U.S. patent application number 16/456453 was filed with the patent office on 2020-04-30 for therapy for kinase-dependent malignancies.
The applicant listed for this patent is CHILDREN'S HOSPITAL MEDICAL CENTER. Invention is credited to Mohammad AZAM, Meenu KESARWANI.
Application Number | 20200129456 16/456453 |
Document ID | / |
Family ID | 64656446 |
Filed Date | 2020-04-30 |
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United States Patent
Application |
20200129456 |
Kind Code |
A1 |
AZAM; Mohammad ; et
al. |
April 30, 2020 |
THERAPY FOR KINASE-DEPENDENT MALIGNANCIES
Abstract
A pharmaceutically acceptable composition and method of therapy
for a kinase-dependent malignancy in a patient in need of such
therapy is provided. The composition contains, as the only active
agents, the combination of (a) an inhibitor of c-Fos, (b) an
inhibitor of Dusp-1, and (c) an inhibitor of a tyrosine kinase. The
composition is administered to the patient in a dosing regimen for
a period sufficient to provide therapy for kinase-dependent
malignancy. Also provided is a method to eradicate leukemia
initiating cells (LIC) or cancer stem cells (CSC) in a patient
being treated with a tyrosine kinase inhibitor.
Inventors: |
AZAM; Mohammad; (Mason,
OH) ; KESARWANI; Meenu; (Mason, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S HOSPITAL MEDICAL CENTER |
Cincinnati |
OH |
US |
|
|
Family ID: |
64656446 |
Appl. No.: |
16/456453 |
Filed: |
June 28, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15900201 |
Feb 20, 2018 |
10342767 |
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16456453 |
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15866544 |
Jan 10, 2018 |
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15900201 |
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14048806 |
Oct 8, 2013 |
9877934 |
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15866544 |
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PCT/US2012/034359 |
Apr 20, 2012 |
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14048806 |
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61477853 |
Apr 21, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/517 20130101;
A61K 2300/00 20130101; A61K 31/506 20130101; A61K 31/135 20130101;
A61K 31/12 20130101; A61K 31/045 20130101; A61K 45/06 20130101;
A61K 31/045 20130101; A61K 2300/00 20130101; A61K 31/12 20130101;
A61K 2300/00 20130101; A61K 31/135 20130101; A61K 2300/00 20130101;
A61K 31/506 20130101; A61K 2300/00 20130101; A61K 31/517 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 31/135 20060101
A61K031/135; A61K 31/045 20060101 A61K031/045; A61K 31/506 20060101
A61K031/506; A61K 31/12 20060101 A61K031/12; A61K 45/06 20060101
A61K045/06; A61K 31/517 20060101 A61K031/517 |
Claims
1. A pharmaceutically acceptable composition comprising at least
one biocompatible excipient and, as the only active agents, (a) a
c-Fos inhibitor, (b) a Dusp-1 inhibitor, and (c) at least one
oncogenic kinase inhibitor, where the oncogenic kinase is selected
from the group consisting of BCR-ABL, BTK, FLT3, MET, KIT, JAK2,
MEK, EGFR, PDGFR, ALK, HER2, B-Raf, FGFR2, RAF, PI3K, and
combinations thereof.
2. The pharmaceutically acceptable composition of claim 1 wherein,
(a) the c-Fos inhibitor is selected from the group consisting of
curcumin, difluorinated curcumin (DFC),
[3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazo-
l-6-yl)methoxy]phenyl}propionic acid] (T5224), nordihydroguaiaretic
acid (NOGA), dihydroguaiaretic acid (DHGA), and
[(E,E,Z,E)-3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1--
yl)-2,4,6,8-nonatetraenoic acid (SR11302); (b) the Dusp-1 inhibitor
is selected from the group consisting of
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI--also known as NSC 150117), TP1-2, TP1-3, and triptolide; and
(c) the tyrosine kinase inhibitor is selected from the group
consisting of lmatinib, Dasatinib, Ponatinib or Nilotinib when the
oncogenic kinase is BCR-ABL; lbrutinib when the oncogenic kinase is
BTK; Ruxolitinib, Crizotinib, or Quizartinib when the oncogenic
kinase is one of FL T3, MET, KIT, or JAK2; Ruxolitinib or
Trametinib when the oncogenic kinase is JAK2 or MEK; Gefitinib or
Axitinib when the oncogenic kinase is one of EGFR, PDGFR, or ALK;
Gefitinib, Axitinib, or dasatinib when the oncogenic kinase is one
of EGFR or PDGFR; Gefitinib or Axitinib when the oncogenic kinase
is one of HER2 or EGFR; Vemurafenib or Sorafenib when the oncogenic
kinase is one of B-Raf or MEK; Crizotinib or Dasatinib when the
oncogenic kinase is one of MET, FGFR2, or HER2; Ceritinib,
Alectinib or Crizotinib when the oncogenic kinase is one of MET,
FGFR2, or HER2; Ceritinib, Alectinib or Crizotinib when the
oncogenic kinase is one of ALK, KIT, or FGFR; and Vemurafenib,
Sorafenib or ldelalisib when the oncogenic kinase is one of RAF or
PI3K.
3. (canceled)
4. A method of treating a kinase-dependent malignancy in a patient,
the method comprising administering to the patient in need thereof
a composition containing at least one biocompatible excipient and,
as the only active agents, a combination of (a) an inhibitor of
c-Fos resulting in inhibition of c-Fos, (b) an inhibitor of Dusp-1
resulting in inhibition of Dusp-1, and (c) at least one inhibitor
of an oncogenic kinase resulting in inhibition of the oncogenic
kinase, wherein the composition is administered to the patient in a
dosing regimen for a period sufficient to provide treatment for the
kinase-dependent malignancy in the patient in need thereof.
5. The method of claim 4, wherein (a) the c-Fos inhibitor is
selected from the group consisting of curcumin, difluorinated
curcumin (DFC),
[3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazo-
l-6-yl)methoxy]phenyl}propionic acid] (T5224), nordihydroguaiaretic
acid (NOGA), dihydroguaiaretic acid (DHGA), and
[(E,E,Z,E)-3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1--
yl)-2,4, 6,8-nonatetraenoic acid (SR11302); and (b) the Dusp-1
inhibitor is selected from the group consisting of
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI--also known as NSC 150117), TP1-2, TP1-3, and triptolide.
6. The method of claim 4, wherein the kinase-dependent malignancy
is: Chronic myeloid leukemia (CIVIL) and the at least one inhibitor
is lmatinib, Dasatinib, Ponatinib and/or Nilotinib; Chronic
lymphocytic leukemia (CLL) and the at least one inhibitor is
lbrutinib; Acute myeloid leukemia (AML) and the at least one
inhibitor is Ruxolitinib, Crizotinib, and/or Quizartinib;
Myeloproliferative Neoplasm (MPN) and the at least one inhibitor is
Ruxolitinib and/or Trametinib; lung cancer and the at least one
inhibitor is Gefitinib and/or Axitinib; brain tumor and the at
least one inhibitor is Gefitinib, Axitinib, and/or Dasatinib;
breast cancer and the at least one inhibitor is Gefitinib and/or
Axitinib; bladder carcinoma and the at least one inhibitor is
Gefitinib and/or Axitinib; melanoma and the at least one inhibitor
is Vemurafenib and/or Sorafenib; pancreatic cancer and the at least
one inhibitor is Crizotinib and/or Dasatinib; colon cancer and the
at least one inhibitor is Ceritinib, Alectinib and/or Crizotinib;
and prostate cancer and the at least one inhibitor is Vemurafenib,
Sorafenib and/or ldelalisib.
7. The method of claim 4, wherein the treatment is curative.
8. A method to eradicate leukemia initiating cells (LIC) or cancer
stem cells (CSC) in a patient being treated with a tyrosine kinase
inhibitor (TKI), the method comprising administering to the patient
in need thereof a composition containing at least one biocompatible
excipient and a combination of (a) an inhibitor of c-Fos resulting
in inhibition of c-Fos, and (b) an inhibitor of Dusp-1 resulting in
inhibition of Dusp-1, the composition administered to the patient
in a dosing regimen for a period sufficient to eradicate the LIC or
CSC cells.
Description
[0001] This application is a continuation-in-part of co-pending
U.S. application Ser. No. 15/866,544 filed Jan. 10, 2018, which is
a continuation of U.S. application Ser. No. 14/048,806 filed Oct.
8, 2013, which is a continuation-in-part of International
Application Serial No. PCT/US2012/034359 filed Apr. 20, 2012, which
claims priority to U.S. Provisional Application Ser. No. 61/477,853
filed Apr. 21, 2011, each of which is expressly incorporated by
reference herein in its entirety.
[0002] In one embodiment, a composition and method of using the
composition to effect therapy for a kinase-dependent malignancy is
provided. In one embodiment, a composition and method of using the
composition to effect therapy for leukemia is provided. In one
embodiment, therapy is for chronic myelogenous leukemia. In one
embodiment, therapy is for acute myelogenous leukemia. Therapy for
targeting cancer stem cells and other leukemias are included. In
one embodiment, the kinase-dependent malignancy is a solid tumor.
As used herein, therapy and treatment are broadly defined to
encompass disease cure, or any lessening of disease presence,
prevalence, severity, symptoms, etc. In one embodiment, therapy
means curative therapy.
[0003] In one embodiment, the composition contains at least one
biocompatible excipient and, as its only active agents, the
combination of at least one inhibitor of c-Fos, at least one
inhibitor of Dusp-1, and at least one inhibitor of an oncogenic
kinase. In one embodiment, the patient is already receiving at
least one inhibitor of an oncogenic kinase for a kinase-dependent
malignancy, and the composition contains at least one biocompatible
excipient and, as its only active agents, the combination of at
least one inhibitor of c-Fos and at least one inhibitor of Dusp-1.
In one embodiment, the oncogenic kinase is at least one of the
tyrosine kinases listed in Table 1, and the at least one inhibitor
of a tyrosine kinase is selected from Imatinib, Dasatinib,
Ponatinib, Nilotinib, Ibrutinib, Ruxolitinib, Crizotinib,
Quizartinib, Trametinib, Gefitinib, Axitinib, Dasatinib,
Vemurafenib, Sorafenib, Ceritinib, Alectinib, Vemurafenib, and
Idelalisib.
TABLE-US-00001 TABLE 1 BCR-ABL A gene formed when pieces of
chromosomes 9 and 22 break off and trade places. The ABL gene from
chromosome 9 joins to the BCR gene on chromosome 22, to form the
BCR-ABL fusion gene. The changed chromosome 22 with the fusion gene
on it is called the Philadelphia chromosome. The BCR-ABL fusion
gene is found in most patients with chronic myelogenous leukemia
(CML), and in some patients with acute lymphoblastic leukemia (ALL)
or acute myelogenous leukemia (AML). Inhibitors-Imatinib,
Dasatinib, Ponatinib and Nilotinib BTK Bruton's tyrosine kinase
(BTK) also known as tyrosine-protein kinase BTK is an enzyme that
in humans is encoded by the BTK gene. BTK is a kinase that plays a
crucial role in B-cell development. FLT3 FMS-like tyrosine kinase 3
(FLT3), which is involved in the formation and growth of new blood
cells. Mutated (changed) forms of the FLT3 gene may cause an
over-active FLT3 protein to be made. This may cause the body to
make too many immature white blood cells. These changes have been
found in some types of leukemia, including acute myeloid leukemia
(AML) and acute lymphoblastic leukemia (ALL). MET a member of the
receptor tyrosine kinase family of proteins and the product of the
proto-oncogene MET. KIT Mast/stem cell growth factor receptor
(SCFR), also known as proto-oncogene c-Kit or tyrosine-protein
kinase Kit or CD117, is a receptor tyrosine kinase protein that in
humans is encoded by the KIT gene. JAK2 Janus kinase 2 (commonly
called JAK2) is a non-receptor tyrosine kinase. It is a member of
the Janus kinase family and has been implicated in signaling by
members of the type II cytokine receptor family (e.g. interferon
receptors), the GM-CSF receptor family (IL-3R, IL-5R and GM-CSF-R),
the gp130 receptor family (e.g., IL-6R), and the single chain
receptors (e.g. Epo-R, Tpo-R, GH-R, PRL-R). JAK2 signaling is
activated downstream from the prolactin receptor. MEK
Mitogen-activated protein kinase kinase (also known as MAP2K, MEK,
MAPKK) is a kinase enzyme which phosphorylates mitogen-activated
protein kinase (MAPK). EGFR The epidermal growth factor receptor
(EGFR; ErbB-1; HER1 in humans) is a transmembrane protein that is a
receptor for members of the epidermal growth factor family (EGF
family) of extracellular protein ligands. The epidermal growth
factor receptor is a member of the ErbB family of receptors, a
subfamily of four closely related receptor tyrosine kinases: EGFR
(ErbB-1), HER2/neu (ErbB-2), Her3 (ErbB-3) and Her4 (ErbB-4). PDGFR
Platelet-derived growth factor receptors (PDGF-R) are cell surface
tyrosine kinase receptors for members of the platelet-derived
growth factor (PDGF) family. PDGF subunits -A and -B are important
factors regulating cell proliferation, cellular differentiation,
cell growth, development and many diseases including cancer. ALK
Anaplastic lymphoma kinase (ALK) also known as ALK tyrosine kinase
receptor or CD246 (cluster of differentiation 246) is an enzyme
that in humans is encoded by the ALK gene. HER2 Receptor
tyrosine-protein kinase erbB-2, also known as CD340 (cluster of
differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2
(human), is a protein that in humans is encoded by the ERBB2 gene.
It is also frequently called HER2 (from human epidermal growth
factor receptor 2) or HER2/neu. HER2 is a member of the human
epidermal growth factor receptor (HER/EGFR/ERBB) family. B-Raf BRAF
is a human gene that encodes a protein called B-Raf. The gene is
also referred to as proto-oncogene B-Raf and v-Raf murine sarcoma
viral oncogene homolog B, while the protein is more formally known
as serine/ threonine-protein kinase B-Raf. The B-Raf protein is
involved in sending signals inside cells which are involved in
directing cell growth. FGFR2 Fibroblast growth factor receptor 2
(FGFR2) also known as CD332 (cluster of differentiation 332) is a
protein that in humans is encoded by the FGFR2 gene residing on
chromosome 10. FGFR2 is a receptor for fibroblast growth factor.
RAF RAF kinases are a family of three serine/ threonine-specific
protein kinases that are related to retroviral oncogenes. RAF is an
acronym for Rapidly Accelerated Fibrosarcoma. RAF kinases
participate in the RAS-RAF-MEK-ERK signal transduction cascade,
also referred to as the mitogen-activated protein kinase (MAPK)
cascade. Activation of RAF kinases requires interaction with
RAS-GTPases. The three RAF kinase family members are A-RAF, B-RAF,
and c-Raf. PI3K Phosphatidylinositol-4,5-bisphosphate 3-kinase
(also called phosphatidylinositide 3-kinases,
phosphatidylinositol-3-kinases, PI 3-kinases, P1(3)Ks, or PI-3Ks)
are a family of enzymes involved in cellular functions such as cell
growth, proliferation, differentiation, motility, survival and
intracellular trafficking, which in turn are involved in cancer.
PI3Ks are a family of related intracellular signal transducer
enzymes capable of phosphorylating the 3 position hydroxyl group of
the inositol ring of phosphatidylinositol (Ptdlns).
[0004] In one embodiment, the composition contains at least one
biocompatible excipient and, as its only active agents, the
combination of at least one inhibitor of c-Fos, at least one
inhibitor of Dusp-1, and at least one inhibitor of BCR-ABL tyrosine
kinase. In one embodiment, the composition contains at least one
biocompatible excipient and, as its only active agents, the
combination of one inhibitor of c-Fos, one inhibitor of Dusp-1, and
one inhibitor of BCR-ABL tyrosine kinase. In the aforementioned
embodiments, the inhibitor may inhibit the gene and/or the protein,
i.e., the c-Fos inhibitor may inhibit the c-Fos gene and/or
protein, the Dusp-1 inhibitor may inhibit the Dusp-1 gene and/or
protein, and the BCR-ABL tyrosine kinase inhibitor may inhibit the
BCR-ABL tyrosine kinase gene and/or protein. Such inhibitors
include commercially available inhibitors and inhibitors under
development. Small molecule inhibitors, such as curcumin,
difluorinated curcumin (DFC),
[3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazo-
l-6-yl) methoxy]phenyl}propionic acid] (T5224, Roche),
nordihydroguaiaretic acid (NDGA), dihydroguaiaretic acid (DHGA),
[(E,E,Z,E)-3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1--
yl)-2,4,6,8-nonatetraenoic acid (SR11302, Tocris Biosciences),
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI), TPI-2, TPI-3, triptolide, Imatinib mesylate (Gleevec.TM.),
Nilotinib, Dasatinib and Ponatinib, are encompassed. In one
embodiment, inhibitors of c-Fos used in the composition are
curcumin, difluorinated curcumin (DFC),
[3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benz-
isoxazol-6-yl) methoxy]phenyl}propionic acid] (T5224, Roche),
nordihydroguaiaretic acid (NDGA), dihydroguaiaretic acid (DHGA),
and
[(E,E,Z,E)-3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1--
yl)-2,4,6,8-nonatetraenoic acid (SR11302, Tocris Biosciences). In
one embodiment, inhibitors of Dusp-1 are
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI), also known as NSC 150117, TPI-2, TPI-3, and triptolide. In
one embodiment, inhibitors of BCR-ABL tyrosine kinase are Imatinib
mesylate (Gleevec.TM.), Nilotinib, Dasatinib and Ponatinib. In one
embodiment, the composition administered is curcumin, BCI, and
Imatinib. In one embodiment, the composition administered is
difluorinated curcumin (DFC), BCI, and Imatinib. In one embodiment,
the composition administered is NDGA, BCI, and Imatinib. In one
embodiment, the composition is T5224, BCI, and Imatinib. In one
embodiment, the composition is administered to the patient at a
concentration of 2 grams per day to 8 grams per day, inclusive, of
the c-Fos inhibitor, 100 mg per day to 600 mg per day, inclusive,
of BCI, and 400 mg to 800 mg per day, inclusive, of the BCR-ABL
tyrosine kinase inhibitor Imatinib mesylate (Gleevec.TM.). The
composition is alkaline, about pH 8.5. In one embodiment, the
composition is administered to the patient for 30 days. The
composition may be administered by any route including but not
limited to intravenous administration. The composition is
preferably administered intravenously, orally, intramuscularly,
transdermally, and/or intraperitoneally. Any biocompatible
excipient may be used in the inventive composition, as known to one
skilled the art. Biocompatible excipients include, but are not
limited to, buffers, tonicity agents, pH modifying agents,
preservatives, stabilizers, penetrant enhances, osmolality
adjusting agents, etc. In one embodiment, the composition
components are administered as individual components by the same
route of administration or by different routes of administration,
with administration of each component or components at
substantially the same time. In one embodiment, the composition
components are formulated into a cocktail, using methods known by
one skilled in the art.
[0005] Cancer can be treated by identifying a molecular defect.
This was demonstrated with chronic myelogenous leukemia (CML), the
first cancer to be associated with a defined genetic abnormality,
BCR-ABL, and the success of the small molecule tyrosine kinase
inhibitor (TKI) Imatinib.
[0006] Despite Imatinib's efficacy in treating CML patients, it
failed to provide a curative response because it preferentially
targets the differentiated and dividing cells, therefore causing
relapse upon Imatinib withdrawal. The major limitation to develop
curative therapy is lack of understanding of the molecular and
patho-physiological mechanisms driving cancer maintenance,
progression, mechanisms of therapeutic response and relapse. As in
the case of CML, differentiated and dividing cells undergo
apoptosis following the acute inhibition of BCR-ABL, termed
"oncogene addiction". In contrast, leukemic stem cells (LSCs) do
not show similar response. Given the intrinsic resistance of LSCs
to TKI therapy in CML, understanding the molecular mechanisms of
oncogene addiction in therapeutically responsive cells would allow
strategies to target the LSCs.
[0007] More specifically, the BCR-ABL tyrosine kinase inhibitor
Imatinib improved the survival of patients with leukemia, but did
not eliminate leukemia initiating cells (LIC). This suggested that
LICs were not addicted to BCR-ABL.
[0008] In one aspect, the inventive method demonstrates that the
down-regulation of c-Fos and Dusp-1 mediate BCR-ABL addiction, and
that inhibition of c-Fos and Dusp-1 together induces apoptosis in
BCR-ABL positive cells following Imatinib treatment. Furthermore,
it has also been found that inhibition of c-Fos and Dusp-1 induces
apoptosis in various oncogenic kinase positive cells following
treatment with an oncogenic kinase inhibitor in various
kinase-dependent maliganacies. The combination of c-Fos and Dusp-1
inhibition has no effect on survival and apoptosis of parental BaF3
cells, a hematopoietic cell line; Dusp-1 and c-Fos knockout mice
are viable and survive without any serious phenotype, suggesting
that these targets are suitable for therapeutic development. In one
aspect, the inventive method assessed effectiveness of targeted
c-Fos and Dusp-1 inhibition in LICs for Imatinib response.
Assessment included both genetic (shRNA) and pharmacological
inhibitors. This provided a basis for clinical application of a
composition containing Imatinib, a c-Fos inhibitor, and a Dusp-1
inhibitor to target leukemic cells, such as CML initiating cells
and AML initiating cells. This finding also provided the basis for
extending the utility of inhibition of c-Fos and Dusp-1 into other
kinase-dependent malignancies, including solid tumors, as described
in detail below.
[0009] Chronic myelogenous leukemia (CML) is a slow-growing bone
marrow cancer resulting in overproduction of white blood cells. CML
is caused by the abnormal phosphorylation of cellular proteins by a
deregulated enzyme, BCR-ABL tyrosine kinase. A small molecule
inhibitor Imatinib mesylate (Gleevec.TM.) was developed to block
aberrant BCR-ABL tyrosine kinase activity. Gleevec.TM. was a major
breakthrough in fighting cancer; Imatinib treatment not only
revolutionized CML management but also paved the way for
development of tyrosine kinase inhibitor therapy for other
diseases.
[0010] However, imatinib treatment is not curative. Many patients
develop resistance despite continued treatment and some patients
simply do not respond to treatment. Evidence suggests that a subset
of cancer cells, termed "cancer stem cells", drive tumor
development and are refractory to most treatments. In other words,
cancer cells that respond to the drug treatment are critically
dependent upon uninterrupted oncogene function, are "addicted to
oncogene", whereas cancer stem cells are not dependent or addicted
to oncogene. Thus, eradication of these cancer stem cells is a
critical part of any successful anti-cancer therapy.
[0011] CML has long served as a paradigm for generating new
insights into the cellular origin, pathogenesis and improved
approaches to treating many types of human cancer. Cancer stem
cells in CML serve as safe reservoir to develop therapeutic
resistance. This emphasizes the need for new agents that
effectively and specifically target CML stem cells.
[0012] In one aspect, the inventive method targeted the CML stem
cells to produce curative therapies that do not require lifelong
treatments. The inventive method served as a paradigm to
investigate other disease models and provided the described
improved strategies for curative therapeutics for kinase-dependent
malignancies.
[0013] Oncogene addiction is the "Achilles' heel" of many cancers.
The major limitation to develop curative cancer therapy has been a
lack of understanding of the molecular and patho-physiological
mechanisms driving cancer maintenance, progression, and mechanisms
of therapeutic response and relapse. In 2002, Bernard Weinstein
proposed the concept that cancer cells acquire abnormalities in
multiple oncogenes and tumor suppressor genes. Inactivation of a
single critical gene can induce cancer cells to differentiate into
cells with normal phenotype, or to undergo apoptosis, which is
popularly known as "oncogene addiction". This dependence or
addiction for maintaining the cancer phenotype provides an Achilles
heel for tumors that can be exploited in cancer therapy. In CML,
differentiated and dividing cells undergo apoptosis following acute
inhibition of BCR-ABL, and are thus "BCR-ABL addicted". However,
CML LICs, as well as kinase-dependent cancers' stem cells, do not
show a similar response and are thus not "addicted" to BCR-ABL
function or oncogenic kinase activity.
[0014] The clinical activity of Imatinib in multiple disease
settings, together with numerous cancer cell line studies
demonstrating an apoptotic response to drug treatment, suggests
that clinical responses are likely to reflect oncogene dependency
on activated kinases for their survival. Likewise, EGFR inhibitors
in the treatment of lung cancer represents another example of
oncogene addiction that has yielded clinical success in a subset of
patients with advanced disease that are otherwise refractory to
conventional chemotherapy treatment. Mutations in the kinase domain
of EGFR are found in a small subset of non-small cell lung cancers
(NSCLC), and clinical responses to EGFR inhibitors, Gefitinib and
Erlotinib, have been well correlated with such mutations. Further,
cancer genome sequencing data have also highlighted the likely role
of "kinase addiction" in a variety of human cancers, e.g.,
activation of MET, BRAF, FGFR2, FGFR3, ALK, AURK and RET kinase in
various different malignancies. Underscoring the importance of
oncogene addiction is the fact that in all of these
kinase-dependent malignancies, acute inactivation of the mutated
kinase by either genetic or pharmacological means results in growth
inhibition or tumor cell death. In sum, the potential and
importance of oncogene addiction in molecularly targeted cancer
therapy highlights the fact that activated oncogenes, especially
kinases, represent cancer culprits that frequently contribute to a
state of oncogene dependency.
[0015] Cell culture models, genetically engineered mice, and
clinical testing of targeted drugs support a widespread role for
oncogene addiction in tumor cell maintenance and response to acute
oncoprotein inactivation. The precise mechanism by which cells
acquire dependency on a single pathway or activated protein is not
clear in most cases, but multiple theories have nonetheless been
put forth; signaling network dysregulation, synthetic lethality
genetic streamlining, and oncogenic shock. However, experimental
evidence to prove these models is generally lacking, and it is
unlikely that a single mechanism accounts for the numerous reported
experimental findings that appear to represent examples of oncogene
dependency, and therefore it represents an important area of
investigation. Additionally, mechanisms governing oncogene
addiction may vary according to the cellular and extracellular
context.
[0016] Given the intrinsic resistance of LICs to TKI therapy in
CML, a detailed understanding of oncogene dependency in
therapeutically responsive cells permits engineering the
therapeutically resistant cells LICs to achieve drug sensitivity.
mRNA and miRNA expression studies were thus performed in BCR-ABL
addicted and non-addicted cells to identify the candidate gene(s)
mediating the drug response.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0018] FIGS. 1A-D demonstrate that growth factor signaling in
leukemic cells abrogates BCR-ABL dependence.
[0019] FIGS. 2A1-F demonstrate that AP-1 transcription factor c-Fos
and dual specificity phosphatase-1 mediate BCR-ABL addiction.
[0020] FIGS. 3A-F schematically demonstrate in vitro and in vivo
evaluation of c-Fos and Dusp-1 to induce BCR-ABL addiction in
leukemic stem cells (LSCs).
[0021] FIG. 4 shows the chemical structure of selected
inhibitors.
[0022] FIG. 5 demonstrates treatment effects for Imatinib,
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI), and curcumin separately and combined.
[0023] FIG. 6 demonstrates efficacy of compositions in curing mice
with leukemia in retroviral-transduction bone marrow
transplantation mouse model of chronic myelogenous leukemia
(CML).
[0024] FIGS. 7A-D demonstrate ability of inventive compositions to
eradicate leukemic stem cells from SCL-BCR/ABL mice.
[0025] FIGS. 8A-J show expression of c-Fos, Dusp1, and Zfp36
constitutes a common signature of imatinib-resistant cells. FIG. 8A
shows immunoblot analysis of BCR-ABL expression in the indicated
cell lines. Doxycycline (Dox) was used to induce BCR-ABL expression
in BaF3-LTBA cells. Actin was used as a loading control. The band
labeled c-Abl represents endogenous c-Abl kinase. FIG. 8B shows
percentage survival of BaF3 or BaF3-LBTA (LTBA) cells treated with
imatinib (3 .mu.M) without or with IL-3. FIG. 8C shows percentage
survival of BaF3 or BaF3-BA cells treated with imatinib (3 .mu.M)
without or with IL-3. FIG. 8D shows immunoblot analysis of BCR-ABL
expression in BaF3-BA cells treated with increasing concentrations
of imatinib with or without IL-3, pBCR-ABL is
phosphorylated-BCR-ABL. FIG. 8E shows percentage survival of K562
cells treated with (3 .mu.M) imatinib alone or with the indicated
cytokines. FIG. 8F shows Venn diagram showing three commonly
expressed genes among four different experiments, described in FIG.
9G. FIG. 8G shows real-time qPCR analysis of Fos, Dusp1, and Zfp36
expression in BaF3 cells, either untreated or 1 h after IL-3 was
withdrawn from IL-3 treated cells (-IL-3), and in BaF3-BA cells
with or without IL-3 treatment or 1 h imatinib (IM) treatment. The
data shown are mean.+-.s.d. from three technical replicates of qPCR
(P values are shown above the compared bars; Student's t-test).
FIG. 8H shows immunoblot analysis and FIG. 8I shows densitometric
quantification from one representative blot of c-Fos, Dusp1, and
Zfp36 expression in BaF3 cells and BaF3-BA cells+/-IL-3. FIG. 8J
shows real-time qPCR analysis of c-FOS, DUSP1, and ZFP36 expression
in primary CML patient-derived peripheral blood mononuclear cells
(except for sample CP4, for which CD34+ cells were analyzed)
normalized to expression in normal donor CD34+ cells (black bar).
Four chronic phase (CP) and three blast-crisis (BC) patients were
analyzed. Data are shown from two independent qPCR analysis
.+-.s.d.
[0026] FIGS. 9A-G show expression of c-Fos, Dusp1, and Zfp36
constitutes a common signature of imatinib-resistant cells. FIG. 9A
shows representative scatter plots of BaF3-BCR-ABL cells stained
with Annexin V and propidium iodide to quantify and sort the live
(pink), early-apoptotic (blue) and dead cells (green), treated with
Imatinib with IL3 (left panel) and without IL3 (right panel). Live
cells from both groups (labeled as A and B), early apoptotic
(labeled as C) and apoptotic or dead cells (labeled as D) from
imatinib treated cells without IL3 were sorted by FACS to determine
differential expression of genes. FIG. 9B shows scatter plots
showing the live and dead K562 cells treated with imatinib +/-Epo.
Live and early apoptotic cells were sorted for gene expression
studies. FIG. 9C shows heat map showing differential expression of
192 genes by BCR-ABL in the presence of IL3. To identify the genes
that are directly modulated by BCR-ABL and IL3, we used doxycycline
inducible BaF3-LTBA cells. As constitutive expression of BCR-ABL
destabilizes the genome by modulating several checkpoint and DNA
repair enzymes causing irreversible genetic and epigenetic changes.
Therefore, it makes difficult to identify genes that are modulated
directly by BCR-ABL. To address this we made BaF3-LTBA using a
third generation Tet-on promoter that lacks basal expression (shown
in FIG. 8A). Total RNA was isolated from the LTBA cells after 12
hrs of doxycycline induction +/-IL3. Likewise, total RNA from the
parental BaF3 cells grown with IL3 and doxycycline was used to
filter out the background noise. FIG. 9D shows heat map showing
differential expression of 308 genes between live cells treated
with imatinib +/-IL3. FIG. 9E shows heat map showing modulation of
1437 genes in K562 cells treated with imatinib +/-erythropoietin.
FIG. 9F shows heat map of expression profiles from CML CD34+ cells
showing differential expression of 85 genes in untreated and after
two weeks of imatinib treatment. FIG. 9G shows a Venn diagram
showing induced expression of three genes (c-Fos, Dusp1 and Zfp36)
by BCR-ABL, IL3 and imatinib.
[0027] FIGS. 10A-G show c-Fos, Dusp1 and Zfp36 is required for
BCR-ABL dependent survival. FIG. 10A shows a cartoon depiction of
retroviral vectors expressing BCR-ABL, c-Fos, Dusp1, and Zfp36
cDNAs with different fluorescent proteins in the BaF3-BCR-ABL
cells. FIG. 10B shows a dose response curve showing overexpression
of all three genes, c-Fos, Dusp1 and Zfp36, confers resistance to
imatinib in the absence of growth factor, IL3. FIG. 10C shows a
Q-PCR analysis showing the relative expression of c-Fos, Dusp1 and
Zfp36 in BaF3-BCRABL cells expressing shRNAs for c-Fos, Dusp1 and
Zfp36, a scrambled SC-shRNA was used as a control. FIG. 10D shows
immunoblots showing reduced protein expression of Fos, Dusp1 and
Zfp36 in BaF3-BA cells expressing gene specific shRNAs in
comparison to control (scrambled shRNA). FIG. 10E shows a cell
proliferation curve of parental BaF3 cells expressing shRNAs for
c-Fos, Dusp1 and Zfp36. Depletion of c-Fos, Dusp1 and Zfp36 did not
show any adverse effect on survival and proliferation of BaF3
cells. FIG. 10F shows cell proliferation curve of BaF3-BA cells,
showing significant reduction in proliferation and survival
(>50%) by genetic depletion or c-Fos, Dusp1 and Zfp36 alone or a
combination of c-Fos+Dusp1 or Dusp1+Zfp36. BaF3 or BaF3-BCRABL
cells expressing shRNAs for c-Fos+Zfp36 or c-Fos+Dusp1+Zfp36 did
not survive, thus precluded further analysis. FIG. 10G shows bar
graph showing c-Fos, and Dusp1 knockdown sensitized the BaF3-BA
cells to imatinib compared to BaF3 cells in the presence of GF,
while depletion of Zfp36 equally sensitized both BaF3 and BaF3-BA
cells. Individual data points are shown as empty circles in all bar
graphs.
[0028] FIGS. 11A-O show genetic deletion of Fos and Dusp1 increases
the response of BCR-ABL-induced leukemia to imatinib. FIG. 11A
shows experimental design of in vitro and in vivo experiments to
analyze BCR-ABL disease using Dusp1.sup.-/-,
ROSACre.sup.ERT2Fos.sup.fl/fl, and ROSACre.sup.ERT2FoS.sup.fl/fl;
Dusp1.sup.-/- mice. Kit+ cells from mouse bone marrow were
transduced with BCR-ABL-IRES-YFP retrovirus. 5,000 GFP.sup.+ cells
were plated for in vitro CFU assays, and 40,000 YFP.sup.+ cells
were transplanted to monitor leukemia development in vivo in
lethally irradiated C57BL/6 mice. FIGS. 11B-D show percentage of
CFUs from Kit.sup.+ cells expressing BCR-ABL in the absence of
Dusp1 (FIG. 11B), Fos (FIG. 11C), and both Fos and Dusp1 (FIG.
11D). The data show the mean colony number .+-.s.d. (n=3; P values
are shown above the compared bars by Student's t-test). FIG. 11E
shows representative photographs of BCR-ABL-positive colonies
described in FIGS. 11B-D. FIGS. 11F, H, J show survival curves of
mice transplanted with BCR-ABL-YFP transduced Kit.sup.+ cells from
WT, Dusp1.sup.-/- mice (FIG. 11F) ROSACre.sup.ERT2Fos.sup.fl/fl
mice (FIG. 11H), and ROSACre.sup.ERT2Fos.sup.fl/flDusp1.sup.-/-
mice (FIG. 11J). Mice were untreated or treated with imatinib. Data
are from two independent transplantation experiments (n=6 mice per
group); log-rank Mantel-Cox test: P<0.0001 between WT and
Dusp1.sup.-/- with or without imatinib (FIG. 11F); n=12 log-ranked
Mantel-Cox test P<0.0001 between WT and Fos.sup.-/- with or
without imatinib (FIG. 11H); n=12 log-rank Mantel-Cox test
P<0.0001 between WT and Fos.sup.-/-Dusp1.sup.-/- with or without
Imatinib (FIGS. 11H, J). In FIGS. 11H, J, transplant recipients
were treated with three doses of 2 mg/kg tamoxifen injection to
delete Fos. FIGS. 11G,I, K show leukemic burden in mice
transplanted with Dusp1.sup.-/- (FIG. 11G), Fos.sup.-/- (FIG. 11I),
and Fos.sup.-/-Dusp1.sup.-/- (FIG. 11K) Kit+ cells, as measured by
the percentage of YFP+ cells in peripheral blood. Dead mice are
represented with an X. FIG. 11I shows primary structure of c-Fos
and its dominant-negative version, c-Fos-.DELTA.RK, which lacks the
DNA-binding domain consisting of a basic-RK motif (amino acid
residues 133-159). FIG. 11M shows tertiary structure of Fos and Jun
bound to AP1 site on DNA, illustrating the homo/heterodimer
assembly of Fos with Jun. FIG. 11N shows percentage of CFUs from
wild-type (WT) BCR-ABL-YFP+Kit+ cells expressing dominant-negative
c-Fos-.DELTA.RK with or without imatinib, as compared to
Fos.sup.-/- BCR-ABL-YFP+Kit+ cells with or without imatinib. FIG.
11O shows percentage of CFUs from Fos.sup.-/-Dusp1.sup.-/- Kit+
cells with retroviral-vector-mediated rescue of c-Fos and Dusp1
expression. Data shown are from two independent experiments
.+-.s.d. (n=3, P values are indicated above the compared bars by
Student's t-test).
[0029] FIGS. 12A-I show reduced expression of c-Fos in
c-Fos.sup.fl/fl/Dusp1.sup.-/- mice prolonged the survival of CML
mice. FIG. 12A shows survival curve of mice transplanted with
BCR-ABL-YFP transduced Kit+ cells from
ROSACre.sup.ERT2c-Fos.sup.fl/fl mice, showing no significant
difference with imatinib treatment compared to wild type (WT) donor
cells. Data shown are from two independent transplant experiments
(n=12). FIG. 12B shows survival curve of mice transplanted with
BCR-ABL-YFP transduced cells from ROSACre.sup.ERT2c
Fos.sup.fl/flDusp1.sup.-/- mice. Data shown are from two
independent transplant experiments (n=12; p=0.017). Note the
leukemia free survival of 30-40% of mice transplanted with
ROSACre.sup.ERT2c-Fos.sup.fl/flDusp1.sup.-/- cells, FIG. 12C,D
shows bar graphs illustrating leukemic burden in mice transplanted
with ROSACre.sup.ERT2c-Foe (FIG. 12C), and
ROSACre.sup.ERT2c-Fos.sup.fl/flDusp1.sup.-/- (FIG. 12D). Leukemic
burden were measured by the level of YFP in peripheral blood as a
surrogate for BCR-ABL expression. Cohorts of mice that died are
represented as X. FIG. 12E shows q-PCR analysis of c-Fos in wild
type (WT) and ROSACre.sup.ERT2c-Fos.sup.fl/flDusp1.sup.-/-
bonemarrow cells showing reduced expression of c-Fos (5 fold) in
ROSACre.sup.ERT2c-Fos.sup.fl/flDusp1.sup.-/- mice, suggesting that
the reduced expression of c-Fos in the absence Dusp1 is sufficient
to reduce the MRD by imatinib treatment in the absence of full
deletion of c-Fos (FIG. 12D), FIG. 12F shows agarose gel showing a
representative PCR analysis of c-Fos gene from the peripheral blood
of ROSACre.sup.ERT2c-Fos.sup.fl/fl or
ROSACre.sup.ERT2c-Fos.sup.fl/flDusp1.sup.-/- mice treated with or
without tamoxifen. Note amplification of c-Fos deletion specific
PCR product (280 bp) by primer P1 and P3 (shown below in a cartoon
representation) after tamoxifen treatment, while non-deleted FOR
product (0.4 kb) amplified by P1 and P2 are present before or
non-tamoxifen treated mice. These mice were monitored for six
months after tamoxifen injection and deletion specific PCR were
performed periodically that showed persistent presence of Fos
deleted cells. Mice were sacrificed after six months, and we did
not observe any defect in blood and organs, suggesting that
therapeutic targeting of these two genes will not have any adverse
effect on normal tissues and organs. FIG. 12G shows bar graph
showing the levels of granulocytes, monocytes, B, and T cells after
two weeks of transplantation from the peripheral blood of mice
transplanted with Kit+ from wild type and
ROSACre.sup.ERT2:c-Fos.sup.fl/fl/Dusp1.sup.-/- mice expressing
vector (pMSCV-Ires-YFP) and BCR-ABL. Expression of BCR-ABL induces
granulocytosis at the expense of B cells in both wild-type and
Fos.sup.fl/fl/Dusp1.sup.-/- recipient mice, Representative data
showing mean values of peripheral blood cells .+-.S.D. (n=5;
**=p<0.01), FIG. 12H shows survival curves of mice transplanted
with vector (MIY) and BCR-ABL-YFP transduced Kit+ cells from wild
type (WT), and ROSACre.sup.ERT2c-Fos.sup.fl/flDusp1.sup.-/-. c-Fos
was deleted by tamoxifen after establishing the CML (after three
weeks of transplantation). FIG. 12I shows graph showing the
leukemic burden in transplanted mice measured by YFP positive cells
in peripheral blood. Note, deletion of both Fos and Dusp1 do not
affect the chimerism of vector (MIY) expressing cells, while their
deletions in leukemic cells show gradual decrease in chimerism and
imatinib treatment completely eradicated the leukemic cells.
individual data points are shown as circles in all bar graphs.
[0030] FIGS. 13A-G show chemical inhibition of c-Fos, Dusp1, and
BCR-ABL eradicates minimal MRD in mice. FIG. 13A shows experimental
design for testing the efficacy of small-molecule inhibitors of
c-Fos (difluorocurumin, DFC) and Dusp1
((E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one,
BCI) in vitro and in vivo in CML mice. FIG. 13B shows percentage of
CFUs from WT and BCR-ABL LSK (Lin-Sca1+Kit+) cells treated with the
indicated drugs. Data shown are the mean colony numbers from two
independent experiments .+-.s.d. (n=3, P values are indicated above
the compared bars by Student's t-test). FIG. 13C shows survival
curve of BCR-ABL-expressing Kit+ cell recipients treated with
vehicle (blue), imatinib (red), or a combination of imatinib with
DFC and BCI (green). The time period during which the drugs were
administered is indicated by light-blue shading. Data shown are
from one of the two independent transplantation experiments with
similar results (n=5 mice per group; P=0.0285). FIG. 13D shows
percentage of YFP+ cells in peripheral blood of mice treated with
imatinib or imatinib+DFC+BCI. FIG. 13E shows schematic structures
of the transgenes used in transgenic mice to drive BCR-ABL
expression in stem cells. Top, Scl-3' enhancer drives expression of
the tetracycline transactivator protein (tTA); bottom, a
tetracycline-responsive promoter (Tet-P) drives BCR-ABL expression.
Transgenic mice are fed doxycycline-containing chow; doxycycline
withdrawal induces expression of BCR-ABL in hematopoietic stem
cells. FIG. 13F shows experimental design for studying the effects
of Dusp1 and c-Fos inhibition in leukemic stem cells. Mice received
a competitive transplant of 3,000-5,000 LSK Scl-BCR-ABL cells in
combination with 500,000 WT total bone marrow cells. Engraftment
was evaluated 1 month after transplantation by flow cytometry
(CD45.1 versus CD45.2); the mice were then treated with imatinib
alone or imatinib+DFC+BCI for 3 months, and the presence of MRD was
evaluated at indicated times. FIG. 13G shows percentage of leukemic
cells (CD45.2) in bone marrow of BoyJ recipients (CD45.1) at the
indicated time points after cell transplantation and treatment with
imatinib or imatinib+DFC+BCI.
[0031] FIGS. 14A-D show chemical inhibition of c-Fos and Dusp1
sensitized leukemic cells to imatinib. FIG. 14A shows chemical
structures of small molecule inhibitors targeting Dusp1
((E)-2-Benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one;
BCI) and Fos (Diflourinated curcumin, DFC; Curcumin and NDGA). FIG.
14B,C show bar graphs showing percent CFU from normal and BCR-ABL
LSK cells (Lin-Sca1+Kit+), with single and combinations of
inhibitors utilizing different c-Fos inhibitors, curcumin (FIG.
14B) and NDGA (FIG. 14C). Representative data shown are the mean
colony number from two independent experiments .+-.S.D. P values
are indicated above the compared bars). FIG. 14D shows survival
curve of mice of two independent experiments transplanted with Kit+
expressing BCR-ABL-YFP. Treatments with single drugs or combination
of two inhibitors are ineffective in treating these mice, most mice
showed a marginal 5-7 days prolongation of their survival except
BCI+ curcumin treated cohort (20 of CML mice survived). Groups of
CML mice treated with triple combinations, imatinib+Curcumin+BCI
and imatinib+NDGA+BCI, showed prolonged survival, 50 and 60%,
respectively. Individual data points are shown as empty circles in
all bar graphs.
[0032] FIGS. 15A-D show inhibition of c-Fos, Dusp1 and BCR-ABL
eradicated the leukemic stem cells. FIG. 15A shows representative
scatter plots showing minimal effect by imatinib treatment on
BCR-ABL (CD45.2) and (BCR-ABL-Lin-Sca+Kit+) cells. FIG. 15B shows
representative scatter plots showing eradication of BCR-ABL
(CD45.2) and (BCR-ABL-Lin-Sca+Kit+) by imatinib+DFC+BCI treatment.
FIG. 15C shows percentage of leukemic cells (CD45.2) in bone marrow
of BoyJ recipients (CD45.1). Imatinib treatment (blue bars) reduces
leukemic burden (month 3=<20%), which after treatment
discontinuation rebounds (month 6=>60%). Treatment with
imatinib+ curcumin+BCI (red bar) and imatinib+NDGA+BCI (purple)
reduces leukemic burden but they relapse after treatment
discontinuation. Treatment with imatinib+DFC+BCI (green bar)
reduces leukemic burden (month 3=<10%), without relapse (month
6=no detection). FIG. 15D shows graphs showing the level of human
leukemic cells in NSG mice at week seven, chimerism at week 2 and 4
are shown in FIG. 16B.
[0033] FIGS. 16A-C show inhibition of c-Fos, Dusp1, and BCR-ABL
selectively eradicates CML cells. FIG. 16A shows experimental
design for the analysis of DUSP1 and c-FOS inhibitor treatment of
patient-derived CML CD34+ cells. Primary CML cells from CP4 were
transplanted into NOD scid-.gamma.C.sup.-/- mice recipients, which
transgenically express human IL-3, IL-6, and GM-CSF (NSGS);
engraftment was assessed 2 weeks after transplanatation, engrafted
mice were treated for 6 weeks with drug combinations, and leukemic
burden was determined at 4 and 7 weeks after transplantation. FIG.
16B shows percentage of human leukemic cells in the bone marrow of
NSGS mice at week 2 (left) and week 4 (right) of treatment. Data
shown are from one of two experiments with similar results (n=6
mice per group. P values are indicated above the compared bars by
Student's t-test). FIG. 16C shows percentage of CFU numbers
determined by LTC-IC assay for samples from two patients with CML,
and a normal donor treated with vehicle or the indicated drug
combinations. Data shown are mean CFU numbers .+-.s.d. (n=3; P
values are indicated above the compared bars).
[0034] FIGS. 17A-C show genetic or chemical inhibition of c-Fos and
Dusp1 downregulates the Fos-Jun network while activating Jun-JunD
target genes. FIG. 17A shows heat map showing commonly modulated
genes in BCR-ABL expressing Kit+ cells with c-Fos and Dusp1
deletion and WT cells treated with Fos (DFC) and Dusp1 (BCI)
inhibitors alone or with imatinib. Genetic and chemical inhibition
resulted in the modulation of 146 genes in common (58 overexpressed
and 88 underexpressed). FIG. 17B,C show netwalker analysis shows
that overexpressed genes are enriched for genes participating in a
Jun-JunD regulated network (FIG. 17B), whereas downregulated genes
are enriched for genes participating in a Fos-Jun regulated network
(FIG. 17C).
[0035] FIGS. 18A-G show inhibition of Dusp1 activates p38. FIG. 18A
shows immunoblot analysis of phospho-p38, total p38, and p-JNK
expression in BaF3 and BaF3-BA cells with or without IL-3, showing
increased p-p38 levels in BCI-treated cells. FIG. 18B shows
immunoblot analysis of the indicated proteins in BaF3 and BaF3-BA
cells, with or without IL-3, expressing Dusp1, Dusp6, and pMSCV
(empty vector). FIG. 18C shows, left, dose-response curve for
survival of BaF3-BA cells at increasing imatinib doses, treated
with vehicle or the p38-specific inhibitor (SB202190, 500 nM) or a
JNK-specific inhibitor (SP600125, 500 nM); right, percentage of
cell survival at 500 nM and 1,000 nM imatinib. The data shown are
mean values .+-.s.d. (n=3; P values are indicated above the
compared bars). FIG. 18D shows CFU numbers from Kit+ cells
coexpressing BCR-ABL (BA) and WT or drug-resistant Dusp1 variants.
The data shown are mean colony number .+-.s.d. (n=3; P values are
indicated above the compared bars). FIG. 18E shows surface
depiction of a structural model of the Dusp1 rhodanese domain,
highlighting amino acids affected by BCI-resistance mutations, as
well as a deletion mutant causing resistance (red). A putative
binding pocket for BCI and kinase-interacting motifs (KIMs) are
indicated. Mutations are clustered together in the structure, and
outline a pocket to which BCI seemingly binds (.DELTA.G=-7.6). FIG.
18F shows ratio of the levels of phospho-p38 to total p38 in
peripheral blood cells before and 6 h after BCI injection into
leukemic mice. Data are shown for three mice (n=3; P=0.04). FIG.
18G shows real-time qPCR analysis showing expression of Bcl2l11,
116, and Lif in mice before and 6 h after DFC+BCI injection. Data
shown are means.+-.s.d. from three mice in triplicates. (P values
are indicated above each comparison; Student's t-test.)
[0036] FIGS. 19A-H show BCI resistant screening identified drug
resistant mutations in the Dusp1. FIG. 19A shows bar graph showing
overexpression of Dusp1, not the Dusp6, confers resistance to BCI
in BaF3-BA cells. Data shown are from two independent experiments
.+-.S.D (n=3; P values are indicated above the compared bars). FIG.
19B shows bar graphs showing CFU numbers derived from Kit+ cells
from WT mouse coexpressing BCR-ABL with either Dusp1 or Dusp6.
Expression of Dusp1 show normal CFU numbers but confers modest
resistance to IM [3 .mu.M]+BCI [0.5 .mu.M] treatment. Surprisingly,
expression of Dusp6 show significantly reduced CFU number and
treatment with IM+BCI did not show any significant change. Data
shown are from two independent experiments .+-.S.D (n=3; P values
are indicated above the compared bars). FIG. 19C shows bar graphs
showing CFU numbers derived from Kit+ cells from wild-type,
Dusp1.sup.-/- and Dusp6.sup.-/- mice expressing BCR-ABL and
BCR-ABL+Dusp6. Unlike Dusp1.sup.-/- cells, Dusp6.sup.-/- cells
expressing BCR-ABL show normal CFU numbers compared to WT, but
conferred drug resistance to IM+BCI treatment. Expression of Dusp6
in Dusp.sup.-/- cells with BCR-ABL partially reduced the CFU
numbers and abrogated the drug resistance. Data shown are from two
independent experiments .+-.S.D (n=3; coexpressing BCR-ABL with
either Dusp1 or Dusp6). FIG. 19D shows a schematic of random
mutagenesis of Dusp1 for in vitro screening of BCI resistant
clones. FIG. 19E shows bar graph showing frequency of resistant
clones per million of BaF3-BA cells expressing randomly mutagenized
Dusp1. FIG. 19F shows bar graph showing BCI resistance conferred by
25 out of 27 clones (except #9 and #16), isolated from the
resistant screen selected at 1.5 .mu.M of BCI. FIG. 19G shows bar
graph showing the frequency of mutations in 25 sequenced resistant
clones. FIG. 19H shows expression of Dusp1 mutants in BaF3-BA cells
conferred resistance to BCI and imatinib+BCI. Note, Dusp1-V83G as a
single mutation conferred significant resistance to both BCI alone
and in combination of imatinib. Individual data points are shown as
empty circles in all bar graphs.
[0037] FIGS. 20A-E show BCI resistant mutations are clustered in
allosteric domain. FIG. 20A shows primary structure of Dusp1 where
catalytic domain lies at the C-terminus of protein. Catalytic
cysteine in catalytic-site is shown in red. The N-terminal
rhodanese domain harboring kinase interaction motif (KIM) required
for binding with MAPKs shown in green. FIG. 20B shows a ribbon
depiction of homology based model of Dusp1 rhodanese domain.
Mapping of BCI resistant mutations identified a single clusture in
the rhodanese domain. Deletion mutations are shown in red while
point mutations are shown in golden. FIG. 20C shows a cartoon
depiction homology based model of Dusp1 catalytic domain. Catalytic
lysine and an inorganic phosphate are shown in red. FIG. 20D shows
surface depiction of Dusp1 rhodanese domain showing the BCI
resistant mutations clustered at the N-terminus of allosteric
domain. Deletion mutations are shown in red while point mutations
are shown in golden. FIG. 20E shows unbiased in silico docking of
BCI revealed a binding pocket to which BCI seemingly binds
(.DELTA.G=-7.6).
[0038] FIGS. 21A-G show deletion of Fos and Dusp1 is synthetic
lethal to B-ALL development. FIG. 21A shows survival curves of mice
transplanted with vector and BCR-ABL-YFP (p190) transduced Kit+
cells from wild type (WT), and
ROSACre.sup.ERT2c-Fos.sup.fl/flDusp1.sup.-/- mice. c-Fos was
deleted by tamoxifen injection (three doses of 2 mg/kg) after two
weeks of transplantation. Mice transplanted with wild-type cells
expressing p190 BCR-ABL developed lethal B-ALL and died within 4-5
weeks, while mice transplanted with Fos.sup.fl/flDusp1.sup.-/-
cells show gradual depletion of BCR-ABL expressing cells (FIG.
21B), and do not develop leukemia determined by WBC count (FIG.
21C). Deletion of Fos accelerates the depletion of BCR-ABL positive
cells compared to Fos non-deleted cells. FIG. 21D shows dose
response analysis of BaF3 cells expressing FLT3-ITD showing
complete resistance to AC220 under growth factor signaling (IL3).
FIG. 21E shows bar graph showing induced expression of c-Fos and
Dusp1 by FLT3-ITD with additional induction by IL3. Data for qPCR
analysis are shown .+-.S.D. (P values are indicated between the
compared bars). FIG. 21F shows dose response analysis of BaF3 cells
expressing Jak2-V617F showing 7-8-fold resistance to ruxolitinib in
the presence of IL3. FIG. 21G shows bar graph showing induced
expression of c-Fos and Dusp1 by JAK2-V617F under growth factor
signaling. Data for qPCR analysis are shown .+-.S.D. (P values are
indicated between the compared bars). Individual data points are
shown as empty circles in all bar graphs.
[0039] FIGS. 22A-I show a model for therapeutic mechanism of TKI
efficacy. FIG. 22A shows graph showing the expressions of c-Fos
(cyan) and Dusp1 (pink) in hematopoietic cells in mouse (left) and
human (right). Each dot in the plot corresponds the expression of
FOS and DUSP1 in a microarray. FIG. 22B shows a cartoon depiction
showing downregulation of c-Fos and Dusp1 with differentiation
during normal hematopoiesis. FIG. 22C shows bar graph showing the
overexpression of c-Fos and Dusp1 in leukemic stem cells of mice
(BCRABL+LSK-Lin-Sca1+Kit+ cells). Representative data shown are
from two independent experiments .+-.S.D. (P values are indicated
above the compared bars). FIG. 22D shows bar graph showing the
overexpression of c-FOS and DUSP1 in human leukemic stem cells
(CD34+CD38-) from CML patients. Each dot in the plot corresponds
the expression of c-FOS and DUSP1 in a microarray (GSE40721). P
values are indicated above the compared samples. FIG. 22E shows
histograms showing the overexpression of cell proliferation genes
(left panel) and anti-apoptotic genes (right panel) in BaF3-LTBA
cells grown with IL-3. FIG. 22F shows bar graph showing q-PCR
analysis of expression of proliferative or survival genes (Id1 and
Ncf4) and anti-apoptotic genes (Aven, SerpinA3G, Bcl2a1a, Bcl2l11
and Xaf1) in BaF3-BCR-ABL cells+IL3 with and without drug
treatments (imatinib, DFC+BCI and DFC+BCI+Imatinib). Note,
treatment with Fos and Dusp1 inhibitor (DFC+BCI) and in combination
with imatinib suppressed their expression suggesting their
regulation by Fos and Dusp1. Representative data shown are the mean
values of qPCR analysis .+-.S.D. (P values are indicated above the
compared bars). FIG. 22G-I shows a model of TKI response in drug
sensitive and leukemic stem cells. Our model suggests that during
normal hematopoiesis c-Fos and Dusp1 are downregualted with
differentiation. In differentiated bulk of leukemia cells which is
sensitive to TKI, expression of an activated kinase induces the
expression of c-Fos and Dusp1, which induces both proliferative and
proapoptotic signal. Therefore, an acute inhibition of activated
oncogene induces oncogenic shock resulting to apoptosis in cells
expressing suboptimal level of c-Fos and Dusp1 (FIG. 22G). In
leukemic stem cells, convergence of oncogenic and growth factor
signaling induces high levels of c-Fos and Dusp1 expression, which
seemingly reprograms transcriptional network to induce pro-survival
and anti-apoptotic genes. Thus, levels of c-Fos and Dusp1
determines the net transcriptional output for
proliferative/pro-apoptotic genes or pro-survival/anti-apoptotic
genes in oncogenic condition. Thus, inhibition of oncogene by TKI
is ineffective against leukemic stem/progenitor cells (FIG. 22H),
failure to induce apoptosis under TKI treatment results into MRD
(FIG. 22I). Individual data points in each bar graphs are shown as
empty circles.
[0040] FIGS. 23A-B show overexpression of DUSP1 not c-FOS in MPN.
FIG. 23A shows overexpression of DUSP1 in MPN patients. CD34+ cells
from six patients representing each subtype were analyzed. P
values: **=>0.001 and *=>0.01. FIG. 23B shows induction of
Dusp1 in MPN cells. Bar graph showing q-PCR analysis of Dusp1 in
Kit+ cells expressing Jak2-V617F, CSF3R-WT, CSF3R-T618 and
MpI-W515L normalized to vector control.
[0041] FIGS. 24A-D show lack of Dusp1 is synthetic lethal to MPN
development in mice. BM derived Kit+ cells from wild type and
Dusp1-/- mice were transduced with retroviruses expressing
CSF3R-T618I, CSF3R-T618I-W791X, MPL-W515L, and Jak2 V617F. FIG. 23A
shows mice transplanted with wild type cells showing robust
leukemia development by CSF3R and MpI mutants, while mice received
Jak2-V617F cells showed mild elevation in WBC, but showed
significant increase in red cells and reticulocytes. FIG. 23B shows
leukemic burden as GFP+ cells over a period of eight weeks. FIG.
23C shows mice that received cells lacking Dusp1 did not show any
signs of leukemia. FIG. 23D shows that all the GFP positive cells
were abolished over the period of seven weeks in oncogenic
conditions, while vector transduced cells have maintained normal
engraftments. These data clearly show that Lack of Dusp1 is
synthetic lethal to MPD development.
[0042] FIGS. 25A-B show induction of FOS and DUSP1 in AML and MPN
confers TKI resistance. Growth factor (GF) signaling abrogates
oncogene dependence and confers TKI resistance. FIG. 25A shows a
dose response curve of BaF3 and BaF3-FLT3ITD cells showing
resistance to Flt3 inhibitor (AC220 or quizartinib) in the presence
of growth factor, IL3. IC50 for AC220 is shown in the parenthesis.
FIG. 25B shows bar graphs showing the induction of c-Fos and Dusp1
by both FLT3ITD and GF signaling.
[0043] FIGS. 26A-B show deletion of FOS and DUSP1 is synthetic
lethal to AML development. c-Fos and Dusp1 constitute non-oncogene
addiction in FLT3ITD:MLLAF9 driven AML. FIG. 26 A shows a scheme to
test the role of Fos and Dusp1 in AML. FIG. 26B shows a bar graph
showing CFU assays using Kit+ cells from the wild type and
Fos-/-/Dusp1-/- mice. CFU assays were performed with and without
Flt3 TKI (5 nM of AC220). Note, cells expressing FLT3ITD and MLLAF9
are resistant to TKI while cells lacking Fos and Dusp1 show
synthetic lethality to oncogene expression, suggesting these genes
are essential for AML development, however, they are indispensable
for normal hematopoiesis because vector transduced cells do not
show any defect in CFU formation (data not presented).
[0044] FIGS. 27A-C show deletion of FOS and DUSP1 is synthetic
lethal to AML development. c-Fos and Dusp1 confer
oncogene-dependence in high-risk FLT3ITD:MLLAF9 driven AML. FIG.
27A shows a humanized AML model. CD34 cells from human cord blood
were transduced by retroviruses expressing FLT3ITD-Ires-Cherry and
MLLAF9-Ires-GFP. Double positive (GFP+Cherry) cells were sorted by
FACS followed with in vitro and in vivo analysis. FIG. 27B shows
histograms showing resistance to AC220 in the presence of GF (IL3,
IL6, SCF and TPO) in in-vitro assay. FIG. 27C shows transplanted
NSGS mice die of leukemia within six weeks and show complete
eradication of leukemic cells when treated with combination of
DFC+BCI+AC220 while AC220 or DFC+BCI alone are ineffective.
[0045] FIGS. 28A-F show inhibition of FOS and DUSP1 with TKI
treatment cured EGFR driven lung cancers. Growth-factor-induced TKI
resistance in solid tumors is mediated by c-FOS and DUSP1. FIG. 28A
shows a dose response curve of the HCC827 cell line (lung
adenocarcinoma; EGFR-DelE746A750) to erlotinib +/- hepatocyte
growth factor (HGF). FIGS. 28B-C show real-time qPCR analysis
illustrating induction of c-FOS (FIG. 28B) and DUSP1 (FIG. 28C)
expression by HGF (indicated times after addition of erlotinib).
FIG. 28D shows cell survival of HCC827 cells (WST assay) when
treated with DFC, BCI and erlotinib alone and in combination. Note
inhibition of DUSP1 alone sensitized the cells for erlotinib, while
concomitant inhibition of both DUSP1 and c-FOS is sufficient to
inhibit proliferation and survival. FIG. 28E shows HCC827 xenograft
growth in recipients treated with erlotinib (red), DFC+BCI (green)
and DFC+BCI+erlotinib (purple). Treatment started after one week of
transplant (n=8 per group, each mouse represented by single dot).
FIG. 28F shows representative images of mouse tumors from cohorts
in panel E.
[0046] FIGS. 29A-F show inhibition of FOS and DUSP1 is sufficient
to cure PDGFR driven lung cancer. FIG. 20A shows a dose response
curve of NCI-H1703 (lung squamous carcinoma; PDGFR amplification)
showing resistance to sunitinib in the presence of epidermal growth
factor and fibroblast growth factor (EGF+FGF). FIGS. 29B-C show
real-time qPCR analysis illustrating induction of FOS (FIG. 29B)
and DUSP1 (FIG. 29C) expression by EGF and FGF (indicated times
after addition of sunitinib). FIG. 29D shows cell survival of
NCI-H1703 cells (WST assay) when treated with DFC, BCI and
sunitinib alone and in combination. Concomitant inhibition of both
DUSP1 and c-FOS is sufficient to inhibit proliferation and
survival. FIG. 29E shows mouse xenografts of NCI-H1703 treated with
sunitinib, DFC+BCI and sunitinib+DFC+BCI (n=5). Treatments were
started two weeks after xenotransplantation. Mice treated with
either DFC+BCI or sunitinib+DFC+BCI showed complete response.
Treatment with sunitinib alone showed initial response but three
mice showed tumor regrowth after three weeks of treatment. FIG. 29F
shows representative images of mouse tumors from cohorts in FIG.
29E.
[0047] FIGS. 30A-D show induction of FOS and DUSP1 in solid tumors
confers TKI resistance. Growth factor induced TKI resistance in
solid tumors is mediated by c-FOS and DUSP1. FIG. 30A shows dose
response curves showing TKI resistance in solid tumor cell lines in
the presence of growth factors. AU565 (breast cancer HER2 amplified
AU565) conferred resistance to lapatinib by growth factor,
neuregulin 1-NRG1. RT4 (bladder carcinoma, EGFR amplified)
conferred resistance to lapatinib in the presence of EGF. SKMEL28
(melanoma, BRAF-V600E) conferred resistance to PLX4720 in the
[presence of HGF. FIGS. 30B-C show real-time qPCR analysis
illustrating induction of c-FOS (FIG. 30B) and DUSP1 (FIG. 30C)
expression by growth factors (indicated times after addition of
erlotinib). Time at 0 hours represents the level of expression
without TKI +/- GF. Note that the growth factors induce higher
expression of c-FOS in all cell lines and DUSP1 in RT4 at 0 hours,
while the addition of both TKI and growth factors induced both
c-FOS and DUSP1. FIG. 30D shows bar graphs showing cell survival by
WST assay when treated with DFC, BCI and TKI alone and in
combination. Note that inhibition of c-FOS and DUSP1 is sufficient
to kill AU565 cells, while their inhibition in RT4 and SKMEL28
cells restored the TKI sensitivity in the presence of growth
factors.
[0048] In one aspect, a pharmaceutically acceptable composition is
provided comprising at least one biocompatible excipient and, as
the only active agents, (a) a c-Fos inhibitor, (b) a Dusp-1
inhibitor, and (c) at least one oncogenic kinase inhibitor, where
the oncogenic kinase is selected from the group consisting of
BCR-ABL, BTK, FLT3, MET, KIT, JAK2, MEK, EGFR, PDGFR, ALK, HER2,
B-Raf, FGFR2, RAF, PI3K, and combinations thereof. In one
embodiment, the c-Fos inhibitor is selected from the group
consisting of curcumin, difluorinated curcumin (DFC),
[3-{5-[4-(cyclopentyloxy)-2-hydroxpenzoyl]-2-[(3-hydroxy-1,2-benzisoxazol-
-6-yl) methoxy]phenyl}propionic acid] (T5224), nordihydroguaiaretic
acid (NDGA), dihydroguaiaretic acid (DHGA), and
RE,E,Z,E)-3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1-y-
l)-2,4,6,8-nonatetraenoic acid (SR11302). In one embodiment, the
Dusp-1 inhibitor is selected from the group consisting of
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI--also known as NSC 150117), TPI-2, TPI-3, and triptolide. In
one embodiment, the tyrosine kinase inhibitor is selected from the
group consisting of Imatinib, Dasatinib, Ponatinib or Nilotinib
when the oncogenic kinase is BCR-ABL; Ibrutinib when the oncogenic
kinase is BTK; Ruxolitinib, Crizotinib, or Quizartinib when the
oncogenic kinase is one of FLT3, MET, KIT, or JAK2; Ruxolitinib or
Trametinib when the oncogenic kinase is JAK2 or MEK; Gefitinib or
Axitinib when the oncogenic kinase is one of EGFR, PDGFR, or ALK;
Gefitinib, Axitinib, or dasatinib when the oncogenic kinase is one
of EGFR or PDGFR; Gefitinib or Axitinib when the oncogenic kinase
is one of HER2 or EGFR; Vemurafenib or Sorafenib when the oncogenic
kinase is one of B-Raf or MEK; Crizotinib or Dasatinib when the
oncogenic kinase is one of MET, FGFR2, or HER2; Ceritinib,
Alectinib or Crizotinib when the oncogenic kinase is one of MET,
FGFR2, or HER2; Ceritinib, Alectinib or Crizotinib when the
oncogenic kinase is one of ALK, KIT, or FGFR; and Vemurafenib,
Sorafenib or Idelalisib when the oncogenic kinase is one of RAF or
PI3K. In one embodiment, a pharmaceutically acceptable composition
is provided comprising at least one biocompatible excipient and, as
the only active agents,
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI), difluorinated curcumin (DFC), and at least one oncogenic
kinase inhibitor selected from the group consisting of Imatinib,
Dasatinib, Ponatinib, Nilotinib, Ibrutinib, Ruxolitinib,
Crizotinib, Quizartinib, Trametinib, Gefitinib, Axitinib,
Dasatinib, Vemurafenib, Sorafenib, Ceritinib, Alectinib,
Vemurafenib, and Idelalisib.
[0049] In one aspect, a method of treating a kinase-dependent
malignancy in a patient is provided. In one embodiment, the method
comprises administering to the patient in need thereof a
composition containing at least one biocompatible excipient and, as
the only active agents, a combination of (a) an inhibitor of c-Fos
resulting in inhibition of c-Fos, (b) an inhibitor of Dusp-1
resulting in inhibition of Dusp-1, and (c) at least one inhibitor
of an oncogenic kinase resulting in inhibition of the oncogenic
kinase, where the composition is administered to the patient in a
dosing regimen for a period sufficient to provide treatment for the
kinase-dependent maliganancy in the patient in need thereof. In one
embodiment, the c-Fos inhibitor is selected from the group
consisting of curcumin, difluorinated curcumin (DFC),
[3-{5-[4-(cyclopentyloxy)-2-hydroxybenzoyl]-2-[(3-hydroxy-1,2-benzisoxazo-
l-6-yl) methoxy]phenyl}propionic acid] (T5224),
nordihydroguaiaretic acid (NDGA), dihydroguaiaretic acid (DHGA),
and
RE,E,Z,E)-3-methyl-7-(4-methylphenyl)-9-(2,6,6-trimethyl-1-cyclohexen-1-y-
l)-2,4,6,8-nonatetraenoic acid (SR11302). In one embodiment, the
Dusp-1 inhibitor is selected from the group consisting of
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI--also known as NSC 150117), TPI-2, TPI-3, and triptolide. In
one embodiment, the kinase-dependent malignancy is chronic myeloid
leukemia (CML) and the at least one inhibitor is Imatinib,
Dasatinib, Ponatinib and/or Nilotinib; chronic lymphocytic leukemia
(CLL) and the at least one inhibitor is Ibrutinib; acute myeloid
leukemia (AML) and the at least one inhibitor is Ruxolitinib,
Crizotinib, and/or Quizartinib; myeloproliferative neoplasm (MPN)
and the at least one inhibitor is Ruxolitinib and/or Trametinib;
lung cancer and the at least one inhibitor is Gefitinib and/or
Axitinib; brain tumor and the at least one inhibitor is Gefitinib,
Axitinib, and/or Dasatinib; breast cancer and the at least one
inhibitor is Gefitinib and/or Axitinib; bladder carcinoma and the
at least one inhibitor is Gefitinib and/or Axitinib; melanoma and
the at least one inhibitor is Vemurafenib and/or Sorafenib;
pancreatic cancer and the at least one inhibitor is Crizotinib
and/or Dasatinib; colon cancer and the at least one inhibitor is
Ceritinib, Alectinib and/or Crizotinib; and prostate cancer and the
at least one inhibitor is Vemurafenib, Sorafenib and/or Idelalisib.
In one embodiment, the treatment is curative.
[0050] In another aspect, a method to eradicate leukemia initiating
cells (LIC) or cancer stem cells (CSC) in a patient being treated
with a tyrosine kinase inhibitor (TKI) is provided. In one
embodiment, the method comprises administering to the patient in
need thereof a composition containing at least one biocompatible
excipient and a combination of (a) an inhibitor of c-Fos resulting
in inhibition of c-Fos, and (b) an inhibitor of Dusp-1 resulting in
inhibition of Dusp-1, where the composition is administered to the
patient in a dosing regimen for a period sufficient to eradicate
the LIC or CSC cells.
[0051] Chronic myelogenous leukemia (CML) initiating cells are
intrinsically resistant to small-molecule kinase inhibitors. This
discovery has prompted interest in developing strategies to more
effectively target CML initiating cells. One line of activity
involves global gene expression analyses. Another line of activity
involves identification of downstream partners essential for
maximum BCR-ABL oncoprotein activity. These have reinforced early
evidence of activation of the JAK/STAT, PI3K/AKT, RAS/MAPK and NFKB
pathways in the primitive CML LIC. These studies have also
identified differentially expressed genes involved in regulation of
DNA repair, cell cycle control, cell adhesion, homing,
transcription factors, and drug metabolism. None of these studies
identified potential therapeutic targets useful to eradicate the
CML LIC. Failure to identify such a target may be due to the fact
that, in many studies, expression profiling was done either on
total bone-marrow samples or CD34+ fractionated cells. Apart from
constitutional BCR-ABL expression that causes genetic instability
in time dependent fashion, CD34+ fractionated cells carry a good
degree of heterogeneity in itself. Thus, variations in patients
sample and use of a heterogeneous cell population obscured
identification of meaningful targets. Based on these observations,
knowing the mechanisms of oncogene addiction in Imatinib sensitive
cells will permit engineering of CML LIC to achieve sensitivity for
kinase inhibitors.
[0052] In one embodiment, the BCR-ABL tyrosine kinase inhibitor is
at least one of Imatinib (Novartis), Nilotinib (Novartis),
Dasatinib (BMS), and Ponatinib (Ariad). In one embodiment, the
BCR-ABL tyrosine kinase inhibitor is Imatinib.
[0053] In one embodiment, the Dusp-1 inhibitor is at least one of
BCI, TPI-2, TPI-3, and triptolide. In one embodiment, the Dusp-1
inhibitor is BCI.
[0054] In one embodiment, the c-Fos inhibitor is at least one of
curcumin, difluorinated curcumin (DFC), T5224, nordihydroguaiaretic
acid (NDGA), dihydroguaiaretic acid (DHGA), and SR11302. In one
embodiment, the c-Fos inhibitor is curcumin. In one embodiment, the
c-Fos inhibitor is difluorinated curcumin (DFC). In one embodiment,
the c-Fos inhibitor is NDGA. In one embodiment, the c-Fos inhibitor
is T5224.
[0055] An unbiased mRNA expression profiling was performed using
BAF3 cells, which requires IL-3 for survival, expressing the
BCR-ABL tyrosine kinase under a Tet-R responsive promoter that
renders them IL-3-independent. BaF3 cells were used because it is
homogeneous in terms of gene expression, and because BCR-ABL
dependence is reversible. Specifically, in the presence of
exogenous IL-3, BAF3 cells no longer depend on BCR-ABL for
survival, as shown in FIG. 1.
[0056] More specifically, FIG. 1 shows that growth factor signaling
in leukemic cells abrogates the BCR-ABL dependence. FIG. 1A shows
conditional expression of BCR-ABL in BaF3 cells; without
doxycycline there is no expression of BCR-ABL in BaF3 cells. FIG.
1B is a Western blot showing the kinase activity of BCR-ABL at
different concentrations of inhibitor. This demonstrated that IL-3
had no effect on mediated kinase inhibition. FIG. 1C shows a dose
response curve for Imatinib on BAF3-BCR-ABL cells, where squares
are BCR-ABL+IL-3, circles are BCR-ABL, and triangles are BAF3. This
demonstrated that Imatinib was no longer effective when cells were
grown with IL-3. FIG. 1D shows cell proliferation assays showing
the abrogation of BCR-ABL addiction K562 cells when grown with
erythropoietin (EPO), while other hematopoietic cytokines did not
have a significant effect.
[0057] This biology is reminiscent of CD34+ CML stem cell behavior.
The data were obtained on freshly made BaF3 cells expressing
BCR-ABL conditionally, because long-term expression of BCR-ABL in
any cell causes severe genomic instability and permanent
irreversible changes in gene expression. This likely would
exacerbate problems identifying the critical gene or genes involved
in BCR-ABL addiction.
[0058] To define the differential expression of gene(s) in BCR-ABL
addicted and non-addicted conditions, expression analysis was
performed using total RNA from BaF3 cells, BaF3 cells expressing
BCR-ABL conditionally in the presence and absence of exogenously
added IL-3 (FIG. 2A1) and BaF3-BCR-ABL cells treated with Imatinib
in the presence and absence of IL-3 (FIG. 21B1).
[0059] AP-1 transcription factor c-Fos and dual specificity
phosphatase-1 mediated the BCR-ABL addiction. Comparative analysis
of gene expression from these two data sets would allow
identification of the sets of genes involved in BCR-ABL addiction,
and identified 331 genes that were differently expressed in these
conditions. Given BCR-ABL addiction in K562 cells and attenuation
of addiction by erythropoietin, similar gene expression analysis in
K562 cells would permit sorting out the false positives and may
corroborate the data sets. Expression profiling of K562 cells
identified 301 differently expressed genes; about one third of the
genes are common to the gene list of BCR-ABL-BaF3 (FIG. 21B1). To
narrow the list to identify clinically significant candidate genes,
these data sets were compared with the expression profiling of
CD34.sup.+ cells from CML patients before and after Imatinib
treatment. Only three genes, Dusp-1, Dusp-10, and c-Fos, were down
regulated in BCR-ABL addicted cells, while they were upregulated to
3-5 fold in non-addicted cells. This suggested their role in
BCR-ABL dependence. The role of these three genes in mediating
BCR-ABL addiction were evaluated; specifically, whether their
down-regulation in non-addicted cells would sensitize them to
Imatinib induced apoptosis. c-Fos, Dusp-1 and Dusp-10 were knocked
down using shRNA hairpin, and cell survival analysis was performed
in the presence of 5 .mu.M Imatinib, which typically kills addicted
cells in 24 hrs at this concentration, and IL-3. Dusp-1 and c-Fos
knockdown alone induced 30% and 40% sensitivity to Imatinib,
respectively. Dusp-10 knock down did not show any significant
sensitivity to Imatinib. This suggested that double knock down of
c-Fos and Dusp-1 may sensitize the BCR-ABL cells fully. To test
this, instead of using shRNA mediated gene knock down of Dusp-1, a
small molecule inhibitor that targets Dusp-I, BCI, was used. In
cell proliferation assays, BaF3-BCR-ABL cells with c-Fos knockdown
were fully sensitive to Imatinib when combined with BCI (FIG. 2F).
The same combinations of drugs had no effect on BCR-ABL positive
and parental BaF3 cells, highlighting the response specificity.
[0060] FIGS. 2A1-2A5 are a heat map of differential gene expression
in BaF3 cells expressing the BCR-ABL grown with and without
exogenously added IL-3. Expression of BCR-ABL was induced by adding
doxycycline in the growth media. This expression profile was
normalized with parental BaF3 cells grown with IL-3. This analysis
identified 809 genes that were differently regulated by BCR-ABL in
the presence of IL-3. FIGS. 2B1-2B12 are a heat map showing that
900 genes were differently expressed in the BCR-ABL-BaF3 cells
treated with Imatinib in the presence and absence of IL-3. Cells
treated with IL-3 and Imatinib are resistant to apoptosis and are
represented as live cells; cells treated with Imatinib in the
absence of IL-3 will apoptose. To identify the critical genes that
mediates resistance or sensitivity to Imatinib in addicted cells,
cells were separated into three distinct sub-populations: live,
early-apoptotic, and late-apoptotic using Annexin V and propidium
iodide staining. Comparing gene lists from A and B identified that
331 genes are common and are differently regulated. FIGS. 2C1-2C2
show expression profiling of K562 cells treated with Imatinib in
the presence and absence of erythropoietin (EPO). This analysis
identified 301 genes that were expressing differently in K562
cells. FIGS. 2D1-2D2 show expression profiling of CD34.sup.+
positive cells from CML patients before and after one week of
Imatinib treatment (gene set enrichment (GSE) 12211) which
identified 87 genes that were differently expressed. FIG. 2E is a
Venn diagram showing overexpression of three genes Dusp-1, Dusp-10,
and c-Fos in BaF3 cells, K562, and CML-CD34.sup.+ cells. FIG. 2F is
a cell proliferation assay of BCR-ABL cells expressing shRNA
hairpins for c-Fos, Dusp1 and Dusp-10 was performed in the presence
of IL-3 with 5 .mu.M Imatinib and 1 .mu.M of the Dusp-1 inhibitor
(E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one
(BCI), alone or in combination. In each group of three, the top bar
indicates Imatinib, the middle bar indicates BCI, and the lower bar
indicates Imitinib+BCI. The results revealed that down regulation
of c-Fos and Dusp-1 together mediated the BCR-ABL addiction.
[0061] Efficacy of Dusp-1 and c-Fos inhibition in mouse model of
CML and CD34.sup.+ cells from CML patients was shown. The BCR-ABL
tyrosine kinase inhibitor Imatinib improves the survival of
patients but does not eliminate LICs. This suggested that these
cells are not addicted to BCR-ABL. The data demonstrated that
downregulation of c-Fos and Dusp-1 mediated BCR-ABL addiction.
Inhibition of c-Fos and Dusp-1 together induced apoptosis in
BCR-ABL positive cells following Imatinib treatment. The same
combination has no effect on survival and apoptosis of parental
BaF3 cells. Dusp-1 and c-Fos knockout mice were viable and survived
without any serious phenotype, suggesting that these targets were
suitable for therapeutic development. The effectiveness of c-Fos
and Dusp-1 inhibition in LICs for Imatinib response was determined
before making any therapeutic utility.
[0062] c-Fos and Dusp-1 were targeted using both genetic (shRNA)
and pharmacological inhibitors to provide a basis for clinical
application to target CML initiating cells. The retroviral bone
marrow transduction transplantation model of BCR-ABL-induced CML
was established. FIG. 3 shows schema for in vitro and in vivo
evaluation of c-Fos and Dusp-1 to induce BCR-ABL addiction in LSCs.
FIG. 3A shows retrovirus and lentivirus constructs for
hematopoietic stem/progenitor transduction. FIGS. 3B, C and D show
bone marrow harvesting and sorting of Kit.sup.+ cells. FIG. 3E
shows viral transduction of K.sup.+L.sup.-S.sup.+ cells with pMIGBA
and pL VIR viruses followed by cell sorting for doubly positive
cells GFPIRFP cells. FIG. 3F shows that these doubly positive cells
will be injected to mice followed with treatment by Imatinib alone
and in combination with Dusp-1 and Fos inhibitors. CFU assays in
the presence and absence of Imatinib, and also in combination with
Dusp-1 and Fos inhibitors are performed.
[0063] Therapeutic response of Imatinib in LICs following the c-Fos
and Dusp-1 knock down using shRNA overexpression was evaluated. As
shown in FIG. 3, flow-sorted Kit.sup.+Lin.sup.-Sca1.sup.+ cells
from C51BU6 mice were transduced with retroviruses expressing
BCR-ABL-Ires-GFP and lentiviruses overexpressing shRNAs for c-Fos
and Dusp-1 with RFP. The transduced cells were sorted again for GFP
and RFP positivity. These doubly positive cells were used for in
vitro and in vivo analysis. As a control, vector containing
scrambled shRNA transduced cells and cells expressing the shRNA for
Dusp-1 and Fos alone in the presence and absence of BCR-ABL were
used. For each condition, 10 mice were injected through tail vein
with 10.sup.4 sorted cells mixed with 5.times.10.sup.5 RBC depleted
total bone marrow. After seven days, mice were subjected to drug,
Imatinib, BCI, treatments. To evaluate the effect of drug
administration on apoptosis of stem cells in vivo, a set of
leukemic mice were sacrificed on day 5 of treatment and apoptosis
in the KLS population was measured by labeling with Annexin V and
DAPI. For in vitro CFU assays, methylcellulose colonogenic assays
are performed by plating 10.sup.3 sorted cells in 0.9% MethoCult
(Stem Cell Technologies) with hematopoietic growth factors in the
presence of Imatinib alone, BCI alone, and in combination of both
inhibitors. Colonies (>100 .mu.m) from primary cells are scored
after 7-15 days. If good transduction efficiency is not achieved
due to use of two different viruses, inducible transgenic
Scl-tTaBCRIABL are used. BM cells are obtained from
Scl-tTa-BCRABL-GFP mice 4 weeks after induction of BCR-ABL
expression by tetracycline withdrawal, and a pure population of
KLS/GFP-expressing cells are sorted by flow cytometry followed with
viral transduction expressing shRNA hairpins for Dusp-1 and c-Fos.
These transduced cells are subjected to in vitro and in vivo
analysis.
[0064] Inhibition of c-Fos and Dusp-1 in primary CML CD34.sup.+
cells was shown to evaluate the inventive composition as a
therapeutic agent on primary human samples. Quiescent CD34.sup.+
CML cells from chronic phase patients are known to be less
sensitive than the bulk of the CD34.sup.+ leukemic cells to the
cytotoxic effects of Imatinib inhibition in vitro. This quiescent
population is enriched in CML stem cells (CD34.sup.+CD38.sup.-
cells), but also typically still contains large numbers of more
mature CD34.sup.+CD38.sup.+ cells.
[0065] To determine the effect of c-Fos and Dusp-1 inhibition with
Imatinib, Lin.sup.-CD34.sup.+CD38.sup.+ primitive CML stem cells
were isolated followed with in vitro colony forming unit (CFU)
assay. Additionally, 50,000 Lin.sup.-CD34.sup.+CD38.sup.- cells
were grown in liquid culture with and without the growth factors
IL-3, IL-6, G-CSF, Flt3-LG, SCF and EPO in the presence of Imatinib
(alone), BCI (alone), and with all compounds in combination. After
72 hrs cells were stained with Annexin V and PI to analyze
apoptosis. Clinical samples from CML patients were tested.
[0066] mRNA expression studies were performed in BCR-ABL addicted
and non-addicted cells to identify the candidate gene or genes
mediating drug response. Of several candidate genes, inhibition of
Dual-specificity phosphatase-1 (Dusp-1) and c-Fos by ShRNA and/or
small molecule inhibitors greatly sensitized the LSCs for Imatinib.
This suggested intrinsic resistance of cancer stem cells could be
targeted and may provide curative benefit.
[0067] To validate the role of Dusp-1 and c-Fos in Imatinib
response and therapeutic targeting of leukemic stem cells in vivo,
a bone marrow transduction transplantation model was used. Bone
marrow cells from normal C57Bl/6 mice were transduced with BCR-ABL
retroviruses expressing GFP and transferred to sub-lethally
irradiated mouse hosts. Such mice develop a reproducible
myeloproliferative disease similar to human CML. Treatment with
BCR-ABL inhibitors Imatinib, Nilotinib and Dasatinib prolonged
survival of these mice for 3-4 weeks and leukemic stem cells in
these mice are resistant to therapy as in human subjects,
suggesting kinase inhibitor therapy is not curative. Groups of mice
(n=6) were treated with Imatinib at a dose of 100 mg/kg/day, BCI at
a dose of 5 mg/kg/day targeting Dusp-1, and curcumin at a dose of
50 mg/kg/day targeting c-Fos by intraperitoneal injection. An
identical dose of combination of drugs, Imatinib and BCI, Imatinib
and curcumin, BCI and curcumin, and Imatinib and BCI and curcumin,
were injected intraperitoneally. Drug treatments were started on
day 8 following the bone marrow transplants. Leukemic burden in
mice was assessed weekly by monitoring the GFP positive cells in
peripheral blood using FACS.
[0068] As shown in FIG. 5. the combination of Imatinib, BCI and
curcumin cured mice from CML. In FIG. 5, from left to right, the
six bars in each of the four groups (1st week, 3rd week, 5th week,
7th week) are, in this order, Imatinib, BCI, curcumin,
curcumin+BCI, curcumin+Imatinib, curcumin+Imatinib+BCI. The
histograms show the percentage of GFP positive cells from
peripheral blood as leukemic burden in mice. Each histogram
represented the average value of GFP positive cells from six mice.
Single drug treatment, or a two drug combination treatment
suppressed most leukemic cells, but there were residual leukemic
cells in circulation at three weeks. However, a combination of
Imatinib, BCI, and curcumin did not show any significant number of
leukemic cells in circulation. Mice treated with the Imatinib, BCI
and curcumin (the rightmost bar in each group) did not relapse
following drug withdrawal. This result suggested these mice were
cured from the disease.
[0069] A way to ascertain that there are no leukemic stem cells in
mice is stop drug treatment and test for disease relapse. Any
leukemic stem cells surviving in bone marrow will repopulate the
disease, while curing the disease will fail to do so. Also as shown
in FIG. 5, drug treatment was thus stopped after the fourth week
for the analysis of disease relapse. Leukemic cell analysis from
peripheral blood in the fifth and seventh week clearly demonstrated
that the mice treated with single and two drugs relapsed, while
triple drug treatment had no sign of leukemic cells in peripheral
blood. These results suggested that mice in this treatment group
were cured of the disease.
[0070] Given the problems associated with curcumin absorption and
bioavailability, other c-FOS inhibitors were evaluated. The c-fos
inhibitors nordihydroguaiaretic acid (NDGA) and difluorinated
curcumin (DFC) were tested in two different mouse models of
leukemia, namely, retroviral-bone marrow transplant model, and a
BCR/ABL transgenic mouse model that allows expression of BCR/ABL
only in primitive and multiprogenitors (MPPs) hematopoietic stem
cells. Assessing efficacy of these drug combinations in transgenic
mouse models permitted analysis of LSC dynamics and survival, and
provided definitive proof for eradication of LSCs.
[0071] The data demonstrated that a combination of DFC, BCI, and
Imatinib was more potent than combinations with curcumin, BCI, and
Imatinib, and with NDGA, BCI, and Imatinib, as shown in FIGS. 6 and
7A-D.
[0072] FIG. 6 shows that the combination of Imatinib, DFC and BCI
was more effective in curing the mice from leukemia in
retroviral-transduction bone marrow-transplantation mouse model of
CML. Briefly, c-Kit positive bone marrow cells were harvested from
wild type mice and transduced with retroviruses expressing BCR/ABL
followed with transplantation of 100,000 transduced cell in each
mice with 1 million normal bone marrow cells. In this model, mice
develop leukemia within two weeks and all mice die within three to
four weeks.
[0073] All three combinations, namely DFC, BCI, and Imatinib;
curcumin, BCI, and Imatinib; and NDGA, BCI, and Imatinib, cured
mice from the disease. DFC, BCI, and Imatinib was most effective in
curing mice from the disease. While not being bound by a single
theory, the greater efficacy of DFC, BCI, and Imatinib was likely
due to DFC's greater bioavailability and binding with c-Fos.
[0074] FIGS. 7A-D show that the combination of Imatinib, DFC and
BCI completely eradicated the leukemic stem cells from the
SCL-BCR/ABL mice. Briefly, bone marrow cells were harvested from
the SCL-BCRABL mice and transplanted in Boy/J mice with equal
amount of BM cells from the Boy/J mice. After one month,
transplantation chimerism was recorded by measuring the percentage
of CD45.2 (BCR/ABL) from the bone marrow aspirates which is labeled
as 0 month. After one-month drug treatments were started, and
leukemic burdens were monitored by measuring the levels of CD45.2.
As shown in FIGS. 7A-D, the combination of Imatinib, DFC and BCI
completely cured the mice.
[0075] In vivo data unequivocally demonstrated that Dusp-1 and
c-Fos mediated BCR-ABL addiction and leukemic stem cell biology.
Dusp-1 and c-Fos inhibitors are thus targets for curative therapy
in CML.
[0076] Tyrosine-kinase inhibitor (TKI) therapy for human cancers is
not curative, and relapse occurs owing to the continued presence of
tumor cells, referred to as minimal residual disease (MRD). The
survival of MRD stem or progenitor cells in the absence of
oncogenic kinase signaling, a phenomenon referred to as intrinsic
resistance, depends on diverse growth factors. Here we report that
oncogenic kinase and growth-factor signaling converge to induce the
expression of the signaling proteins FBJ osteosarcoma oncogene
(c-FOS, encoded by Fos) and dual-specificity phosphatase 1 (DUSP1).
Genetic deletion of Fos and Dusp1 suppressed tumor growth in a
BCR-ABL fusion protein kinase-induced mouse model of chronic
myeloid leukemia (CML). Pharmacological inhibition of c-FOS, DUSP1
and BCR-ABL eradicated MRD in multiple in vivo models, as well as
in mice xenotransplanted with patient-derived primary CML cells.
Growth-factor signaling also conferred TKI resistance and induced
FOS and DUSP1 expression in tumor cells modeling other types of
kinase-driven leukemias. Our data demonstrate that c-FOS and DUSP1
expression levels determine the threshold of TKI efficacy, such
that growth-factor-induced expression of c-FOS and DUSP1 confers
intrinsic resistance to TKI therapy in a wide-ranging set of
leukemias, and might represent a unifying Achilles' heel of
kinase-driven cancers.
[0077] Protein kinases are frequently activated in a variety of
human cancers and represent attractive drug targets. In this
regard, chronic myeloid leukemia (CML) represents an important
paradigm, given that the success of imatinib in treating patients
with CML provided proof of concept for targeted anti-kinase therapy
and paved the way for the development of TKI therapy for several
solid tumor types (Daley, G. Q., Van Etten, R. A. & Baltimore,
D. Induction of chronic myelogenous leukemia in mice by the
P210bcr/abl gene of the Philadelphia chromosome. Science 247,
824-830 (1990); Druker, B. J. et al. Effects of a selective
inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl
positive cells. Nat. Med. 2, 561-566 (1996)). Despite the
impressive response to TKI therapy in the clinic, it is not
curative because a small population of cancer cells are insensitive
to treatment, manifesting as minimal residual disease (MRD)
(O'Hare, T., Zabriskie, M. S., Eiring, A. M. & Deininger, M. W.
Pushing the limits of targeted therapy in chronic myeloid
leukaemia. Nat. Rev. Cancer 12, 513-526 (2012)). The cells
responsible for MRD in CML are referred to as leukemia-initiating
cells (LICs), whereas those responsible for MRD in solid tumors are
referred to as cancer stem cells (CSCs). In .about.50-60% of
patients with CML, continuous drug treatment is needed to prevent
MRD cells from reinstating the disease (Rousselot, P. et al.
Imatinib mesylate discontinuation in patients with chronic
myelogenous leukemia in complete molecular remission for more than
2 years. Blood 109, 58-60 (2007); Mahon, F. X. et al.
Discontinuation of imatinib in patients with chronic myeloid
leukaemia who have maintained complete molecular remission for at
least 2 years: the prospective, multicentre Stop Imatinib (STIM)
trial. Lancet Oncol. 11, 1029-1035 (2010); Ross, D. M. et al.
Safety and efficacy of imatinib cessation for CML patients with
stable undetectable minimal residual disease: results from the
TWISTER study. Blood 122, 515-522 (2013)). MRD cells serve as a
reservoir that can develop TKI resistance by acquiring mutations or
by activating alternative survival mechanisms (Chu, S. et al.
Detection of BCR-ABL kinase mutations in CD34+ cells from chronic
myelogenous leukemia patients in complete cytogenetic remission on
imatinib mesylate treatment. Blood 105, 2093-2098 (2005); Savona,
M. & Talpaz, M. Getting to the stem of chronic myeloid
leukaemia. Nat. Rev. Cancer 8, 341-350 (2008); Azam, M., Latek, R.
R. & Daley, G.Q. Mechanisms of autoinhibition and
STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL.
Cell 112, 831-843 (2003)). Even the most potent kinase inhibitors
are ineffective against LICs that are present in MRD (O'Hare, T.,
Zabriskie, M. S., Eiring, A. M. & Deininger, M. W. Pushing the
limits of targeted therapy in chronic myeloid leukaemia. Nat. Rev.
Cancer 12, 513-526 (2012); Krause, D. S. & Van Etten, R. A.
Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med.
353, 172-187 (2005)).
[0078] Oncogene addiction refers to the exquisite dependence of
transformed cells on a single mutant protein or signaling pathway
for survival and proliferation (Weinstein, I. B. Cancer. Addiction
to oncogenes--the Achilles heal of cancer. Science 297, 63-64
(2002)). The therapeutic response to TKIs is mediated by oncogene
addiction to mutant tyrosine-kinase oncoproteins (Weinstein, I. B.
Cancer. Addiction to oncogenes--the Achilles heal of cancer.
Science 297, 63-64 (2002); Sawyers, C. L. Shifting paradigms: the
seeds of oncogene addiction. Nat. Med. 15, 1158-1161 (2009);
Pagliarini, R., Shao, W. & Sellers, W. R. Oncogene addiction:
pathways of therapeutic response, resistance, and road maps toward
a cure. EMBO Rep. 16, 280-296 (2015)). Multiple theories, including
signaling-network dysregulation, synthetic lethality (Reddy, A.
& Kaelin, W. G., Jr. Using cancer genetics to guide the
selection of anticancer drug targets. Curr. Opin. PharmacoL 2,
366-373 (2002); Kaelin, W. G., Jr. The concept of synthetic
lethality in the context of anticancer therapy. Nat. Rev. Cancer 5,
689-698 (2005)), genetic streamlining (Kamb, A. Consequences of
nonadaptive alterations in cancer. Mol. Biol. Cell 14, 2201-2205
(2003); Mills, G. B., Lu, Y. & Kohn, E. C. Linking molecular
therapeutics to molecular diagnostics: inhibition of the
FRAP/RAFT/TOR component of the PI3K pathway preferentially blocks
PTEN mutant cells in vitro and in vivo. Proc. Natl. Acad. Sci. USA
98, 10031-10033 (2001)), and oncogenic shock (Sharma, S. V. &
Settleman, J. Exploiting the balance between life and death:
targeted cancer therapy and "oncogenic shock". Biochem. Pharmacol.
80, 666-673 (2010); Sharma, S. V. & Settleman, J. Oncogene
addiction: setting the stage for molecularly targeted cancer
therapy. Genes Dev. 21, 3214-3231 (2007)), have attempted to
explain how cells become oncogene addicted and how acute inhibition
of an oncoprotein induces cell death. However, it is still not
understood how MRD cells that do not respond to TKI therapy escape
addiction to the driver oncogene. Recent studies have revealed that
growth-factor signaling mediates resistance to TKI therapy in both
leukemia and solid organ tumors (Corbin, A. S. et al. Human chronic
myeloid leukemia stem cells are insensitive to imatinib despite
inhibition of BCR-ABL activity. J. Clin. Invest. 121, 396-409
(2011); Straussman, R. et al. Tumour micro-environment elicits
innate resistance to RAF inhibitors through HGF secretion. Nature
487, 500-504 (2012); Wilson, T. R. et al. Widespread potential for
growth-factor-driven resistance to anticancer kinase inhibitors.
Nature 487, 505-509 (2012)), but it remains to be determined
whether intrinsic resistance conferred by a diverse set of growth
factors utilizes distinct or shared molecular pathways. For
instance, interleukin (IL)-3, IL-6, stem cell factor (SCF),
fms-like tyrosine kinase 3 ligand (FLT3L) and granulocyte
colony-stimulating factor (G-CSF) signaling in CML progenitor cells
confer intrinsic resistance to imatinib. Similarly, hepatocyte
growth factor (HGF) and neuregulin 1 (NRG1) signaling confer
intrinsic resistance to protooncogene protein B-raf (BRAF) and
epidermal growth factor receptor (EGFR) inhibitors in solid tumors
(Corbin, A. S. et al. Human chronic myeloid leukemia stem cells are
insensitive to imatinib despite inhibition of BCR-ABL activity. J.
Clin. Invest. 121, 396-409 (2011); Straussman, R. et al. Tumour
micro-environment elicits innate resistance to RAF inhibitors
through HGF secretion. Nature 487, 500-504 (2012); Wilson, T. R. et
al. Widespread potential for growth-factor-driven resistance to
anticancer kinase inhibitors. Nature 487, 505-509 (2012)).
Growth-Factor-Induced Expression of c-FOS and DUSP1 Confers TKI
Resistance
[0079] To understand how growth-factor signaling induces intrinsic
resistance to TKI treatment, we modeled
growth-factor-induced-mitigation of TKI response using the
IL-3-dependent BaF3 mouse cell line. We generated BaF3 cells with
tetracycline-inducible expression of BCRABL (BaF3-LTBA; FIG. 8A),
as well as cells with constitutive BCR-ABL expression (BaF3-BA9).
Imatinib treatment of both BaF3-LTBA cells and BaF3-BA cells caused
cell death, whereas the addition of IL-3 conferred resistance to
imatinib, even in the case of sustained inhibition of BCR-ABL
enzymatic activity (FIGS. 8B-D and FIG. 9A). Similarly,
erythropoietin treatment conferred imatinib resistance in the human
BCR-ABL+ cell line K562 (an erythromyeloblastoid leukemia cell line
derived from a patient with blast-crisis CML; FIG. 8E and FIG. 9B).
Thus, we were able to recapitulate cytokine and/or
growth-factor-induced resistance to imatinib in vitro.
[0080] We hypothesized that expression of the critical genes
mediating TKI resistance would be modulated by BCR-ABL,
growth-factor signaling, and TKI treatment. We therefore compared
the expression profiles of BCR-ABL-induced BaF3-LTBA cells with and
without IL-3 treatment (192 genes were differentially expressed;
FIG. 9C), as well as imatinib-treated BaF3-BA cells with and
without IL-3 (308 genes were differentially expressed; FIG. 9D).
Next, we evaluated erythropoietin-modulated gene expression in
imatinib-treated K562 cells (1,338 genes were differentially
expressed; FIG. 9E). Finally, we analyzed existing gene-expression
profiles from primary bone marrow (BM)-derived BCR-ABL+CD34+ cells
collected from patients with CML before and after 1 week of
imatinib treatment (Bruennert, D. et al. Early in vivo changes of
the transcriptome in Philadelphia chromosome-positive CD34+ cells
from patients with chronic myelogenous leukaemia following imatinib
therapy. Leukemia 23, 983-985 (2009)), and we identified genes that
were differentially expressed in the surviving marrow cells (85
genes were differentially expressed; FIG. 9F). When these four data
sets were compared, only three differentially expressed genes were
common to all comparisons: FOS (also known as c-FOS),
dual-specificity phosphatase-1 (DUSP1) and ZFP36 (FIG. 8F and FIG.
9G). c-Fos belongs to the family of activator protein 1 (AP1)
transcription factors implicated in the regulation of cell
proliferation, survival, apoptosis, transformation, and oncogenesis
(Eferl, R. & Wagner, E. F. AP-1: a double-edged sword in
tumorigenesis. Nat. Rev. Cancer 3, 859-868 (2003)). DUSP1 is a
nuclear protein that provides feedback regulation to MAPK signaling
by inactivating MAPKs25 and has been implicated in the regulation
of inflammation, immune regulation, and chemoresistance in cancer
(Lawan, A., Shi, H., Gatzke, F. & Bennett, A. M. Diversity and
specificity of the mitogen-activated protein kinase phosphatase-1
functions. Cell. MoL Life Sci. 70, 223-237 (2013); Jeffrey, K. L.,
Camps, M., Rommel, C. & Mackay, C. R. Targeting
dual-specificity phosphatases: manipulating MAP kinase signalling
and immune responses. Nat. Rev. Drug Discov. 6, 391-403 (2007)).
ZFP36 is an RNA-binding protein that has been implicated in cancer
development, inflammation, and immune functions (Brooks, S. A.
& Blackshear, P. J. Tristetraprolin (TTP): interactions with
mRNA and proteins, and current thoughts on mechanisms of action.
Biochim. Biophys. Acta 1829, 666-679 (2013)).
[0081] In support of the hypothesis that oncogenic and
growth-factor signaling modulate Fos, Dusp1, and Zfp36 expression,
we found that both BCR-ABL and imatinib induced expression of these
genes in BaF3-BA cells (FIG. 8G-I). Similarly, expression analysis
of patient samples from chronic and blast-phase CML revealed higher
(2-10-fold) expression of FOS, DUSP1, and ZFP36 as compared to that
in normal CD34+ cells (FIG. 8J; the CML patient samples used in
this study are described in Table 2). Whereas treatment with
imatinib alone downregulated the expression of these genes,
treatment with imatinib plus growth factor (IL-3) resulted in
4-5-fold higher expression of these genes, as compared to BaF3-BA
plus imatinib (FIG. 8G). In the absence of IL-3, ectopic expression
of either c-Fos or DUSP1 led to modest resistance to imatinib,
whereas ectopic expression of ZFP36 did not have an effect (FIG.
10A,B). However, similarly to IL-3, ectopic expression of Fos,
Dusp1, and Zfp36 together in BaF3-BA cells impaired the inhibitory
effect of imatinib on cell survival (FIG. 10B). Conversely, the
depletion of Fos, Dusp1, and Zfp36 (either each alone or in
combination) by shRNA-mediated knockdown reduced BCR-ABL-dependent
proliferation and survival in BaF3-BA cells, whereas parental BaF3
cells were not affected (FIG. 10C-F). These results suggest that
Fos, Dusp1, and Zfp36 are functional mediators of
growthfactor-induced imatinib resistance. Furthermore, knockdown of
Fos and Dusp1 alone or together sensitized BaF3-BA cells to
imatinib, even in the presence of IL-3 (FIG. 10G). However, the
depletion of Zfp36 sensitized parental BaF3 and BaF3-BA cells
equally to imatinib, which suggests that, unlike Fos and Dusp1,
Zfp36 is not differentially required by BCR-ABL-expressing cells.
Therefore, we focused subsequent analyses on Fos and Dusp1.
TABLE-US-00002 TABLE 2 Description of CML patient samples Sample
FISH Karyotype BCR-ABL1 ID Diagnosis Treatment Age Sex (% Ph+)
comments Origin sequencing CP1 CML- Treated 40 Male 98 46, XY, t(9;
22) Peripheral WT Chronic with Blood Phase Hydrea (1000 mg) CP2
CML- Untreated 30 Female 13 46, XX, t(9; 22) Bone WT Chronic Marrow
Phase CP3 CML- Untreated 30 Female 64 46, XX, t(9; 22) Bone WT
Chronic Marrow Phase CP4 CML- Untreated 35 Male 99.2 46, XY, t(9;
22) Peripheral WT Chronic Blood Phase BC1 CML- Untreated 38 Male
72.5 46, XY, t(9; 22) Peripheral WT Blast Blood Crisis BC2 CML-
Untreated 59 Female 13.5 46, XX, t(9; 22) Peripheral WT Blast Blood
Crisis BC3 CML- Untreated 34 Male 64 46, XY, t(9; 22). Peripheral
WT Blast Mutation in Blood Crisis ASXL1.
Deletion of Fos and Dusp1 abrogates intrinsic TKI resistance
[0082] To determine the roles of c-Fos and DUSP1 in BCR-ABL-induced
leukemogenesis, we determined the effects of the deletion of either
Dusp1.sup.-/- (Dorfman, K. et al. Disruption of the erp/mkp-1 gene
does not affect mouse development: normal MAP kinase activity in
ERP/MKP-1-deficient fibroblasts. Oncogene 13,925-931 (1996)) or
Fos.sup.fl/fl (ROSACre.sup.ERT2Fos.sup.fl/fl) (Zhang, J. et al.
c-fos regulates neuronal excitability and survival. Nat. Genet. 30,
416-420 (2002)), or both genes
(ROSACre.sup.ERT2Fos.sup.fl/fl;Dusp1.sup.-/-). We used
hematopoietic Kit+ cells transduced with BCR-ABL-Ires-YFP
retroviruses for in vitro colony-forming unit (CFU) assays and for
generating an in vivo model of CML (FIG. 11A). Genetic deletion of
Fos or Dusp1 significantly reduced (by 50%) the number of CFUs
generated by BCR-ABL-expressing Kit+ cells, whereas CFU generation
by control cells (Kit+ cells transduced with a MSCV-Ires-YFP virus)
was not affected (FIG. 11B,C). The deletion of Fos and Dusp1 alone
sensitized BCR-ABL-expressing Kit+ cells to imatinib treatment
(.about.80% reduction, as compared to 50% reduction in wild-type
(WT) controls). Strikingly, the deletion of both Fos and Dusp1
suppressed the number of CFUs generated by BCR-ABL expressing cells
(.about.90%), and treatment with imatinib completely eradicated
BCR-ABL-positive colonies (FIG. 11D,E). For analysis in vivo, mice
were transplanted with 40,000 Kit+BM cells expressing BCR-ABL and
YFP. Recipient mice developed fatal leukemia with a disease latency
of 2-3 weeks (Zhao, C. et al. Hedgehog signalling is essential for
maintenance of cancer stem cells in myeloid leukaemia. Nature 458,
776-779 (2009)). Imatinib treatment did not result in a significant
reduction in leukemic burden as compared to vehicle treatment, and
all mice died in 3-4 weeks (FIG. 11F,G). By contrast, the deletion
of Dusp1 delayed BCR-ABL-induced leukemia by 1 week, and deletion
of Fos led to a disease latency of 7-8 weeks (FIG. 11F-G). Notably,
imatinib treatment of mice transplanted with c-Fos-deficient cells
for 1 month led to a significant reduction in leukemic burden (to
0.5-4%); .about.50% of mice survived, and treatment discontinuation
did not result in disease relapse (FIG. 11H,I), which suggests that
TKI resistant MRD was eliminated. In control experiments in which
Fos was not deleted (using non-tamoxifen-treated
ROSACreERT2Fosfl/fl donor cells), recipients showed disease-latency
periods similar to those observed in WT mice in both
imatinib-treated and untreated groups (FIG. 12A,C).
[0083] The deletion of both Fos and Dusp1 had a greater effect than
deletion of either gene alone, rescuing .about.60% of the mice from
leukemic death and significantly reducing leukemic burden (FIG.
11H,K). Control ROSACreERT2Fosfl/fl;Dusp1-/- mice, not treated with
tamoxifen, showed modestly prolonged survival as compared to WT
controls (FIG. 12B,D), which was correlated with lower Fos mRNA
expression (FIG. 12E). Deletion of both Fos and Dusp1, when
combined with a 5-week course of imatinib treatment, eradicated all
leukemic cells from peripheral blood and bone marrow, and
discontinuation of treatment did not result in disease relapse, as
assessed by YFP+ cell burden in blood and bone marrow at the end of
the experiment, 4 months after tumor cell transplantation (FIG.
11J,K). Thus, genetic deletion of Fos and Dusp1 sensitizes
BCR-ABL-expressing cells (but not WT cells) to imatinib treatment
and eliminates TKI-resistant MRD. Moreover, the deletion of Fos and
Dusp1 in ROSACreERT2Fosfl/fl;Dusp1-/- mice by tamoxifen injection
did not show any apparent hematopoietic defects, and c-Fos- and
Dusp1-deficient cells were maintained in the peripheral blood and
bone marrow (FIG. 12F), which supports the concept that these genes
are required for BCR-ABL-induced transformation and leukemia
development but are dispensable for normal hematopoiesis.
[0084] To test whether c-Fos is required for the maintenance of
disease, we deleted Fos after the onset of disease (3 weeks after
tumor cell transplantation). At 2 weeks after transplantation, both
WT and ROSACreERT2Fosfl/fl;Dusp1-/- mice showed an increase in
granulocytes (.about.70-80% of peripheral blood cells, as compared
to 32% in WT controls) at the expense of B cells (FIG. 12G). As
shown above, the deletion of both Fos and Dusp1 at week 3 rescued
.about.60% of the mice from leukemia (FIG. 12H). Imatinib treatment
rescued mice transplanted with Fos- and Dusp1-deleted cells
expressing BCR-ABL from leukemia and led to rapid clearance of the
leukemic cells. Whereas non-imatinib-treated mice transplanted with
cells expressing BCR-ABL in which Fos and Dusp1 had been deleted
showed gradual depletion of leukemic cells, vector-only control
cells not expressing BCR-ABL with the same deletion showed stable
engraftment (FIG. 12H,I). Taken together, these data suggest that
genetic loss of Fos and Dusp1 together sensitizes leukemic cells to
TKI in CML and confers synthetic lethality to BCR-ABLtransformed
cells, given that leukemic cells are gradually depleted even
without TKI treatment.
[0085] To rule out potential nonspecific effects of Fos or Dusp1
deletion on BCR-ABL-induced leukemia, we performed two additional
experiments. First, we expressed a dominant-negative c-FOS (lacking
the DNA-binding domain (Ransone, L. J., Visvader, J., Wamsley, P.
& Verma, I. M. Trans-dominant negative mutants of Fos and Jun.
Proc. Natl. Acad. Sci. USA 87, 3806-3810 (1990)); FIG. 11L,M)
together with BCR-ABL in WT C57BL/6 bone marrow cells. Expression
of dominant-negative c-FOS had effects that were similar to those
observed for Fos deletion; i.e., >50% reduction in the number of
BCR-ABL-dependent CFUs (FIG. 11N). Second, expression of WT c-FOS
partially rescued the phenotype, whereas expression of both c-FOS
and Dusp1 using a monocistronic vector (P2A peptide cleavage)
restored CFU numbers to normal levels (FIG. 11O).
Inhibition of c-Fos, Dusp1, and BCR-ABL by Small-Molecule
Inhibitors Cures Mice of CML
[0086] To test the potential of targeting c-Fos and Dusp1 for
therapy, we performed in vitro and in vivo experiments using
small-molecule inhibitors of c-Fos and Dusp1 (FIG. 13A). CFU
analysis of BCR-ABLLSK cells (bone-marrow-derived
Lin-Sca1+Kit+(LSK) cells represent hematopoietic stem and
progenitor cells) treated with c-Fos and Dusp1 inhibitors
recapitulated the genetic data. Specifically, combined treatment
with a Dusp1 and Dusp6 inhibitor
((E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one;
BCI) (Molina, G. et al. Zebrafish chemical screening reveals an
inhibitor of Dusp6 that expands cardiac cell lineages. Nat. Chem.
Biol. 5, 680-687 (2009)), a c-Fos inhibitor (curcumin) (Huang, T.
S., Lee, S. C. & Lin, J. K. Suppression of c-Jun/AP-1
activation by an inhibitor of tumor promotion in mouse fibroblast
cells. Proc. Natl. Acad. Sci. USA 88, 5292-5296 (1991)) and a
BCR-ABL inhibitor (imatinib) completely suppressed CFU formation
(imatinib+ curcumin+BCI; FIG. 14A,B). To address the possibility of
off-target effects of curcumin, we tested two other chemically
distinct compounds targeting c-Fos: nordihydroguaiaretic acid
(Park, S., Lee, D. K. & Yang, C. H. Inhibition of fos-jun-DNA
complex formation by dihydroguaiaretic acid and in vitro cytotoxic
effects on cancer cells. Cancer Lett. 127, 23-28 (1998)) (NDGA) and
difluorinated curcumin (Padhye, S. et al. Fluorocurcumins as
cyclooxygenase-2 inhibitor: molecular docking, pharmacokinetics and
tissue distribution in mice. Pharm. Res. 26, 2438-2445 (2009))
(DFC). Both NDGA and DFC (imatinib+NDGA+BCI and imatinib+DFC+BCI)
were effective at suppressing BCR-ABL-dependent CFU formation (FIG.
13B and FIG. 14C), which suggests that c-Fos is the relevant target
of these compounds. Next, we examined the efficacy of these
compounds in vivo by using a retroviral bone marrow transduction
transplantation leukemogenesis model. A 1-month course of treatment
with individual compounds with imatinib (BCI, curcumin, and NDGA)
or without imatinib (BCI and curcumin) did not effectively treat
leukemogenesis; however, treatment with imatinib+ curcumin+BCI and
imatinib+NDGA+BCI rescued 50% and 60% of the recipient mice,
respectively (FIG. 14D). By contrast, a 1-month course of treatment
with imatinib+DFC+BCI rescued .about.90% of mice, and we were
unable to detect MRD by flow cytometry (FIG. 13C,D).
[0087] Given the lack of MRD in the treated mice, we next wished to
test the effectiveness of c-FOS and Dusp1 inhibition in a tumor
model in which leukemic stem cells drive leukemogenesis. To this
end, we used transgenic mice in which the 3' enhancer of the Scl
gene drives expression of the tetracycline transactivator (tTA) to
regulate tet-O-BCR-ABL transgene expression in hematopoietic stem
and progenitor cells (Koschmieder, S. et al. Inducible chronic
phase of myeloid leukemia with expansion of hematopoietic stem
cells in a transgenic model of BCR-ABL leukemogenesis. Blood 105,
324-334 (2005)) (FIG. 13E). We transplanted 3,000-5,000 bone marrow
LSK cells into irradiated BoyJ mice, which developed leukemia
within 4-6 weeks. 4 weeks after transplantation, drug treatment was
started (FIG. 13F). Treatment with imatinib alone for 3 months
resulted in a reduction (.about.60%) of CML cells in the bone
marrow; however, a substantial percentage of BCR-ABL-positive cells
were present in the bone marrow (FIG. 13G). Moreover, when
treatment was discontinued, disease relapse occurred in all mice,
replicating the clinical course of TKI therapy in CML (FIG. 13G).
By contrast, not only did treatment with imatinib+DFC+BCI for 3
months eradicate the donor-derived BCR-ABL-positive leukemic cells
(CD45.2) in the bone marrow, but no relapse occurred when treatment
was discontinued (FIG. 13G and FIG. 15A-C). Similarly to the
retroviral CML model (FIG. 13C), treatment with imatinib+
curcumin+BCI or imatinib+NDGA+BCI resulted in effective eradication
of CML stem progenitor cells, but a few recipient mice in these
treatment groups relapsed once treatment was discontinued (FIG.
15C). Taken together, these results suggest that treatment with
imatinib+DFC+BCI is more efficient than the combinations (imatinib
+BCI+ curcumin or imatinib+BCI+NDGA) at targeting leukemic stem
cells that persist in MRD, perhaps owing to superior
pharmacokinetics of DFC over curcumin (Padhye, S. et al.
Fluorocurcumins as cyclooxygenase-2 inhibitor: molecular docking,
pharmacokinetics and tissue distribution in mice. Pharm. Res. 26,
2438-2445 (2009)).
Inhibition of c-FOS and DUSP1 Sensitizes Patient-Derived LICs to
Imatinib
[0088] To extend these findings to human cells, we tested the
effect of c-FOS and DUSP1 inhibition on the survival of primary CML
patient samples (Table 2). Mice transplanted with 3,000 CD34+ cells
from the primary patient sample CP4 showed robust cell engraftment
within 2 weeks (FIG. 16A,B). Drug treatment was started 2 weeks
after transplantation and continued for a period of 4-6 weeks,
using imatinib alone, DFC and BCI, or imatinib+DFC+BCI. As
expected, treatment with imatinib+DFC+BCI for 3 weeks resulted in
effective inhibition of leukemic cells, whereas treatment with
imatinib alone or DFC+BCI was ineffective (FIG. 16B). As previously
reported (Li, L. et al. Activation of p53 by SIRT1 inhibition
enhances elimination of CML leukemia stem cells in combination with
imatinib. Cancer Cell 21, 266-281 (2012)), leukemic engraftment in
this model is not stable, and most mice lost grafts within 7-8
weeks after transplantation (FIG. 15D), which precluded further
investigation of the effect of drug treatment on MRD.
[0089] Next, to test the efficacy of these drugs on primitive LSCs,
we performed long-term-culture-initiating cell (LTC-IC) assays, a
stringent in vitro assay for the detection of primitive
hematopoietic or leukemic stem cells, using mononuclear cells from
the CP1 sample, CD34+ cells from the CP4 sample, and CD34+ cells
from a healthy donor as a control. As expected, LTC-IC activity was
increased in imatinib-treated CP1 cells as compared to the
vehicle-treated controls (FIG. 16C), in agreement with previous
studies (Copland, M. et al. BMS-214662 potently induces apoptosis
of chronic myeloid leukemia stem and progenitor cells and
synergizes with tyrosine kinase inhibitors. Blood 111, 2843-2853
(2008)). However, imatinib treatment of sample CP4 showed a
significant decrease in LTC-IC activity as compared to the vehicle
treated controls (FIG. 16C). Although the basis for the sensitivity
of the CP4 sample to imatinib is unknown--perhaps underlying
patient specific genetic or epigenetic changes confer TKI
sensitivity--the difference in the responses between these two
samples underscores the heterogeneous nature of LSCs in the context
of TKI therapy. Notably, treatment with DFC+BCI did not affect
LTC-IC activity in leukemic or normal cells (FIG. 16C). As
expected, treatment with DFC+BCI+ imatinib selectively eradicated
the LTC-IC activity of leukemic cells (FIG. 16C); however, as
opposed to the results with genetic deletion, this drug treatment
partially inhibited normal cell growth, perhaps owing to off-target
toxicity. Taken together, these results provide evidence that a
combination of DFC+BCI+ imatinib selectively targets CML stem and
progenitor cells but spares normal CD34+ cells.
c-Fos and Dusp1 Deficiency Alters the AP1-Regulated Networks
[0090] Given that curcumin and its analogs target many different
proteins in addition to c-Fos, we compared the effects of chemical
inhibition and genetic loss of c-Fos on gene expression. We
performed whole-genome RNA-seq analysis on wild-type Kit+ cells
expressing BCR-ABL treated with imatinib, DFC+BCI, DFC+BCI+
imatinib and nontreated controls. Similarly, BCR-ABL-expressing
Kit+ cells lacking c-Fos and DUSP1, treated with and without
imatinib, were subjected to RNA-seq analysis. We compared gene
expression in these samples to that of Kit+ cells transduced with
the control vector (pMSCV-Ires-GFP) from WT mice. Consistent with
the notion that DFC+BCI treatment inhibits c-Fos and Dusp1, we
found a striking similarity between DFC+BCI-treated WT cells and
Fos- and Dusp1-double-knockout BCR-ABL-expressing cells: 146 genes
were regulated in common (58 upregulated and 88 downregulated)
relative to those in untreated BCR-ABL-expressing cells (FIG. 17A).
Further analysis of these differentially expressed genes, using
Netwalker, suggests that the inhibition of c-Fos and Dusp1 in the
context of BCR-ABL expression leads to both downregulation of a
Fos-Jun-associated gene network and the induction of a Jun-JunD
associated gene network (FIG. 17B,C).
[0091] The AP1 transcription factor is a dimeric complex that
contains members of the JUN (JUN, JUNB, and JUND) and FOS (FOS,
FOSB, FRA1, and FRA2) protein families (Angel, P. & Karin, M.
The role of Jun, Fos and the AP-1 complex in cell-proliferation and
transformation. Biochim. Biophys. Acta 1072, 129-157 (1991)). The
final outcome of AP1 activity is dependent on AP1 dimer
composition, as well as on the cellular and genetic context (Eferl,
R. & Wagner, E. F. AP-1: a double-edged sword in tumorigenesis.
Nat. Rev. Cancer 3, 859-868 (2003)). Our data show that, in the
context of BCRABL expression, the absence of c-Fos and Dusp1
results in a net AP1 transcriptional output that is
anti-proliferative (reduced expression of Gfi1, Il6, Lif, and
Cited2, and overexpression of JunD) and pro-apoptotic
(overexpression of BCL2L11) (FIG. 17B,C). This analysis suggests
that acute inhibition of BCR-ABL in c-Fos- and Dusp1-inhibited or
c-Fos- and Dusp1-deficient cells undergo apoptotic shock
(Pagliarini, R., Shao, W. & Sellers, W. R. Oncogene addiction:
pathways of therapeutic response, resistance, and road maps toward
a cure. EMBO Rep. 16, 280-296 (2015); Sharma, S. V. &
Settleman, J. Oncogene addiction: setting the stage for molecularly
targeted cancer therapy. Genes Dev. 21, 3214-3231 (2007)) owing to
elevated expression of pro-apoptotic genes.
Dusp6 Deficiency Confers Imatinib Resistance
[0092] Because BCI inhibits both Dusp6 and Dusp1 (Molina, G. et al.
Zebrafish chemical screening reveals an inhibitor of Dusp6 that
expands cardiac cell lineages. Nat. Chem. Biol. 5, 680-687 (2009)),
to determine whether the therapeutic efficacy of BCI is due to the
inhibition of Dusp1 or Dusp6, we tested the effects of BCI on MAPK
signaling in BaF3 and BaF3-BA cells overexpressing Dusp1 or Dusp6.
Although all Dusp family members have the ability to
dephosphorylate MAPKs, each Dusp shows a high degree of specificity
toward its specific substrates (Lawan, A., Shi, H., Gatzke, F.
& Bennett, A. M. Diversity and specificity of the
mitogen-activated protein kinase phosphatase-1 functions. Cell. MoL
Life Sci. 70, 223-237 (2013); Jeffrey, K. L., Camps, M., Rommel, C.
& Mackay, C. R. Targeting dual-specificity phosphatases:
manipulating MAP kinase signalling and immune responses. Nat. Rev.
Drug Discov. 6, 391-403 (2007); Owens, D. M. & Keyse, S. M.
Differential regulation of MAP kinase signalling by
dual-specificity protein phosphatases. Oncogene 26, 3203-3213
(2007); Boutros, T., Chevet, E. & Metrakos, P.
Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase
regulation: roles in cell growth, death, and cancer. Pharmacol.
Rev. 60, 261-310 (2008)). For instance, Dusp6 and Dusp9 show a
preference for dephosphorylating ERK2 over p38 or JNK (Groom, L.
A., Sneddon, A. A., Alessi, D. R., Dowd, S. & Keyse, S. M.
Differential regulation of the MAP, SAP and RK/p38 kinases by
Pyst1, a novel cytosolic dual-specificity phosphatase. EMBO J. 15,
3621-3632 (1996); Fjeld, C. C., Rice, A. E., Kim, Y., Gee, K. R.
& Denu, J. M. Mechanistic basis for catalytic activation of
mitogen-activated protein kinase phosphatase 3 by extracellular
signal-regulated kinase. J. Biol. Chem. 275, 6749-6757 (2000)),
whereas Dusp8, Dusp10 and Dusp16 specifically dephosphorylate JNK
and p38 kinases (Jeffrey, K. L., Camps, M., Rommel, C. &
Mackay, C. R. Targeting dual-specificity phosphatases: manipulating
MAP kinase signalling and immune responses. Nat. Rev. Drug Discov.
6, 391-403 (2007)). Similarly, studies from mice lacking Dusp1 have
revealed that it preferentially targets p38 and JNK (Dorfman, K. et
al. Disruption of the erp/mkp-1 gene does not affect mouse
development: normal MAP kinase activity in ERP/MKP-1-deficient
fibroblasts. Oncogene 13, 925-931 (1996); Zhao, Q. et al. MAP
kinase phosphatase 1 controls innate immune responses and
suppresses endotoxic shock J. Exp. Med. 203, 131-140 (2006)).
Overall, Dusp-mediated MAPK regulation is dependent on the
cellular, genetic, and signaling contexts (Lawan, A., Shi, H.,
Gatzke, F. & Bennett, A. M. Diversity and specificity of the
mitogen-activated protein kinase phosphatase-1 functions. Cell. MoL
Life Sci. 70, 223-237 (2013); Jeffrey, K. L., Camps, M., Rommel, C.
& Mackay, C. R. Targeting dual-specificity phosphatases:
manipulating MAP kinase signalling and immune responses. Nat. Rev.
Drug Discov. 6, 391-403 (2007); Hirsch, D. D. & Stork, P. J.
Mitogen-activated protein kinase phosphatases inactivate
stress-activated protein kinase pathways in vivo. J. Biol. Chem.
272, 4568-4575 (1997)).
[0093] In accord with this complexity, BCI treatment resulted in
enhanced phospho-p38 levels in both BaF3 and BaF3-BA cells and
decreased phospho-JNK in BaF3-BA cells; the effects in BaF3-BA
cells were observed with or without IL-3 co-treatment (FIG. 18A).
Ectopic expression of Dusp1 in BaF3 and BaF3-BA cells reduced
phospho-p38 levels, whereas the expression of Dusp6 did not modify
the levels of phospho-ERK1/2, phospho-p38 or phospho-JNK in either
cell type (FIG. 18B). Furthermore, overexpression of Dusp1, but not
Dusp6, conferred resistance to BCI in BaF3-BA cells (FIG. 19A).
These data support the hypothesis that BCI-mediated inhibition of
Dusp1 activates p38 to induce TKI sensitivity and suggests that p38
inhibition would confer resistance to imatinib. Accordingly, we
found that inhibition of p38 (using SB202190, a derivative of
SB20358046), but not inhibition of JNK (using SP600125, which is
100-fold more selective for inhibition of JNK as compared to p38
(Bennett, B. L. et al. SP600125, an anthrapyrazolone inhibitor of
Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 98, 13681-13686
(2001)), conferred imatinib resistance in BaF3-BA cells (FIG. 18C).
Notably, coexpression of WT Dusp6 with BCR-ABL in WT bone
marrow-derived Kit+ cells resulted in a significant reduction in
CFU formation (.about.55%, P=0.001), but did not affect the cells
expressing either vector or Dusp1 (FIG. 19B). Importantly,
overexpression of Dusp1 but not of Dusp6 conferred resistance to
drug (imatinib+BCI) treatment (FIG. 19B). Notably, unlike Dusp1-/-
cells, Dusp6-/- cells were resistant to drug (imatinib+BCI)
treatment and displayed normal CFU activity in comparison to
untreated WT cells (FIG. 19C). Moreover, ectopic expression of
Dusp6 in Dusp6-/- cells abolished drug resistance and restored TKI
sensitivity to a normal level (FIG. 19C). Previous work has shown
that Dusp6 expression might be a requirement for oncogenic
transformation in B-ALL but not CML (Shojaee, S. et al. Erk
negative feedback control enables pre-B cell transformation and
represents a therapeutic target in acute lymphoblastic leukemia.
Cancer Cell 28, 114-128 (2015)), and loss of Dusp6 expression in
lung cancer confers TKI resistance (Hrustanovic, G. et al. RAS-MAPK
dependence underlies a rational polytherapy strategy in
EML4-ALK-positive lung cancer. Nat. Med. 21, 1038-1047 (2015)).
Taken together with these previous results, our data suggest that
the inhibition of Dusp6 favors cancer cell survival upon TKI
treatment, and conversely, that the inhibition of Dusp1 modulates
p38 activity to promote TKI-induced cell death in LICs.
Mutations Affecting the Allosteric Domain of Dusp1 Confer
Resistance to BCI
[0094] To determine the relevant target of BCI more conclusively,
we performed in vitro drug-resistant screening to select for
BCI-resistant mutations in Dusp1. BaF3-BA cells transfected with an
expression library of randomly mutagenized Dusp1 construct were
used for the selection of mutations conferring resistance to
different concentrations of BCI (FIG. 19D). At 1 .mu.M and 1.5
.mu.M BCI, resistant clones emerged with a frequency of 1,200
clones and 27 clones per million cells, respectively, whereas
selection at 2 .mu.M of BCI did not yield any resistant clones
(FIG. 19E). 27 clones that emerged in 1.5 .mu.M of BCI were
randomly selected and further analyzed. Dose-response analysis
confirmed their resistance to BCI (FIG. 19F). Sequencing of the
Dusp1 gene in these clones identified four different substitution
mutations (E19R, C24G, V83G, and E112R) and two deletion mutations
at the N terminus (.DELTA.2-8 and .DELTA.2-28; FIG. 19G). Because
most resistant clones carried two or more mutations, we generated
clones with individual mutations for further analysis. Expression
of these resistant variants in BaF3-BA cells conferred resistance
to BCI (FIG. 19H). Notably, the Dusp1-V83G variant demonstrated
higher resistance than other tested variants, which potentially
explains why it was more frequently observed in the screen (FIG.
19H). Similarly, expression of these resistant variants in BCR-ABL
expressing BM-derived Kit+ cells conferred resistance to BCI
treatment, and the Dusp1-V83G variant conferred greater resistance
than the other variants tested (FIG. 18D). We mapped these
resistant mutations on a homology-based structural model of Dusp1
and found that they clustered at the N terminus of the allosteric
domain (rhodanese domain) rather than at the catalytic domain (FIG.
20). Furthermore, an unbiased docking of BCI using a structural
model of the Dusp1 rhodanese domain identified the putative site to
which BCI binds, with a predicted free energy of .DELTA.G=-7.63
(FIG. 18E). Taken together, these data provide clear evidence that
BCI-induced cell death is mediated by the inhibition of Dusp1
(rather than of Dusp6).
In Vivo Inhibition of c-Fos and Dusp1 Targets by DFC and BCI
[0095] To model the therapeutic potential of c-Fos and Dusp1
inhibition in vivo, we performed a pharmacodynamic analysis for BCI
and DFC. We obtained peripheral blood mononuclear cells 6 h before
and after BCI injection, and measured the levels of phospho-p38 and
of the c-Fos-target genes Bcl2l11, 116, and Lif (FIG. 18F,G). As
expected, cells from mice injected with BCI showed an increase (by
4-8 fold) in phospho-p38 levels (FIG. 18F). Moreover, treatment
with DFC+BCI induced Bcl2l11 expression while dampening the
expression of Lif and Il6 (FIG. 18G). Notably, elevated serum IL-6
levels have been reported to be required for leukemic disease
development (Zhang, B. et al. Altered microenvironmental regulation
of leukemic and normal stem cells in chronic myelogenous leukemia.
Cancer Cell 21, 577-592 (2012); Reynaud, D. et al. IL-6 controls
leukemic multipotent progenitor cell fate and contributes to
chronic myelogenous leukemia development. Cancer Cell 20, 661-673
(2011)), and IL-6-neutralizing antibodies can suppress disease
development (Weiner, R. S. et al. Treatment of chronic myelogenous
leukemia by blocking cytokine alterations found in normal stem and
progenitor cells. Cancer Cell 27, 671-681 (2015)). Our data suggest
that these markers (phospho-p38, IL6, and Lif) could potentially
help to test the efficacy of c-Fos and Dusp1 inhibition and
evaluate disease progression.
Deletion of Fos and Dusp1 Blocks BCR-ABL-Induced B-ALL
Development
[0096] Given that growth-factor signaling mediates intrinsic
resistance to TKI therapy in both leukemia and solid organ tumors
(Corbin, A. S. et al. Human chronic myeloid leukemia stem cells are
insensitive to imatinib despite inhibition of BCR-ABL activity. J.
Clin. Invest. 121, 396-409 (2011); Straussman, R. et al. Tumour
micro-environment elicits innate resistance to RAF inhibitors
through HGF secretion. Nature 487, 500-504 (2012); Wilson, T. R. et
al. Widespread potential for growth-factor-driven resistance to
anticancer kinase inhibitors. Nature 487, 505-509 (2012)), we
reasoned that c-FOS and DUSP1 might have crucial roles in other
types of kinase-driven leukemia. First, we tested the roles of Fos
and Dusp1 in BCR-ABL-induced B-ALL, which, similarly to
BCR-ABL-induced CML, is driven by a diverse spectrum of oncogenic
tyrosine kinases and cytokine receptors (Roberts, K. G. et al.
Genetic alterations activating kinase and cytokine receptor
signaling in high-risk acute lymphoblastic leukemia. Cancer Cell
22, 153-166 (2012)); moreover, similarly to BCR-ABL-induced CML,
most patients with BCR-ABL-induced B-ALL relapse under TKI
treatment (Druker, B. J. et al. Activity of a specific inhibitor of
the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid
leukemia and acute lymphoblastic leukemia with the Philadelphia
chromosome. N. Engl. J. Med. 344, 1038-1042 (2001)). To model B-ALL
in vivo in mice, we used bone marrow-derived mononuclear cells
(MNCs) from WT and ROSACreERT2Fosfl/fl;Dusp1-/- mice transduced
with BCR-ABL-Ires-YFP (P190) retroviruses (Chang, K. H. et al. Vav3
collaborates with p190-BCR-ABL in lymphoid progenitor
leukemogenesis, proliferation, and survival. Blood 120, 800-811
(2012)). Fos was deleted after 2 weeks of transplantation by
tamoxifen injection. Recipients of WT BM-derived cells developed
lethal leukemia with a disease latency of 4-5 weeks; by contrast,
the deletion of both Fos and Dusp1 led to complete suppression of
disease development and the eradication of leukemic cells within 3
weeks after tamoxifen injection (FIG. 21A-C). These data provide
evidence that loss of c-Fos and Dusp1 together results in synthetic
lethality in BCR-ABL expressing B-ALL cells. Surprisingly, mice
that received BCR-ABL transduced ROSACreERT2Fosfl/flDusp1-/- cells,
but that were not treated with tamoxifen, did not develop leukemia;
leukemic cells disappeared from these mice, although with a delayed
latency when compared to tamoxifen-treated mice (FIG. 21A-C), most
likely owing to lower Fos mRNA expression in these mice as compared
to WT mice (FIG. 12E). Taken together, these data suggest that,
unlike in CML, deletion of Fos and Dusp1 are sufficient to
completely eradicate BCR-ABL-induced B-ALL.
Induction of c-Fos and Dusp1 by the Oncogenic Kinases FLT3-ITD and
JAK2-V617F
[0097] Next, we analyzed whether growth-factor signaling can confer
resistance to the inhibitors of the oncogenic kinases Flt3 and
Jak2. As expected, growth-factor signaling conferred resistance to
the Flt3 inhibitor AC220 and the Jak2 inhibitor ruxolitinib in
BaF3-FLT3-ITD and BaF3-Jak2-V617F cells, respectively (FIG. 21D,F).
Similarly to BCR-ABL, the expression of FLT3-ITD and JAK2-V617F
induced the expression of c-Fos and Dusp1 (FIG. 21E,G). This
induction suggests a more general role for c-Fos and Dusp1 in TKI
resistance.
c-Fos and Dusp1 in Normal Hematopoietic Cells
[0098] Both HSCs and LSCs (BCR-ABL-expressing HSCs) are dependent
on growth factor-signaling, which suggests that elevated Fos and
Dusp1 expression in these cells confer intrinsic resistance to TKI.
As expected, we found that HSCs have higher levels of FOS and DUSP1
in both humans and mice (Bagger, F. O. et al. BloodSpot: a database
of gene expression profiles and transcriptional programs for
healthy and malignant haematopoiesis. Nucleic Acids Res. 44D1,
D917-D924 (2016)) (FIG. 22A, B). Expression of BCR-ABL in mouse
hematopoietic stem and progenitor cells (LSK cells) induced
expression of c-Fos and Dusp1, as compared to vector transduced LSK
cells (FIG. 22C), perhaps owing to the convergence of oncogenic and
growth-factor signaling at these signaling nodes. Similarly, LSCs
(CD34+ and CD38- cells) from patients with CML showed higher
expression of FOS and DUSP1 as compared to normal HSCs (CD34+ and
CD38- cells) (FIG. 22D).
Growth Factor Signaling Induces Pro-Survival and Anti-Apoptotic
Genes
[0099] Expression analysis of BaF3-LTBA cells treated with IL-3
showed that higher Fos and Dusp1 expression, as compared to
untreated BaF3-LTBA cells (FIG. 9C), is correlated with induced
expression of pro-survival and anti-apoptotic genes (FIG. 22E),
which might represent a protective mechanism. Notably, expression
of these pro-survival and anti-apoptotic genes is dependent upon
Fos and Dusp1 function, as shown using FOS and DUSP1 inhibitors
(FIG. 22E,F). Taken together, these data provide evidence that
higher levels of Fos and Dusp1 are required to maintain growth
factor-induced expression of pro-survival and anti-apoptotic
genes.
[0100] Initial excitement over the targeting of BCR-ABL with
imatinib, a small-molecule TKI, has been tempered by the
observation that LSCs from patients with CML can survive TKI
treatment. Although this resistance was first thought to be due
perhaps to incomplete inhibition of kinase activity, given that
LSCs under imatinib treatment displayed residual kinase activity,
later studies showed that LSCs survive even when treated with a
next-generation BCR-ABL inhibitor, such as nilotinib, which fully
quenches kinase activity in vivo (Jorgensen, H. G., Allan, E. K.,
Jordanides, N. E., Mountford, J. C. & Holyoake, T. L. Nilotinib
exerts equipotent antiproliferative effects to imatinib and does
not induce apoptosis in CD34+ CML cells. Blood 109, 4016-4019
(2007)). One could conclude from these data that LSCs are not
oncogene dependent. According to our model (FIG. 22G-I), the
expression of an activated kinase such as BCR-ABL usurps c-Fos- and
Dusp1-mediated regulation of cell proliferation and survival. TKI
treatment downregulates c-Fos and Dusp1 expression, leading to
apoptosis in the bulk tumor; however, within TKI resistant cells
(LSCs or BaF3-BA cells treated with IL3), growth-factor signaling
can rescue c-Fos and Dusp1 expression, leading to sustained
expression of pro-survival and anti-apoptotic genes and TKI
resistance. Previous work showing a lack of addiction to BCR-ABL in
LSCs from patients with CML prompted many researchers to identify
pathways and potential therapeutic targets in these LSCs, such as
phosphoinositide 3-kinase (PI3K)-AKT serine/threonine kinase 1
(AKT1); transforming growth factor (TGF)-.beta.-Forkhead box O
(FoxO), Hedgehog, and Wnt-.beta.-catenin pathways (Holyoake, T. L.
& Vetrie, D. The chronic myeloid leukemia stem cell: stemming
the tide of persistence. Blood
https://doi.org/10.1182/blood-2016-09-696013 (2017)). Although
inhibition of these targets either alone or in combination with
kinase inhibitors inhibits the survival of LSCs, such therapy has
been shown to be detrimental to normal HSCs, because of the
requirement of the targeted pathways for cell survival and
self-renewal pathways in normal HSCs.
[0101] Our study shows that c-FOS and DUSP1 are activated by both
kinase oncoproteins and growth factors, and that their expression
levels are uniquely critical for the maintenance of growth-factor
mediated rescue of MRD in mouse models of TKI-treated leukemia.
Moreover, our data show that normal HSCs do not have a critical
requirement for c-Fos and Dusp1, given that BM-derived cells
lacking Fos and Dusp1 do not show any functional impairment, as
assessed by transplantation experiments. We speculate that
kinase-driven LSCs differ from normal HSCs because they have
adapted to chronic kinase signaling and have become addicted to
elevated c-Fos and Dusp1 activity, a growth-factor-induced
signaling node. The levels of c-Fos and Dusp1 might dictate the
threshold of TKI efficacy, such that lower levels confer
sensitivity, whereas higher levels drive intrinsic resistance that
leads to MRD in leukemia, and as described below, in solid organ
cancers. Thus, these proteins may represent a unifying Achilles'
heel of kinase-driven cancers. Our findings provide proof of
principle that MRD can be treated through the inhibition of a
convergent signaling node that mediates growth-factor-dependence in
kinase-induced leukemia.
Methods
[0102] Mice. All mice were housed in the barrier facility at
Cincinnati Children's Hospital (CCHMC). All experiments were
performed under an IACUC approved protocol of the Cincinnati
Children's Hospital in accordance with accepted national standards
and guidelines. To generate conditional Fos mice, Fosfl/fl mice
(Zhang, J. et al. c-fos regulates neuronal excitability and
survival. Nat. Genet. 30, 416-420 (2002)) were crossed with
ROSACreERT2 mice (Jackson laboratory, Bar Harbor, Me.) to generate
ROSACreERTFosfl/fl mice. To create the doubleknockout mice,
ROSACreERT2Fosfl/fl mice were bred with Dusp1-/- mice (Dorfman, K.
et al. Disruption of the erp/mkp-1 gene does not affect mouse
development: normal MAP kinase activity in ERP/MKP-1-deficient
fibroblasts. Oncogene 13, 925-931 (1996)) to generate
ROSACreERT2Fosfl/fl;Dusp1-/- mice. BoyJ mice were purchased from
the mouse core facility at CCHMC. C57BL6 mice were purchased from
Jackson Laboratory. Scl-tTA transgenic mice were obtained from the
lab of C. Huettner. Mouse genotypes were confirmed by PCR analysis
using gene-specific primers (Dorfman, K. et al. Disruption of the
erp/mkp-1 gene does not affect mouse development: normal MAP kinase
activity in ERP/MKP-1-deficient fibroblasts. Oncogene 13, 925-931
(1996); Zhang, J. et al. c-fos regulates neuronal excitability and
survival. Nat. Genet. 30, 416-420 (2002)). Athymic Ncr nu/nu,
NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) and hSCF, hIL-3, and
h-GM-CSF transgenic NSG-derived (NSGS) mice were purchased from the
CCHMC mouse core. 6-8-week-old mice were used in all experiments.
Mice with the indicated genotypes were included in the study
without any further preselection or formal randomization, and both
male and female mice were used; we used age- and gender-matched
mice. The investigators were not blinded to genotype group
allocations.
[0103] Human specimens. Umbilical cord blood (UCB) cells, normal
BM, CML (p210-BCR-ABL+) and blastic-phase leukemia specimens were
obtained through Institutional Review Board--approved protocols
(Institutional Review Board: Federalwide Assurance #00002988
Cincinnati Children's Hospital Medical Center) and donor-informed
consent from CCHMC and University of Cincinnati. The patient
samples are described in Table 1.
[0104] Plasmids and constructs. BCR-ABL was cloned into the
pLVX-puro and pLVX-Tet-On-Puro (Clontech, USA) plasmids to yield
pLVBA and pLTBA for constitutive and inducible expression,
respectively. The plasmid pEYKBA9 was digested with EcoRI to
release the BCR-ABL fragment that was purified and ligated to
EcoRI-digested pLVX-puro and pLVX-Tet-On-Puro to generate the
plasmids pLVBA and pLTBA, respectively. To create the
dominant-negative c-Fos (c-Fos-.DELTA.RT), the basic region of FOS
DNA-binding domain (amino acid residues 133-159) was deleted by PCR
using primers (c-FOS-DRK-FP and c-FOS-DRK-RP) by QuikChange
lightning multi-site directed mutagenesis kit (Agilent
Technologies). The PCR reaction was carried out using template DNA
(pDonor201-FOS obtained from PlasmID at Harvard cat. #
HsCD00001156). Subsequently, these entry vectors were used to
develop retroviral expression clones using destination vector
(pMSCV-Ires-GFP.GW) by recombination cloning using LR clonase from
Invitrogen. Similarly, retroviral expression vectors for Dusp1 and
Dusp6 (pMSCV-Dusp1-Myc-Ires-cherry and pMSCV-Dusp6-Ires-GFP) were
created by recombination cloning using entry clones (pENTR-Dusp1,
pENTR-Dusp6 obtained from PlasmID at Harvard). BCI-resistant
mutations of Dusp1 were created by site-directed mutagenesis kit,
as described above, using pMSCV-Dusp1-Myc-Ires-cherry as template.
All pENTR clones were confirmed by sequencing. Retroviral
expression vectors (pMSCV-Fos-P2A-Dusp1) expressing Fos, and Dusp1
as a polycistronic construct, were cloned by recombination cloning
using plasmids (pENTR-FOS/HA, pENTR-Dusp1/Myc). The retroviral
vector pMSCV-BCR-ABL-IRES-YFP, a gift from T. Reya (Zhao, C. et al.
Hedgehog signalling is essential for maintenance of cancer stem
cells in myeloid leukaemia. Nature 458, 776-779 (2009)), was used
to express BCR-ABL in primary mouse bone marrow cells for
colony-formation cell assays and for in vivo transplantation and
leukemia development.
[0105] Chemical reagents and cytokines. Kinase inhibitor imatinib
was purchased from LC laboratories (Woburn, Mass.). Inhibitors for
c-Fos, diflouro-curcumin (DFC) and curcumin, were purchased from
LKT laboratories. The Dusp1 inhibitor BCI was synthesized by
Chemzon Scientific (Montreal, Canada). Mouse cytokines (IL-3, SCF,
IL-6 and Flt3L) were purchased from Peprotech, NJ, USA. Human
cytokine erythropoietin was purchased from Amgen, Calif. NDGA,
tamoxifen, and 4-hydroxy tamoxifen were purchased from
Sigma-Aldrich. Hydrocortisone was purchased from STEMCELL
technologies.
[0106] Cell culture. BaF3, K562, and HEK293T cells were obtained
from G. Daley's lab. MS5 was a gift. BaF3 and K562 were cultured
and maintained in RPMI supplemented with 10% FBS and 100 IU/ml
penicillin, 100 .mu.g/ml streptomycin, and 2 mM I-glutamine.
HEK293T cells were maintained in DMEM supplemented with 10% FBS and
100 IU/ml penicillin, 100 .mu.g/ml streptomycin, and 2 mM
I-glutamine. BaF3 parental cells were grown in RPMI with 10% WEHI
conditioned media, used as a source of IL-3. BAF3 cells with
BCR-ABL were maintained in RPMI without IL-3 supplementation.
[0107] Generation of stable cell lines. Cells stably expressing the
BCR-ABL, Fos, Dusp1, and Dusp6 were generated by transducing with
high-titer retroviruses, as described earlier (Kesarwani, M. et al.
Targeting substrate-site in Jak2 kinase prevents emergence of
genetic resistance. Sci. Rep. 5, 14538 (2015)). Inducible
expression of BCR-ABL in BaF3 cells was achieved by transducing
these cells with pLVX-Tet-On-Hygro viruses (Clontech), followed by
selection for hygromycin resistance (selected at 600 .mu.g/ml).
Finally, BaF3 cells were transduced with pLTBA-puro viruses. Cells
were selected for puromycin resistance at 3 .mu.g/ml, generating
the inducible-expression cell line BaF3-LTBA.
[0108] Cell-proliferation assay. 1.times.10.sup.4 cells were seeded
in 96-well plates in 100 .mu.l of media with or without growth
factors (50 ng/ml) and appropriate drug concentrations. The cells
were incubated for 60 h. Cell viability was assessed with the WST-1
reagent (Roche) according to the manufacturer's recommendations,
and read with a 96-well plate reader at 450 nm. All assays were
performed in triplicate, and readings were averaged. A
dose-response analysis to determine half-maximal inhibitory
concentration (1050) values was performed by sigmoidal curve
fitting in GraphPad6, as described previously (Kesarwani, M. et al.
Targeting substrate-site in Jak2 kinase prevents emergence of
genetic resistance. Sci. Rep. 5, 14538 (2015)).
[0109] Apoptosis assay. BaF3-BA and K562 cells constitutively
expressing BCR-ABL were grown with or without growth factors to the
logarithmic phase. The cells from each group were treated with
imatinib (5 .mu.M) for 6 hours. The cells were then stained with
APC-conjugated Annexin V (BD Biosciences), according to the
supplier's instructions. 5 .mu.l of propidium iodide (P1) was added
to each sample after annexin-V staining, and the cells were
subjected to FACS analysis (BD Canto II). Single annexin-V-positive
cells were considered to be early apoptotic cells, whereas PI and
Annexin-V-double-positive cells were considered to be late
apoptotic or dead cells.
[0110] RNA isolation and gene-expression profiling. 5-6 million
BaF3 and BaF3-LTBA cells grown with or without doxycycline (500
ng/ml) and IL-3 from the logarithmic phase were collected and
resuspended in Qiazol for RNA isolation. Similarly, BaF3-BA cells
(with constitutive expression of BCR-ABL) were grown with or
without IL-3 and treated with imatinib (3 .mu.M) for 6 h.
Similarly, K562 cell lines were grown with or without
erythropoietin (100 U/ml). After 6 h of imatinib (3 .mu.M)
treatment, cells were stained with annexin V and PI to quantify the
levels of apoptotic cell death. The three separate populations of
cells (double-negative cells (live cells), APC, or
annexin-V-positive cells (early apoptotic cells) and
double-positive cells (late apoptotic cells)) were sorted in PBS
from each of the cell lines. 2 million sorted cells from each
condition were immediately pelleted and frozen in 700 .mu.l of
Qiazol for RNA extraction. Total RNA was quantified, and 1 .mu.g of
RNA was used for expression profiling on the Mouse ST_1.0 Gene Chip
Array (Affymetrix) for BaF3 cells and ExonExprChip.
HuGene-1_0-st-v1 (Affymetrix) for K562 cells, at the Cincinnati
Children's Gene Expression Core. The data were collected and .cel
files were generated in the MASS suite (Affymetrix). The .cel files
were imported into GeneSpring-GX 12.6.1 (Agilent Technologies) and
analyzed using the latest annotation available. All biological
replicates were averaged. For the first experiment (experiment 1;
conditional expression of BCR-ABL in the presence or absence of
IL-3, with the aim of determining changes in gene expression that
are modulated by IL-3 in an oncogenic condition), the data were
normalized to the median of parental BaF3 cells. After
normalization, the genes were filtered on the basis of expression,
and genes with probe-intensity values less than the 20th percentile
in at least one condition were eliminated. For the second
experiment (experiment 2; imatinib-induced changes in gene
expression in BaF3-BA cells treated with or without IL-3) and the
third experiment (experiment 3; imatinib-induced changes in gene
expression in K562 cells treated with or without EPO), the data
were normalized to the median of all samples followed by filtering
on the basis of expression, and genes with probe-intensity values
less than the 40th percentile in at least one condition were
eliminated. Lists of genes that are differentially expressed were
created using filtered genes with fold-change analysis between the
cells grown with or without the growth factors for all three of the
experiments. The fold-change cut-off was set to 1.5 for experiment
1 and was set to 2.0 for experiments 2 and 3. A published data set
(GSE12211) that describes gene expression of CML-CD34+ cells during
imatinib therapy (Bruennert, D. et al. Early in vivo changes of the
transcriptome in Philadelphia chromosome-positive CD34+ cells from
patients with chronic myelogenous leukaemia following imatinib
therapy. Leukemia 23, 983-985 (2009)) was similarly analyzed using
GeneSpring GX software. The samples were normalized to the median
of control sample and filtered on the basis of expression, and
genes with probe intensity values below the 50th percentile in at
least one condition were eliminated. Gene lists were created
containing genes that were differentially expressed by more than
2.0-fold between imatinib-treated and untreated samples. Finally,
to identify commonly regulated genes in all four data sets, data
sets were analyzed in GeneSpring GX, and the results are presented
as a Venn diagram.
[0111] Real-time qPCR analysis. Candidate genes picked by
microarray analysis were validated by real time qPCR. Total RNA was
isolated as described above. The RNA was first subjected to DNase
treatment using DNA-free DNase kit (Ambion, Life technologies). 2
.mu.g of total RNA was converted to cDNA with Superscript III
first-strand synthesis kit (Life technologies). qPCR reactions were
performed with the human gene-specific primers using the SYBR green
method and using a Mastercycler RealPlex2 instrument (Eppendorf).
All PCR reactions were performed in triplicate and the real-time
data was normalized to .beta.-actin expression.
[0112] Western blotting. 4-6 million cells were collected, and
whole-cell extracts were prepared using lysis buffer supplemented
with a protease-inhibitor cocktail (Roche) and
phosphatase-inhibitor cocktail 2 (Sigma-Aldrich), as described
previously (Azam, M., Seeliger, M. A., Gray, N. S., Kuriyan, J.
& Daley, G.Q. Activation of tyrosine kinases by mutation of the
gatekeeper threonine. Nat. Struct. Mol. Biol. 15, 1109 1118
(2008)). The proteins were resolved on 10% SDS-PAGE gels and
transferred to nitrocellulose membranes (Bio-Rad). Membranes were
blocked in TBST with 5% nonfat milk and probed with appropriate
antibodies as indicated. Densitometry was carried out using ImageJ
software.
[0113] Colony-forming cell assays. Kit+ cells from the BM of WT,
Dusp1-/-, ROSACreERT2Fosfl/fl, or ROSACreERT2Fosfl/fl;Dusp1-/- mice
were isolated using the CD117 MicroBead Kit (Miltenyi biotec),
according to the manufacturer's instructions. The cells were
incubated overnight in IMDM media supplemented with 10% FBS and a
cytokine cocktail with FLT3 (20 ng/ml), IL-6 (10 ng/ml), IL-3 (10
ng/ml), and mSCF (50 ng/ml). After 12 h of stimulation, the cells
were transduced with BCR-ABL-IRES-YFP virus using retronectin
(Takara). 5,000 YFP-positive cells (isolated by FACS) were plated
on MethoCult GF M3434 (STEMCELL technologies containing imatinib (3
.mu.M), DFC (0.2 .mu.M), and BCI (0.5 .mu.M)) alone or in
combinations on three replicate plates. Similarly, curcumin (5
.mu.M) or NDGA (5 .mu.M) was used alone or in combination with
imatinib (3 .mu.M) and BCI (0.5 .mu.M). Colony numbers were
recorded after 1 week of plating. To delete Fos, the cells were
plated on MethoCult GF M3434 containing 4-hydroxy tamoxifen (1
.mu.g/ml).
[0114] BM cell transduction transplantation model of CML. Kit+
cells from the BM of 6-8-week-old WT C57BL/6, Dusp1-/-,
ROSACreERT2Fosfl/fl, or ROSACreERT2 c-Fosfl/fl;Dusp1-/- mice were
isolated and transduced with MSCV-BCR-ABLIRES-YFP, as described
above. The transduced cells were cultured overnight. The percentage
of BCR-ABL-positive cells was determined by measuring the level of
YFP-positive cells after 20 h of viral transduction using flow
cytometry (Fortessa I). 40,000 YFP-positive cells with 0.3 million
normal BM-derived cells as carriers were transplanted into each
mouse through tail-vein injection. After 1 week of transplantation,
engraftment was determined by analyzing the YFP-positive cells from
the peripheral blood using FACS. Transplanted mice that showed
10-40% YFP-positive cells in the peripheral blood were used for the
experiment. Mice that had less than 2% YFP-positive cells were
discarded from the study. To delete the Fosfl/fl allele, tamoxifen
(100 mg/kg in corn oil) was i.p. injected into mice 1 week of
transplantation, every day for three consecutive days. After
tamoxifen treatment, where appropriate, the mice were grouped for
drug treatments (n=5 per group). Mice were monitored for leukemia
progression and survival, and the leukemic burden (YFP-positive
cells) was determined weekly for up to 8 weeks in surviving mice.
Animal numbers were chosen on the basis of previous experience and
published data (Zhao, C. et al. Hedgehog signalling is essential
for maintenance of cancer stem cells in myeloid leukaemia. Nature
458, 776-779 (2009)) for the transplantation of BCR-ABL-positive
cells.
[0115] BM cell transduction transplantation model of B-ALL. BM
cells were harvested from WT and ROSACreERT2Fosfl/fl ;Dusp1-/-
mice. Total mononuclear cells (TMNCs) were isolated by gradient
centrifugation using Ficoll. Cells were washed and resuspended in 1
ml IMDM media+10% FBS supplemented with SCF (50 ng/ml; Prospec) and
IL-7 (20 ng/ml; Peprotech). The cells were transduced with
pMSCV-BCRABL(p190)-Ires-GFP virus. After 8 h of transduction, the
cells were washed and injected (2.times.106 cells) into tail veins
by i.v. into lethally irradiated C57BL/6 mice. After 2 weeks of
transplantation, mice were injected with tamoxifen (100 mg/kg once
per day for 3 d) to delete Fos. Peripheral blood from the
transplanted mice was used to determine the leukemic burden (%
GFP-positive cells), and the levels of white blood cells by
complete blood counter (Hemavet, Drew Scientific, Oxford,
Conn.).
[0116] BCR-ABL transgenic mouse model. Total BM cells from Scl-tTA
transgenic mice (Koschmieder, S. et al. Inducible chronic phase of
myeloid leukemia with expansion of hematopoietic stem cells in a
transgenic model of BCR-ABL leukemogenesis. Blood 105, 324-334
(2005)) were isolated and followed with lineage depletion using
lineage antibody cocktail from Milteny biotec, according to the
manufacturer's instructions. Isolated Lin_cells were labeled with
anti-Kit and anti-Sca1 antibodies to isolate LSK (Lin-Sca1+Kit+)
cells by FACS. 3,000-5,000 BCR-ABL-LSK cells with 0.3 million
helper bone marrow cells from WT BoyJ mice were injected via the
tail vein into lethally irradiated BoyJ mice. 4 weeks
post-transplantation, recipient mice were analyzed for CD45.1- and
CD45.2-positive cells by FACS analysis to determine leukemic
engraftment and chimerism. Mice were grouped into four groups (n=6
per group). Mice were treated with imatinib (75 mg/kg twice daily)
alone and in combination with DC+BCI (both drugs were given at a
dose of 10 mg/kg twice daily). Similarly, other groups were treated
with combinations of imatinib (75 mg/kg)+ curcumin (150 mg/kg)+BCI
(10 mg/kg), and imatinib (75 mg/kg)+NDGA (100 mg/kg)+BCI (10
mg/kg), for 3 months twice a day by i.p. injection. The mice were
analyzed for leukemic chimerism by determining the percentage of
CD45.2-positive cells once a month for 6 months.
[0117] RNAseq analyses of Kit+ cells from WT and Fos- and
Dusp1-knockout mice. Kit+ cells from the BM of WT and
ROSACreERT2Fosfl/fl ;Dusp1-/- mice were isolated using the CD117
MicroBead Kit (Miltenyi biotec), according to the manufacturer's
instructions. These cells were incubated overnight in IMDM media
supplemented with 10% FBS and a cytokine cocktail of FLT3 (50
ng/ml), IL-6 (10 ng/ml), IL-3 (10 ng/ml), and mSCF (50 ng/ml).
After 12 h of stimulation, the cells were transduced with
BCR-ABL-IRES-YFP virus using retronectin (Takara). 1-2 million
positive cells (isolated by FACS) from each group was treated for 4
h with imatinib alone, DFC+BCI, or imatinib+DFC+BCI (3 .mu.M
imatinib, 0.2 .mu.M DFC and 0.5 .mu.M BCI alone or in combinations.
The treatments were done in two replicates. To delete FOS,
4-hydroxy tamoxifen (1 .mu.g/ml) was added to the media where
applicable. Total RNA was isolated, and RNA-seq (20 million reads
with paired ends) was performed at the DNA-sequencing core of
Cincinnati Children's Hospital. Genes with an absolute log 2 change
of 1 in BCR-ABL-expressing Fos-/-;Dusp1-/- cells (Fos was deleted
by adding 4-hydroxy tamoxifen (1 .mu.g/ml) treated with DFC+BCI, as
compared to BCR-ABL WT cells (680 genes), were selected. From this
list of genes, genes with similar profiles (146) in both
Fos-/-;Dusp1-/- expressing BCR-ABL and WT cells expressing BCR-ABL
that were treated with DFC+BCI were selected. To build a gene
network, downregulated or upregulated genes were used as seeds to
build a coherent network using the GeneConnector functionality in
NetWalker suite (Komurov, K., Dursun, S., Erdin, S. & Ram, P.
T. NetWalker: a contextual network analysis tool for functional
genomics. BMC Genomics 13, 282 (2012)).
[0118] Mouse models of CML with patient-derived cells. 3 million
CD34+ cells from CML patient CP4 (described in Table 1) were
transplanted into sublethally irradiated 8-week-old NSG mice. 2
weeks after transplantation, leukemic engraftment in bone marrow
was determined by FACS using mouse and human specific antibodies
against CD45. Mice were grouped into four different cohorts
(n=6/group) for treatment with vehicle, imatinib (75 mg/kg),
DFC+BCI (both at 10 mg/kg), and imatinib (75 mg/kg)+DFC+BCI (both
at 10 mg/kg). Drugs were diluted in PBS (vehicle) and administered
by intraperitoneal injection twice daily. Mice were treated for 6
weeks, and the leukemic burden was determined every 2 weeks, until
week 8 after transplantation.
[0119] LTC-IC assay. The LTC-IC assay was performed according to
the instruction manual of StemCell Technologies. 5,000 CD34+ cells
from patient CP4 or 1 million total MNCs from patient CP1 were
cultured in StemSpan SFEM medium containing 50 ng/ml SCF, 5 ng/ml
IL-3, 20 ng/ml IL-6, 50 ng/ml Flt3L, and 100 ng/ml GM-CSF for 24 h
in the following conditions: untreated; imatinib (3 .mu.M), DFC
(200 nM)+BCI (500 nM), and imatinib (3 .mu.M)+DFC (200 nM)+BCI (500
nM). After 24 h, the cells were washed in human long-term culture
medium (HLTM; MyeloCult H5100 media containing 1 .mu.M
hydrocortisone) and were plated on irradiated MS-5 stromal cells.
Cultures were maintained for 5 weeks with weekly half-medium
changes. Cells were then harvested, counted, and transferred to
methylcellulose-containing media (MethoCult Express, StemCell
Technologies) for colony-forming assays. At the end of week 5,
adherent and nonadherent cells were isolated and plated in
methylcellulose (METHOCULT H4434 classic, stem cell technology) for
CFU analysis in triplicate. Plates were incubated at 37.degree. C.,
and colonies were scored 2 weeks after plating.
[0120] Pharmacodynamic analysis of Dusp1 and c-Fos targets.
Phospho-p38 analysis. Three leukemic mice (8-12 weeks old), which
had received transplants of BCR-ABL-expressing Kit+ cells, were
injected with BCI (10 mg/kg) by intraperitoneal injection 4 weeks
after transplantation. Phospho-p38 levels were quantified in
peripheral blood MNCs using the Phosflow kit (BD Biosciences),
according to the supplier's instructions. In brief, blood was
collected from each mouse before and 6 hours after drug injection.
Mononuclear cells were isolated by RBC depletion: RBCs were lysed
twice using 4 ml Pharmlyse solution (BD Biosciences) per 100 .mu.l
of peripheral blood by mixing and incubated on ice for 5 min. After
the second lysis step, cell pellets were washed with 1 ml 2% BSA in
PBS followed by fixation using 100 .mu.l of fixation and
permeabilization solutions (BD Biosciences) for 20 min at 4.degree.
C. in the dark. The pellets were washed with 1 ml of 1.times. BD
Perm/wash buffer. After fixation, the cells were blocked using 300
.mu.l 2% BSA in Perm/wash buffer (BD Biosciences) at room
temperature for 20 min. The cells were divided into three equal
aliquots, 100 .mu.l each (.about.1 million), and incubated with 1
.mu.l total p-38 antibody, 1 .mu.l phospho-p38 antibody, or 1 .mu.l
isotype IgG control, overnight at 4.degree. C. The cells were then
washed and incubated with secondary antibody (1 .mu.l of
AlexaFluor-488-conjugated secondary antibody) for 1 h at room
temperature. Cells were washed once again and suspended in 200
.mu.l PBS. Data were acquired by FACS on Fortessa instrument, and
the data were analyzed by FlowJo software. The mean fluorescence
intensity (MFI) of the IgG control was deducted from the MFI of the
experimental samples. The MFI values of phospho-p38 were normalized
to those of total p38 to determine the phospho-p38 levels after BCI
injection. Quantitative gene-expression analysis of target genes.
Three mice (8-12 weeks old) with leukemia, which had received
transplants of BCR-ABL expressing Kit+ cells, were injected with
DFC+BCI (10 mg/kg each). Peripheral blood was collected from each
mouse before and 6 h after drug injection. Mononuclear cells were
isolated by RBC depletion, as above. MNCs were pelleted and
resuspended in Qiazol lysis buffer (Qiagen). Total RNA was
extracted, followed by cDNA synthesis and qPCR analysis of Bcl2l11,
Lif, and Il6 using gene-specific primers.
[0121] Drug preparation. All drugs were prepared as 10 mM stocks in
DMSO and stored at -20.degree. C. until use. For in vivo injection,
the stocks of imatinib and BCI were diluted in PBS, whereas the DFC
stock was diluted in alkaline PBS containing 15 mM of sodium
hydroxide. All drugs were injected into mice via i.p injection.
[0122] FACS analysis. Peripheral blood (PB) cells were collected
from transplant-recipient mice once per week via tail bleeding. 20
.mu.l of blood were lysed using Pharmlyse solution (BD
Biosciences), and the remaining mononucleated cells were pelleted
by centrifugation. The cell pellets were washed once with cold PBS.
The percentage of leukemic chimerism was determined by quantifying
the levels of YFP-positive cells (BCR-ABL-Ires-YFP), analyzed by
FACS. BM cells from mice transplanted with Scl-ttA-BCR-ABL cells or
patient-derived cells were aspirated from the mouse femurs to
determine the levels of CD45.2 and human CD45. These BM cells were
blocked with FcR block (BD bioscience) followed by staining with
anti-mouse FITC labeled CD45.1 and anti-mouse PE CD45.2. The FACS
analysis was performed on an LSRII instrument, and the data were
analyzed using FACSDIVA software. For the analysis of human grafts,
bone marrow cells were aspirated every 2 weeks from femurs of the
transplanted mice. RBCs were lysed using RBC lysis buffer as above,
and the total MNCs were pelleted by centrifugation. The pellet was
washed once with cold 1.times.PBS. The cells were blocked with
human FcR block and mouse FcR Block (Miltenyi Biotec), followed by
staining with anti-human CD45 FITC and anti-mouse CD45 APC Cy7
overnight at 4.degree. C. The FACS analysis was performed on an
LSRII instrument, and the data were analyzed using FACSDIVA
software. For differential analysis of peripheral blood (PB) cells,
20 .mu.l of blood was lysed using RBC lysis buffer and the TMNCs
were pelleted by centrifugation and washing as described above. The
cells were then blocked for 10 min at room temperature using mouse
FcR blocking reagent (Miltenyi Biotec) followed by staining with
the following antibodies for 30 min at 4.degree. C.: anti-CD11 b
(recognizes monocytes), anti-CD3 (recognizes T cells), anti-B220
(recognizes B cells), and anti-Gr1 (recognizes granulocytes). The
FACS analysis was performed on an LSRII instrument, and the data
were analyzed using FloJo software.
[0123] Random mutagenesis and screening of Dusp1 mutants. A Gateway
entry clone containing mouse Dusp1 complementary DNA was purchased
from the Harvard PalsmiD repository (cat. # MmCD00312825). The
Dusp1 coding region was transferred into the retroviral gateway
vector pMSCV-Ires-GFP.GW59 by recombination cloning. Mutagenesis
and resistance screening were performed as described previously
(Azam, M., Latek, R. R. & Daley, G.Q. Mechanisms of
autoinhibition and STI-571/imatinib resistance revealed by
mutagenesis of BCR-ABL. Cell 112, 831-843 (2003)). Mutants isolated
in the screen were engineered into the pMSCV-Dusp1-Ires-GFP vector,
using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent).
The sequence of each point mutation was confirmed by sequence
analysis.
[0124] Dusp1 structural modeling and inhibitor docking. Structural
models of Dusp1 domains were built by homology-based modeling using
SWISS-MODEL software, and crystal structures, as described
previously (Kesarwani, M. et al. Targeting substrate-site in Jak2
kinase prevents emergence of genetic resistance. Sci. Rep. 5, 14538
(2015)). A model of the Dusp1 phosphatase domain was built using
the crystal structures of Dusp4 (PDB: 3EZZ; Dusp4 has 85% sequence
identity with Dusp1 and Dusp6 (PDB: 1MKP; Dusp6 has 48% sequence
identity with Dusp1. The structure of the N-terminal rhodanese
domain of Dusp1 was built using the crystal structure of Dusp16
(PDB: 2VSW; the N-terminal domain of Dusp16 has 26% sequence
identity to Dusp1). Only three structures of a Dusp allosteric
domain are available in the database (for Dusp6, Dusp10, and
Dusp16), and a sequence analysis did not show sufficient sequence
identity of the Dusp1 rhodanese domain with the N-terminal domains
of Dusp6 and Dusp10 (less than 17%); we therefore used only the
Dusp16 structure (PDB: 2VSW) to build the model. Blind docking of
BCI to the rhodanese domain model of Dusp1 was performed using
SwissDock software (Kesarwani, M. et al. Targeting substrate-site
in Jak2 kinase prevents emergence of genetic resistance. Sci. Rep.
5, 14538 (2015)). Modules with the most favorable energies were
clustered. For each cluster, binding modules with the lowest energy
(i.e., the most likely to represent true binding) were selected to
validate the model by site-directed mutagenesis, given that
mutation V83G conferred resistance to BCI. Figures were generated
using PyMol software.
[0125] Statistical analysis. Unless otherwise specified, results
are depicted as the mean.+-.s.d. Statistical analyses were
performed using one-tailed Student's t test using GraphPad Prism
(v6 GraphPad). Mantel-Cox test was used to perform Kaplan-Meier
survival analysis in GraphPad Prism (v6, GraphPad).
[0126] Myeloproliferative Neoplasms (MPNs) are blood cancers that
occur when the body makes too many white or red blood cells, or
platelets. This overproduction of blood cells in the bone marrow
can create problems for blood flow and lead to various symptoms.
MPNs were called Myeloproliferative Diseases until 2008 when the
World Health Organization reclassified them as cancers and renamed
them Myeloproliferative Neoplasms. There are three main types of
MPNs: Polycythemia vera (PV), Essential thrombocythemia (ED, and
Myelofibrosis (MF). With the discovery of specific gene mutations
in MPN, medications were designed to inhibit the abnormal proteins
related to these mutations. The drug imatinib (Gleevec) was
developed because it can inhibit the abnormal BCR-ABL protein in
chronic myeloid leukemia cells. Ruxolitinib (Jakafi) is a JAK1/JAK2
inhibitor, and it is used to treat intermediate-to-high risk
myelofibrosis (including primary myelofibrosis and myelofibrosis
related to polycthemia vera or essential thrombocythemia).
[0127] As shown in FIG. 23, DUSP1 but not c-FOS is overexpressed in
MPN. As shown in FIG. 23A, CD34+ cells from six patients
representing each subtype were analyzed and showed overexpression
of DUSP1 in the 3 forms of MPN as compared to healthy donor. As
shown in FIG. 23B, induction of Dusp1 in MPN cells is shown by qPCR
analysis of Dusp1 in Kit+ cells expressing Jak2-V617F, CSF3R-WT,
CSF3R-T618 and MpI-W515L normalized to vector control. As shown in
FIG. 24, lack of Dusp1 is synthetic lethal to MPN development in
mice. BM derived Kit+ cells from wild type and Dusp1-/- mice were
transduced with retroviruses expressing CSF3R-T618I,
CSF3R-T618I-W791X, MPL-W515L, and Jak2 V617F. As shown in FIG. 24A,
mice transplanted with wild type cells showing robust leukemia
development by CSF3R and MpI mutants, while mice received
Jak2-V617F cells showed mild elevation in WBC, but showed
significant increase in red cells and reticulocytes. As shown in
FIG. 24B, leukemic burden as GFP+ cells over a period of eight
weeks is shown. FIG. 24C shows that mice that received cells
lacking Dusp1 did not show any signs of leukemia. FIG. 24D shows
that all the GFP positive cells were abolished over the period of
seven weeks in oncogenic conditions, while vector transduced cells
have maintained normal engraftments. These data clearly show that
the lack of Dusp1 is synthetic lethal to MPN development.
[0128] Acute myeloid leukemia (AML) is a cancer of the myeloid line
of blood cells, characterized by the rapid growth of abnormal cells
that build up in the bone marrow and blood and interfere with
normal blood cells. Although progress has been made in treating
many types of cancers during recent years, AML remains a deadly
disease with survival rate lagging behind other blood cancers. A
combination of toxic chemotherapies has been the standard AML
treatment for more than 40 years. With efforts to define the
pathogenesis of AML, therapeutic drugs targeting key molecular
defects in AML are being used. Mutated in nearly 30% of AML,
FMS-like tyrosine kinase 3 (FLT3) represents one of the most used
targets. FLT3 mutants resulted from either internal tandem
duplication (ITD) or point mutations possess enhanced kinase
activity and cause constitutive activation of signaling. To date,
several small molecule inhibitors of FLT3 have been developed but
their clinical efficacy is limited due to a lack of potency and the
generation of drug resistance. KIT is mutated in 8.0% of acute
myeloid leukemia (AML). Oncogenic KIT mutations occur primarily in
core binding factor (CBF). KIT mutations occur primarily in exon 17
and affect the activation loop of the kinase domain. These changes
result in improved survival and growth of tumor cells. Induction of
FOS and DUSP1 in AML and MPN confers tyrosine kinase inhibitor
(TKI) resistance. FIG. 25 shows that growth factor (GF) signaling
abrogates oncogene dependence and confers TKI resistance. FIG. 25A
shows a dose response curve of BaF3 and BaF3-FLT3ITD cells showing
resistance to Flt3 inhibitor (AC220 or quizartinib) in the presence
of growth factor, IL3. IC50 for AC220 is shown in the parenthesis.
FIGS. 25B-C show bar graphs showing the induction of c-Fos and
Dusp1 by both FLT3ITD and GF signaling. FIG. 26 shows that deletion
of FOS and DUSP1 is synthetic lethal to AML development. c-Fos and
Dusp1 constitute non-oncogene addiction in FLT3ITD:MLLAF9 driven
AML. FIG. 26A shows a scheme to test the role of Fos and Dusp1 in
AML. FIG. 26B shows a bar graph showing CFU assays using Kit+ cells
from the wild type and Fos-/-/Dusp1-/- mice. CFU assays were
performed with and without Flt3 TKI (5 nM of AC220). Note, cells
expressing FLT3ITD and MLLAF9 are resistant to TKI while cells
lacking Fos and Dusp1 show synthetic lethality to oncogene
expression, suggesting these genes are essential for AML
development, however, they are indispensable for normal
hematopoiesis because vector transduced cells do not show any
defect in CFU formation (data not presented). FIG. 27 shows that
c-Fos and Dusp1 confer oncogene-dependence in high-risk
FLT3ITD:MLLAF9 driven AML. FIG. 27A shows a humanized AML model.
CD34 cells from human cord blood were transduced by retroviruses
expressing FLT3ITD-Ires-Cherry and MLLAF9-Ires-GFP. Double positive
(GFP+Cherry) cells were sorted by FACS followed with in vitro and
in vivo analysis. FIG. 27B shows histograms showing resistance to
AC220 in the presence of GF (IL3, IL6, SCF and TPO) in in-vitro
assay and FIG. 27C shows transplanted NSGS mice die of leukemia
within six weeks and show complete eradication of leukemic cells
when treated with combination of DFC+BCI+AC220 while AC220 or
DFC+BCI alone are ineffective.
[0129] Lung cancer is the leading cause of cancer-related mortality
in both men and women. Although chemotherapy recently has shown
promising results in the adjuvant clinical setting and there has
been some progress in the treatment of locally advanced and
advanced disease, treatment outcomes for non-small cell lung cancer
(NSCLC) patients are in general disappointing. Somatic, activating
mutations in EGFR identify a significant minority of patients with
non-small cell lung cancer (NSCLC). Although these mutations are
associated with an approximately 70% response rate to some EGFR
tyrosine kinase inhibitors (gefitinib, erlotinib, and afatinib),
patients develop resistance (i.e., "acquired resistance") after a
median of 9 to 12 months. As shown in FIG. 28, inhibition of FOS
and DUSP1 with TKI treatment cured EGFR driven lung cancers.
Growth-factor-induced TKI resistance in solid tumors is mediated by
c-FOS and DUSP1. FIG. 28A shows a dose response curve of the HCC827
cell line (lung adenocarcinoma; EGFR-DelE746A750) to erlotinib +/-
hepatocyte growth factor (HGF). FIGS. 28B-C show real-time qPCR
analysis illustrating induction of c-FOS (FIG. 28B) and DUSP1 (FIG.
28C) expression by HGF (indicated times after addition of
erlotinib). FIG. 28D shows cell survival of HCC827 cells (WST
assay) when treated with DFC, BCI and erlotinib alone and in
combination. Note inhibition of DUSP1 alone sensitized the cells
for erlotinib, while concomitant inhibition of both DUSP1 and c-FOS
is sufficient to inhibit proliferation and survival. FIG. 28E shows
HCC827 xenograft growth in recipients treated with erlotinib (red),
DFC+BCI (green) and DFC+BCI+erlotinib (purple). Treatment started
after one week of transplant (n=8 per group, each mouse represented
by single dot). FIG. 28F shows representative images of mouse
tumors from cohorts in FIG. 28E.
[0130] Platelet-derived growth factor receptors (PDGFRs), including
PDGFR.alpha. and PDGFR.beta., belong to the family of cell surface
type III receptor tyrosine kinases (RTKs). Upon binding of the
ligands, platelet-derived growth factors (PDGFs), the receptor
complex is activated and the cytosolic domains serve as docking
sites for coactivators and subsequently initiate downstream
signaling cascades such as MAPK, PI3K, and STAT3 pathways. PDGFR
signaling regulates a variety of biological processes, including
cellular growth, cellular differentiation, cell migration, and
angiogenesis. Deregulated PDGFR signaling has been implicated in
the pathogenesis of several human diseases and malignancies. For
example, in patients with gastrointestinal stromal tumors, chronic
myelomonocytic leukemia, glioblastoma multiforme, and lung cancer,
mutations have been identified in the genes encoding PDGFR, which
results in constitutive activation of the kinase activity,
overstimulation of signal transduction, interaction with adjacent
stroma and vasculature, and eventually tumor cell growth. Current
NSCLC therapies include several agents involved directly or
indirectly platelet-derived growth factors (PDGFs) and its
receptors (PDGFRs), e.g., sorafenib, sunitunib, imatinib, and
bevacizumab. As shown in FIG. 29, inhibition of FOS and DUSP1 is
sufficient to cure PDGFR driven lung cancer. As shown in FIG. 29A,
dose response curve of NCI-H1703 (lung squamous carcinoma; PDGFR
amplification) showing resistance to sunitinib in the presence of
epidermal growth factor and fibroblast growth factor (EGF+FGF).
FIGS. 29B-C show real-time qPCR analysis illustrating induction of
FOS (FIG. 29B) and DUSP1 (FIG. 29C) expression by EGF and FGF
(indicated times after addition of sunitinib). FIG. 29D shows cell
survival of NCI-H1703 cells (WST assay) when treated with DFC, BCI
and sunitinib alone and in combination. Concomitant inhibition of
both DUSP1 and c-FOS is sufficient to inhibit proliferation and
survival. FIG. 29E shows mouse xenografts of NCI-H1703 treated with
sunitinib, DFC+BCI and sunitinib+DFC+BCI (n=5). Treatments were
started two weeks after xenotransplantation. Mice treated with
either DFC+BCI or sunitinib+DFC+BCI showed complete response.
Treatment with sunitinib alone showed initial response but three
mice showed tumor regrowth after three weeks of treatment. FIG. 29F
shows representative images of mouse tumors from cohorts in FIG.
29E.
[0131] Growth factor induced TKI resistance in solid tumors is
mediated by c-FOS and DUSP1. ERBB2 is a transmembrane tyrosine
kinase receptor and a member of the ErbB protein family (ie, the
epidermal growth factor receptor [EGFR] family). ERBB2 is most
commonly known as HER2 and sometimes also as NEU. HER2 gene product
is overexpressed in 18-20% of invasive breast cancers. Lapatinib is
an orally active drug for breast cancer and other solid tumours. It
is a dual tyrosine kinase inhibitor which interrupts the HER2/neu
and epidermal growth factor receptor (EGFR) pathways. It is used in
combination therapy for HER2-positive breast cancer. It is used for
the treatment of patients with advanced or metastatic breast cancer
whose tumors overexpress HER2 (ErbB2). Lapatinib is used as a
treatment in patients who have HER2-positive advanced breast cancer
that has progressed after previous treatment with other
chemotherapeutic agents, such as anthracycline, taxane-derived
drugs, or trastuzumab (Herceptin).
[0132] Muscle invasive bladder cancer (MIBC) is a highly aggressive
disease, with a 5 year survival rate post-diagnosis of
approximately 50%. Although the implementation of neoadjuvant
chemotherapy extended overall patient survival prior to the recent
advent of immune checkpoint inhibitors, no relevant new therapies
have been introduced in the last 3 decades. This is in stark
contrast to several other major cancers. MIBC has the third highest
rate of ERBB2 amplification (after breast and gastric cancer) and
demonstrates frequent Her2 overexpression. Even so, anti-Her2
treatments in MIBC have not been as encouraging.
[0133] Melanoma is an increasingly common cancer and a major cause
of cancer-related death. Metastatic melanoma has a low survival
rate and few effective treatments, thus new targets are needed for
effective therapy. A molecule implicated in metastasis of cancer in
general is c-Met, a receptor tyrosine kinase (RTK) involved in cell
proliferation, migration, and invasion. c-Met and its ligand,
hepatocyte growth factor (HGF), are upregulated in metastatic
melanoma and are implicated in invasion and clinical disease
progression. The discovery that .about.50-60% of melanomas carry
BRAF.sup.V600E point mutations prompted the generation of compounds
specifically targeting this hyperactive mutated kinase. One such
compound, PLX4032, has shown therapeutic efficacy in clinical
trials and was therefore FDA-approved for clinical therapy under
the name vemurafenib. Despite its remarkable efficacy, almost all
patients receiving BRAF inhibitor treatment relapsed after weeks to
months of therapy.
[0134] As shown in FIG. 30, growth factor induced TKI resistance in
solid tumors is mediated by c-FOS and DUSP1. FIG. 30A shows dose
response curves showing TKI resistance in solid tumor cell lines in
the presence of growth factors. AU565 (breast cancer HER2 amplified
AU565) conferred resistance to lapatinib by growth factor,
neuregulin 1-NRG1. RT4 (bladder carcinoma, EGFR amplified)
conferred resistance to lapatinib in the presence of EGF. SKMEL28
(melanoma, BRAF-V600E) conferred resistance to PLX4720 in the
presence of HGF. FIGS. 30B-C show real-time qPCR analysis
illustrating induction of c-FOS (FIG. 30B) and DUSP1 (FIG. 30C)
expression by growth factors (indicated times after addition of
erlotinib). Time at 0 hours represents the level of expression
without TKI+/-GF. Note that the growth factors induce higher
expression of c-FOS in all cell lines and DUSP1 in RT4 at 0 hours,
while the addition of both TKI and growth factors induced both
c-FOS and DUSP1. FIG. 30D shows bar graphs showing cell survival by
WST assay when treated with DFC, BCI and TKI alone and in
combination. Note inhibition of c-FOS and DUSP1 is sufficient to
kill AU565 cells, while their inhibition in RT4 and SKMEL28 cells
restored the TKI sensitivity in the presence of growth factors.
[0135] Chronic lymphocytic leukemia (CLL) is characterized by
constitutive activation of the B-cell receptor (BCR) signaling
pathway, but variable responsiveness of the BCR to antigen
ligation. Bruton's tyrosine kinase (BTK) shows constitutive
activity in CLL and is the target of irreversible inhibition by
ibrutinib, an orally bioavailable kinase inhibitor that has shown
outstanding activity in CLL. However, early clinical results in CLL
with other reversible and irreversible BTK inhibitors have been
less promising.
[0136] Each of the following references is expressly incorporated
by reference herein in its entirety: [0137] Aikawa et al.
"Treatment of arthritis with a selective inhibitor of
c-Fos/activator protein-1," Nature Biotechnology, vol. 26, no. 7
(2008), pp. 817-823. [0138] Day et al., "Small Molecule Inhibitors
of DUSP6 and Uses Therefor," WO2010/108058, Sep. 23, 2010. [0139]
Park et al., "Inhibition of fos-jun-DNA complex formation by
dihydroguaiaretic acid and in vitro cytotoxic effects on cancer
cells," Cancer Letters, vol. 127 (1998), pp. 23-28. [0140] Padhye
S, et al. New difluoro Knoevenagel condensates of curcumin, their
Schiff bases and copper complexes as proteasome inhibitors and
apoptosis inducers in cancer cells. Pharm Res 2009; 26:1874-80.
[0141] Padhye S et al. Fluorocurcumins as cyclooxygenase-2
inhibitor: molecular docking, pharmacokinetics and tissue
distribution in mice. Pharm Res. 2009 November; 26(11):2438-45.
[0142] Daley, G.Q., Van Etten, R. A. & Baltimore, D. Induction
of chronic myelogenous leukemia in mice by the P210bcr/abl gene of
the Philadelphia chromosome. Science 247, 824-830 (1990). [0143]
Druker, B. J. et al. Effects of a selective inhibitor of the Abl
tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med.
2,561-566 (1996). [0144] O'Hare, T., Zabriskie, M. S., Eiring, A.
M. & Deininger, M. W. Pushing the limits of targeted therapy in
chronic myeloid leukaemia. Nat. Rev. Cancer 12,513-526 (2012).
[0145] Rousselot, P. et al. Imatinib mesylate discontinuation in
patients with chronic myelogenous leukemia in complete molecular
remission for more than 2 years. Blood 109,58-60 (2007). [0146]
Mahon, F. X. et al. Discontinuation of imatinib in patients with
chronic myeloid leukaemia who have maintained complete molecular
remission for at least 2 years: the prospective, multicentre Stop
Imatinib (STIM) trial. Lancet Oncol. 11,1029-1035 (2010). [0147]
Ross, D. M. et al. Safety and efficacy of imatinib cessation for
CML patients with stable undetectable minimal residual disease:
results from the TWISTER study. Blood 122,515-522 (2013). [0148]
Chu, S. et al. Detection of BCR-ABL kinase mutations in CD34+ cells
from chronic myelogenous leukemia patients in complete cytogenetic
remission on imatinib mesylate treatment. Blood 105,2093-2098
(2005). [0149] Savona, M. & Talpaz, M. Getting to the stem of
chronic myeloid leukaemia. Nat. Rev. Cancer 8, 341-350 (2008).
[0150] Azam, M., Latek, R. R. & Daley, G.Q. Mechanisms of
autoinhibition and STI-571/imatinib resistance revealed by
mutagenesis of BCR-ABL. Cell 112,831-843 (2003). [0151] Krause, D.
S. & Van Etten, R. A. Tyrosine kinases as targets for cancer
therapy. N. Engl. J. Med. 353,172-187 (2005). [0152] Weinstein, I.
B. Cancer. Addiction to oncogenes--the Achilles heal of cancer.
Science 297,63-64 (2002). [0153] Sawyers, C. L. Shifting paradigms:
the seeds of oncogene addiction. Nat. Med. 15,1158-1161 (2009).
[0154] Pagliarini, R., Shao, W. & Sellers, W. R. Oncogene
addiction: pathways of therapeutic response, resistance, and road
maps toward a cure. EMBO Rep. 16,280-296 (2015). [0155] Reddy, A.
& Kaelin, W. G., Jr. Using cancer genetics to guide the
selection of anticancer drug targets. Curr. Opin. Pharmacol.
2,366-373 (2002). [0156] Kaelin, W. G., Jr. The concept of
synthetic lethality in the context of anticancer therapy. Nat. Rev.
Cancer 5,689-698 (2005). [0157] Kamb, A. Consequences of
nonadaptive alterations in cancer. Mol. Biol. Cell 14,2201-2205
(2003). [0158] Mills, G. B., Lu, Y. & Kohn, E. C. Linking
molecular therapeutics to molecular diagnostics: inhibition of the
FRAP/RAFT/TOR component of the PI3K pathway preferentially blocks
PTEN mutant cells in vitro and in vivo. Proc. Natl. Acad. Sci. USA
98,10031-10033 (2001). [0159] Sharma, S. V. & Settleman, J.
Exploiting the balance between life and death: targeted cancer
therapy and "oncogenic shock". Biochem. Pharmacol. 80,666-673
(2010). [0160] Sharma, S. V. & Settleman, J. Oncogene
addiction: setting the stage for molecularly targeted cancer
therapy. Genes Dev. 21,3214-3231 (2007). [0161] Corbin, A. S. et
al. Human chronic myeloid leukemia stem cells are insensitive to
imatinib despite inhibition of BCR-ABL activity. J. Clin. Invest.
121,396-409 (2011). [0162] Straussman, R. et al. Tumour
micro-environment elicits innate resistance to RAF inhibitors
through HGF secretion. Nature 487,500-504 (2012). [0163] Wilson, T.
R. et al. Widespread potential for growth-factor-driven resistance
to anticancer kinase inhibitors. Nature 487,505-509 (2012). [0164]
Bruennert, D. et al. Early in vivo changes of the transcriptome in
Philadelphia chromosome-positive CD34+ cells from patients with
chronic myelogenous leukaemia following imatinib therapy. Leukemia
23,983-985 (2009). [0165] Eferl, R. & Wagner, E. F. AP-1: a
double-edged sword in tumorigenesis. Nat. Rev. Cancer 3,859-868
(2003). [0166] Lawan, A., Shi, H., Gatzke, F. & Bennett, A. M.
Diversity and specificity of the mitogen-activated protein kinase
phosphatase-1 functions. Cell. Mol. Life Sci. 70,223-237 (2013).
[0167] Jeffrey, K. L., Camps, M., Rommel, C. & Mackay, C. R.
Targeting dual-specificity phosphatases: manipulating MAP kinase
signalling and immune responses. Nat. Rev. Drug Discov. 6,391-403
(2007). [0168] Brooks, S. A. & Blackshear, P. J.
Tristetraprolin (TTP): interactions with mRNA and proteins, and
current thoughts on mechanisms of action. Biochim. Biophys. Acta
1829,666-679 (2013). [0169] Dorfman, K. et al. Disruption of the
erp/mkp-1 gene does not affect mouse development: normal MAP kinase
activity in ERP/MKP-1-deficient fibroblasts. Oncogene 13,925-931
(1996). [0170] Zhang, J. et al. c-fos regulates neuronal
excitability and survival. Nat. Genet. 30,416-420 (2002). [0171]
Zhao, C. et al. Hedgehog signalling is essential for maintenance of
cancer stem cells in myeloid leukaemia. Nature 458,776-779 (2009).
[0172] Ransone, L. J., Visvader, J., Wamsley, P. & Verma, I. M.
Trans-dominant negative mutants of Fos and Jun. Proc. Natl. Acad.
Sci. USA 87,3806-3810 (1990). [0173] Molina, G. et al. Zebrafish
chemical screening reveals an inhibitor of Dusp6 that expands
cardiac cell lineages. Nat. Chem. Biol. 5,680-687 (2009). [0174]
Huang, T. S., Lee, S. C. & Lin, J. K. Suppression of c-Jun/AP-1
activation by an inhibitor of tumor promotion in mouse fibroblast
cells. Proc. Natl. Acad. Sci. USA 88,5292-5296 (1991). [0175] Park,
S., Lee, D. K. & Yang, C. H. Inhibition of fos-jun-DNA complex
formation by dihydroguaiaretic acid and in vitro cytotoxic effects
on cancer cells. Cancer Lett. 127,23-28 (1998). [0176] Padhye, S.
et al. Fluorocurcumins as cyclooxygenase-2 inhibitor: molecular
docking, pharmacokinetics and tissue distribution in mice. Pharm.
Res. 26,2438-2445 (2009). [0177] Koschmieder, S. et al. Inducible
chronic phase of myeloid leukemia with expansion of hematopoietic
stem cells in a transgenic model of BCR-ABL leukemogenesis. Blood
105,324-334 (2005). [0178] Li, L. et al. Activation of p53 by SIRT1
inhibition enhances elimination of CML leukemia stem cells in
combination with imatinib. Cancer Cell 21,266-281 (2012). [0179]
Copland, M. et al. BMS-214662 potently induces apoptosis of chronic
myeloid leukemia stem and progenitor cells and synergizes with
tyrosine kinase inhibitors. Blood 111, 2843-2853 (2008). [0180]
Angel, P. & Karin, M. The role of Jun, Fos and the AP-1 complex
in cell-proliferation and transformation. Biochim. Biophys. Acta
1072,129-157 (1991). [0181] Owens, D. M. & Keyse, S. M.
Differential regulation of MAP kinase signalling by
dual-specificity protein phosphatases. Oncogene 26,3203-3213
(2007). [0182] Boutros, T., Chevet, E. & Metrakos, P.
Mitogen-activated protein (MAP) kinase/MAP kinase phosphatase
regulation: roles in cell growth, death, and cancer. Pharmacol.
Rev. 60, 261-310 (2008). [0183] Groom, L. A., Sneddon, A. A.,
Alessi, D. R., Dowd, S. & Keyse, S. M. Differential regulation
of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic
dualspecificity phosphatase. EMBO J. 15, 3621-3632 (1996). [0184]
Fjeld, C. C., Rice, A. E., Kim, Y., Gee, K. R. & Denu, J. M.
Mechanistic basis for catalytic activation of mitogen-activated
protein kinase phosphatase 3 by extracellular signal-regulated
kinase. J. Biol. Chem. 275,6749-6757 (2000). [0185] Zhao, Q. et al.
MAP kinase phosphatase 1 controls innate immune responses and
suppresses endotoxic shock. J. Exp. Med. 203,131-140 (2006). [0186]
Hirsch, D. D. & Stork, P. J. Mitogen-activated protein kinase
phosphatases inactivate stress-activated protein kinase pathways in
vivo. J. Biol. Chem. 272,4568-4575 (1997). [0187] Young, P. R. et
al. Pyridinyl imidazole inhibitors of p38 mitogen-activated protein
kinase bind in the ATP site. J. Biol. Chem. 272,12116-12121 (1997).
[0188] Bennett, B. L. et al. SP600125, an anthrapyrazolone
inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA
98,13681-13686 (2001). [0189] Shojaee, S. et al. Erk negative
feedback control enables pre-B cell transformation and represents a
therapeutic target in acute lymphoblastic leukemia. Cancer Cell
28,114-128 (2015). [0190] Hrustanovic, G. et al. RAS-MAPK
dependence underlies a rational polytherapy strategy in
EML4-ALK-positive lung cancer. Nat. Med. 21,1038-1047 (2015).
[0191] Zhang, B. et al. Altered microenvironmental regulation of
leukemic and normal stem cells in chronic myelogenous leukemia.
Cancer Cell 21,577-592 (2012). [0192] Reynaud, D. et al. IL-6
controls leukemic multipotent progenitor cell fate and contributes
to chronic myelogenous leukemia development. Cancer Cell 20,661-673
(2011). [0193] Weiner, R. S. et al. Treatment of chronic
myelogenous leukemia by blocking cytokine alterations found in
normal stem and progenitor cells. Cancer Cell 27,671-681 (2015).
[0194] Roberts, K. G. et al. Genetic alterations activating kinase
and cytokine receptor signaling in high-risk acute lymphoblastic
leukemia. Cancer Cell 22,153-166 (2012). [0195] Druker, B. J. et
al. Activity of a specific inhibitor of the BCR-ABL tyrosine kinase
in the blast crisis of chronic myeloid leukemia and acute
lymphoblastic leukemia with the Philadelphia chromosome. N. Engl.
J. Med. 344, 1038-1042 (2001). [0196] Chang, K. H. et al. Vav3
collaborates with p190-BCR-ABL in lymphoid progenitor
leukemogenesis, proliferation, and survival. Blood 120, 800-811
(2012). [0197] Bagger, F. O. et al. BloodSpot: a database of gene
expression profiles and transcriptional programs for healthy and
malignant haematopoiesis. Nucleic Acids Res. 44D1, D917-D924
(2016). [0198] Jorgensen, H. G., Allan, E. K., Jordanides, N. E.,
Mountford, J. C. & Holyoake, T. L. Nilotinib exerts equipotent
antiproliferative effects to imatinib and does not induce apoptosis
in CD34+ CML cells. Blood 109, 4016-4019 (2007). [0199] Holyoake,
T. L. & Vetrie, D. The chronic myeloid leukemia stem cell:
stemming the tide of persistence. Blood
https://doi.org/10.1182/blood-2016-09-696013 (2017). [0200]
Kesarwani, M. et al. Targeting substrate-site in Jak2 kinase
prevents emergence of genetic resistance. Sci. Rep. 5, 14538
(2015). [0201] Azam, M., Seeliger, M. A., Gray, N. S., Kuriyan, J.
& Daley, G.Q. Activation of tyrosine kinases by mutation of the
gatekeeper threonine. Nat. Struct. Mol. Biol. 15, 1109-1118 (2008).
[0202] Komurov, K., Dursun, S., Erdin, S. & Ram, P. T.
NetWalker: a contextual network analysis tool for functional
genomics. BMC Genomics 13, 282 (2012).
[0203] Other variations or embodiments will be apparent to a person
of ordinary skill in the art from the above description. Thus, the
foregoing embodiments are not to be construed as limiting the scope
of the claimed invention.
* * * * *
References