotic catastrophe in human breast cancer cells, Molecular
Cancer Therapies (10) 1495 (2005).
[0049] Mitotic catastrophe can occur via the caspase cascade. In
particular, one or more of the initiator caspases can be activated
in mitotic catastrophe to trigger cell death (e.g., apoptosis). In
a particular embodiment, caspase 2 can be activated to trigger the
caspase cascade to lead to cell death, which may include
apoptosis.
[0050] The term "derived from" refers to the origin or source, and
may include naturally occurring, recombinant, unpurified, or
purified molecules. The proteins and molecules of the present
invention may be derived from human or non-human molecules.
[0051] The term "nucleic acid" refers to a polymeric form of
nucleotides of any length, either ribonucleotides or
deoxyribonucleotides, or analogs thereof. This term refers to the
primary structure of the molecule, and thus includes double- and
single-stranded DNA, as well as double- and single-stranded RNA. It
also includes modified nucleic acids such as methylated and/or
capped nucleic acids, nucleic acids containing modified bases,
backbone modifications, and the like. The terms "nucleic acid" and
"nucleotide sequence" are used interchangeably. In some particular
aspects of the invention, caspase 8 comprises the nucleotide
sequence of SEQ ID NO. 1, caspase 9 comprises the nucleotide
sequence of SEQ ID NO. 2, and p53 comprises the nucleotide sequence
of SEQ ID NO. 3.
[0052] The term "gene" refers to a nucleic acid or portion of a
nucleic acid comprising a sequence that encodes a protein. It is
understood in the art that a gene also comprises non-coding
sequences, such as 5' and 3' flanking sequences (such as promoters,
enhancers, repressors, and other regulatory sequences) as well as
introns.
[0053] The terms "polynucleotide", "polynucleotide sequence",
"nucleic acid sequence", "nucleic acid fragment", and "isolated
nucleic acid fragment" are used interchangeably herein. These terms
encompass nucleotide sequences and the like. A polynucleotide may
be a polymer of RNA or DNA that is single- or double-stranded, that
optionally contains synthetic, non-natural or altered nucleotide
bases. A polynucleotide in the form of a polymer of DNA may be
comprised of one or more segments of cDNA, genomic DNA, synthetic
DNA, or mixtures thereof. Nucleotides (usually found in their
5'-monophosphate form) are referred to by a single letter
designation as follows: "A" for adenylate or deoxyadenylate (for
RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate,
"G" for guanylate or deoxyguanylate, "U" for uridylate, "T" for
deoxythymidylate, "R" for purines (A or G), "Y" for pyrimidines (C
or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and
"N" for any nucleotide.
[0054] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. These terms also include proteins that are
post-translationally modified through reactions that include
glycosylation, acetylation and phosphorylation. In some particular
aspects of the invention, caspase 8 comprises the amino acid
sequence of SEQ ID NO. 4, caspase 9 comprises the amino acid
sequence of SEQ ID NO. 5, and p53 comprises the nucleotide sequence
of SEQ ID NO. 6.
[0055] The term "nucleotide" is defined as a modified or naturally
occurring deoxyribonucleotide or ribonucleotide. Nucleotides
typically include purines and pyrimidines, which include thymidine,
cytidine, guanosine, adenine and uridine.
[0056] The term "at least a portion" of a nucleic acid or
polypeptide means a portion having the minimal size characteristics
of such sequences, or any larger fragment of the full length
molecule, up to and including the full length molecule. For
example, a portion of a nucleic acid may be 12 nucleotides, 13
nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17
nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 22
nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30
nucleotides, 32 nucleotides, 34 nucleotides, 36 nucleotides, 38
nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 55
nucleotides, and so on, going up to the full length nucleic acid.
Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino
acids, 6 amino acids, 7 amino acids, and so on, going up to the
full length polypeptide. The length of the portion to be used will
depend on the particular application. A portion of a nucleic acid
useful as hybridization probe may be as short as 12 nucleotides; in
one embodiment, it is 20 nucleotides. A portion of a polypeptide
useful as an epitope may be as short as 4 amino acids. A portion of
a polypeptide that performs the function of the full-length
polypeptide would generally be longer than 4 amino acids.
[0057] Determination of marker expression may be performed by one
or more of the methods known to one of ordinary skill in the art.
For example, gene expression levels may be determined by detection
of (a) a polypeptide encoded by a marker gene, (b) mRNA encoding a
marker protein or marker polypeptide, (c) a portion of DNA which
constitutes a marker gene, or (d) any combination thereof.
[0058] Levels of marker expression can be detected, for example, by
measuring levels of protein using specific binding compositions.
The detection of protein levels may be carried out using any of the
methods known to one of ordinary skill in the art including, but
not limited to, chemiluminescence methods, histochemical staining
or biochemical detection (i.e., immuno-histochemistry assays),
Western Blot analysis, flow cytometry, immuno-precipitation (or the
equivalent thereof for non-antibody agents), Plasmon resonance
absorbance measurement, and the like. In one embodiment of the
invention, the method of detecting protein levels is an immunoassay
(such as an ELISA), which includes the use of at least one
antibody. In yet another embodiment of the invention, protein
staining, in tissue sample for example, formalin-fixed,
paraffin-embedded tissue sections can be carried out by
immuno-histochemistry using an antibody, and determining the
expression of the gene.
[0059] One embodiment of the invention is performed using an IHC
kit which uses a primary mouse monoclonal antibody, a secondary
anti-mouse IgG antibody, a peroxidase blocker to quench the
endogenous peroxidase activity and a chromogenic substrate.
Measurement of the polypeptide encoded by a marker may include
measurements of fragments of the polypeptide, wherein the fragments
arise from transcriptional or translational variants of the marker;
or alternatively, differently sized polypeptides arise as a result
of post translational modifications including proteolysis of a
larger portion of a polypeptide.
[0060] Detection of levels of mRNA may also serve as an indicator
of marker expression. The methods used to detect mRNA levels are
well known in the art, and include, for example, the detection of
hybridization or amplification with the mRNA encoding a gene
product. This detection may be carried out by analysis of mRNA
either in vitro or in situ (e.g., in a tissue sample) using one of
the methods known to one of ordinary skill in the art as
exemplified in the Current Protocols in Molecular Biology (John
Wiley & Sons, 1999); in U.S. Pat. No. 5,882,864; and the like.
An mRNA detected will be any RNA transcript of a specific gene, or
fragment thereof.
[0061] Some embodiments of the invention may include assessing,
determining, quantifying, or altering the expression of a marker.
As used herein, expression encompasses any and all processes
through which material derived from a nucleic acid template may be
produced. Expression thus includes RNA transcription, mRNA
splicing, protein translation, protein folding, post-translational
modification, membrane transport, associations with other
molecules, addition of carbohydrate moieties to proteins,
phosphorylation, protein complex formation and any other process
along a continuum that results in biological material derived from
genetic material. Expression also encompasses all processes through
which the production of material derived from a nucleic acid
template may be actively or passively suppressed. Such processes
include all aspects of transcriptional and translational
regulation. Examples include heterochromatic silencing,
transcription factor inhibition, any form of RNAi silencing,
microRNA silencing, small interfering RNA silencing, alternative
splicing, protease digestion, posttranslational modification, and
alternative protein folding.
[0062] Expression may be assessed by any number of methods used to
detect material derived from a nucleic acid template used currently
in the art and yet to be developed. Examples of such methods
include any nucleic acid detection method including the following
nonlimiting examples, microarray analysis, RNA in situ
hybridization, RNAse protection assay, Northern blot, reverse
transcriptase PCR, quantitative PCR, quantitative reverse
transcriptase PCR, quantitative real-time reverse transcriptase
PCR, reverse transcriptase treatment followed by direct sequencing,
or any other method of detecting a specific nucleic acid now known
or yet to be disclosed. Other examples include any process of
assessing expression that uses an antibody including the following
nonlimiting examples, flow cytometry, immunohistochemistry, ELISA,
Western blot, and immunoaffinity chromatography. Antibodies may be
monoclonal, polyclonal, or any antibody fragment including an Fab,
F(ab)2, Fv, scFv, phage display antibody, peptibody, multispecific
ligand, or any other reagent with specific binding to a marker.
Such methods also include direct methods used to assess protein
expression including the following non-limiting examples: HPLC,
mass spectrometry, protein microarray analysis, PAGE analysis,
isoelectric focusing, 2-D gel electrophoresis, and enzymatic
assays. Samples from which expression may be detected include
single cells, whole organs or any fraction of a whole organ,
whether in vitro, ex vivo, in vivo, or post-mortem.
[0063] Other methods used to assess expression include the use of
natural or artificial ligands capable of specifically binding one
or more markers, including a protein, carbohydrate, fat, nucleic
acid, catalytic site, or any combination of these such as an
enzyme, glycoprotein, cell membrane, virus, cell, organ, organelle,
or any uni- or multimolecular structure that constitutes a marker
that may be specifically bound by a ligand. Such ligands include
antibodies, antibody complexes, conjugates, natural ligands, small
molecules, nanoparticles, or any other molecular entity capable of
specific binding to a marker. Ligands may be associated with a
label such as a radioactive isotope or chelate thereof, dye
(fluorescent or non-fluorescent) stain, enzyme, metal, or any other
substance capable of aiding a machine or a human eye from
differentiating a cell expressing a marker from a cell not
expressing a marker. Additionally, expression may be assessed by
monomeric or multimeric ligands associated with substances capable
of killing the cell. Such substances include protein or small
molecule toxins, cytokines, pro-apoptotic substances, pore forming
substances, radioactive isotopes, or any other substance capable of
killing a cell.
[0064] Positive expression encompasses any difference between a
cell expressing markers and a cell that does not express one or
more of the markers. The exact nature of positive expression varies
by the method, but is well known to those skilled in the art of
practicing a particular method. Positive expression may be assessed
by a detector, an instrument containing a detector, or by aided or
unaided human eye. Examples include but are not limited to specific
staining of cells expressing a target in an IHC slide, binding of
RNA from a sample to a microarray and detection of binding through
the use of said microarray, a particular rate of dye incorporation
in real-time RT-PCR measured in .DELTA.Ct or alternatively in the
number of PCR cycles necessary to reach a particular optical
density at a wavelength at which a double stranded DNA binding dye
(e.g., SYBR Green) incorporates, through release of label from a
previously labeled reporter probe used in a real-time RT-PCR
reaction, detection of fluorescence on a cell expressing a target
by a flow cytometer, the presence of radiolabeled bands on film in
a Northern blot, detection of labeled blocked RNA by RNAse
protection assay, cell death measured by apoptotic markers, cell
death measured by shrinkage of a tumor, or any other signal for the
expression of a marker in existence now or yet to be developed.
[0065] Reduced expression encompasses any reduction (i.e., partial
or complete reduction) in the expression of one or more markers on
or in a cell from a subject relative to the expression of the same
markers in or on any other cell (e.g., a control cell). In some
aspects of the invention, reduced expression includes no detectable
expression. However, the concept of reduced expression further
encompasses insufficient expression to reach or exceed a threshold,
cutoff, or level that has been previously shown to result in a
particular cellular or physiological response. Reduced expression
may include similar expression relative to a control that has been
previously determined not to express the marker(s) or similar
expression to a control that has been previously determined not to
exhibit the response. In this case, even though expression may be
detectable, it still constitutes reduced expression. In some
aspects of the invention, an expression level of a marker in a
control known to have a reduced or increase risk of recurrence is
predetermined and expression similar to that level is correlated
with reduced or increase risk of recurrence. Increased or reduced
expression includes expression that is 75% 50%, 25%, 10%, 5%, 1%,
0.1%, greater or less of that of a control cell or a median level
of expression in a population. Reduced expression may also include
greater than or less than 1.times.10.sup.-5 greater or less
expression normalized to the expression of a housekeeping gene.
[0066] Assessing the risk of a particular disease outcome includes
the performing of any type of test, assay, examination, result,
readout, or interpretation that correlates with an increased or
decreased probability that an individual has had, currently has, or
will develop a particular disease, disorder, symptom, syndrome, or
any condition related to health or bodily state. Examples of
disease outcomes include, but need not be limited to survival,
death, progression of existing disease, remission of existing
disease, initiation of onset of a disease in an otherwise
disease-free subject, or the continued lack of disease in a subject
in which there has been a remission of disease. Assessing the risk
of a particular disease encompasses diagnosis in which the type of
disease afflicting a subject is determined. Assessing the risk of a
disease outcome also encompasses the concept of prognosis. A
prognosis may be any assessment of the risk of disease outcome in
an individual in which a particular disease has been diagnosed.
Assessing the risk further encompasses prediction of therapeutic
response in which a treatment regimen is chosen based on the
assessment. Assessing the risk also encompasses a prediction of
overall survival after diagnosis.
[0067] Some embodiments of the invention may comprise the use of
one or more methods of amplifying a nucleic acid-based starting
material (i.e., a template). Nucleic acids may be selectively and
specifically amplified from a template nucleic acid contained in a
sample. In some nucleic acid amplification methods, the copies are
generated exponentially. Examples of nucleic acid amplification
methods known in the art include: polymerase chain reaction (PCR),
ligase chain reaction (LCR), self-sustained sequence replication
(3SR), nucleic acid sequence based amplification (NASBA), strand
displacement amplification (SDA), amplification with 013 replicase,
whole genome amplification with enzymes such as qp29, whole genome
PCR, in vitro transcription with T7 RNA polymerase or any other RNA
polymerase, or any other method by which copies of a desired
sequence are generated.
[0068] An oligonucleotide may be any polynucleotide of at least 2
nucleotides. Oligonucleotides may be less than 10, 15, 20, 30, 40,
50, 75, 100, 200, or 500 nucleotides in length. While
oligonucleotides are often linear, they may assume a circular or
other two dimensional structure. Oligonucleotides may be chemically
synthesized by any of a number of methods including sequential
synthesis, solid phase synthesis, or any other synthesis method now
known or yet to be disclosed. Alternatively, oligonucleotides may
be produced by recombinant DNA based methods. In some aspects of
the invention, an oligonucleotide may be 2 to 1000 bases in length.
In other aspects, it may be 5 to 500 bases in length, 5 to 100
bases in length, 5 to 50 bases in length, or 10 to 30 bases in
length. One skilled in the art would understand the length of
oligonucleotide necessary to perform a particular task.
Oligonucleotides may be directly labeled, used as primers in PCR or
sequencing reactions, or bound directly to a solid substrate as in
oligonucleotide arrays.
[0069] In addition to genomic DNA, any oligonucleotide or
polynucleotide sequence can be amplified with an appropriate set of
primer molecules. In particular, the amplified segments created by
the PCR process itself are, themselves, efficient templates for
subsequent PCR amplifications.
[0070] PCR generally involves the mixing of a nucleic acid sample,
two or more primers that are designed to recognize the template
DNA, a DNA polymerase, which may be a thermostable DNA polymerase
such as Taq or Pfu, and deoxyribose nucleoside triphosphates
(dNTP's). Reverse transcription PCR, quantitative reverse
transcription PCR, and quantitative real time reverse transcription
PCR are other specific examples of PCR. In general, the reaction
mixture is subjected to temperature cycles comprising a
denaturation stage (typically 80-100.degree. C.), an annealing
stage with a temperature that is selected based on the melting
temperature (Tm) of the primers and the degeneracy of the primers,
and an extension stage (for example 40-75.degree. C.). In real-time
PCR analysis, additional reagents, methods, optical detection
systems, and devices known in the art are used that allow a
measurement of the magnitude of fluorescence in proportion to
concentration of amplified DNA. In such analyses, incorporation of
fluorescent dye into the amplified strands may be detected or
measured.
[0071] Alternatively, labeled probes that bind to a specific
sequence during the annealing phase of the PCR may be used with
primers. Labeled probes release their fluorescent tags during the
extension phase so that the fluorescence level may be detected or
measured. Generally, probes are complementary to a sequence within
the target sequence downstream from either the upstream or
downstream primer. Probes may include one or more label. A label
may be any substance capable of aiding a machine, detector, sensor,
device, or enhanced or unenhanced human eye from differentiating a
labeled composition from an unlabeled composition. Examples of
labels include but are not limited to: a radioactive isotope or
chelate thereof, dye (fluorescent or nonfluorescent) stain, enzyme,
or nonradioactive metal. Specific examples include, but are not
limited to: fluorescein, biotin, digoxigenin, alkaline phosphatese,
biotin, streptavidin, .sup.3H, .sup.14C, .sup.32P, .sup.3S, or any
other compound capable of emitting radiation, rhodamine,
4-(4'-dimethylamino-phenylazo) benzoic acid ("Dabcyl");
4-(4'-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)
("Dabsyl"); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid
("EDANS"); Psoralene derivatives, haptens, cyanines, acridines,
fluorescent rhodol derivatives, cholesterol derivatives;
ethylenediaminetetraaceticacid ("EDTA") and derivatives thereof or
any other compound that may be differentially detected. The label
may also include one or more fluorescent dyes optimized for use in
genotyping. Examples of dyes facilitating the reading of the target
amplification include, but are not limited to: CAL-Fluor Red 610,
CAL-Fluor Orange 560, dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET,
dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, and LIZ.PCR
facilitating the reading of the target amplification.
[0072] Either primers or primers along with probes allow a
quantification of the amount of specific template DNA present in
the initial sample. In addition, RNA may be detected by PCR
analysis by first creating a DNA template from RNA through a
reverse transcriptase enzyme. The marker expression may be detected
by quantitative PCR analysis facilitating genotyping analysis of
the samples.
[0073] An illustrative example, using dual-labeled oligonucleotide
probes in PCR reactions is disclosed in U.S. Pat. No. 5,716,784 to
DiCesare. In one example of the PCR step of the multiplex Real
Time-PCR/PCR reaction of the present invention, the dual-labeled
fluorescent oligonucleotide probe binds to the target nucleic acid
between the flanking oligonucleotide primers during the annealing
step of the PCR reaction. The 5' end of the oligonucleotide probe
contains the energy transfer donor fluorophore (reporter fluor) and
the 3' end contains the energy transfer acceptor fluorophore
(quenching fluor). In the intact oligonucleotide probe, the 3'
quenching fluor quenches the fluorescence of the 5' reporter fluor.
However, when the oligonucleotide probe is bound to the target
nucleic acid, the 5' to 3' exonuclease activity of the DNA
polymerase, e.g., Taq DNA polymerase, will effectively digest the
bound labeled oligonucleotide probe during the amplification step.
Digestion of the oligonucleotide probe separates the 5' reporter
fluor from the blocking effect of the 3' quenching fluor. The
appearance of fluorescence by the reporter fluor is detected and
monitored during the reaction, and the amount of detected
fluorescence is proportional to the amount of fluorescent product
released. Examples of apparatus suitable for detection include,
e.g. Applied Biosystems.TM.7900HT real-time PCR platform and
Roche's 480 LightCycler, the ABI Prism 7700 sequence detector using
96-well reaction plates or GENEAMP PC System 9600 or 9700 in 9600
emulation mode followed by analysis in the ABA Prism Sequence
Detector or TAQMAN LS-50B PCR Detection System. The labeled probe
facilitated multiplex Real Time-PCR/PCR can also be performed in
other real-time PCR systems with multiplexing capabilities.
[0074] "Amplification" is a special case of nucleic acid
replication involving template specificity. Amplification may be a
template-specific replication or a non-template-specific
replication (i.e., replication may be specific template-dependent
or not). Template specificity is here distinguished from fidelity
of replication (synthesis of the proper polynucleotide sequence)
and nucleotide (ribo- or deoxyribo-) specificity. Template
specificity is frequently described in terms of "target"
specificity. Target sequences are "targets" in the sense that they
are sought to be sorted out from other nucleic acid. Amplification
techniques have been designed primarily for this sorting out.
[0075] The term "template" refers to nucleic acid originating from
a sample that is analyzed for the presence of a molecule of
interest. In contrast, "background template" or "control" is used
in reference to nucleic acid other than sample template that may or
may not be present in a sample. Background template is most often
inadvertent. It may be the result of carryover, or it may be due to
the presence of nucleic acid contaminants sought to be purified out
of the sample. For example, nucleic acids from organisms other than
those to be detected may be present as background in a test
sample.
[0076] In addition to primers and probes, template specificity is
also achieved in some amplification techniques by the choice of
enzyme. Amplification enzymes are enzymes that, under the
conditions in which they are used, will process only specific
sequences of nucleic acid in a heterogeneous mixture of nucleic
acid. Other nucleic acid sequences will not be replicated by this
amplification enzyme. Similarly, in the case of T7 RNA polymerase,
this amplification enzyme has a stringent specificity for its own
promoters (Chamberlin et al. (1970) Nature (228):227). In the case
of T4 DNA ligase, the enzyme will not ligate the two
oligonucleotides or polynucleotides, where there is a mismatch
between the oligonucleotide or polynucleotide substrate and the
template at the ligation junction (Wu and Wallace (1989) Genomics
(4):560). Finally, Taq and Pfu polymerases, by virtue of their
ability to function at high temperature, are found to display high
specificity for the sequences bounded and thus defined by the
primers; the high temperature results in thermodynamic conditions
that favor primer hybridization with the target sequences and not
hybridization with non-target sequences (H. A. Erlich (ed.) (1989)
PCR Technology, Stockton Press).
[0077] The term "amplifiable nucleic acid" refers to nucleic acids
that may be amplified by any amplification method. It is
contemplated that "amplifiable nucleic acid" will usually comprise
"sample template." The terms "PCR product," "PCR fragment," and
"amplification product" refer to the resultant mixture of compounds
after two or more cycles of the PCR steps of denaturation,
annealing and extension. These terms encompass the case where there
has been amplification of one or more segments of one or more
target sequences.
[0078] In some forms of PCR assays, quantification of a target in
an unknown sample is often required. Such quantification is often
in reference to the quantity of a control sample. The control
sample DNA may be co-amplified in the same tube in a multiplex
assay or may be amplified in a separate tube. Generally, the
control sample contains DNA at a known concentration. The control
sample DNA may be a plasmid construct comprising only one copy of
the amplification region to be used as quantification reference. To
calculate the quantity of a target in an unknown sample, various
mathematical models are established. Calculations are based on the
comparison of the distinct cycle determined by various methods,
e.g., crossing points (CP) and cycle threshold values (Ct) at a
constant level of fluorescence; or CP acquisition according to
established mathematic algorithm.
[0079] The algorithm for Ct values in real time-PCR calculates the
cycle at which each PCR amplification reaches a significant
threshold. The calculated Ct value is proportional to the number of
target copies present in the sample, and the Ct value is a precise
quantitative measurement of the copies of the target found in any
sample. In other words, Ct values represent the presence of
respective target that the primer sets are designed to recognize.
If the target is missing in a sample, there should be no
amplification in the Real Time-PCR reaction.
[0080] Alternatively, the Cp value may be utilized. A Cp value
represents the cycle at which the increase of fluorescence is
highest and where the logarithmic phase of a PCR begins. The
LightCycler 480 Software calculates the second derivatives of
entire amplification curves and determines where this value is at
its maximum. By using the second-derivative algorithm, data
obtained are more reliable and reproducible, even if fluorescence
is relatively low.
[0081] The various and non-limiting embodiments of the PCR-based
method detecting marker expression level as described herein may
comprise one or more probes and/or primers. Generally, the probe or
primer contains a sequence complementary to a sequence specific to
a region of the nucleic acid of the marker gene. A sequence having
less than 60% 70%, 80%, 90%, 95%, 99% or 1000/identity to the
identified gene sequence may also be used for probe or primer
design if it is capable of binding to its complementary sequence of
the desired target sequence in marker nucleic acid.
[0082] Some embodiments of the invention may include a method of
comparing a marker in a sample relative to one or more control
samples. A control may be any sample with a previously determined
level of expression. A control may comprise material within the
sample or material from sources other than the sample.
Alternatively, the expression of a marker in a sample may be
compared to a control that has a level of expression predetermined
to signal or not signal a cellular or physiological characteristic.
This level of expression may be derived from a single source of
material including the sample itself or from a set of sources.
[0083] The sample in this method is preferably a biological sample
from a subject. The term "sample" or "biological sample" is used in
its broadest sense. Depending upon the embodiment of the invention,
for example, a sample may comprise a bodily fluid including whole
blood, serum, plasma, urine, saliva, cerebral spinal fluid, semen,
vaginal fluid, pulmonary fluid, tears, perspiration, mucus and the
like; an extract from a cell, chromosome, organelle, or membrane
isolated from a cell; a cell; genomic DNA, RNA, or cDNA, in
solution or bound to a substrate; a tissue; a tissue print, or any
other material isolated in whole or in part from a living subject.
Biological samples may also include sections of tissues such as
biopsy and autopsy samples, and frozen sections taken for
histologic purposes such as blood, plasma, serum, muscle, sputum,
stool, tears, mucus, hair, skin, and the like. Biological samples
also include explants and primary and/or transformed cell cultures
derived from patient tissues. In some embodiments, the sample can
comprise tissue obtained from an adrenal cortex of a subject,
including a living or a deceased subject.
[0084] Some embodiments of the invention may include a method of
comparing a marker in a sample relative to one or more control
samples. A control may be any sample with a previously determined
level of expression. A control may comprise material within the
sample or material from sources other than the sample.
Alternatively, the expression of a marker in a sample may be
compared to a control that has a level of expression predetermined
to signal or not signal a cellular or physiological characteristic.
This level of expression may be derived from a single source of
material including the sample itself or from a set of sources.
[0085] The invention may further comprise sequencing nucleic acids
from a sample, including sequencing a subject's entire genome,
exome, or transcriptome. Methods of sequencing include but need not
be limited to any form of DNA sequencing including Sanger,
next-generation sequencing, pyrosequencing, SOLID sequencing,
massively parallel sequencing, pooled, and barcoded DNA sequencing
or any other sequencing method now known or yet to be
disclosed.
[0086] In Sanger Sequencing, a single-stranded DNA template, a
primer, a DNA polymerase, nucleotides and a label such as a
radioactive label conjugated with the nucleotide base or a
fluorescent label conjugated to the primer, and one chain
terminator base comprising a dideoxynucleotide (ddATP, ddGTP,
ddCTP, or ddTTP), are added to each of four reaction (one reaction
for each of the chain terminator bases). The sequence may be
determined by electrophoresis of the resulting strands. In dye
terminator sequencing, each of the chain termination bases is
labeled with a fluorescent label of a different wavelength that
allows the sequencing to be performed in a single reaction.
[0087] In pyrosequencing, the addition of a base to a
single-stranded template to be sequenced by a polymerase results in
the release of a pyrophosphate upon nucleotide incorporation. An
ATP sulfuryrlase enzyme converts pyrophosphate into ATP that in
turn catalyzes the conversion of luciferin to oxyluciferin which
results in the generation of visible light that is then detected by
a camera or other sensor capable of capturing visible light.
[0088] In SOLID sequencing, the molecule to be sequenced is
fragmented and used to prepare a population of clonal magnetic
beads (in which each bead is conjugated to a plurality of copies of
a single fragment) with an adaptor sequence and alternatively a
barcode sequence. The beads are bound to a glass surface.
Sequencing is then performed through 2-base encoding.
[0089] In massively parallel sequencing, randomly fragmented
targeted DNA is attached to a surface. The fragments are extended
and bridge amplified to create clusters, each with a plurality of
copies of a single fragment sequence, within flow cell lanes. The
templates are sequenced by synthesizing the fragments in parallel.
Bases are indicated by the release of a fluorescent dye correlating
to the addition of the particular base to the fragment. Nucleic
acid sequences may be identified by the IUAPC letter code which is
as follows: A--Adenine base; C--Cytosine base; G--guanine base; T
or U--thymine or uracil base. M--A or C; R-A or G; W-A or T; S-C or
G; Y-C or T; K-G or T; V-A or C or G; H-A or C or T; D A or G or T;
B-C or G or T; N or X-A or C or G or T. Note that T or U may be
used interchangeably depending on whether the nucleic acid is DNA
or RNA. A sequence having less than 60%, 70%, 80%, 90%, 95%, 99% or
100% identity to the identifying sequence may still be encompassed
by the invention if it is able of binding to its complimentary
sequence and/or facilitating nucleic acid amplification of a
desired target sequence. In some embodiments, the method may
include the use of massively parallel sequencing, as detailed in
U.S. Pat. Nos. 8,431,348 and 7,754,429, which are hereby
incorporated by reference in their entirety.
[0090] Cancer cells include any cells derived from a tumor,
neoplasm, cancer, precancer, cell line, malignancy, or any other
source of cells that have the potential to expand and grow to an
unlimited degree. Cancer cells may be derived from naturally
occurring sources or may be artificially created. Cancer cells may
also be capable of invasion into other tissues and metastasis.
Cancer cells further encompass any malignant cells that have
invaded other tissues and/or metastasized. One or more cancer cells
in the context of an organism may also be called a cancer, tumor,
neoplasm, growth, malignancy, or any other term used in the art to
describe cells in a cancerous state.
[0091] Examples of cancers that could serve as sources of cancer
cells include solid tumors such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endothelio sarcoma, lymphangiosarcoma,
lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal
cancer, kidney cancer, pancreatic cancer, bone cancer, breast
cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach
cancer, oral cancer, nasal cancer, throat cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, uterine cancer, testicular cancer, small
cell lung carcinoma, bladder carcinoma, lung cancer, epithelial
carcinoma, glioma, glioblastoma multiforme, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
skin cancer, melanoma, neuroblastoma, and retinoblastoma.
[0092] Additional cancers that may serve as sources of cancer cells
include blood bome cancer, such as acute lymphoblastic leukemia
("ALL,"), acute lymphoblastic B-cell leukemia, acute lymphoblastic
T-cell leukemia, acute myeloblastic leukemia ("AML"), acute
promyelocytic leukemia ("APL"), acute monoblastic leukemia, acute
erythroleukemic leukemia, acute megakaryoblastic leukemia, acute
myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute
undifferentiated leukemia, chronic myelocytic leukemia ("CML"),
chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, multiple
myeloma, lymphoblastic leukemia, myelogenous leukemia, lymphocytic
leukemia, myelocytic leukemia, Hodgkin's disease, non-Hodgkin's
Lymphoma, Waldenstrom's macroglobulinemia, Heavy chain disease, and
Polycythemia vera.
[0093] In some embodiments, cancer may comprise a cancer of the
endocrine system of the subject. For example, the endocrine cancer
can comprise a cancer of an adrenal gland of the subject. In some
embodiments, the adrenal gland cancer can be a malignant cancer. In
a preferred embodiment, the malignant adrenal gland cancer can be
adrenocortical carcinoma.
[0094] The term "endocrine system" refers to a subject's collection
of glands, tissues, cells, and other bodies and organs within the
animal that produce and/or secrete hormones. The endocrine system
includes adrenal glands, the hypothalamus, ovaries, testicles,
pancreas, parathyroid glands, pineal body, pituitary gland, thymus
gland, and thyroid gland. Moreover, "cancer of the endocrine
system" or "endocrine cancer" refers to partially or completely
unregulated growth of one or more cellular components of the
endocrine system. For example, endocrine cancers can include a
cancer of one or more of the adrenal glands, carcinoid tumors,
parathyroid tumors, pituitary gland tumors, and thyroid tumors.
[0095] Some endocrine cancers, such as a cancer of the adrenal
gland may comprise a malignant cancer or a benign cancer. The term
"malignant" refers to a type of cancer that has the potential to
invade and/or damage proximal or distal tissues and organs.
Malignant also includes the ability to metastasize or
separate/detach from an initial site of cancerous growth, enter a
circulatory and/or a lymphatic system of an animal, and form
additional cancerous growths in other locations within the animal.
The term "benign" refers to a type of cancer that generally does
not metastasize to other locations within the animal. For example,
malignant cancer of the adrenal gland can be adrenocortical
carcinoma.
[0096] The term "PLK1-mediated condition", "polo-like kinase 1
mediated disorder" or any other variation thereof, as used herein
means any disease or other condition in which PLK1 is known to play
a role, or a disease state that is associated with elevated
activity or expression of PLK1. For example, a "PLK1-mediated
condition" may be relieved by inhibiting PLK1 activity. Such
conditions include various cancers, including bladder, thyroid,
ovarian, pancreatic, breast, endometrial, prostate, colorectal,
lung (e.g. non-small cell lung cancer), head and neck, gastric,
oropharyngeal, and esophageal cancers, glioma, glioblastoma,
papillary carcinoma, hepatoma, melanoma, lymphomas (e.g.
non-Hodgkins lymphoma, Hodgkin's lymphoma), leukemias (e.g. chronic
myeloid leukemia, acute myeloid leukemia), adrenocortical
carcinoma, advanced metastatic cancers, and advanced solid
tumors.
[0097] The term "subject" is used in its broadest sense. "Subject"
may also be used interchangeably with the term "patient." In a
preferred embodiment, the subject is a mammal. Non-limiting
examples of mammals include humans, dogs, cats, horses, cows,
sheep, goats, and pigs. Preferably, a subject includes any human or
non-human mammal, including for example: a primate, cow, horse,
pig, sheep, goat, dog, cat, or rodent, capable of developing cancer
including human patients that are suspected of having cancer, that
have been diagnosed with cancer, or that have a family history of
cancer (e.g., adrenocortical carcinoma).
[0098] Some embodiments of the invention may include the
administration of a pharmaceutical composition or more than one
pharmacological composition to a subject that has been diagnosed
with cancer. Such pharmaceutical compositions may take any physical
form necessary depending on a number of factors including the
desired method of administration and the physicochemical and
stereochemical form taken by the compound or pharmaceutically
acceptable salts of the compound. Such physical forms include a
solid, liquid, gas, sol, gel, aerosol, or any other physical form
now known or yet to be disclosed.
[0099] The concept of a pharmaceutical composition encompasses a
compound or a pharmaceutically acceptable salt thereof with or
without any other additive. The physical form of the invention may
affect the route of administration and one skilled in the art would
know to choose a route of administration that takes into
consideration both the physical form of the compound and the
disorder to be treated. Pharmaceutical compositions that include
the compound may be prepared using methodology well known in the
pharmaceutical art. A pharmaceutical composition that includes the
disclosed compound may include a second effective compound of a
distinct chemical formula from the disclosed compound. This second
effective compound may have the same or a similar molecular target
as the target or it may act upstream or downstream of the molecular
target of the compound with regard to one or more biochemical
pathways.
[0100] Pharmaceutical compositions include materials capable of
modifying the physical form of a dosage unit. In one non-limiting
example, the composition includes a material that forms a coating
that contains the compound.
[0101] Materials that may be used in a coating, include, for
example, sugar, shellac, gelatin, or any other inert coating
agent.
[0102] Pharmaceutical compositions including the disclosed compound
may be prepared as a gas or aerosol. Aerosols encompass a variety
of systems including colloids and pressurized packages. Delivery of
a composition in this form may include propulsion of a
pharmaceutical composition including the disclosed compound through
use of liquefied gas or other compressed gas or by a suitable pump
system. Aerosols may be delivered in single phase, bi-phasic, or
multi-phasic systems.
[0103] In some aspects of the invention, the pharmaceutical
composition including the disclosed compound is in the form of a
solvate. Such solvates are produced by the dissolution of the
disclosed compound in a pharmaceutically acceptable solvent.
Pharmaceutically acceptable solvents include any mixtures of one or
more solvents. Such solvents may include pyridine, chloroform,
propan-1-ol, ethyl oleate, ethyl lactate, ethylene oxide, water,
ethanol, and any other solvent that delivers a sufficient quantity
of the disclosed compound to treat the indicated condition.
[0104] Pharmaceutical compositions may also include at least one
pharmaceutically acceptable carrier. Carriers include any substance
that may be administered with the disclosed compound with the
intended purpose of facilitating, assisting, or helping the
administration or other delivery of the compound. Carriers include
any liquid, solid, semisolid, gel, aerosol or anything else that
may be combined with the disclosed compound to aid in its
administration. Examples include diluents, adjuvants, excipients,
water, and oils (including petroleum, animal, vegetable or
synthetic oils.) Such carriers include particulates such as a
tablet or powder, liquids such as oral syrup or injectable liquid,
and inhalable aerosols.
[0105] Further examples include saline, gum acacia, gelatin, starch
paste, talc, keratin, colloidal silica, and urea. Such carriers may
further include binders such as ethyl cellulose,
carboxymethylcellulose, microcrystalline cellulose, or gelatin;
excipients such as starch, lactose or dextrins; disintegrating
agents such as alginic acid, sodium alginate, Primogel, and corn
starch; lubricants such as magnesium stearate or Sterotex; glidants
such as colloidal silicon dioxide; sweetening agents such as
sucrose or saccharin, a flavoring agent such as peppermint, methyl
salicylate or orange flavoring, or coloring agents. Further
examples of carriers include polyethylene glycol, cyclodextrin,
oils, or any other similar liquid carrier that may be formulated
into a capsule. Still further examples of carriers include sterile
diluents such as water for injection, saline solution,
physiological saline, Ringer's solution, isotonic sodium chloride,
fixed oils such as synthetic mono or digylcerides, polyethylene
glycols, glycerin, cyclodextrin, propylene glycol or other
solvents; antibacterial agents such as benzyl alcohol or methyl
paraben; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid; buffers
such as acetates, citrates or phosphates and agents for the
adjustment of tonicity such as sodium chloride or dextrose,
thickening agents, lubricating agents, and coloring agents.
[0106] The pharmaceutical composition may take any of a number of
formulations depending on the physicochemical form of the
composition and the type of administration. Such forms include
solutions, suspensions, emulsions, tablets, pills, pellets,
capsules, capsules including liquids, powders, sustained-release
formulations, directed release formulations, lyophylates,
suppositories, emulsions, aerosols, sprays, granules, powders,
syrups, elixirs, or any other formulation now known or yet to be
disclosed. Additional examples of suitable pharmaceutical carriers
and formulations are well known in the art.
[0107] Methods of administration include, but are not limited to,
oral administration and parenteral administration. Parenteral
administration includes, but is not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, sublingual, intramsal, intracerebral,
iratraventricular, intrathecal, intravaginal, transdermal, rectal,
by inhalation, or topically to the ears, nose, eyes, or skin. Other
methods of administration include but are not limited to infusion
techniques including infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings such as oral mucosa,
rectal and intestinal mucosa. Compositions for parenteral
administration may be enclosed in ampoule, a disposable syringe or
a multiple-dose vial made of glass, plastic or other material.
[0108] Administration may be systemic or local. Local
administration is administration of the disclosed compound to the
area in need of treatment. Examples include local infusion during
surgery; topical application, by local injection; by a catheter; by
a suppository; or by an implant. Administration may be by direct
injection into the central nervous system by any suitable route,
including intraventricular and intrathecal injection.
Intraventricular injection can be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. Pulmonary administration may be
achieved by any of a number of methods known in the art. Examples
include the use of an inhaler or nebulizer, formulation with an
aerosolizing agent, or via perfusion in a fluorocarbon or synthetic
pulmonary surfactant. Compounds may be delivered in the context of
a vesicle such as a liposome or any other natural or synthetic
vesicle. Additional examples of suitable modes of administration
are well known in the art.
[0109] A pharmaceutical composition formulated to be administered
by injection may be prepared by dissolving the disclosed compound
with water so as to form a solution. In addition, a surfactant may
be added to facilitate the formation of a homogeneous solution or
suspension.
[0110] Surfactants include any complex capable of non-covalent
interaction with the disclosed compound so as to facilitate
dissolution or homogeneous suspension of the compound.
[0111] Pharmaceutical compositions may be prepared in a form that
facilitates topical or transdermal administration. Such
preparations may be in the form of a solution, emulsion, ointment,
gel base, transdermal patch or iontophoresis device. Examples of
bases used in such compositions include opetrolatum, lanolin,
polyethylene glycols, beeswax, mineral oil, diluents such as water
and alcohol, and emulsifiers and stabilizers, thickening agents, or
any other suitable base now known or yet to be disclosed.
[0112] Determination of an effective amount of the disclosed
compound is within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein. The
effective amount of a pharmaceutical composition used to affect a
particular purpose as well as its toxicity, excretion, and overall
tolerance may be determined in vitro, or in vivo, by pharmaceutical
and toxicological procedures either known now by those skilled in
the art or by any similar method yet to be disclosed. One example
is the in vitro determination of the IC.sub.50 (half maximal
inhibitory concentration) of the pharmaceutical composition in cell
lines or target molecules. Another example is the in vivo
determination of the LD.sub.50 (lethal dose causing death in 50% of
the tested animals) of the pharmaceutical composition. The exact
techniques used in determining an effective amount will depend on
factors such as the type and physical/chemical properties of the
pharmaceutical composition, the property being tested, and whether
the test is to be performed in vitro or in vivo. The determination
of an effective amount of a pharmaceutical composition will be well
known to one of skill in the art who will use data obtained from
any tests in making that determination. Determination of an
effective amount of disclosed compound for administration also
includes the determination of an effective therapeutic amount and a
pharmaceutically acceptable dose, including the formulation of an
effective dose range for use in vivo, including in humans.
[0113] Treatment of a condition or disease is the practice of any
method, process, or procedure with the intent of halting,
inhibiting, slowing or reversing the progression of a disease,
disorder or condition, substantially ameliorating clinical symptoms
of a disease disorder or condition, or substantially preventing the
appearance of clinical symptoms of a disease, disorder or
condition, up to and including returning the diseased entity to its
condition prior to the development of the disease. Generally, the
effectiveness of treatment is determined by comparing treated
groups with non-treated groups.
[0114] The addition of a therapeutically effective amount of a
compound encompasses any method of dosing of a compound. Dosing of
the disclosed compounds may include single or multiple
administrations of any of a number of pharmaceutical compositions
that include the disclosed compound as an active ingredient.
Examples include a single administration of a slow release
composition, a course of treatment involving several treatments on
a regular or irregular basis, multiple administrations for a period
of time until a diminution of the disease state is achieved,
preventative treatments applied prior to the instigation of
symptoms, or any other dosing regimen known in the art or yet to be
disclosed that one skilled in the art would recognize as a
potentially effective regimen. A dosing regimen including the
regularity of and mode of administration will be dependent on any
of a number of factors including but not limited to the subject
being treated; the severity of the condition; the manner of
administration, the stage of disease development, the presence of
one or more other conditions such as pregnancy, infancy, or the
presence of one or more additional diseases; or any other factor
now known or yet to be disclosed that affects the choice of the
mode of administration, the dose to be administered and the time
period over which the dose is administered.
[0115] "Therapeutically effective amount" means that amount of a
compound, material, or composition of the present invention, which
is effective for producing a desired therapeutic effect, at a
reasonable benefit/risk ratio applicable to any medical treatment.
For example, a "therapeutically effective amount" is an amount
effective to reduce or lessen at least one symptom of the disease
or condition being treated or to reduce or delay onset of one or
more clinical markers or symptoms associated with the disease or
condition, or to modify or reverse the disease process.
[0116] "Treatment" or "treating" when referring to a disease or
condition, means producing a desired therapeutic effect. Exemplary
therapeutic effects include delaying onset or reducing at least one
symptom associated with the disease, positively affecting (e.g.,
reducing or delaying onset) of a clinical marker associated with
the disease and slowing or reversing disease progression.
[0117] "Pharmaceutically acceptable" refers to those properties
and/or substances that are acceptable to a patient (e.g., human
patient) from a toxicological and/or safety point of view.
[0118] Pharmaceutical compositions may be administered prior to,
concurrently with, or after administration of additional or second
pharmaceutical compositions. Concurrent administration means
compositions are administered within about one minute of each
other. If not administered concurrently, the additional or second
pharmaceutical compositions may be administered a period of one or
more minutes, hours, days, weeks, or months before or after the
pharmaceutical composition that includes the currently disclosed
compound. Alternatively, a combination of pharmaceutical
compositions may be cyclically administered. Cycling therapy
involves the administration of one or more pharmaceutical
compositions for a period of time, followed by the administration
of one or more different pharmaceutical compositions for a period
of time and repeating this sequential administration. Cycling
therapy may be used, for example, to reduce the development of
resistance to one or more of the compositions, to avoid or reduce
the side effects of one or more of the compositions, and/or to
improve the efficacy of the treatment.
[0119] "Polo-like Kinase 1 Inhibitor" or "PLK1 inhibitor" refers to
a compound or pharmaceutical composition that is capable of
inhibiting the expression and/or the activity of the polo-like
kinase 1 protein. For example, administration of a PLK1 inhibitor
may partially or completely interrupt the physical, chemical,
electrostatic or any other interactions between PLK1 protein and
MDM2 and/or p53 protein. Some non-limiting examples PLK1 inhibitors
include BI-2536, cyclapolin 9, GW 843682X, TC-S 7005, Wortmannin,
NMS-P937, GSK461364A, any compounds disclosed in any of the
following patent references, U.S. Pat. No. 7,517,873, U.S. Pat. No.
7,977,336, U.S. Pat. No. 8,318,727, or any combination thereof. For
example, a PLK1 inhibitor can treat one or more symptoms or causes
of PLK1-mediated conditions, such as adrenocortical carcinoma. In
some aspects of the invention, the PLK1 inhibitor can be
administered with one or more other pharmaceutical compounds. In
some embodiments, a PLK1 inhibitor can comprise a composition that
augments expression of the PLK1 protein, such as the addition of
small interfering RNAs ("siRNAs").
[0120] "MDM2 inhibitor" refers to a compound or pharmaceutical
composition that is capable of inhibiting the expression and/or the
activity of the MDM2 protein. For example, an MDM2 inhibitor may
comprise a compound or composition that is capable of partially or
completely interrupting the physical, chemical, electrostatic or
any other interactions between MDM2 protein and p53 protein. Some
non-limiting examples of MDM2 inhibitors include any nutlin (e.g.,
nutlin-3), caylin-1, HLI 373, caylin-2, JNJ 26854165, NSC 66811,
trans-4-lodo. 4'-boranyl-chalcone, any compounds disclosed in any
of the following patent references, U.S. Pat. No. 8,569,341, U.S.
Pat. No. 7,851,626, U.S. Patent Publication No. 2011/0251252, or
any combination thereof.
[0121] In some aspects of the invention, the MDM2 inhibitor can be
administered with one or more other pharmaceutical compounds (e.g.,
a PLK2 inhibitor). For example, the MDM2 and PLK1 inhibitors can be
administered in a single dose/pharmaceutical composition to the
subject receiving the treatment. In other aspects, the MDM2 and
PLK1 inhibitors can be administered in two separate
doses/pharmaceutical compositions that are provided to the subject
at the same time, sequentially, or at regular or irregular
intervals. In a preferred embodiment of the invention, the MDM2 and
PLK1 inhibitors are administered together, either simultaneously or
sequentially.
[0122] The present invention provides a kit to determine the levels
of marker expression in the sample. Such a kit will comprise a
reagent for detecting the mRNA encoding a marker, the corresponding
polypeptide, or any combination or fragment thereof. The reagent
will comprise one or more molecules capable of specifically binding
a nucleic acid sequence (DNA or RNA) encoding a gene, or the
corresponding polypeptide.
[0123] "Mitotic catastrophe inducing composition" or "mitotic
catastrophe inducing compound" refers to one or more pharmaceutical
compositions that are able to trigger mitotic catastrophe. For
example, mitotic catastrophe inducing compounds may be any
composition that is able to activate an initiator caspase (e.g.,
caspase 2) to trigger the caspase cascade to trigger apoptosis via
mitotic catastrophe. In some embodiments, a mitotic catastrophe
inducing compound does not activate or cause to be activated
caspases 8 and 9 and does activate or cause to be activated caspase
2. In a preferred embodiment, a mitotic catastrophe inducing
composition or compound is any active ingredient that can activate
or cause to be activated caspase 2. Some non-limiting examples of a
mitotic catastrophe inducing compound in include PLK1 inhibitors,
MDM2 inhibitors, docetaxel, paclitaxel, moscatilin, S23806,
radiation (e.g., ionizing radiation), subamolide A, doxorubicin,
nocodazole, or any combination thereof.
[0124] The kit may comprise one or more nucleic acid reagents for
the detection of mRNA encoding a gene (either sense or antisense).
The one or more nucleic acid reagents may be used for hybridization
and/or amplification of the mRNA encoding the gene. The kit may
comprise one or more pairs of primers for amplifying the mRNA
encoding the gene. The kit may further comprise samples of total
mRNA derived from tissue of various physiological states, such as
normal/non-diseased, and diseased (e.g., from a subject with
cancer), for example, to be used as controls. The kit may also
comprise buffers, nucleotide bases, and other compositions to be
used in hybridization and/or amplification reactions. Each solution
or composition may be contained in a vial or bottle and all vials
held in close confinement in a box for commercial sale. Another
embodiment of the present invention encompasses a kit for use in
detecting mRNA encoding a gene in a biological sample comprising
oligonucleotide probes effective to bind with elevated affinity to
mRNA encoding the gene in vitro or in situ and containers for each
of these probes.
[0125] In a further embodiment, the invention encompasses a kit for
use in determining the level of marker expression in a biological
sample comprising one or more agents, such as, for example, one or
more antibodies, specific for one or more polypeptides or
fragments. In one particular embodiment, the kit will comprise one
or more agents and one or more nucleic acid markers wherein the
agents and nucleic acid markers are modified in a fashion
appropriate for carrying out immuno-polymerase chain reaction
assays.
[0126] One preferred embodiment of the invention is directed to a
kit for determining the levels of gene expression in a mammalian
biological sample, wherein said levels of marker expression is an
indicator of the prognosis or diagnosis of cancer, said kit
comprising: a) an antibody that specifically binds to a gene
product or an antigen binding fragment thereof, b) a reagent useful
for detecting the extent of interaction between said antibody and
the marker; c) a reagent or solution useful for antigen retrieval;
and c) positive and/or negative control samples. Said antibody may
be directly linked to an indicator reagent, wherein said indicator
reagent is selected from the group consisting of fluorescent,
colorimetric, immunoperoxidase and isotopic reagents.
Alternatively, the kit may further include a second indicator
antibody linked to an indicator reagent, wherein said indicator
reagent is selected from the group consisting of fluorescent,
calorimetric, immunoperoxidase and isotopic reagents.
[0127] In one embodiment, the kit contains at least one primary
antibody, at least one labeled secondary antibody, and at least one
substrate (e.g., TMB). Alternatively, the kits can contain
radiolabeled secondary antibody in place of the secondary antibody
labeled with an enzyme. The kits may also contain disposable
supplies for carrying out detection assays (e.g., microtiter
plates, pipettes).
EXAMPLES
Materials and Methods
[0128] Clinical Samples
[0129] Three ACC specimens were collected as part of an ongoing
Adrenocortical Carcinoma Biorespository. The study was conducted
under Western Institutional Review Board (WIRB) approved protocol
#20051769 with written consent. Tumor samples were flash frozen
within 30 minutes of collection and stored at -80.degree. C.
Peripheral blood was collected by venipuncture in sodium heparin
vacutaners and kept at -80.degree. C. until DNA extraction.
[0130] Next Generation Sequencing:
[0131] Genomic and exome (tumor and normal), and whole
transcriptome (tumor only) libraries were generated and paired-end
sequenced on the Illumina HiSeq2000. A pool of four normal adrenal
glands from different individuals was RNA sequenced as a
control.
[0132] Whole Genome Library Preparation
[0133] Genomic DNA samples were fragmented on the Covaris E210
96-well high-throughput sonicator to a target size of 1000 bp.
Libraries were prepared and indexed using Illumina's TruSeq DNA
Sample Preparation Kits. Ligation products 1100-1200 bp were size
selected on a 2% TAE gel, purified, and enriched and amplified with
10 cycles of PCR using the TruSeq kit's PCR Master Mix and primer
cocktail. Final libraries were quantified using the Agilent
Bioanalyzer and Qubit and insert sizes verified using the
Bioanalyzer.
[0134] Exome Library Preparation
[0135] 1.1 .mu.g of genomic DNA was used to prepare each library
using the Illumina TruSeq DNA Sample Prep Kit and TruSeq Exome
Enrichment Kit. Samples were fragmented to a target size of 200-300
bp using sonication and fragmentation was verified on a 2% TAE gel.
Samples were then end repaired, purified, and adenylated. Adapter
indexes were ligated onto adenylated molecules, purified, and
amplified using PCR. 500 ng of each sample was combined to create a
pool for exome capture. The pooled samples underwent a 24-hour
hybridization, and were then washed and targets were eluted. This
step was repeated for a total of two hybridizations. Final eluted
targets were PCR amplified and evaluated using the Agilent
Bioanalyzer.
[0136] RNAseq Library Preparation
[0137] 10 ng of total RNA was used to generate whole transcriptome
libraries for RNA sequencing. Using the Nugen Ovation RNA-Seq
System v2, total RNA was used to generate double stranded cDNA,
which was subsequently amplified using Nugen's SPIA linear
amplification process. Amplified products were cleaned using
Qiagen's QIAquick PCR Purification Kit and quantitated using
Invitrogen's Quant-iT Picogreen. 1 .mu.g of amplified cDNA was
fragmented on the Covaris E210 to a target size of 300 bp.
Illumina's TruSeq DNA Sample Preparation Kit was used for preparing
libraries from amplified cDNA. 1 .mu.g of amplified cDNA was
fragmented to a target insert size of 300 bp, end repaired, and
cleaned using Ampure XP beads. Samples were then adenylated at the
3' end and indexed paired end adapters with T overhangs are ligated
onto the A-tailed inserts. Ligation products were run on a 2% TAE
gel and size selected at 450 bp. Ligation products were isolated
from gel punches using Bio-Rad Freeze 'n Squeeze columns and Ampure
XP beads. Cleaned ligation products were then PCR amplified and
cleaned using Ampure XP beads, and quantified using the Agilent
Bioanalyzer and Qubit.
[0138] Paired End Sequencing
[0139] Libraries with a 1% phiX spike-in were used to generate
clusters on HiSeq Paired End v3 flowcells on the Illumina cBot
using Illumina's TruSeq PE Cluster Kit v3. Clustered flowcells were
sequenced by synthesis on the Illumina HiSeq 2000 using paired-end
technology and Illumina's 200 cycle TruSeq SBS Kit, extending to
104 bp for each of two reads for whole genome libraries and 75 bp
83 bp for each of two reads for exome and RNAseq libraries.
[0140] Bioinformatic Analyses:
[0141] Converted FASTQ files were aligned with BWA (Burrows-Wheeler
Aligner) against Grch 37.62 and processed to produce BAM files
without duplicate reads and re-aligned to facilitate tumor-normal
comparisons. Somatic variant (SNV) calls were made with Seurat and
annotated with snpEff. Copy number analysis of copy number variants
(CNVs) was completed by determining the log 2 difference of the
normalized physical coverage for germline and tumor samples across
a sliding 2 kb window of the mean. Translocations were called with
both svdetect and breakdancer. TopHat, Cufflinks, and Cuffdiff
(Center for Bioinformatics and Computational Biology, University of
Maryland) were used to align RNA reads and compute differential
gene expression. Pathway enrichment was evaluated in MetaCore
(Thompson-Reuters).
[0142] Tissue Culture
[0143] ACC cell lines SW-13 and H295R were obtained from ATCC and
authenticated by STR analysis on Aug. 24, 2012. BD140 lines (A-C)
represent distinct subclones established from the tumor of ACC140.
BD167 was also established. They are positive for DHEAS expression,
confirming adrenocortical origin. SW-13 and the BD140-B were grown
in DMEM supplemented with 10% FBS, 1% L-glutamine, 1%
inulin-transferin-selenium (ITS), and penicillin-streptomycin.
BD140-A, BD140-B and BD167 were grown in RPMI supplemented with 10%
FBS, 1% L-glutamine, 1% ITS, and penicillin-streptomycin. H295R was
grown in DMEM:F12 supplemented with 2.5% NuSerum, 1% L-glutamine,
1% ITS, and penicillin-streptomycin. All cells lines were grown at
37.degree. C. in a 5% CO.sub.2 atmosphere.
[0144] Caspase 3/7 Glo Assay for Apoptosis
[0145] ACC cell lines were plated and 24 hours later, 0.5 .mu.M, 1
.mu.M, 2.5 .mu.M, and 5 .mu.M of doxorubicin was added to the
cultures. The activity of caspases 3 and 7 was assessed using
Caspase 3/7 Glo (Promega, Madison, Wis.) at 8, 24, and 48 hours
after drug addition according to manufacturer's instructions. Data
was normalized to cells alone, then to vehicle alone and expressed
as a percentage of vehicle alone.
[0146] Induction of apoptosis after treatment with BI-2536 and
nutlin-3 alone or together in combination was determined using the
Caspase 3/7 Glo assay (Promega, Madison, Wis.). H295R cells were
plated at a density of 1750 cells/well in 40 .mu.l of medium and
SW-13 was plated at 1250 cells/wells in 40 .mu.l DMEM with 2% FBS.
24 hours after plating, the cells were treated with 100 .mu.M, 33.3
.mu.M, 11.1 .mu.M, 3.703 .mu.M and 1.234 .mu.M of BI-2536 and
nutlin-3 in a 10 .mu.l volume. Induction of apoptosis was
determined by the cleavage of caspase 3/7 using the Caspase 3/7 Glo
assay (Promega, Madison, Wis.) at 8, 16, 24 and 48 hours after
compound addition. Percent caspase 3/7 activity was normalized to
cells and to DMSO control. Induction of apoptosis with double
compounds was determined as above except IC.sub.25 concentrations
(0.006841M for SW-13 and 0.0374 .mu.M for H295R) of BI-2536 were
kept constant and nutlin-3 was evaluated in serial dilution. 51M
Doxorubicin (Tocris, Minneapolis, Minn.) was used as a positive
control for induction of apoptosis. In those cases, percent caspase
3/7 activity was also normalization to the median caspase 3/7
activity of BI-2536 alone.
[0147] In Vitro Drug Dose-Response Curves
[0148] Sensitivity to BI-2536 and nutlin-3 (Tocris, Minneapolis,
Minn.), was tested as follows. H295R cells were plated at a density
of 1750 cells/well in 40 .mu.l of medium and SW-13 cells were
plated at 1250 cells/wells in 40 .mu.l DMEM with 2% FBS in white
384-well plates and allowed to attach for 24 hours. The cells were
dosed with 3-fold dilutions of BI-2536 and nutlin-3 in 10 .mu.l of
medium and cell viability was assessed after 96 hours for SW-13 and
120 hours for H295R cells by CellTiter Glo (Promega, Madison,
Wis.). Cell viability was normalized to cells alone and DMSO
controls. Double compound studies were conducted as above except
IC.sub.25 concentrations (0.00684 .mu.M for SW-13 and 0.0374 .mu.M
for H295R) of BI-2536 were kept constant and nutlin-3 was evaluated
in a serial dilution, where, cell viability was normalized to
cells, to DMSO controls and then to the median viability of BI-2536
alone.
[0149] Dose response curves and IC.sub.50 values for cell survival
in the presence of the drugs were calculated using Prism software
(GraphPad) using the log(inhibitor) vs. response--4 parameter
function which fits the following equation:
Y=Bottom+(Top-Bottom)/[1+10 (X-Log IC.sub.50)] where X is the
logarithm of concentration and Y is the percent cell survival. Y
starts at the top and goes to bottom with a sigmoid shape as X
increases. All experiments were done at least three technical
replicates and values are represented as averages with standard
error.
[0150] siRNA Knockdown of PLK-1
[0151] 2.times.10.sup.5 SW-13 and 3.5.times.10.sup.5 H295R cells
were plated in 6-well plates in their respective media without
antibiotics and allowed to attach overnight. The next day, for
SW-13, Lipofectamine2000 reagent (Invitrogen, Carlsbad, Calif.) and
for H295R TransIT-siQUEST reagent (Mirus Bio, Madison, Wis.) was
used to transfect in 20 nM PLK-1 siRNA (Qiagen, Valencia, Calif.),
an all-stars negative siRNA (Qiagen, Valencia, Calif.) as a
negative control, and a universally lethal positive-control siRNA
directed against ubiquitin B (UBBs1) (Qiagen, Valencia, Calif.)
using the manufacturer's recommended protocols respectively. For
transfection of SW-13 cells, 5 .mu.l of Lipofectamine2000 and 20 nM
PLK-1, negative-control, or UBBs1 siRNA were mixed together in
equal volumes, in serum free media (SFM) and allowed to incubate at
room temperature for 30 minutes. All the media from the SW-13 wells
was aspirated off and 500 .mu.l of the siRNA--Lipofectamine2000 mix
was added to each well along with 1.5 ml of media without
antibiotics. Cells were assayed for PLK-1 protein expression 72
hours after transfection. For transfection of H295R cells, 4 .mu.l
of TransIT-siQUEST reagent was diluted in SFM. 20 nM PLK-1,
negative-control, or UBBs1 siRNA was added to the diluted
transfection reagent and allowed to incubate at room temperature
for 20 minutes. 250 .mu.l of the siRNA--TransIT-siQUEST mix was
added to each well containing 1.25 ml of media without antibiotics.
Cells were assayed for PLK-1 protein expression by western blot 72
hours after transfection.
[0152] To determine cell viability after PLK-1 transfection, SW-13
cells were reverse transfected with siRNA to PLK-1 or control siRNA
and assayed for viability after 96 hours. Briefly, 384 well plates
were printed with 20 nM of Hs_PLK-1.sub.--7 siRNA, UBBs1, negative
control siRNAs, including a non-silencing scrambled siRNA or a
siRNA directed against green fluorescent protein (GFP). A total of
20 .mu.l of diluted Lipofectamine2000 solution was added to each
well. After 30 minutes, 1200 cells for SW-13 in 20 .mu.l of medium
were added per well and then cultured at 37.degree. C. After 96
hours, viability was assessed by CellTiter Glo following the
manufacturer's protocol. Relative luminescence values were
normalized to cells and to cells with transfection agent to get
normalized percent viability.
[0153] To determine viability of H295R cells after PLK-1
transfection, H295R were transfected with PLK-1, negative-control
or UBBs1 siRNA and assayed for viability after 72 hours. Briefly,
20,000 H295R cells were plated in 80 .mu.l in 96 well plates and
allowed to attach overnight. The next day 20 .mu.l of
TransIT-siQUEST reagent and 20 nM PLK-1, negative-control or UBBs1
siRNA in SFM were added to each well containing 80 .mu.l of media
without antibiotics. Cells were assayed for viability using
CellTitre Glo 72 hours after transfection as per the manufacturer's
protocol. Relative luminescence values were normalized to cells
alone and then cells with transfection agent alone to get
normalized percent viability.
[0154] Total RNA Extraction
[0155] 6.times.10.sup.5 SW-13 and 7.5.times.10.sup.5 H295R cells
were plated in 10 cm.sup.2 dishes in 10 ml of media. Cells were
allowed to adhere for 24 hours and were then treated with their
respective BI-2536 IC.sub.10, IC.sub.25 and IC.sub.50 doses
including DMSO controls. Total RNA was extracted 24 hours later
using the mirVana miRNA Isolation Kit (Ambion, Inc, Grand Island,
N.Y.) as per the manufacturer's protocol.
[0156] RT-qPCR Validation of p53 and p21 mRNA Levels
[0157] Total RNA was reverse transcribed utilizing both random
hexamer and oligo-dT primers and the RT.sup.2 First Strand cDNA
Synthesis Kit (SABiosciences, Valencia, Calif.). The resulting cDNA
was amplified on the iQ5 Real-Time PCR Detection System (Bio-Rad
Laboratories, Inc, Hercules, Calif.) using primer sets for TP53
(p53), CDKN1A (p21) and ACTB (.beta.-actin) and RT.sup.2 SYBR Green
Master Mix (all from SABiosciences, Valencia, Calif.) and run
according to manufacturer's instructions. Melting curve analysis
was performed to evaluate primer set specificity. Fold difference
relative to 1-actin, which was used as the reference gene, was
calculated using the Pfaffl method taking into account reaction
efficiencies.
[0158] Expression of Caspases Using RT-qPCR
[0159] Total RNA from tumors was extracted using the mirVana miRNA
Isolation Kit (Ambion Inc., Grand Island, N.Y.) as per the
manufacturer's protocol. 1 .mu.g of total RNA was used to
synthesize cDNA with oligo-dT and random hexamer primers from the
RT2 First Strand cDNA Synthesis Kit (SABiosciences, Valencia,
Calif.). RT-qPCR reactions were carried out with 1 .mu.l of cDNA
mix and 1 .mu.l of primer sets for Caspases 1 (PPH00105C), 2
(PPH0011A), 3 (PPH00107C), 4 (PPH00366F), 6 (PPH00109B), 7
(PPH00110C), 8 (PPH00359F), 9 (PPH353B), 10 (PPH00106F) and
.beta.-Actin (PPH00073G) with the RT2 SYBR Green Master Mix (all
from SABiosciences, Valencia, Calif.) as per the recommended
manufacturer's protocol. A melting curve analysis was also
performed to evaluate specificity of the primer sets. Fold change
in the expression of the caspases was calculated using the Pfaffl
method (Pfaffl MW, A new mathematical model for relative
quantification in real-time RT-PCR Nucleic Acids Res. (29) e45
(2001)) using calculated primer efficiencies and using .beta.-Actin
as the reference gene.
[0160] Western Blot Analysis
[0161] 6.times.10.sup.5 SW-13 and 7.5.times.10.sup.5 H295R cells
were plated in 10 cm.sup.2 dishes in 10 ml of media. Cells were
allowed to attach for 24 hours after which they were treated with
IC.sub.10, IC.sub.25, IC.sub.50 concentrations of BI-2536 and DMSO
vehicle control for 24 hours. Cells were lysed with RIPA buffer
with protease and phosphatase inhibitors, and the resulting protein
lysate quantitated by BCA (Pierce, Thermo Scientific, Pittsburgh,
Pa.). Thirty micrograms of protein were loaded onto 4-12% Bis-Tris
pre-cast gels (Invitrogen, Carlsbad, Calif.) and allowed to
separate at 150V for 1 hour. The gels were then transferred onto
PVDF membranes for 7 minutes using the iBLOT western transfer
system (Invitrogen, Carlsbad, Calif.) at room temperature.
Following the transfer of proteins, the membranes were blocked in
5% blocking solution made from, non-fat dry milk dissolved in
1.times.TBST (50 mM Tris.HCl, pH 7.4, 150 mM NaCl+0.1% Tween 20)
for 1 hour. Primary PLK-1 antibody (Cell Signaling, Technology,
Danvers, Mass.) at a dilution of 1:500 was added to membranes in 5%
BSA (1.times.TBST+5% BSA) overnight at 4.degree. C. Primary
antibodies to MDM2 (AbCam, Cambridge, Mass.) and p53 (AbCam,
Cambridge, Mass.) at a dilution of 1:500 were added to the
membranes in 1% blocking solution overnight at 4.degree. C. The
next day the membranes were washed with 1.times.TBST twice for 10
minutes and appropriate HRP labeled secondary anti-rabbit (Cell
Signaling Technology, Danvers, Mass.) for PLK-1 and anti-mouse
(Cell Signaling Technology, Danvers, Mass.) for MDM2 and p53
antibodies were added to the blots at a dilution of 1:1000 for 2
hours at room temperature. The blots were then washed in
1.times.TBST four times for 10 minutes. The membranes were
developed using the SuperSignal West Femto Chemiluminescent
Substrate (Pierce, Thermo Scientific, Pittsburgh, Pa.) and were
visualized and quantitated with the Bio Spectrum 500 Imaging System
(UVP, Cambridge, UK).
[0162] The blots were processed as described above for the
detection of .beta.-Actin (AbCam, Cambridge, Mass.) in 5% blocking
solution which was used as an internal loading control. The
.beta.-Actin antibody was used at a dilution of 1:1000 along with
the anti-rabbit secondary antibody also at a dilution of 1:1000.
The actin membranes were also detected using the SuperSignal West
Femto Chemiluminescent Substrate (Pierce, Thermo Scientific,
Pittsburgh, Pa.) and were visualized and quantitated with the Bio
Spectrum 500 Imaging System (UVP, Cambridge, UK) and relative
amounts of PLK-1, MDM2 and p53 protein are reported relative to
.beta.-Actin. All experiments were done in three technical
replicates and are represented as averages with standard error.
[0163] To determine PLK-1 protein expression after siRNA knockdown,
blots for PLK-1 and .beta.-Actin were processed as described above.
Relative amounts of the PLK-1 protein after siRNA knockdown are
reported relative to .beta.-Actin. All experiments were done in
three technical replicates and are represented as averages with
standard error.
[0164] Statistical Analysis
[0165] Statistical assessment of caspase expression and apoptosis
was done in GraphPad Prism 6 (GraphPad Software, Inc., La Jolla,
Calif.) using 2-way ANOVA followed by Sidak's multiple comparisons
test (RT-qPCR) or Dunnett's multiple comparison test (apoptosis).
p-values below 0.05 were considered to be significant.
Results
Example 1
[0166] Patient clinical information is listed in Table 1. These
tumors were characterized by the presence of shared genomic
alterations as well as tumor specific events that converged on
common pathways.
TABLE-US-00001 TABLE 1 Clinical Description of ACC Patient and
Tumor Characteristics Tumor Age Survival Tumor Tumor Weiss Hormone
Sample* (years) (years) Stage Grade Score Status ACC 129 55 0.583 2
4 4 cortisol, DHEAS ACC 132 51 6 Re- 2 NA non- currence functional
ACC 140 63 2.08 4 4 4 non- functional
Example 2
Differential Caspase Expression in ACC Samples
[0167] Differential gene expression was evaluated relative to a
pool of 4 normal adrenals through pathway enrichment in GeneGo and
analyzed the involvement of pathways involved in ACC pathogenesis,
including p53 responses and IGF2 signaling. GeneGo analysis
identified commonly affected pathways, including loss of caspase 9
(CASP9) expression.
[0168] In ACC 129, IGF2 signaling appeared to be directed towards
suppression of apoptosis through 14-3-3. In ACC 132 and ACC 140,
signaling was repressed, and instead, ACC 132 and ACC 140 have
evidence of MET amplification and signaling. All tumors also
under-expressed beta-catenin. p53 transcriptional response was
disrupted in all tumors, with the two wild-type p53 tumors (ACC 129
and ACC132) over-expressing MDM2.
[0169] Because sporadic adult ACC is usually wild-type for p53 yet
highly resistant to chemotherapy, cell death response pathways were
studied, including the caspase cascade, which is central to both
apoptosis and necroptosis. It was observed that all three tumors
had a reduction in CASP9 expression. Each tumor harbored additional
alterations in caspase signaling, most commonly resulting in loss
of initiator caspase expression, as illustrated in FIG. 1.
Example 3
Confirmation of Differential Caspase Expression Via Quantitative
RT-PCR
[0170] As shown in FIG. 2, the expression of multiple caspases was
altered, with CASP9 being significantly under-expressed in all
three tumors and in the ACC cell lines H295R and SW-13. The primary
tumors (panel A) and the cell lines, H295R, and SW-13 (panel B),
show increased expression of caspase 2, with over-expression being
statistically significant in ACC140 and H295R. Additionally, two of
the tumors over-express one or more of the effector caspases.
Example 4
Reduced Caspase Expression Correlates with Reduced Apoptotic
Response
[0171] A time series was used for assaying caspase 3/7 activation
following doxorubicin exposure using clinically relevant
concentrations. FIG. 3 shows that apoptosis is delayed in the H295R
cell line (panel B). There appears to be no impact on SW-13
response, with a significant induction within 8 hours (panel A).
The BD140 lines (three different clones derived from ACC140) have
different responses, but fall between H295R and SW-13, with
moderate induction of caspase 3/7 activity only seen at 2.5 .mu.M
and 5 .mu.M concentrations by 24 hours. At the doses of doxorubicin
commonly used to assess caspase activation (0.5 .mu.M and 1 .mu.M),
induction of caspase 3/7 activity was not seen until 48 hours in
the BD140 lines.
Example 5
Inhibition of Polo-Like Kinase 1 (PLK-1) Reduces the Viability of
ACC Cell Lines
[0172] Examination of previous gene expression data (Demeure, M. J.
et al., PTTG1 overexpression in adrenocortical cancer is associated
with poor survival and represents a potential therapeutic target
Surgery (154) 1405-1416 (2013)) showed that PLK1 was over-expressed
in a subset of tumors with a mean fold-change relative to normal
adrenal of 1.4. Since increased PLK-1 expression has been
correlated to poor prognosis and aggressiveness of cancers, the
effect of down-regulating PLK-1 on the ACC cell lines was
investigated. Knocking down expression of PLK-1 using siRNA not
only reduced the amount of PLK-1 protein (FIG. 4, panel A and B),
but also decreased the viability of both H295R and SW-13 cell lines
to levels close to that of the positive control, UBB1, after siRNA
knockdown (FIG. 4, panel C), which indicates that the ACC cell
lines were sensitive to the loss of PLK-1.
[0173] Exposure to the PLK-1 inhibitor, BI-2536, showed that both
H295R and SW-13 cell lines were also sensitive to PLK-1 inhibition
(FIG. 4, panel D and E). At 96 hours following addition of drug,
H295R had an IC.sub.50 value of 0.063 .mu.M and SW-13 had with an
IC.sub.50 value of 0.0095 .mu.M (FIG. 4, panel D and E, Table 2).
These values are below the concentrations achieved clinically
(31.2-55.2 .mu.M) following a single intravenous injection of 50-70
mg of BI-2536. Frost, A. et al., Phase i study of the Plk1
inhibitor BI 2536 administered intravenously on three consecutive
days in advanced solid tumors Curr. Oncol. (19) 28-35 (2012).
TABLE-US-00002 TABLE 2 BI-2536 inhibitory concentrations BI-2536
(.mu.M) Cell line IC.sub.10 IC.sub.25 IC.sub.50 H295R 0.00565
0.00684 0.0095 SW-13 0.0222 0.0374 0.0628
Example 6
Inhibition of PLK-1 Reduces Levels of Mutant p53 Protein but not
Wild Type p53
[0174] PLK-1 is a negative modulator of p53 activity but does not
affect its expression levels. Ando, K. et al., Polo-like kinase 1
(Plk1) inhibits p53 function by physical interaction and
phosphorylation J. Biol. Chem. (279) 25549-25561 (2004). p53
protein levels were examined by immunoblot using an antibody that
recognizes both mutant and wild-type p53 isoforms. A dose-dependent
decrease of mutant p53 protein levels was observed in SW-13 cells
but not in the wild-type p53 protein levels in H295R cells (FIG. 5,
panels A-C). Treatment with BI-2536 did not significantly affect
the levels of p53 transcript in either of the two cell lines (FIG.
5, panel D).
Example 7
Inhibition of PLK-1 Restores Functioning of Wild Type 53
[0175] Inhibition of PLK-1 with BI-2536 resulted in a general trend
of increased transcription of CDKN1A, a gene that encodes the p21
protein, in H295R cells with wild type p53 (FIG. 6, panel A). A
decrease in mutant p53 protein in SW-13 cells after exposure to
BI-2536 also reduced the amount of CDNK1A message (FIG. 6, panel
B). Treatment with BI-2536 resulted in a robust induction of
apoptosis after treatment with BI-2536 in H295R cells (FIG. 6,
panel C) and in SW-13 cells with mutant p53 (FIG. 6, panel D).
Maximum induction of apoptosis in SW-13 cells was observed at 48
hrs, while only high concentrations of BI-2536 resulted in an
increase in apoptosis at early time points (FIG. 6, panels C and
D).
Example 8
Synergy of PLK-1 Inhibition by BI-2536 with MDM2 Inhibition by
Nutlin-3
[0176] Although both PLK-1 and MDM2 regulate p53 activity
independently, these two molecules also cooperate to modify p53
function. PLK-1 phosphorylates MDM2 at serine 260, stimulating the
activity of MDM2 and increasing the turnover of p53. Dias, S. S. et
al., Polo-like kinase-1 phosphorylates MDM2 at Ser260 and
stimulates MDM2-mediated p53 turnover FEBS Lett. (583) 3543-3548
(2009). Inhibition of PLK-1 by BI-2536 does not affect the levels
of the MDM2 protein (FIG. 7, panels A and B). Inhibiting MDM2 with
the MDM2-specific inhibitor, nutlin-3, reduced viability of ACC
cells through the restoration of p53 function. Treating both H295R
and SW-13 cells with nutlin-3 decreased their cell viability (FIG.
7, panel C) and restored the apoptotic response in H295R cells,
which have wild type p53 (FIG. 7, panels D and E).
[0177] Given that PLK-1 directly modulates p53 functions as well as
indirectly modulating MDM2 functions, the potential role of PLK-1
inhibition in sensitizing ACC cells to the effects of nutlin-3 was
investigated. A decrease in the IC.sub.50 values of nutlin-3 was
observed when combined with the IC.sub.25 concentration of BI-2536
(FIG. 8, panels A and B; Table 3). This effect was independent of
p53 mutation status, as both ACC cell lines responded in a similar
fashion. Combined inhibition of PLK-1 and MDM2 did not increase
apoptotic response of SW-13 over that seen with BI-2536 alone,
whereas an additive apoptotic response was observed in H295R cells
with wild-type p53, which is responsive to MDM2 inhibition (FIG. 8,
panels C and D; Table 3).
TABLE-US-00003 TABLE 3 IC.sub.50 values of BI-2536 and nutlin-3
alone and in combination. Compound H295R (IC.sub.50 Value in .mu.M)
SW-13 (IC.sub.50 Value in .mu.M) BI-2536 0.0628 0.0094 nutlin-3
12.75 19.78 nutlin-3 + BI-2536 2.838 6.502 Combination Index 0.22
0.33
Sequence CWU 1
1
612750DNAHomo sapiens 1gtgctctgag tttttggttt ctgtttcacc ttgtgtctga
gctggtctga aggctggttg 60ttcagactga gcttcctgcc tgcctgtacc ccgccaacag
cttcagaaga aggagcagcc 120cctgggtgcg tccactttct gggcacgtga
ggttgggcct tggccgcctg agcccttgag 180ttggtcactt gaaccttggg
aatattgaga ttatattctc ctgcctttta aaaagatgga 240cttcagcaga
aatctttatg atattgggga acaactggac agtgaagatc tggcctccct
300caagttcctg agcctggact acattccgca aaggaagcaa gaacccatca
aggatgcctt 360gatgttattc cagagactcc aggaaaagag aatgttggag
gaaagcaatc tgtccttcct 420gaaggagctg ctcttccgaa ttaatagact
ggatttgctg attacctacc taaacactag 480aaaggaggag atggaaaggg
aacttcagac accaggcagg gctcaaattt ctgcctacag 540ggtcatgctc
tatcagattt cagaagaagt gagcagatca gaattgaggt cttttaagtt
600tcttttgcaa gaggaaatct ccaaatgcaa actggatgat gacatgaacc
tgctggatat 660tttcatagag atggagaaga gggtcatcct gggagaagga
aagttggaca tcctgaaaag 720agtctgtgcc caaatcaaca agagcctgct
gaagataatc aacgactatg aagaattcag 780caaaggggag gagttgtgtg
gggtaatgac aatctcggac tctccaagag aacaggatag 840tgaatcacag
actttggaca aagtttacca aatgaaaagc aaacctcggg gatactgtct
900gatcatcaac aatcacaatt ttgcaaaagc acgggagaaa gtgcccaaac
ttcacagcat 960tagggacagg aatggaacac acttggatgc aggggctttg
accacgacct ttgaagagct 1020tcattttgag atcaagcccc acgatgactg
cacagtagag caaatctatg agattttgaa 1080aatctaccaa ctcatggacc
acagtaacat ggactgcttc atctgctgta tcctctccca 1140tggagacaag
ggcatcatct atggcactga tggacaggag gcccccatct atgagctgac
1200atctcagttc actggtttga agtgcccttc ccttgctgga aaacccaaag
tgttttttat 1260tcaggcttgt cagggggata actaccagaa aggtatacct
gttgagactg attcagagga 1320gcaaccctat ttagaaatgg atttatcatc
acctcaaacg agatatatcc cggatgaggc 1380tgactttctg ctggggatgg
ccactgtgaa taactgtgtt tcctaccgaa accctgcaga 1440gggaacctgg
tacatccagt cactttgcca gagcctgaga gagcgatgtc ctcgaggcga
1500tgatattctc accatcctga ctgaagtgaa ctatgaagta agcaacaagg
atgacaagaa 1560aaacatgggg aaacagatgc ctcagcctac tttcacacta
agaaaaaaac ttgtcttccc 1620ttctgattga tggtgctatt ttgtttgttt
tgttttgttt tgtttttttg agacagaatc 1680tcgctctgtc gcccaggctg
gagtgcagtg gcgtgatctc ggctcaccgc aagctccgcc 1740tcccgggttc
aggccattct cctgcctcag cctcccgagt agctgggact acaggggccc
1800gccaccacac ctggctaatt ttttaaaaat atttttagta gagacagggt
ttcactgtgt 1860tagccagggt ggtcttgatc tcctgacctc gtgatccacc
cacctcggcc tcccaaagtg 1920ctgggattac aggcgtgagc caccgcgcct
ggccgatggt actatttaga tataacacta 1980tgtttattta ctaattttct
agattttcta ctttattaat tgttttgcac ttttttataa 2040gagctaaagt
taaataggat attaacaaca ataacactgt ctcctttctc ttatgcttaa
2100ggctttggga atgtttttag ctggtggcaa taaataccag acacgtacaa
aatccagcta 2160tgaatataga gggcttatga ttcagattgt tatctatcaa
ctataagccc actgttaata 2220ttctattaac tttaattctc tttcaaagct
aaattccaca ctaccacatt aaaaaaatta 2280gaaagtagcc acgtatggtg
gctcatgtct ataatcccag cactttggga ggttgaggtg 2340ggaggattgc
ttgaacccaa gaggtcaagg ctgcagtgag ccatgttcac accgctgcac
2400tcaagcttgg gtgacagaac aagaccccgt ctcaaaaaaa attttttttt
taataaaaca 2460aaatttgttt gaaatctttt aaaaattcaa atgattttta
caagttttaa ataagctctc 2520cccaaacttg ctttatgcct tcttattgct
tttatgatat atatatgctt ggctaactat 2580atttgctttt tgctaacaat
gctctggggt ctttttatgc atttgcattt gctctttcat 2640ctctgcttgg
attattttaa atcattagga attaagttat ctttaaaatt taagtatctt
2700ttttcaaaaa cattttttaa tagaataaaa tataatttga tcttattaaa
275023038DNAHomo sapiens 2ttggccctgg gggcggggcg aggcgcagag
gtgcgtcctg agggcggggc ggtgacgcaa 60gagcgactcc tgggggcggg gcgaggcctt
ggagatgcgt ccggaggcgg tggggagcga 120agactgaccc ggggccgtga
cgcgggggca ggccctgggg cgggggcggg tcctggggac 180tggggcgggc
ggccgaggcc cggaagcgga ctgaggcggc ctggagtctt agttggctac
240tcgccatgga cgaagcggat cggcggctcc tgcggcggtg ccggctgcgg
ctggtggaag 300agctgcaggt ggaccagctc tgggacgccc tgctgagccg
cgagctgttc aggccccata 360tgatcgagga catccagcgg gcaggctctg
gatctcggcg ggatcaggcc aggcagctga 420tcatagatct ggagactcga
gggagtcagg ctcttccttt gttcatctcc tgcttagagg 480acacaggcca
ggacatgctg gcttcgtttc tgcgaactaa caggcaagca gcaaagttgt
540cgaagccaac cctagaaaac cttaccccag tggtgctcag accagagatt
cgcaaaccag 600aggttctcag accggaaaca cccagaccag tggacattgg
ttctggagga tttggtgatg 660tcggtgctct tgagagtttg aggggaaatg
cagatttggc ttacatcctg agcatggagc 720cctgtggcca ctgcctcatt
atcaacaatg tgaacttctg ccgtgagtcc gggctccgca 780cccgcactgg
ctccaacatc gactgtgaga agttgcggcg tcgcttctcc tcgctgcatt
840tcatggtgga ggtgaagggc gacctgactg ccaagaaaat ggtgctggct
ttgctggagc 900tggcgcagca ggaccacggt gctctggact gctgcgtggt
ggtcattctc tctcacggct 960gtcaggccag ccacctgcag ttcccagggg
ctgtctacgg cacagatgga tgccctgtgt 1020cggtcgagaa gattgtgaac
atcttcaatg ggaccagctg ccccagcctg ggagggaagc 1080ccaagctctt
tttcatccag gcctgtggtg gggagcagaa agaccatggg tttgaggtgg
1140cctccacttc ccctgaagac gagtcccctg gcagtaaccc cgagccagat
gccaccccgt 1200tccaggaagg tttgaggacc ttcgaccagc tggacgccat
atctagtttg cccacaccca 1260gtgacatctt tgtgtcctac tctactttcc
caggttttgt ttcctggagg gaccccaaga 1320gtggctcctg gtacgttgag
accctggacg acatctttga gcagtgggct cactctgaag 1380acctgcagtc
cctcctgctt agggtcgcta atgctgtttc ggtgaaaggg atttataaac
1440agatgcctgg ttgctttaat ttcctccgga aaaaactttt ctttaaaaca
tcataaggcc 1500agggcccctc accctgcctt atcttgcacc ccaaagcttt
cctgccccag gcctgaaaga 1560ggctgaggcc tggactttcc tgcaactcaa
ggactttgca gccggcacag ggtctgctct 1620ttctctgcca gtgacagaca
ggctcttagc agcttccaga ttgacgacaa gtgctgaaca 1680gtggaggaag
agggacagat gaatgccgtg gattgcacgt ggcctcttga gcagtggctg
1740gtccagggct agtgacttgt gtcccatgat ccctgtgttg tctctagagc
agggattaac 1800ctctgcacta ctgacatgtg gggccaggtc accctttgct
gtgaggctgt cctgtacatt 1860gtgggatgtt cagcactgtc ccttgcctca
atgccagtaa cgcgtcttcc tgagtggtgc 1920caaacaaaaa ggttctcagg
tgttgccaaa tatgtcctgg ggtataaaac tttcctcgcc 1980tgacaaccac
tggtctgtag ggatttttgg ctacacacaa accagtatcg ctcatagatc
2040agcaaaccgg ggcctactag agtctgaaca gctgtaatct atgaattcta
agtgaaattt 2100taaaaattgt taatttttcc tatattgcat taattttaaa
aaataaatct gaggcaaata 2160tggactctct tttgcctatt tcttccctca
ttttgctcca actctttctt cttccttaca 2220aaagagactt ttgctttttt
cgaaacattt ccccatgttt ttctggggtc tcgctatgtt 2280gcccaggctg
gtctcaaact cctgggctca agtgaccctc ccaagtagct cttactacag
2340gcgtgcacca ttgcacccag ccccatttat tcatgtctta tttcacttga
tccttatccc 2400atcccaggaa ggcaacaagg gtgagaaccc tgtgctcagg
gaggttaggt ctcttgtcca 2460agggaaaacg attatccaga gaagagacct
ggccagaacc tgggtcccct gagtcctagc 2520catgcttccc atgtgcctta
cttgctgaag cacccccgga ctgcagtgtg aacgtgctgt 2580gcaatagtga
cacgctgggc ttccccacaa ggctccaccc tgaggtcttt taagctgtcc
2640ttatgccagc ctatttcttg ttttttgggc cttttttttt ggagataggg
tctcactctg 2700tcgcccaggc tggagtgcaa tgacgcaatc ttggcttatt
gcagtctcga cctcctgggc 2760tcaagagatc cttccacctc agccacctga
gtagcttgga ctacaggtgt gcaccacctc 2820tcccagttaa tttttgtatt
tttagtagag acagagttat gccatgttac tcaggctggt 2880cttgaactcc
tggactcaag cgatcagcct gccttagcct cccaaagtgc aggggttaca
2940ggcttgagcc attgcgcctg acctatttct ggttcttagg gccctggatg
ttaggatgga 3000tttctgaatt aataataata ataaaaccct catcaaga
3038332772DNAHomo sapiens 3ctccttggtt caagtaattc tcctgcctca
gactccagag tagctgggat tacaggcgcc 60cgccaccacg cccagctaat tttttgtatt
tttaatagag atggggtttc atcatgttgg 120ccaggctggt ctcgaactcc
tgacctcagg tgatccacct gcctcagcct cccaaagtgc 180tgggattaca
ggagtcagcc accgcaccca gccccaacta atttttgtat ttgagtagag
240acagggtttt accatgttgg ccaggctggt ctaaaactct tcacctcagg
tgatccaccc 300atctcagcct cccaaagtgt tgggattaca ggcgtgagcc
accgtgcctg gccctggatt 360tcactcttgc ccacccataa accattcact
cttctgtttt aaaactctct tggccgggcg 420cagtggctca tgcctgtaat
cccagcactt tgggaggcca aggtgggcag atcacaaggt 480caggagttcg
agaccagcct ggccaatatg atgaaccccc catctctact aaaaaaatac
540aaaaaaatta gccgggtgtg gtggcacatg cctgtaatcc cagctactcg
gaaggttgag 600gcaggagaat cacttaaacc tgggaggcgg aggttgcggt
gagctgagat ggtgccactg 660cactccagcc tggacaacag agcaagactc
tgtctcaaac aaacaaacaa aaaaaacctc 720tctcatggcc tggcatggtg
gctcacgcct gtaatcctag cactttggaa ggctgaggca 780ggtggatcac
ctgaggtcag gagtttgaga ccagccaggc caacgtggca aaacctgtct
840ctactaaaaa tacaaaaatt aagccaggcg cggtggctca tgcctataat
cccagcactt 900tgggaggccg agaccggcgg atcaaatgtc aggagtacga
gaccatcctg gccaatatgg 960taaaaccccg tctctattta aaaaaataca
aaaattagct gggcatggtg gcgggtgcct 1020gtaatcccag ctactcggga
ggctgaggca ggagaatcgc ttgaatccag gaggcagagg 1080ttgcagtgag
ccgagatcac gccattgcac tccagcctgg gcgacagagc aagactcgtc
1140tcaaaaaaaa aaaaaaaaca cctctctcat gacttcccaa ataaactcca
aatgccttac 1200ccataagaac caacacgacg tggctgctct tctctgtcct
cacccctctg tccccctcac 1260ttgcctcagt ctggctatac cagcattctg
ggttttttgt tttgtgtttg tttgtttgtt 1320tgtttgtttg tttgtttgtt
ttgggatgga gtctcactct gtcacccagg ctggagtgca 1380gtgacatgat
cttggctcac tgcaacctcc atctcctggg ttcaaatgat tcttctgctt
1440cagcctctca agtagctggg attacaggca cccaccatca cacccagcta
atttttgtat 1500ttttgtagag atgaggtttt gccatgttgg ccaggctggt
ctcaaactct tgacctcagg 1560tgatctgccc acctcagcct cccaaagtgc
tgggattaca ggtgtgagcc actgagccca 1620gtctgttttg ttgttttttg
agatggagtc tcactctgtc gcccaggctg gagtgcagtg 1680gtacaatttt
gtctgactgc agcctccacc tcctgagttt aagagattct cctgcctcag
1740ccacacaagt agctgggatt acaggcgcgt gccaccacat gcgtctaatt
ttggtatttt 1800tagtagcgat ggggttttgc catattggcc aggctggtct
cgaactcctg gcctcaagtg 1860atctgcacac ctcggcctcc tgagtagctg
ggattacagg cgtgagccac catgcagggc 1920ctttctggtc ttttaacgca
caaagctatt tcccagttct gggtgtttat agtatcattg 1980ccagttcctg
gaaagctctt tccagaagcc ttcacatgac tgatccctta tcctccttca
2040ggtcttagct caaatgcctc cttttcagag agctccttcc tgaccatttt
atgcaatgtg 2100ctttcctcta gttagtctct cttcattctg tgtatttcct
tcagagcatt tatcatcatc 2160ttggtcctga tgcttgctgg tttacatgtt
tgttgtctgt ctcccacaca gaaagcaaac 2220cagcccttca tctgtcttgt
ccaccacttt atcccagcac agtgactaac atatatcagt 2280agtcaatcaa
taaatagcta taagccaggc acggtgactc acacctgtga tccccgcact
2340ttgggaggcc aaggtgggcg gatcctctga ggtcaggaat ttgatatcag
cctgcccaac 2400atggcgaaac cccgtctcta ctaaaactat aaaaattagt
cgagtgtgat ggcgtgcgcc 2460tgtaatacca gctacttgtg aggctgaggc
aggagaatcg cttgaacccg ggaggcagag 2520gttgcagtga gccgagatca
caccacagca ctccagcctg ggtgacagag caagaccttg 2580tctcaaaaaa
ataagaaata gaaatatata tatgctgcca caagaaattc actacttttt
2640agcaaaaaac acagattcct aattaaagaa aggggaaacc tctctctaaa
taacttcaga 2700attcggggca gaaaacgcta catgtggaaa ctcgtcttga
aaaacagtcc cgtttgtgaa 2760caggaaacac ttggactaca ctttttccat
ctccacgaag gacttaaatg tgcagcattg 2820atgaagaggc agttagcccc
agtccttacc atttctgcat attccacctg ctccccctca 2880tggtacagct
ctgggggcag gttataaatt cgcaagatgt tatcagcact attggtcaag
2940atgcaggaac cgtcaggagc cctagaaaca ggggagagtt agaaagctgg
ccagacctat 3000gcttttcaag tgtagggcta gggctgagcc tgcctctggg
gtaggtaagc ccccctgaat 3060ccttgaggga agtagaagac acaaactgct
agataaaatg taagctcagt ctaaaagggc 3120tacgtgccgc ttctcccagc
tctggggcat ccctctccta gaaaactgga ctgttttaca 3180gtgaaaatct
cgggggtggt cagctccctg ccccgttgtt atccttacca cttacagcct
3240ttcaagaagt tctcaggttg ggtgctgaac tctgaccagg aaccactgag
aaatcgaggc 3300agctgggaga agctgtagtt ccaagcgctg aaaggaagat
gggggacaat aaacctgggt 3360cgccaagcaa agggggcaga ggcctggaga
agtgggtctc aggaccagag gacagatcga 3420cctcacactt catctcccca
gactccacac tccactgcca tcaccactta cgtgtctccc 3480tcgtcctctg
cagcgggttc cccagaggta tcttccatgg cttttccaga ccccaactct
3540ggcccgttcg cttcttcttc agaaaggctc ccgtttgctt cttctgcagg
aaggcttgta 3600ttttcagaaa gttcttgctc ctcgattcga ggactcaact
cactagggga accaaactct 3660gtttccaggg gagtggagag agaaactggg
tccccctccc gtagctcctg ggacacagct 3720gagccagcca caggatctgg
ggacaaccgg ggcggatccc ccctttcggg aggcggtggc 3780atcagttcag
agtccgcatt tttattcatc ggggaagcgt ggggagaagg atgggctgga
3840gctgggtcct ggtctgaagg acagcagtcc ggagctaacg gttgagtctc
caaagtcttc 3900atactgcaga ggaagcacag cggagattag cctcagccag
gatggcttcg aagttctcag 3960ggatccgacg cagagctaaa gaaacccacc
tgtgcttccc tcctcttctg ggagtaggca 4020gaagactccc gggaggagag
gcgaacagcg gacgccaatt cttttgaaag cactgtgttc 4080cttagcaccg
cgggtcgcta cgggcctctt gctgtcgcgg gatttcggtc caccttccga
4140ttgggccgcc gcatcccgga tcagatttcg cgggcgaccc acggaacccg
cggagccggg 4200acgtgaaagg ttagaaggtt tcccgttccc atcaagccct
agggctcctc gtggctgctg 4260ggagttgtag tctgaacgct tctatcttgg
cgagaagcgc ctacgctccc cctaccgagt 4320cccgcggtaa ttcttaaagc
acctgcaccg cccccccgcc gcctgcagag ggcgcagcag 4380gtcttgcacc
tcttctgcat ctcattctcc aggcttcaga cctgtctccc tcattcaaaa
4440aatatttatt atcgagctct tacttgctac ccagcactga tataggcact
caggaataca 4500acaatgaata agatagtaga aaaattctat atcctcataa
ggcttacgtt tccatgtact 4560gaaagcaatg aacaaataaa tcttatcaga
gtgataaggg ttgtgaagga gattaaataa 4620gatggtgtga tataaagtat
ctgggagaaa acgttagggt gtgatattac ggaaagcctt 4680cctaaaaaat
gacattttaa ctgatgagaa gaaaggatcc agctgagagc aaacgcaaaa
4740gctttcttcc ttccaccctt catatttgac acaatgcagg attcctccaa
aatgatttcc 4800accaattctg ccctcacagc tctggcttgc agaattttcc
accccaaaat gttagtatct 4860acggcaccag gtcggcgaga atcctgactc
tgcaccctcc tccccaactc catttccttt 4920gcttcctccg gcaggcggat
tacttgccct tacttgtcat ggcgactgtc cagctttgtg 4980ccaggagcct
cgcaggggtt gatgggattg gggttttccc ctcccatgtg ctcaagactg
5040gcgctaaaag ttttgagctt ctcaaaagtc tagagccacc gtccagggag
caggtagctg 5100ctgggctccg gggacacttt gcgttcgggc tgggagcgtg
ctttccacga cggtgacacg 5160cttccctgga ttgggtaagc tcctgactga
acttgatgag tcctctctga gtcacgggct 5220ctcggctccg tgtattttca
gctcgggaaa atcgctgggg ctgggggtgg ggcagtgggg 5280acttagcgag
tttgggggtg agtgggatgg aagcttggct agagggatca tcataggagt
5340tgcattgttg ggagacctgg gtgtagatga tggggatgtt aggaccatcc
gaactcaaag 5400ttgaacgcct aggcagagga gtggagcttt ggggaacctt
gagccggcct aaagcgtact 5460tctttgcaca tccacccggt gctgggcgta
gggaatccct gaaataaaag atgcacaaag 5520cattgaggtc tgagactttt
ggatctcgaa acattgagaa ctcatagctg tatattttag 5580agcccatggc
atcctagtga aaactggggc tccattccga aatgatcatt tgggggtgat
5640ccggggagcc caagctgcta aggtcccaca acttccggac ctttgtcctt
cctggagcga 5700tctttccagg cagcccccgg ctccgctaga tggagaaaat
ccaattgaag gctgtcagtc 5760gtggaagtga gaagtgctaa accaggggtt
tgcccgccag gccgaggagg accgtcgcaa 5820tctgagaggc ccggcagccc
tgttattgtt tggctccaca tttacatttc tgcctcttgc 5880agcagcattt
ccggtttctt tttgccggag cagctcacta ttcacccgat gagaggggag
5940gagagagaga gaaaatgtcc tttaggccgg ttcctcttac ttggcagagg
gaggctgcta 6000ttctccgcct gcatttcttt ttctggatta cttagttatg
gcctttgcaa aggcaggggt 6060atttgttttg atgcaaacct caatccctcc
ccttctttga atggtgtgcc ccaccccgcg 6120ggtcgcctgc aacctaggcg
gacgctacca tggcgtgaga cagggaggga aagaagtgtg 6180cagaaggcaa
gcccggaggt attttcaaga atgagtatat ctcatcttcc cggaggaaaa
6240aaaaaaagaa tgggtacgtc tgagaatcaa attttgaaag agtgcaatga
tgggtcgttt 6300gataatttgt cggaaaaaca atctacctgt tatctagctt
tgggctaggc cattccagtt 6360ccagacgcag gctgaacgtc gtgaagcgga
aggggcgggc ccgcaggcgt ccgtgtggtc 6420ctccgtgcag ccctccggcc
cgagccggtt cttcctggta ggaggcggaa ctcgaattca 6480tttctcccgc
tgccccatct cttagctcgc ggttgtttca ttccgcagtt tcttcccatg
6540cacctgccgc gtaccggcca ctttgtgccg tacttacgtc atctttttcc
taaatcgagg 6600tggcatttac acacagcgcc agtgcacaca gcaagtgcac
aggaagatga gttttggccc 6660ctaaccgctc cgtgatgcct accaagtcac
agaccctttt catcgtccca gaaacgtttc 6720atcacgtctc ttcccagtcg
attcccgacc ccacctttat tttgatctcc ataaccattt 6780tgcctgttgg
agaacttcat atagaatgga atcaggctgg gcgctgtggc tcacgcctgc
6840actttgggag gccgaggcgg gcggattact tgaggatagg agttccagac
cagcgtggcc 6900aacgtggtga atccccgtct ctactaaaaa atacaaaaat
tagctgggcg tggtgggtgc 6960ctgtaatccc agctattcgg gagggtgagg
caggagaatc gcttgaaccc gggaggcaga 7020ggttgcagtg agccaagatc
gtgccactac actccagcct gggcgacaag aacgaaactc 7080cgtctcaaaa
aaaagggggg aatcatacat tatgtgctca tttttgtcgg gcttctgtcc
7140ttcaatgtac tgtctgacat tcgttcatgt tgtatatatc agtattttgc
tccttttcat 7200ttagtatagt ccatcgattg tatatccgtc cttttgatgg
ccttttgagt tgtttcccat 7260ttgcggttat gaaataaagc tgctataaac
attcttgtac aattcttttt gtgatcatat 7320gttttcgtgt ttcttggaga
aatacttagg aggggaattg cgagtttgga agtaaaaagt 7380agctgtattt
tgaacttttt cagaagctct gagttttcca gagcggttgt accattttac
7440actccaacta gcaaggtatg ggagttatta tggttgtgcc acagccttcc
ggacattagg 7500tattgtcagt ctttctaatg tggtatatcc ttgtggttgt
aatttacagt tctctattga 7560ctaaggatgt tcagcatttt ttcatgtgcc
tattggccat tcgtattttg tttgtaaagt 7620agctcttcga gtcttttacc
tgttattttg gttttttgtt tgtttttatt gttcagttgt 7680gggactgctt
tatacattct ggatacaagt cctttatcag atccatgtgt cgtgaatgtt
7740ttcttctgat ctgttgcttg cctatttgtt tgctttacag agtttacagt
atcttaagag 7800gagtggattt atctttttta tgttcagtat ttgccttgtc
ctgtttagga catctttttt 7860ttttttttta accccagggt catgaagata
ttatcttaca ttttctttta ggacctttat 7920ggttgtaagt tttacagtaa
ggtccttgag ccattaatta attcttaaaa ttaattgttt 7980atggtgtgag
gtgtaggagt cagtctctgg tatctttcct gtatggaaat ccagttattc
8040tgtctccact tgttgaaata ggcttccttt ctctactgaa tgcttttaat
tttaattatt 8100ttacagttgg agtatagggc taccatttta gtgctatttt
ctttttttct ttgttaattt 8160ttgagacagg gactcacact gttgcccagg
ctagagtaca atggcacaat caaggcttac 8220tgcagcctcg aacccctggg
ctcaagcagt cctctagcag cctcacgagt agctgggatt 8280actccaccac
acccagctaa ctattttatt tttttgtatt gacaggatct cactatgttg
8340cccaggctgg tctcaaactg ctggcctcaa gctttcatcc catctcggcc
tcccaaagtg 8400ctgggattac aggtgtgagc caccatgcct gacctcttag
tgctattttc tatttatctc 8460ctctgttctc tgctctcttt aaacgttgga
ggaagaaaca gtacccatct tacacaaact 8520cttcagaaaa cagaggaaca
gactgggcgc ggtggctcat acctgtaatc tcagcacttt 8580ggtacgctga
ggcaggggat catttgaggt cgggagttcg agaccagcct ggccaacacg
8640gcgaaacccc atctctacta aaaatacaaa aagtagctag gcgtggtgac
acatacctgt 8700aatgccagtt actcaggagg ctgaggcaca agaatccctt
gaacctggga agcggaggtt 8760gcagtgagcc gagattgcgc cactgcactc
cagcctgggc aacagagtga gaccctgtct 8820cagaaaaaaa aagaaagaaa
gaaaaaatag aggaatattt cccaacttgt tttcgaagcc 8880agcataatcc
tggtaccaaa accaaacaag gacattataa gaaaagaaaa tatagaccaa
8940tattcctgtt agcatagaca tgcaacagct aaccaatttt agcaaaccaa
acctggtaat 9000atagaaaaaa ggataaatag gccagtcgcg gtggctcacg
cctgtaatcc cagcactttg 9060ggaggctgag gcaggcagat cacttgaggt
caggagtttg agaccagcct gaccaacatg 9120gtgaaacccc gtttctaata
aaaatacaaa aatcaggctg ggcacggtgg ctcacgcctg
9180taatcccagc actttgggag gccgaggtgg gcagatcacg aggtcaggag
ttcaagacca 9240gcctgaccaa tgtggtgaaa cgccatctct actaaaaata
caaaaatcag ccggtgtggt 9300ggcacctgcc tgtaatccca gctactcagg
aggctgaggc agaattgctt gaacccggga 9360ggcagaggtt gcagtgagcc
aagatcgtgc cactgcactc cagcctgggc gacagagcaa 9420gacttcatct
caaaaaaaaa aaaaaattag ctgggcatgg tggtgggcac ctgaaatccc
9480agctactcgg gagtctgagg caggagaatc gcttgaaccc aggaggcaga
agttgcactg 9540agctgggatc acaccattgc actccagcct gggcaacaga
gtgagactcc atctcaaaaa 9600aagaaaaaga aaaaggataa atacattcta
accaaataat gtttatctca tgattgtagc 9660tgattcaaca ttcaaaaatt
ggcctggtgc agtagctcag gcctgtaatc ccaacatttt 9720aggaggctga
ggcaggaaga tctcttgagc ccaggatttc aagaccagcc tgggcaacat
9780agtcagactg gtctttactg gggggaaaaa aatcagtctg tgtaattcac
cacattaaca 9840aagggaaaca taaaaaccct atgatcattt caacagatgt
agcaaaagca gttaatgata 9900ttcaacacat atgcatgatt acaaaccaac
caacctccta gcaaactagg gaaaggaaac 9960ttaacctagt ttgataacag
ggcgtccaca gtcggagttc cactagcagc atacataatg 10020gtagaaaact
cagtgctgcc gggcgcggtg gctcacgcct gtaatgccag cactttggga
10080ggcctaggcg ggcggatcac gaggtcagga gatcgagact gtcctgacta
gcatgctgaa 10140accccgtctc tactaaaaat acaaaaacaa aaaattagcc
gggcatggtg gcgggcgcct 10200atagtcccag ctactcggga ggctgaggcg
agagaatggc gtgaacccgg gaggcggagc 10260ttgcagagcc tagatcgtgc
cactgcactc cagcctgggt gacagagtga gacttcgtct 10320caaaaaaaaa
aaaaaaaaaa aaagaaaaga aaactcaacg ctttttcctc taagatcagg
10380aactagaaaa ggatttgact ctcacaacgt tgataccata ctggaggttt
taaccaggca 10440agaaaaagaa ataatgaggg ccgggtgcgg tggctcaggc
ctgtaatccc agcactttgg 10500gaagccgaga cgggtggatc acgaggtcag
gagatcgaga ccatcctggc taacacggtg 10560aaaccctgtc tctactaaat
atacaaaaaa ttagccgggc gtagtggcgg gcgcctgtag 10620tcccagctac
tcgggaggct gaggcaggag aatggcgtga actcaggggg cggagcttgc
10680agtgagctga gatcgagcca ctgcactcca gcctgggcga cagagcaaga
ctgtgtctca 10740aaaaaaaaaa aagaaaaaga aataatgatt agtggcccga
tgtctcacgc ctataatccc 10800agcactttgg gaggccgagg tgggcagatc
acctgaggtc tggagttgga gaccagcctg 10860acaaagatgg tgaaacctcg
tctctattaa aatattaaaa aaatagccag gcgttggccg 10920ggtacagtgg
ctcatgcctg taatcccagc actttgggag gccgaggtgg gtggatcacc
10980tgaggtcagg agttcaacac cagcctggcc aacatggtga aaccccatct
ctactaaaaa 11040tacaaaaatt agccgggcgt agtggcgggc gcctgtaatc
ccagctactt gggaggctta 11100ggcaggagaa tcgcttgaac ctgggaggcg
gaggttgtag tgagccgaga ttgcaccatt 11160gcactccagc ctgggtgaca
aaagcaaaaa ctccgtctca aaaaaaaaag aattagccag 11220gggtagtggt
gaacgcctgt agtcccagct actcaggagg cagaggcagg agaatcactt
11280gaacccagga ggcagaggtt gcagtgagcc gagattgtcc cattgcactc
cagcctaggc 11340gacaagagca aaattccatg tcaaaaaaaa aaaaaaaaaa
ggaaagaaaa aaaataacga 11400ttagaaagga agaaataaaa cacattcaca
gccagtatga ttctatacat acatgtccta 11460atggggccag gcgtggtggc
tcatgcctgt aatcctagca cttttaggag gctgaggcag 11520gtggcttccc
tgggaccagc ctggccaaca tggtgaaacc ccaactctaa taaaaataca
11580aaaaatcagc caggcgtggt gacgggcacc tctaatccca gctactcagg
aggctgaggc 11640aggagaattg cttggacctg ggaggcagag gttgcagtga
gccgagatcg cgctattgca 11700ctccagcctg ggcaacaaga gtgaaactcc
ggcagggtgt ggtggcttac gcctgtaatc 11760ccagcacttc gggaggctga
ggcaggccga tcacctgagg tcaggagttt gagaccaacc 11820taacatggtg
aaaccccgtc tctactaaaa atacaagaat tagctgggtg tagtggtggg
11880cgcctgtaat cccagctact tgggaggctg agacagaaga attgcttgaa
cccaggaggt 11940ggaggttgca gtgagctgag atcatgccat tgcacaccac
gccgggcaac agagcgagat 12000tccgtctcaa aaaaaaaaaa aaagagtgaa
actctatctc aaaaaaaaaa aaaagtccta 12060atggaaaatc cataaaaagc
taccaaaact aataaataaa tatagcaggg ttgcaggtta 12120cagggcaata
tagttatccc tctatctgta ggggcttggt tctgggactc ctcacacacc
12180aaacccacag atgtctaagt cccatatata agacggtata gtatttggat
ttaacctaca 12240catatcctcc catatagttt aaattatctc tagattactt
acattacccc catacaatga 12300aaatgctaat gtacatgcaa gtatgtatgt
aagtacttgt actatattgt ttagggaatc 12360actggacata taggccttca
agactgatac cagcagccac tgttaagatt ctggtcaggc 12420ctgcccctgt
ttggggtctc agttgatctc attgccttcc cacccagcca agggcacctg
12480catttctctt ggctccctgg ccatttggaa ggcctagttc agcctggcac
atttgtatcc 12540tggcccactg atgctggtac ccctgggaag gtcctgctct
gaaaaacacg gagattttag 12600ttgctactga agatttgaga gataaagaca
gggagacctg tctgtagacc tgtgtccctc 12660caagtgggat tgagactttg
ggccccccat ttcaggacag cacctcctgg cctgttgact 12720gaatagatcc
ctgaaggagg tgtacttgca ttaatggagt gggggtggga gcagtaccac
12780agatccgcac taacaatcac acagttctct ctagaataat aatatagaac
aagtgaaata 12840gaacaattgc agaaagagct aacctttgtt gagctcttac
tgtgtgccca gcactttcct 12900caactctaca tttcccataa tacacagagt
actaggtagg ccaggcttgg tggctcacgc 12960ctgtaatccc agcactttag
gaggccaagg ggggtggatc acctgaggtc gggagttcaa 13020gaccagcctg
accaacatgg tgaaaccccg tctctactag aagtacaaaa ttagccaggt
13080gtggtggcac atgcttgtag tcctagctac tcagcaggct gaggcaggag
aatcatttga 13140atccgggagg aggttgcagt aagcggagat agtgccactg
tactccagcc tgggcaataa 13200gagctgagac tccgtctcaa aataaaataa
aataaaataa aataaaataa aataaaataa 13260aaaaagaaaa gagcctgcca
ttaaaggagc tgtttggtag gggatgtttt gtcagtgcaa 13320acaacagaaa
agtgggctgg gcacagtggt tcatgcctgt aatcccagca ctttgggagg
13380ccaaggcggg cggatcacct gaagttggga gttcaagacc agcctgacca
atatggagaa 13440accccgtctc tactaaaaat acaaaattag ccgggcgcag
tggcgcatgc ctgtaatccc 13500agctactcgg gaggctgagg caggagaatc
gcttgaacct gggaggcaga ggttgcggtg 13560agccgagatc gcaccattgc
actccagcct ggacgagagc aaaactctgt ctcaaaaaaa 13620aaaaaaaaca
gaaaagtgta acaaacactt acagtaggca tgtttcttag caaatctgat
13680gacaaatttg gcataaagaa agagagcatc cctgaaaaaa aaaaaaagaa
aaagaaagag 13740agcatcctgc ctgggcaaca tagtgaaacc ctgcctctac
aaaaaaactc aaaaattggc 13800cgggtgcagt ggctcacacc tgtaatccca
gcactttggg agtcggaggc gggaggatca 13860cctgaggtca ggagttcgaa
accagcctgg ccaacatggc aaaaccccat ctctactaaa 13920aatacaaaaa
attaatcagg cgcattggtg ggcgcctgta atcccagcta ctcaggaagt
13980tgaggcaaga ggatcgcttg aatctgggag gtggaggtta cagtgagtcg
agatcacacc 14040actgcactct agcctgggtg acagggcgag actccgtctc
caaaaaaaaa aagaaaaaga 14100aaaagactaa aaaattagcc aggcaggcct
ctgtggtccc agctacttgg gaggctgagg 14160caggagaatc actgagccca
ggagtccgag gctgtagtga gccatgattg caccactgta 14220ccctagcttg
ggcaacaaag caagaccctg cctcaaaaga aaaaagaaag aaagaaagaa
14280catggcgggc caggcacagt ggctcacacc tgtaatccca gcgctttgag
aggccgaggc 14340aggtggatca caaggtcagg agttccacac cagcctggcc
aacatggtga aaccctgtct 14400ctactaaaaa tacaaaaaat cagccaggca
tggtggcagg ggcctgtaat cccagctact 14460cgggaggctg aggcaggaga
attgcttgaa accagaaggc agaggttgca gtgagcctag 14520actgcaccac
tgcactccag cctgggcgaa aagagccaaa ctccatctca aaaaacaaac
14580aaaaaaacaa aacaaaagaa aacatggcaa agcctttgaa agcttgtctg
ggagaaggtg 14640cgatgatagt tgcataactt cgtgcaagat gctggtccac
acaggggctg ccccttgctc 14700tttctcgctc tcttaacctc tcatataaca
ggcttgtgtg ttattcacat ttattgagcc 14760caagcaggtg caaggcattg
tgatctaata ctttggtcag caagacaaca agatagatca 14820ctgccctgcc
cttaggaagt gtatatgcta ttagaggaaa cagataaaat aaacaaggaa
14880aagtatcaga caatgtaagt gctatgagaa tgcaaatgag gtgatgtgaa
ttaaaatagg 14940atgacttaaa gtctgcacgg gaaggagcct acccccatgt
tcctggctag ccaaggaacc 15000accagttgat tagcagagaa gggcagccag
tctagctaga gcttttgggg aagagggagt 15060ggttgttaag agatgagatt
aaagaagccg agacgggcca ttcgtgaggg gtttgtaatg 15120cagggctgag
gagtgtccga agagaatggg caggtgagcg gtgagacagt tgttcttcca
15180gaagctttgc agtgaaagga atcaaagaaa tggagccgtg tatcaggtgg
ggaagggtgg 15240gggccaaggg ggtgtccttc cccatacaga gattgcaggc
tgagaatgac tatatccttg 15300ttaacaggag gtgggagcag ggcacggtag
ctcacacctg taatcttggc actttaggag 15360gctgaggcgg gccgatcacc
tgaagtaagg agttcgagac cagcctggcc aacatgcaaa 15420gccctgtctc
tactaaaaat acaaaaatta gctgggtgtg gtggtactcg cctgtaatcc
15480cagctactcg ggagactgag gcaggagaat ggcttgaacc cggaaggtag
aggttgcagt 15540gagctgagat catgccactg tgctccagcc taggtgacag
agagagactc catctcaaaa 15600aaaaaaaaaa aatacaggaa gggagttggg
aatagggtgc acatttagga agtcttgggg 15660atttagtggt gggaaggttg
gaagtccctc tctgattgtc ttttcctcaa agaagtgcat 15720ggctggtgag
gggtggggca ggagtgcttg ggttgtggtg aaacattgga agagagaatg
15780tgaagcagcc attcttttcc tgctccacag gaagccgagc tgtctcagac
actggcatgg 15840tgttggggga gggggttcct tctctgcagg cccaggtgac
ccagggttgg aagtgtctca 15900tgctggatcc ccacttttcc tcttgcagca
gccagactgc cttccgggtc actgccatgg 15960aggagccgca gtcagatcct
agcgtcgagc cccctctgag tcaggaaaca ttttcagacc 16020tatggaaact
gtgagtggat ccattggaag ggcaggccca ccacccccac cccaacccca
16080gccccctagc agagacctgt gggaagcgaa aattccatgg gactgacttt
ctgctcttgt 16140ctttcagact tcctgaaaac aacgttctgg taaggacaag
ggttgggctg gggacctgga 16200gggctgggga cctggagggc tggggggctg
gggggctgag gacctggtcc tctgactgct 16260cttttcaccc atctacagtc
ccccttgccg tcccaagcaa tggatgattt gatgctgtcc 16320ccggacgata
ttgaacaatg gttcactgaa gacccaggtc cagatgaagc tcccagaatg
16380ccagaggctg ctccccccgt ggcccctgca ccagcagctc ctacaccggc
ggcccctgca 16440ccagccccct cctggcccct gtcatcttct gtcccttccc
agaaaaccta ccagggcagc 16500tacggtttcc gtctgggctt cttgcattct
gggacagcca agtctgtgac ttgcacggtc 16560agttgccctg aggggctggc
ttccatgaga cttcaatgcc tggccgtatc cccctgcatt 16620tcttttgttt
ggaactttgg gattcctctt caccctttgg cttcctgtca gtgttttttt
16680atagtttacc cacttaatgt gtgatctctg actcctgtcc caaagttgaa
tattcccccc 16740ttgaatttgg gcttttatcc atcccatcac accctcagca
tctctcctgg ggatgcagaa 16800cttttctttt tcttcatcca cgtgtattcc
ttggcttttg aaaataagct cctgaccagg 16860cttggtggct cacacctgca
atcccagcac tctcaaagag gccaaggcag gcagatcacc 16920tgagcccagg
agttcaagac cagcctgggt aacatgatga aacctcgtct ctacaaaaaa
16980atacaaaaaa ttagccaggc atggtggtgc acacctatag tcccagccac
ttaggaggct 17040gaggtgggaa gatcacttga ggccaggaga tggaggctgc
agtgagctgt gatcacacca 17100ctgtgctcca gcctgagtga cagagcaaga
ccctatctca aaaaaaaaaa aaaaaaagaa 17160aagctcctga ggtgtagacg
ccaactctct ctagctcgct agtgggttgc aggaggtgct 17220tacgcatgtt
tgtttctttg ctgccgtctt ccagttgctt tatctgttca cttgtgccct
17280gactttcaac tctgtctcct tcctcttcct acagtactcc cctgccctca
acaagatgtt 17340ttgccaactg gccaagacct gccctgtgca gctgtgggtt
gattccacac ccccgcccgg 17400cacccgcgtc cgcgccatgg ccatctacaa
gcagtcacag cacatgacgg aggttgtgag 17460gcgctgcccc caccatgagc
gctgctcaga tagcgatggt gagcagctgg ggctggagag 17520acgacagggc
tggttgccca gggtccccag gcctctgatt cctcactgat tgctcttagg
17580tctggcccct cctcagcatc ttatccgagt ggaaggaaat ttgcgtgtgg
agtatttgga 17640tgacagaaac acttttcgac atagtgtggt ggtgccctat
gagccgcctg aggtctggtt 17700tgcaactggg gtctctggga ggaggggtta
agggtggttg tcagtggccc tccaggtgag 17760cagtaggggg gctttctcct
gctgcttatt tgacctccct ataaccccat gagatgtgca 17820aagtaaatgg
gtttaactat tgcacagttg aaaaaactga agcttacaga ggctaagggc
17880ctcccctgct tggctgggcg cagtggctca tgcctgtaat cccagcactt
tgggaggcca 17940aggcaggcgg atcacgaggt tgggagatcg agaccatcct
ggctaacggt gaaaccccgt 18000ctctactgaa aaatacaaaa aaaaattagc
cgggcgtggt gctgggcacc tgtagtccca 18060gctactcggg aggctgagga
aggagaatgg cgtgaacctg ggcggtggag cttgcagtga 18120gctgagatca
cgccactgca ctccagcctg ggcgacagag cgagattcca tctcaaaaaa
18180aaaaaaaaaa ggcctcccct gcttgccaca ggtctcccca aggcgcactg
gcctcatctt 18240gggcctgtgt tatctcctag gttggctctg actgtaccac
catccactac aactacatgt 18300gtaacagttc ctgcatgggc ggcatgaacc
ggaggcccat cctcaccatc atcacactgg 18360aagactccag gtcaggagcc
acttgccacc ctgcacactg gcctgctgtg ccccagcctc 18420tgcttgcctc
tgacccctgg gcccacctct taccgatttc ttccatacta ctacccatcc
18480acctctcatc acatccccgg cggggaatct ccttactgct cccactcagt
tttcttttct 18540ctggctttgg gacctcttaa cctgtggctt ctcctccacc
tacctggagc tggagcttag 18600gctccagaaa ggacaagggt ggttgggagt
agatggagcc tggtttttta aatgggacag 18660gtaggacctg atttccttac
tgcctcttgc ttctcttttc ctatcctgag tagtggtaat 18720ctactgggac
ggaacagctt tgaggtgcgt gtttgtgcct gtcctgggag agaccggcgc
18780acagaggaag agaatctccg caagaaaggg gagcctcacc acgagctgcc
cccagggagc 18840actaagcgag gtaagcaagc aggacaagaa gcggtggagg
agaccaaggg tgcagttatg 18900cctcagattc acttttatca cctttccttg
cctctttcct agcactgccc aacaacacca 18960gctcctctcc ccagccaaag
aagaaaccac tggatggaga atatttcacc cttcaggtac 19020taagtcttgg
gacctcttat caagtggaaa gtttccagtc taacactcaa aatgccgttt
19080tcttcttgac tgttttacct gcaattgggg catttgccat cagggggcag
tgatgcctca 19140aagacaatgg ctcctggttg tagctaacta acttcagaac
accaacttat accataatat 19200atattttaaa ggaccagacc agctttcaaa
aagaaaattg ttaaagagag catgaaaatg 19260gttctatgac tttgcctgat
acagatgcta cttgacttac gatggtgtta cttcctgata 19320aactcgtcgt
aagttgaaaa tattgtaagt tgaaaatgga tttaatacac ctaatctaag
19380gaacatcata gcttagccta gcctgctttt tttttttttt tttttggaga
cagagtctca 19440ctctgtcacc caggctggag tgcagtggcg ggatctcggc
tcactgcaac ctccgccttc 19500tgggttcaag cgattctcct gcctcagccc
actgagtagc tgggattaca ggcacctgcc 19560ccgacgccca gctaattttt
tgttatttat ttattttttt ttttagtaga gatgaggttt 19620caccatgttg
gccaggctag tctcgaactc ctgaccttgt gatctgcctg ccttggcctc
19680ccaaagtgct gggattacag gcgtgagcca ccgcacccgg cctgcctagc
ctacttttat 19740tttattttta atggagacag catcttgctc tgttgcccag
gctggattac agtgatgtga 19800tcatagctca ttataccctc ctgggctcaa
gcaatccccc taactctgcc tccccagtag 19860ctaggaccac aggcatacac
caccataccc agctaatttt taaaattttt tgtagataga 19920tagagtctca
ctatgttgcc caggctggtc tctagcctac ttttttgaga caaggtcttg
19980ctctgtcacc caggctggat agagtgcagt agtgcagtca cagctcactg
cagcctccac 20040ctcccaggct ccatccatcc tcccagctca gcctcccaag
ttgcttcaac tacaggcctg 20100caccaccatg cctggctaat ttttatttat
ttatttttat tttattttat tttatttttt 20160tgagactcag tctcactctg
tcgcccaggc tggagtgcag tggcatgatc tcggctcact 20220gcaacctctg
cctcctgggt tcaagtgatt ctcctgcctc agcctcccga atagctagga
20280ctacaagcgc ctgctaccac gcccagctaa tttttgtatt tttagtagag
acagggtttc 20340accatgttgg ccaggctggt ctcgaacttc tgaccatgtg
atccgcccgc ctcggcctcc 20400caaagtgctg ggattacagg tgtgagccac
cacgcccggc taatttttat ttatttattt 20460aaagacagag tctcactctg
tcactcaggc tagagtgcag tggcaccatc tcagctcact 20520gcagccttga
cctccctggg ctccggtgat ttcaccctcc caagtagcta ggactacagg
20580cacatgccac gacacccagc taatttttta ttttctgtga agtcaaggtc
ttgctacgtt 20640gcccatgctg gtatcaaacc cctgggctca atcaatcctt
ccacctcagc ctccccaagt 20700attggggtta caggcatgag ctaccacact
cagccctagc ctacttgaaa cgtgttcaga 20760gcatttaagt taccctacag
ttgggcaaag tcatctaaca caaagccctt tttatagtaa 20820taaaatgttg
tatatctcat gtgatttatt gaatattgtt actgaaagtg agaaacagca
20880tggttgcatg aaaggaggca cagtcgagcc aggcacagcc tgggcgcaga
gcgagactca 20940aaaaaagaaa aggccaggcg cactggctca cgcctgtaat
cccagcattt cgggaggctg 21000aggcgggtgg atcacctgag gtcaggagtt
caagaccagc ctagccaaca tggtgaaacc 21060ccgtctctac taaaatacaa
aaattaaccg ggcgtgatgg caggtgcctg taatcccagc 21120tacttgggag
gctgaggcag gagaatcgct tgaaccagga ggcggaggtt gcagggagcc
21180aagatggcgc cactgcactc cagcctgggc gatagagtga gactccgtct
cagaaaaaaa 21240agaaaagaaa cgaggcacag tcgcatgcac atgtagtccc
agttacttga gaggctaagg 21300caggaggatc tcttgagccc aagagtttga
gtccagcctg aacaacatag caagacatca 21360tctctaaaat ttaaaaaagg
gccgggcaca gtggctcaca cctgtaatcc cagcactttg 21420ggaggtggag
gtgggtagat cacctgacgt caggagttgg aaaccagcct ggctaacatg
21480gtgaagcccc atctctacta aaaacacaaa aattagccag gtgtggtagc
acacgcctgt 21540agtcccagct actcgggagg ctgaggcaca agaatcactt
gaaccccaga ggcggagatt 21600gcaatcagcc aagattgcac cattgcactc
ccgcctgggc aacagagtga gaccccatct 21660caaaataaat aaataaatat
ttttaaaagt cagctgtata ggtacttgaa gtgcagtttc 21720tactaaatgc
atgttgcttt tgtaccgtca taaagtcaaa caattgtaac ttgaaccatc
21780ttttaactca ggtactgtgt atatacttac ttctccccct cctctgttgc
tgcagatccg 21840tgggcgtgag cgcttcgaga tgttccgaga gctgaatgag
gccttggaac tcaaggatgc 21900ccaggctggg aaggagccag gggggagcag
ggctcactcc aggtgagtga cctcagcccc 21960ttcctggccc tactcccctg
ccttcctagg ttggaaagcc ataggattcc attctcatcc 22020tgccttcatg
gtcaaaggca gctgacccca tctcattggg tcccagccct gcacagacat
22080ttttttagtc ttcctccggt tgaatcctat aaccacattc ttgcctcagt
gtatccacag 22140aacatccaaa cccagggacg agtgtggata cttctttgcc
attctccgca actcccagcc 22200cagagctgga gggtctcaag gaggggccta
ataattgtgt aatactgaat acagccagag 22260tttcaggtca tatactcagc
cctgccatgc accggcaggt cctaggtgac ccccgtcaaa 22320ctcagtttcc
ttatatataa aatggggtaa gggggccggg cgcagtggct cacgaatccc
22380acactctggg aggccaaggc gagtggatca cctgaggtcg ggagtttgag
cccagcctga 22440ccaacatgga gaaaccccat ctctactaaa aatacaaaag
tagccgggcg tggtgatgca 22500tgcctgtaat cccagctacc tactcgggag
gctgaggcag gagaatcgct tgaacccggg 22560aggcagaggt tgcggtgagc
tgagatctca ccattacact ccagcctggg caacaagagt 22620gaaactccgt
ctcaaaaaag ataaataaag taaaatgggg taagggaaga ttacgagact
22680aatacacact aatactctga ggtgctcagt aaacatattt gcatggggtg
tggccaccat 22740cttgatttga attcccgttg tcccagcctt aggcccttca
aagcattggt cagggaaaag 22800gggcacagac cctctcactc atgtgatgtc
atctctcctc cctgcttctg tctcctacag 22860ccacctgaag tccaaaaagg
gtcagtctac ctcccgccat aaaaaactca tgttcaagac 22920agaagggcct
gactcagact gacattctcc acttcttgtt ccccactgac agcctcccac
22980ccccatctct ccctcccctg ccattttggg ttttgggtct ttgaaccctt
gcttgcaata 23040ggtgtgcgtc agaagcaccc aggacttcca tttgctttgt
cccggggctc cactgaacaa 23100gttggcctgc actggtgttt tgttgtgggg
aggaggatgg ggagtaggac ataccagctt 23160agattttaag gtttttactg
tgagggatgt ttgggagatg taagaaatgt tcttgcagtt 23220aagggttagt
ttacaatcag ccacattcta ggtaggggcc cacttcaccg tactaaccag
23280ggaagctgtc cctcactgtt gaattttctc taacttcaag gcccatatct
gtgaaatgct 23340ggcatttgca cctacctcac agagtgcatt gtgagggtta
atgaaataat gtacatctgg 23400ccttgaaacc accttttatt acatggggtc
tagaacttga cccccttgag ggtgcttgtt 23460ccctctccct gttggtcggt
gggttggtag tttctacagt tgggcagctg gttaggtaga 23520gggagttgtc
aagtctctgc tggcccagcc aaaccctgtc tgacaacctc ttggtgaacc
23580ttagtaccta aaaggaaatc tcaccccatc ccacaccctg gaggatttca
tctcttgtat 23640atgatgatct ggatccacca agacttgttt tatgctcagg
gtcaatttct tttttctttt 23700tttttttttt ttttcttttt ctttgagact
gggtctcgct ttgttgccca ggctggagtg 23760gagtggcgtg atcttggctt
actgcagcct ttgcctcccc ggctcgagca gtcctgcctc 23820agcctccgga
gtagctggga ccacaggttc atgccaccat ggccagccaa cttttgcatg
23880ttttgtagag atggggtctc acagtgttgc ccaggctggt ctcaaactcc
tgggctcagg 23940cgatccacct gtctcagcct cccagagtgc tgggattaca
attgtgagcc accacgtcca 24000gctggaaggg tcaacatctt ttacattctg
caagcacatc tgcattttca ccccaccctt 24060cccctccttc tcccttttta
tatcccattt ttatatcgat ctcttatttt acaataaaac 24120tttgctgcca
cctgtgtgtc tgaggggtga acgccagtgc aggctactgg ggtcagcagg
24180tgcaggggtg agtgaggagg tgctgggaag cagccacctg agtctgcaat
gagtgtgggc
24240tggggggccc agtgcccggg ttccgggagg ggaacaaagg ctggagactg
ggtcagtctg 24300cgggctgcat gacaacaagg gagggggtgg ctccattcat
aactcaggaa ccaaccgtcc 24360ctcctcccct ccggccacgg ctggcacaag
gttctctccc tcccctgctt ctaggactgg 24420gctgcttccc cctcggcagc
ctctcaccaa ggattacggg atttaaatgt ctgatttagc 24480aaggctgagc
ctccagggtg gccatctgct ccatcagaaa gtggcaggat acctgggttc
24540ccaaggggaa caggggtggg tgctactgga tggagagagg ccagtgggag
gcctgctagc 24600cagggtccca ggaaagtggg ggcagctaag gtaagagtag
gggtgtgggg ctaggtcctt 24660cccagcatcc cctcatcctg ggcctcatgc
caggtagctg aatgaattga agctttaaac 24720tctgccagga aaacctttca
aagggcttct tgggataggg aggagagtcg ggttgaggag 24780ctcagtactg
cctgcccatg ctcctcaggg ctgctggctc ccagggaggg gggctgggag
24840caggcaggct cttccccatc acccactgct ctcttggagc cagtgcttga
aggggcagtc 24900agacatggct tgcccttcct cctccctggt ggtggagatg
ggtgttaggg tccagtgggt 24960gctactgtcc aggggggctt ctggggccac
cagcctgtca gctcatcaac caggctgaag 25020gtgcaagcag gagccccttg
ccttgcccca aggatcccag acagctatga agccaccagc 25080cttcctgacc
tcaagaccac cttttttttt tctctttctt actagggaat gccaaacact
25140ctccccagga gatccagacc cgcctctttc agagactttt aacttaaaca
tctgtcccta 25200cccagcaggc aaactagagc tcctgaagct cagtccctgt
ccttgcctct gtagacaggt 25260caccttgatg agcttccttt tttttttttt
aatttttttt tattttaggc tttattgggg 25320cataattgat cccccaaaat
tgcatacatt caaggtatgc agtgtgatga tttgatatgg 25380gggtatattg
tgaaaccatt accacaatca aattaatcag cacgtccatc atcacacaca
25440gttaccattt gtgtgtgtgc acgtgtgttc acctacgacg aggacacttg
gacctactct 25500gcagatctca agtaaacaga aaatctccct ttttgacaac
catcctccac cctttcaatc 25560ccaacctttt cctagattat gtccctagct
ctgtttttat ttctgctgtg ctgcttcaga 25620tccattctga ctctgccaaa
cccttctttg tgagctgata gattgctgga ttgagaatta 25680cagctgggcg
cggtggctca cgcctgtaat cccaacactg tgggaggcca aggccggcgg
25740atcacttgag gtcaggagtt ggagaccagc ctgaccaaca agatgaaacc
ccatctctac 25800taaaaataca aaattagctg ggcatggtgg tgcacgcctg
taatctcatc ttcttgggag 25860gctgaggcag gagaattgct tgaacccggg
aggtggaggt tgcagtgagc caagatcctg 25920ccattgcact ccagcctggg
caacaacagt gaagctccat ctcaaaacac acaaaaaaaa 25980gaagtacaaa
gtctgagact tcaggccagc tctgctacac tatatactct aacctctctg
26040gtcctacttg gtgacttctt tccctctggt cgtgttcaag ttcccgtccc
atccagtcaa 26100gcaggtactc attggtacct taccctgtgc caggagctgt
tctaggccct ggaaacctat 26160ggcagacatg ttccctaccc tcccactcaa
agagcccagg ccttatccta atgagatctg 26220aaatcaaatc tcccaatttc
ctcatggctt cagtctaaac ttgtaattca caaccttaaa 26280tcaatatgtt
ctattttttt atttagaaaa catttccggc caggcacggt ggatcacacc
26340tgtaatccca gctactcggg aggctgaggc aggagaatcg cttgaaccca
ggaggcagag 26400ggttgcagtg agccgagatt gcgccattgc actctagcct
gggcaacaga gcaagactcc 26460atctcaaaaa agaaaaaaaa atggaagaaa
aaaaaatttc cccctcattt taggaacacg 26520aggtctccaa atctaaaatt
cgtactctga ggagattgaa tagccttaaa tgctttcatc 26580attaaaaaga
aaagaaagga acctggtatg catcctaaaa atgaaaaata tacctacctg
26640taatcccagc acacagcaca ttgggaggct aaagcaggag gataacttga
ggccaggagt 26700ttcagatcag cctgggcaac atagcaacac cccatttctt
tttcttttct tttttttttg 26760gagacacagt ctcgctctgt tactcaggct
ggagtgcagt ggctcaatct cagctcactg 26820caagctctgc ctcccaggtt
catgccattc tcctgcctca gcctcccgag tagctgggac 26880tacaggcgcc
cgccaccacg cctggctaat tttttgtatt tttagtagag acagggtttc
26940accgtgttag ccaggatggt ctcgatctcc tgacctcgtg atccgccagc
cttggcctcc 27000taaagtactg ggattacagg cgtgagccac tgcgcctggc
cacaacaccc catttctatt 27060ttaataaaat aaaatactgt gaaaaacatt
tacaattttt aaattttaat tttaaaatta 27120aacttatatt tattcatttg
tgtgtgtggg tttttttttt tttttttttt tgcttttttt 27180ttgagatgga
gtgtcactct gtcacccagg ctggagtgca gtggcgtgat ctctgcctcc
27240cggttcaagt gattctcctg ccatagcctc ccaagtagct gggactacag
gtacacgcca 27300ccacgccggg ttaatttttg tatttttagt agagacagga
tttcactgtg tcgccaggct 27360agcctcgaac tcctgacctc aggtgattcg
cccaccttgg cctcccaaag tgctgtgatt 27420acaagcgtga gccaccgtgc
ccagcccaaa gttggtttta atagcagaaa atctatcaac 27480ataattcaat
atattaaatt tagaaagaaa aattatctat catatcaaca gatactgaaa
27540ggaatttgat taaatttcag tagccatttc cttaaaaaag aaaacacttt
aacacagtaa 27600tagactgata atggaatacc aattttccta ataagttaaa
cattaagata atttcaatta 27660aggtcaagag ctgggccagg tgcagtggct
cacacctgta atcccaacac tttggaggcc 27720aaggtgggtg gatcacctga
ggtcaggagt ggagaccagc ctggctgaca atagtgaaat 27780cctgcctcta
ctaaaaacac aaaaaattag ctgggcatgg tggtgggcac ctataatccc
27840agctactggg aaggctgaga caggagaatt gcttgaacct gggaggcgga
ggttgcagtg 27900agcaaagatc acaccattgc actccagcct gggcgacaga
gccagagtca gtctcaaaaa 27960aaaaaagagg tggccacacc tataatccaa
acattttgtg aggccaaggc aggagaattg 28020cttcaggcca agagttgaac
acctcgtcaa catagccaga cctctctcta gatagataga 28080tagatgatag
atagagagat agatagatga tagatagaga gatagataga tgatagatag
28140atagatagat agatagatag atagatagat agatagatag atagatagat
aatctggccg 28200ggtgtggagg ctcacgcctg taatcccagc actttgggag
gctgaggcgg gcagatcacg 28260aggacaagag attgaaacca tcctggctaa
caaggtgaaa ccccgtctct actaaaaata 28320caaaaaatta ggcgggtgtg
gtggcacgcg cctgtagtcc tagctattca ggaggctgag 28380acaggagaat
tgcttgaatc cgaaaggcgg aggttgcagc gagccgagat cgtgccactg
28440cactccagcc tgggtgacag agcaagactc catctcaaaa taaataaata
aataatcaag 28500aacagtataa ggggctgtat ggtggctcat gcctgtgatc
ccagcacttt gggaggccaa 28560ggtgggagga tcccttgaga ccagcccagg
caacagagaa agaccctgtc tctatttaaa 28620aaaattaaaa actggccggg
cacggtggct cacgcctgta attccagcgc ttgggaggcc 28680aaggcaggca
catcaggagg tcaggagttc gagaccagcc tggccaacgt ggtgaaaccc
28740cgtctctact aaaaatacaa aaagtagcta ggcgtggtgg caggcacctg
taatcccagc 28800tacttgggag gctgaggcag gagaatcgct tgaacccagg
aggcggaggt tgcagtgggc 28860aaagatcgtg ccattgcact cagcctgggt
gacagggcaa gactccatct caaaataaat 28920aaacaaagta attaattaat
taaattaaaa actgtgggga tatagactta ctctggtttt 28980attttttctt
ttcttttctt ttcttttttc tgagacggag tctcgctctg ttgcccaggc
29040tggagtacag tggcgtggtt tctgttctct gcaacctcca cctcccggat
tcaagcgatt 29100ctcttgcctc agcctcttga atacctggaa ttacaggtgc
ctgccaccac ccccggctaa 29160ttttttgtat ttttagtaga gacagggttt
caccatgttg gccaagctgg tctcgaactc 29220ctgacctcat gatccacccg
cctctgcctc ccaaagcact gagactacag gagtgagcca 29280ctgtgcccag
cctactctgg ttttagtgca ttcaagagga acaaaaaagg aagaaaatca
29340ctagtaaata tacctctttc tggttagagt ggatgtttgg aaattatata
tatattatat 29400tatattatat atattatata tatacacaaa cacgtacata
catgcacaca catatatgcc 29460tttttgatta taggatagta taccaaaact
cagaaatatt atggaattaa cagaatttag 29520taaggcagat aagtagtagg
tagaaaaata ttaattttat cttccagcag aagcactgtg 29580aaaaattaga
caacaagaaa acattccatt caaaataatg acaataaggc cgggcatggt
29640ggctcacacc tgtaatccca gcactttggg aggctgaggc aggaggatca
tctgaggtca 29700agtttgagat cagcctggcc aacatggtga caccctgtct
ctactgaaaa tacaaaaatc 29760agccagctat ggtagtgtaa gcctgtaatt
ccagctactc gggaggtcga agcagaagaa 29820tcacttgaac ccaggaggca
gagattgcag tgagccaaga tcctgccagt gctttccagc 29880ctgggcaaca
gtgtgaggct ccatctcaaa aaaaaaaaaa aaaaaaagac aatagcaata
29940aacattaaga aatgtgtaat aggaatggca cacacaaaga aggaatggca
cagagcctgt 30000atgcagaaga ccacaaaccc ttatttaacg acgtaagcca
agatccaaag aaaatgatag 30060attctcagat gggaaaacta aaaaaataag
aaaaatcaat tatctcgaga taaatataat 30120ataatgcaat ttcaattaga
atcccaaatt ttcattgtgt gtgtgtgtga gttgggtaaa 30180tttatcataa
atgtatagga acgagtaagt gtcactagtt gtttaaataa atactggatt
30240tgggccaggc atggtggctc acgcctctaa tcccagcact ttgggagacc
gaggcgggca 30300gatcatgagg tcaggagatc gagaccatct ggccaacata
gtgaaaactc gtctctacta 30360aagatacaaa aaattagctg ggcatggtgg
cacgtgcctg tagttccagc tactctggag 30420gctgaggcag gagagttgct
tgaacccggg aggtggaggt tgcaatgagc cgagatcctg 30480tcactgcact
ccaccctggc gacaaagtga gactccgtct ctctctctct ctttaggcca
30540aggcaggtgg atcacctgag gtcaggagtt caagacagcc tggccaacat
agcgaaatcc 30600catctctact aaaaatacaa aaattagcct ggcagtggtg
gcccacgcct gtaatcccag 30660ctactaaggg ggctgaggca ggaggatctc
ttaaccaggg aggaggaggt tgcagtgagc 30720agagattgtg ccactgcact
ccagcctgtg caacagagtg agactctgtc tcaaaaaaaa 30780taaataaaca
aaatactgga ggccgggcac ggtggctccc gcctgtaatc ccagcacttt
30840gggaggccaa ggcgggtgaa tcgctttcag ctcaggagtt ccggaccagt
ctgggcaaca 30900tggcaaaacc ccgtctatac taaaaataca aaacttagcc
aggcgtggta gtgcatacct 30960ataatcccag ctactcgaga ggctgaggca
ggagaatccc ttgaaaccgg gaggcagagg 31020ttgcagtaag ctgaaatcgt
gccactgcac tccagcctgg acgacacagc gggagactgt 31080ctcaataaat
aaaataaata atataaaata acataaataa taaaattgta aataataagt
31140aaataataag caacagaatg gagagggggt cctatttgcc ttgccagatt
ttagagaact 31200tagtataggc taggcaggtg cagtggctca cgcctgtaat
cccagcactt tgggagtcca 31260aggcagtgga tcacatgagg tcaggagttc
aagaccagcc tgaccaacat ggtgaaaccc 31320catctctact aaaaatacaa
aattagccag gcatggtggc acatgcctgt aatcccagct 31380acttgggagg
ctgaggcagg agaatcactt gaactcgaga ggtggaagct gcagcgagct
31440gagatcacgc tactgtactc cagcctgggc aacaagagtg agactccatc
aaaaaaaaaa 31500aaaaagaaac gagaaaaaaa aaaagaaaac ttactatata
actgctggaa tgaggtggat 31560gtgacatatg gagtggtaga caaaccaagg
aaagcaagta acaaatacag gagatgttct 31620catacaggag aattattaca
tggccaaaat tagcatttca atttaattgg aacacattag 31680atatatttgt
tcttttgttc tttttgttat cttttttttt tttttttttt tttttttttt
31740gagacagagt ttcgctctca ttgcccaggc tggagtgtaa tggtgtgatc
ttgactcacc 31800gcaacctccg cttcctacgt tgaagtgatt ctcctgcctc
agcctcccga gtagccggga 31860ttacaggcat acgccaccac acctggctaa
ttttttttgc attttcagta gagacagggt 31920ttctccatgt tgatcaggct
ggtttcgaac tcctgagctt aggtgatccg cccacctcag 31980cctcccaaag
tgctgggatt acaggcgtga gccactgtgc ccggccccta tctctttttt
32040tgtttgtttg ttttctgaga tggaatctgg ctctctcacc caggctagag
tgcaatggca 32100cgatgttggc tcactgcaac atccacgtcc cgggttcaag
cgattcttct gcctcagcct 32160cccaagtagc tgggattaca ggtgcctgtc
gccacatcca gctaattttt tgtattttta 32220gtagagacag ggtttcaccg
tgttccccag gctggtctca aactcctgag ctccggcaat 32280ccacccgcct
cggcttccca aagcgctagg attacaggcg tgagccaccg cacctggccc
32340ctatctctta aaaatatatt tttttgccca acacacattt ccaagttgcc
ttgggggaaa 32400aaaaataaat gaagctggca caactgaaaa aataaaactg
gggccttggc caggcacagt 32460ggctcaggcc tataatccca gcactttggg
aagctgaggt gggaggatca cttgaggtca 32520ggagttcgag accagtctgg
ctaacacggt gaaaaccctt ctctactaaa aatacaaaag 32580tcagccaggc
gtagtggtgt gcacctgtaa tcccagctac tcaggtagct gaggcatgag
32640aatcacatga acctggaaag tggaggttgc agtgagccga gattgcacca
ctgcactcca 32700gcctgggtaa ggaaatgaga ctctgtctcc aaaaaaaaaa
aaaagatact acaaagtcaa 32760gagacaaaca at 327724479PRTHomo sapiens
4Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser 1
5 10 15 Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro
Gln 20 25 30 Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe
Gln Arg Leu 35 40 45 Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu
Ser Phe Leu Lys Glu 50 55 60 Leu Leu Phe Arg Ile Asn Arg Leu Asp
Leu Leu Ile Thr Tyr Leu Asn 65 70 75 80 Thr Arg Lys Glu Glu Met Glu
Arg Glu Leu Gln Thr Pro Gly Arg Ala 85 90 95 Gln Ile Ser Ala Tyr
Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val 100 105 110 Ser Arg Ser
Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile 115 120 125 Ser
Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile 130 135
140 Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu
145 150 155 160 Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys
Ile Ile Asn 165 170 175 Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Ser
Ser Leu Glu Gly Ser 180 185 190 Pro Asp Glu Phe Ser Asn Gly Glu Glu
Leu Cys Gly Val Met Thr Ile 195 200 205 Ser Asp Ser Pro Arg Glu Gln
Asp Ser Glu Ser Gln Thr Leu Asp Lys 210 215 220 Val Tyr Gln Met Lys
Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn 225 230 235 240 Asn His
Asn Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His Ser 245 250 255
Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr 260
265 270 Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pro His Asp Asp Cys
Thr 275 280 285 Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu
Met Asp His 290 295 300 Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Leu
Ser His Gly Asp Lys 305 310 315 320 Gly Ile Ile Tyr Gly Thr Asp Gly
Gln Glu Ala Pro Ile Tyr Glu Leu 325 330 335 Thr Ser Gln Phe Thr Gly
Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro 340 345 350 Lys Val Phe Phe
Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly 355 360 365 Ile Pro
Val Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met Asp 370 375 380
Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe Leu 385
390 395 400 Leu Gly Met Ala Thr Val Asn Asn Cys Val Ser Tyr Arg Asn
Pro Ala 405 410 415 Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser
Leu Arg Glu Arg 420 425 430 Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile
Leu Thr Glu Val Asn Tyr 435 440 445 Glu Val Ser Asn Lys Asp Asp Lys
Lys Asn Met Gly Lys Gln Met Pro 450 455 460 Gln Pro Thr Phe Thr Leu
Arg Lys Lys Leu Val Phe Pro Ser Asp 465 470 475 5416PRTHomo sapiens
5Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu 1
5 10 15 Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser
Arg 20 25 30 Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg
Ala Gly Ser 35 40 45 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile
Ile Asp Leu Glu Thr 50 55 60 Arg Gly Ser Gln Ala Leu Pro Leu Phe
Ile Ser Cys Leu Glu Asp Thr 65 70 75 80 Gly Gln Asp Met Leu Ala Ser
Phe Leu Arg Thr Asn Arg Gln Ala Ala 85 90 95 Lys Leu Ser Lys Pro
Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg 100 105 110 Pro Glu Ile
Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro 115 120 125 Val
Asp Ile Gly Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser 130 135
140 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys
145 150 155 160 Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg
Glu Ser Gly 165 170 175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys
Glu Lys Leu Arg Arg 180 185 190 Arg Phe Ser Ser Leu His Phe Met Val
Glu Val Lys Gly Asp Leu Thr 195 200 205 Ala Lys Lys Met Val Leu Ala
Leu Leu Glu Leu Ala Gln Gln Asp His 210 215 220 Gly Ala Leu Asp Cys
Cys Val Val Val Ile Leu Ser His Gly Cys Gln 225 230 235 240 Ala Ser
His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys 245 250 255
Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys 260
265 270 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys
Gly 275 280 285 Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr
Ser Pro Glu 290 295 300 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp
Ala Thr Pro Phe Gln 305 310 315 320 Glu Gly Leu Arg Thr Phe Asp Gln
Leu Asp Ala Ile Ser Ser Leu Pro 325 330 335 Thr Pro Ser Asp Ile Phe
Val Ser Tyr Ser Thr Phe Pro Gly Phe Val 340 345 350 Ser Trp Arg Asp
Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp 355 360 365 Asp Ile
Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 370 375 380
Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385
390 395 400 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys
Thr Ser 405 410 415 6393PRTHomo sapiens 6Met Glu Glu Pro Gln Ser
Asp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 10 15 Glu Thr Phe Ser
Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 25 30 Ser Pro
Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 35 40 45
Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro 50
55 60 Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala
Pro 65 70 75 80 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu
Ser Ser Ser
85 90 95 Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg
Leu Gly 100 105 110 Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys
Thr Tyr Ser Pro 115 120 125 Ala Leu Asn Lys Met Phe Cys Gln Leu Ala
Lys Thr Cys Pro Val Gln 130 135 140 Leu Trp Val Asp Ser Thr Pro Pro
Pro Gly Thr Arg Val Arg Ala Met 145 150 155 160 Ala Ile Tyr Lys Gln
Ser Gln His Met Thr Glu Val Val Arg Arg Cys 165 170 175 Pro His His
Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln 180 185 190 His
Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp 195 200
205 Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu
210 215 220 Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys
Asn Ser 225 230 235 240 Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile
Leu Thr Ile Ile Thr 245 250 255 Leu Glu Asp Ser Ser Gly Asn Leu Leu
Gly Arg Asn Ser Phe Glu Val 260 265 270 Arg Val Cys Ala Cys Pro Gly
Arg Asp Arg Arg Thr Glu Glu Glu Asn 275 280 285 Leu Arg Lys Lys Gly
Glu Pro His His Glu Leu Pro Pro Gly Ser Thr 290 295 300 Lys Arg Ala
Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys 305 310 315 320
Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu 325
330 335 Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys
Asp 340 345 350 Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His
Ser Ser His 355 360 365 Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg
His Lys Lys Leu Met 370 375 380 Phe Lys Thr Glu Gly Pro Asp Ser Asp
385 390
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