CDC25A INHIBITOR FOR THE TREATMENT OF DRUG RESISTANT CANCER OR FOR THE PREVENTION OF TUMOR RELAPSE
20170247706 · 2017-08-31
Inventors
Cpc classification
A61K31/713
HUMAN NECESSITIES
A61K31/7088
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
International classification
C12N15/113
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
The present invention concerns a CDC25A phosphatase inhibitor for use in the treatment of a drug resistant cancer and/or in prevention of tumor relapse in a patient suffering or having suffered from cancer.
Claims
1. A method of treating drug resistant acute myeloid leukemia (AML) and/or for preventing tumor relapses in a patient suffering or having suffered from acute myeloid leukemia, comprising administering to the patient an effective amount of a CDC25A phosphatase inhibitor.
2. The method according to claim 1, wherein said CDC25A phosphatase inhibitor is selected from the group consisting of i. Quinone derivatives and maleimide derivatives and ii. an inhibitor of CDC25A phosphatase expression.
3. The method of claim 1, wherein said AML is acute myeloid leukemia associated with a mutated FLT3-ITD.
4. The method of claim 1, wherein said inhibitor of CDC25A phosphatase expression is a siRNA, a ribozyme, or an antisense oligonucleotide.
5. The method of claim 1, wherein said CDC25A phosphatase inhibitor is IRC 083864.
6. A pharmaceutical composition comprising: i. a CDC25A phosphatase inhibitor; ii. a pharmaceutically acceptable carrier; and iii. optionally a chemotherapeutic drug.
7. The pharmaceutical composition according to claim 6 wherein said CDC25A phosphatase inhibitor is IRC 083864.
8-9. (canceled)
10. The method of claim 1, further comprising administering to the patient a chemotherapeutic drug.
11. The method of claim 10, wherein the chemotherapeutic drug is an anti-mitotic agent.
12. The pharmaceutical composition according to claim 6, wherein the chemotherapeutic drug is an anti-mitotic agent.
Description
FIGURES
[0094]
[0095] (a) MV4-11 (left panel) and MOLM-14 (right panel) FLT3-ITD expressing cells were treated for 2 hours with FLT3 inhibitor III (100 nM), and the protein levels of CDC25A, CDC25B, CDC25C, Cyclin A, Cyclin D1 and p27Kip1 were analyzed by western blot. (b) MOLM-14 (FLT3-ITD postive), KG1 and HL-60 (FLT3 wt positive) and K562 (FLT3 negative) cell lines were treated for 2 hours with FLT3 inhibitor III and the level of CDC25A was analyzed by western blot. Actin was used as a loading control. These results are representative of three independent experiments. ns: non specific.
[0096]
[0097] (a) MV4-11 (left panel) and MOLM-14 (right panel) cells were treated for 2 hours with STAT5 inhibitor (100 nM). CDC25A, Pim1, and STAT5 protein and phosphorylation levels were analyzed by western blot. (b) MV4-11 and MOLM-14 cells were transfected for 24 hours with STAT5A/B siRNA and the impact on CDC25A protein level was analyzed by western blot. These results are representative of three independent experiments. Actin was used as a loading control in these experiments. ns: non specific.
[0098]
[0099] (a) MV4-11 cells were first treated by cycloheximide (50 μg/mL) for 90 minutes in order to completely down regulate CDC25A. Cells were then placed in normal culture conditions in the presence (lower panel) or the absence (upper panel) of FLT3 inhibitor (100 nM), and western blot analysis of CDC25A was performed at the indicated times. The FLT3 inhibitor was added 30 minutes before medium wash. This western blot is representative of three independent experiments. The right panel shows the quantification of three independent experiments. (b) CDC25A mRNA expression was measured by quantitative RT-PCR after FLT3 inhibition for 2 hours (100 nM). The graph shows the mean+/−SEM of CDC25A mRNA expression in untreated and treated cells, in three independent experiments. (c) MV4-11 cells were treated with cycloheximide (50 μg/mL) for the indicated times. FLT3 inhibitor III (100 nM) was added 30 minutes before cycloheximide treatment, and left in the medium. This western blot is representative of three independent experiments. The right panel shows the quantification of three independent experiments. ns: non specific.
[0100]
[0101] (a) MV4-11 and MOLM-14 FLT3-ITD positive cells, KG1, HL-60 and TF-1 FLT3 wild type cells, and K562 FLT3 negative cells were cultured in the presence of the CDC25 inhibitor IRC-083864 (200 nM). Cells were harvested each day and counted after trypan blue coloration. The graph represents three independent experiments.
[0102] (b) MOLM-14 and MV4-11 cells were transfected with CDC25A siRNA for 24 hours, and cells were counted after trypan blue coloration (upper panel). The efficiency of CDC25A siRNA was estimated by western blot analysis (lower panel). ns: non specific.
[0103] (c) Primary cells from patients were cultured in semi-solid medium to estimate their clonogenic potential as described in the Methods section, in the presence or the absence of IRC-083864 (100 and 200 nM). 6 FLT3-ITD positive (upper panel) and 4 FLT3-wild type (lower panel) AML primary samples were used for these experiments. Leukemic colonies were scored under an inverted microscope at day 7.
[0104] (d) MOLM-14, and MOLM-14 TKD cells, were grown in the presence of AC-220 1 nM (upper panel) or IRC-083864 200 nM (lower panel). Cells were harvested each day and counted after trypan blue staining. The graphs represent three independent experiments.
[0105]
[0106] (a-b) MOLM-14 and MV4-11 cells were treated for different times with IRC-083864 (100 nM and 200 nM) and the expression of the cell surface markers CD11b, CD14 and CD15 were followed by flow cytometry analysis. (c) MOLM-14 and MV4-11 cells were treated for different times with IRC-083864 (100 nM) and morphological changes were estimated by microscopy after cells were cytospun and stained at days 8 and 13. Original magnification ×100. (d) MOLM-14 cells were treated for different times with IRC-083864 (100 nM) and c-myc expression as well as C/EBPα phosphorylation on serine 21 were analyzed by western blot at day 1, and C/EBPε expression after 6 days of treatment. (e) MOLM-14 cells were transfected with CDC25A shRNA The expression of the cell surface markers CD11b, CD14 and CD15 were followed by flow cytometry analysis at day 3 and 6 (upper panel), and their morphology was analyzed by microscopy after 6 days (lower panel). Original magnification ×100. (f) MOLM-14 cells were transfected with CDC25A siRNA, and their morphology was analyzed by microscopy after 3 days (upper panel). Original magnification ×100. The efficiency of CDC25A siRNA was assessed by western blot analysis (lower panel). These results are representative of three independent experiments. ns: non specific
[0107]
[0108] (a) 4 primary samples from patients carrying the FLT3-ITD mutation (ITD #1-4) were treated for 6 days with IRC-083864 (100 nM), and the expression of CD14 (upper panel) and CD15 (lower panel) was followed by flow cytometry analysis. (b) Quantification (mean±SEM, n=4) of CD14 and CD15 expression in response to IRC-083864. (c) The morphology of primary cells from patients used in (a) was analyzed by microscopy after 6 days and 9 days of treatment with IRC-083864. Original magnification ×100.
[0109]
[0110] (a) NSG mice were injected with 2 millions MOLM-14 cells, and treated with IRC-083864 at 5 mg/kg/week i.p. (n=4) or with vehicle buffer (n=4) as described in the Methods section. Bone marrow cells were extracted after dissection after two weeks and the expression of CD14 and CD15 on human blasts was followed by flow cytometry analysis. (b) Quantification (mean±SEM, n=4) of CD14 and CD15 expression in response to IRC-083864. (c) Morphological analyses of human cells used in (a). Original magnification ×100.
EXAMPLE
[0111] Methods
[0112] Cell Lines and Reagents.
[0113] Human acute myeloid leukemia cell lines MOLM-14 (Matsuo Y et al, Leukemia 1997) (kindly provided by Martin Carroll, University of Pennsylvania, Philadelphia, Pa., USA) MV4-11, K562 (ACC-102 and ACC-10, DSMZ, Braunschweig, Germany) and FLT3-ITD-expressing murine BaF3 cells were cultured in RPMI 1640 medium (Gibco, Life Technologies, Carlsbad, Calif., USA) supplemented with 10% fetal bovine serum (Sigma, Saint Louis, Calif., USA). TF-1 (ATCC-CRL2003) cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and GM-CSF 2 ng/ml. Concerning MOLM-14 cell line, the presence of a monoallelic 21 bp FLT3-ITD mutation and of an MLL-containing ins(11; 9)(q23;p22p23) were verified (Hematology laboratory of Toulouse University Hospital, Prof E. Delabesse and Dr I. Luquet). KG1 and HL-60 (ACC-14 and ACC-3, DSMZ) cell lines were grown in Iscove's Modified Dulbecco's Medium (IMDM, Gibco) plus 20% FBS. All cells were grown in the presence of 100 units/ml of penicillin and streptomycin (Invitrogen) at 37° C. and 5% CO2.
[0114] The FLT3 inhibitor III (Furet P et al, J Med Chem 2006), Akt inhibitor VIII and STAT5 inhibitor were purchased from Calbiochem (San Diego, Calif., USA). The FLT3 inhibitor quizartinib AC220 and the MEK inhibitor PD0325901 were purchased from Selleck Chemicals (Houston, Tex., USA). The CDC25 inhibitor IRC-084864 (Brezak M C et al, Int J Cancer 2009) was kindly provided by IPSEN laboratory (Marie-Odile Contour-Galcera, Les Ulis, France) and NSC-95397 was purchased from Enzo Life Sciences (Farmingdale, N.Y., USA) (Lazo J S et al, Mol Pharmacol 2002). The translation inhibitor cycloheximide was purchased from Sigma (Saint Louis, Mo., USA). The proteasome inhibitor bortezomib was kindly provided by Clement Larrue (CRCT Team 18, Toulouse, France). hFLT3 ligand, hGM-CSF, hG-CSF and hIL-3 were purchased from R&D Systems Inc (Minneapolis, Minn., USA).
[0115] Patient Samples.
[0116] Patient AML samples were obtained after informed consent in accordance with the Declaration of Helsinki and stored at the HIMIP collection. According to the French law, HIMIP collection has been declared to the Ministry of Higher Education and Research (DC 2008-307 collection 1) and obtained a transfer agreement (AC 2008-129) after approbation by ethical committees (Comité de Protection des Personnes Sud-Ouest et Outremer II and APHP ethical committee). Clinical and biological annotations of the samples have been declared to the CNIL (Comité National Informatique et Libertés i.e. Data processing and Liberties National Committee). Frozen cells were thawed in IMDM medium with 20% FBS and immediately processed for treatment. All patients were diagnosed at the Department of Hematology of Toulouse University Hospital. Their characteristics are summarized in Table 1.
[0117] Co-Cultures.
[0118] Patient samples, containing at least 80% of blasts, were co-cultured with murine stromal cells (MS-5 (ACC-441) kindly provided by Helena Boutzen, CRCT Team 18, Toulouse, France) in IMDM (Gibco) supplemented with 15% BIT (Stem Cell Technologies, Vancouver, BC, Canada), 100 units/ml penicillin and streptomycin (Invitrogen), 5 μM β-mercaptoethanol (Invitrogen), 1 mM pyruvate (Sigma), MEM 1× (Sigma), 100 ng/mL DNase (MP Biomedicals, Solon, Ohio, USA), 10 ng/mL hIL-3, 100 ng/mL hSCF, and 10 ng/ml hTPO (all from R&D Systems Inc, Minneapolis, Minn., USA). All the samples were then processed for treatment with the different reagents (IRC-083864 or NSC-95397).
[0119] siRNA.
[0120] The MV4-11 cell line was transfected with the Amaxa nucleofection technology (Lonza, Koeln, Germany). Cells (2×106) were resuspended in 100 μL of Amaxa solution L. 300 nM of specific STAT5A and STAT5B siRNA (ON-TARGETplus SMARTpool, human STAT5A and STAT5B, Dharmacon) or total CDC25A siRNA (Hs_CDC25A_9, Qiagen, Hilden, Germany) or 3′UTR CDC25A siRNA (CDC25A 2943, Sigma) or negative control (si genome control pool non targeting #2, or ON-TARGETplus control pool (Dharmacon)) were added, and cells were transfected with the nucleofector device (program Q-001; solution V and program O-017 for MOLM-14; solution R and program V-001 for KG1). Cells were subsequently resuspended in normal culture medium at a concentration of 5×105 cells/mL. Twenty-four or forty-eight hours after transfection, cells were counted (trypan blue staining), and western blotting was performed.
[0121] Lentiviral Infections.
[0122] To generate lentiviral vectors expressing CDC25A protein, sequences were cloned into the pTrip-TAL-Ires-GFP lentiviral vector. We used 293-T packaging cells, co-transfected with lentiviral protein (GAG, POL, and ENV) encoding plasmids, and plasmids containing a control or CDC25A genes, separately. Supernatants containing lentivirus were collected 48 h after transfection, during 3 consecutive days. MOLM-14 cells were plated at 5×105 cells in 200 μl in serum-free medium and 5 μl of lentiviral supernatant was added during 3 h. Cells were then grown in 10% FBS RPMI medium.
[0123] Western Blot.
[0124] 2×106 cells were usually lysed in 100 μL of NuPAGE® LDS Sample Buffer (Novex, Life Technologies, Carlsbad, Calif., USA), sonicated for 15 seconds, and boiled for 3 minutes. Proteins were then resolved on NuPAGE® 4-12% Bis-Tris Gels and transferred to nitrocellulose membrane. Saturation of the membrane was done for 1 hour in Tris Buffer Saline with Tween 0.1% (TBS-T) containing 5% non-fat milk or 5% bovine serum albumin. Membranes were blotted with proper antibodies overnight at 4° C., washed thrice with TBS-T, and incubated for 30 minutes with HRP-conjugated secondary antibody (Promega, Madison, Wis., USA). After three additional washes, detection was achieved with Supersignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Rockford, Ill., USA). The antibodies used were: monoclonal anti-CDC25A (F-6), anti-Cyclin D1 (HD11), anti-Pim1 (12H8), anti-c-myc (9E10), and polyclonal anti-CDC25B (C-20), anti-CDC25C (C-20), anti-Cyclin A (C-19), and anti-Akt1/2/3 (H-136), from Santa Cruz Biotechnology (Santa Cruz, Calif., USA), monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) and polyclonal anti-phospho-STAT5 (Tyr 694), anti-STAT5, anti-p44/42 MAPK, anti-phospho-Ser473 Akt (D9E) XP, anti-phospho-Cdc2 (Tyr15), anti-phospho-C/EBPα (Ser21), anti-C/EBPα (p42) and anti-C/EBPε (C-22) from Cell Signaling Technology (Beverly, Mass., USA), anti-p27KIP1 from BD Biosciences (San Diego, Calif., USA); anti-β-actin and anti-α-tubulin from Sigma.
[0125] Quantitative RT-PCR.
[0126] Total RNA was extracted by RNeasy Kit (Qiagen) according to the manufacturer. RNA quality and purity was assessed by using the Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, Calif., USA). cDNA was generated with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) following the manufacturer instructions. The PCR was performed with TaqMan® Gene Expression Master Mix (Applied Biosystems, Foster City, Calif., USA) with 1 μl of cDNA on a LightCycler®480 (Roche). The primer used was Hs00947994_m1 (Applied Biosystem) for CDC25A. GUSB (Hs00939627_m1) and B2M (Hs00984230_m1) were used as housekeeping genes. Results were analyzed with the LightCycler®480 software release 1.5.0 SP4 using the conventional ΔΔCt method.
[0127] Flow Cytometry.
[0128] Apoptotic cells were detected with Annexin V-FITC detection kit from BD Pharmingen (San Diego, Calif., USA) according to the manufacturer instructions. To evaluate AML cell differentiation, cells were stained for 30 min with the following anti-human monoclonal antibodies: CD11b-PE (Beckman Coulter), CD14-FITC (BD Biosciences), CD15-APC (BD Biosciences). For patient samples, additional hCD45-APC-H7 (BD Biosciences), Annexin V-Pacific Blue (BioLegend, San Diego, Calif., USA) and 7-AAD (Sigma) were used; and for bone marrow mice cells, additional CD33-FITC, CD44-PE Cy7, mCD45-PerCP Cy 5.5, CD14-Alexa 700 were used. Data were collected on a LSRII or a LSRFortessa cytometer (BD Biosciences), and analyzed with FlowJo software. A minimum of 10,000 events was collected.
[0129] Clonogenic Assay.
[0130] AML cells (106/mL) were grown in duplicate in H4230 methylcellulose medium (Stem Cell Technologies) supplemented with 10% 5637-conditionned medium as described (Récher C et al, Blood 2005). IRC-083864 was added at increasing concentrations in the culture medium. Cells were incubated for 7 days in a humidified CO2 incubator. Leukemic colonies were then scored under an inverted microscope.
[0131] Morphological Examination.
[0132] 105 cells were spun at 500 rpm for 5 minutes onto glass slides and May-Grünwald-Giemsa stained at the Toulouse University Hospital hematology laboratory.
[0133] In Vivo Experiments.
[0134] Animals were used in accordance to a protocol reviewed and approved by the Institutional Animal Care and User Ethical Committee of the UMS006 and Region Midi-Pyrénées (Approval#13-U1037-JES08). NOD/LtSz-scid IL-2Rγchainnull (NSG) mice (Sanchez P V et al) were produced at the Genotoul Anexplo platform in Toulouse (France) using breeders obtained from The Charles River Laboratory. Mice were housed in sterile conditions using HEPA filtered micro-isolators and fed with irradiated food and acidified water. Transplanted mice were treated with antibiotics (enrofloxacin) for the duration of the experiment. Only female mice were considered for this experiment because of higher toxicity of IRC-083864 in males NSG mice in preliminary toxicity assays. Adult mice (6 or 7-week old) were treated with 20 mg/kg Busulfan (Busilvex, Pierre Fabre, France) by i.p. administration 24 hours before injection of leukemic cells. Cultured MOLM-14 cells were washed in PBS and cleared of aggregates and debris using a 0.2 mm cell filter, and suspended in PBS at a final concentration of 2 million cells per 200 μL of PBS per mouse for i.v. injection. IRC-083864 (5 mg/kg/week) was then administrated to leukemic mice by i.p. injection. Daily monitoring of mice for symptoms of disease (ruffled coat, hunched back, weakness and reduced motility) determined the time of animal euthanasia. Cell differentiation was monitored after two IRC-083864 injections: mice were humanely killed in accordance with IACUC protocols. Bone marrow (mixed from tibias and femurs) were dissected, flushed in PBS and made into single cell suspensions for analysis by flow cytometry and cytospins.
[0135] Statistics.
[0136] Experiments in cell lines were performed at least 3 times. Results are expressed as mean value+/−SEM. Statistical analysis of the data was performed by the Mann-Whitney U test and two-way Anova or Kruskal-Wallis test for multiple comparisons using GraphPad Prism software, version 5.0 (GraphPad Software Inc., La Jolla, Calif.). Differences were considered as significant for p values <0.05; *p<0.05, ***p<0.001.
[0137] Results
[0138] CDC25A is an Early Target Downstream of FLT3-ITD
[0139] In order to identify links between the FLT3-ITD mutated receptor and cell cycle progression, we investigated the expression of cell cycle regulating proteins upon FLT3-ITD inhibition in MV4-11 and MOLM-14, two AML cell lines carrying FLT3-ITD mutation. Two unrelated pharmacological inhibitors of FLT3 (FLT3 inhibitor III and the potent new generation inhibitor AC220, quizartinib) induced CDC25A down-regulation in these cell lines (
[0140] Mechanisms of CDC25A Regulation Downstream of FLT3-ITD
[0141] We then aimed at determining which signaling pathway was involved in CDC25A regulation. As shown in
[0142] To verify the involvement of STAT5 in CDC25A regulation, we performed RNA interference experiments, which also induced CDC25A protein down-regulation in MOLM-14 and MV4-11 cells, confirming the results observed with pharmacological inhibition (
[0143] In order to further precise CDC25A regulation mechanisms, we then asked whether stability or synthesis of the protein were modified in response to FLT3-ITD inhibition. First, we performed time course experiments of CDC25A protein accumulation in response to proteasome inhibition (
[0144] CDC25A is an Important Determinant of FLT3-ITD Cells Proliferation
[0145] We then asked whether FLT3-ITD cells are dependent on CDC25A activity for their proliferation. First we used IRC-083864, a potent pharmacological inhibitor of CDC25 (A, B and C) previously characterized in vitro and in vivo in different cancer models (Brezak M C et al, Int J Cancer 2009). IRC-083864 induced a robust inhibition of MV4-11 and MOLM-14 cell lines proliferation, but did not decrease the proliferation of KG1, HL-60, TF-1 and K562 control cells (
[0146] To extend these data, we then measured the impact of CDC25 inhibition on the proliferation of primary AML cells. The main biological characteristics of these samples are depicted in Table 1. The clonogenic potential of FLT3-ITD positive primary cells in semi-solid 5637 conditioned culture medium was significantly reduced in the presence of IRC-083864 in a dose-dependent manner (
[0147] FLT3-ITD/TKD Expressing Cells are Resistant to AC220 but Remain Sensitive to CDC25 Inhibition
[0148] In recent clinical trials performed with pharmacological FLT3 inhibitors such as quizartinib or sorafenib (Pratz K W et al Curr Drug Targets 2010), heterogeneous mechanisms were suggested to contribute to FLT3 inhibitors resistance, and among them FLT3 kinase domain mutations were the most frequently reported (Alvarado Y et al. Cancer. 2014). We consequently developed a cellular model consisting of MOLM-14 cells transfected with a FLT3-ITD mutant with a D835Y amino-acid substitution within the FLT3 kinase domain (FLT3-ITD-D835Y; FLT3-ITD/TKD). In MOLM-14 and in MOLM-14 expressing FLT3-ITD/TKD, treatment with 1 nM AC220 induced low levels of cell death (not shown), and as expected, ITD/TKD cells were resistant to AC220 by comparison with parental MOLM-14 (
[0149] CDC25A Level Predicts Clonogenic Capacity of FLT3-ITD Primary Cells
[0150] We then investigated the expression level of CDC25A mRNA in a cohort of 188 non promyelocytic AML young patients (aged 18-65) treated by intensive chemotherapy in Toulouse University Hospital in the 2000-2010 period. CDC25A mRNA expression was divided into low expression and high expression according to the median value of the entire cohort. We observed no difference in the expression of CDC25A between FLT3-wt and FLT3-ITD patients. FLT3-ITD allelic ratio or insertion length were also not determinants for CDC25A expression. Since CDC25A appears as an important actor of cell proliferation in FLT3-ITD cells (see
[0151] CDC25A is Involved in FLT3-ITD AML Cells Differentiation Arrest
[0152] Involvement of the cyclin-dependent kinase CDK1 in FLT3-ITD positive cells differentiation arrest was recently reported (Radomska H S et al, JCI 2012). Since CDK1 is a major substrate of CDC25 phosphatases during mitosis, we reasoned that CDC25A could be a master regulator of leukemic cells differentiation through its CDK1 activating function. To test this hypothesis, MV4-11 and MOLM-14 cells were treated with IRC-083864, and the differentiation state of the cells was followed by cell surface markers expression and by morphological examination at different times. As shown in
[0153] In order to further confirm the role of CDC25A in this differentiation arrest, we performed RNA interference mediated down-regulation of the protein. First, we used lentiviral infection to perform shRNA-mediated CDC25A down-regulation in MOLM-14 cells. As shown in
[0154] We then asked whether CDC25 inhibition could induce differentiation of primary AML samples expressing either FLT3-ITD or wild-type FLT3. As shown in
[0155] CDC25 Inhibition Induces FLT3-ITD AML Cells Differentiation In Vivo
[0156] In order to establish if CDC25 inhibition could also drive monocytic differentiation in vivo, we used the NSG mice model of xenograft. NSG mice were injected with 2 millions of MOLM-14 cells after busulfan injection, and then treated with IRC-083864 at 5 mg/kg/week by intra-peritoneal administration. Mice were dissected after two weeks of treatment, and human myeloid cells present in the bone marrow were analyzed for the expression of CD11b, CD14 and CD15 surface proteins. At this time point, no change of CD11b expression could be observed, but as shown in
[0157] Altogether these data demonstrate that inhibiting CDC25A reduces proliferation and induces monocytic differentiation of FLT3-ITD-positive AML cells in vitro and in vivo, and they argue for a central function of this phosphatase in the hematopoietic differentiation arrest of these cells.
DISCUSSION
[0158] Because of high frequency and poor prognosis of FLT3-ITD mutation, improving the knowledge of this AML subgroup pathophysiology appears as an essential task for the next years. Major signaling pathways activated by FLT3-ITD have been identified, but downstream effectors, as well as their respective involvements in cell proliferation and drug resistance remain to be specified. Large phosphoproteomic analysis performed in FLT3-ITD expressing cells identified a panel of potential downstream targets of Pim and Akt, two well recognized players of FLT3-ITD oncogenic potential (Choudhary C et al, Mol Cell 2009), but functional importance of these proteins in FLT3-ITD AML remained elusive. In this work, we demonstrate that the dual specificity phosphatase CDC25A, a major activator of cyclin-dependent kinases during different phases of the cell cycle, is regulated very early downstream of FLT3-ITD. Our data argue for a STAT5-dependent transcriptional mechanism being at the origin of this regulation, but we cannot rule out that CDC25A protein level is also governed at the translational level, as we recently observed down-stream of JAK2 V617F, another oncogenic tyrosine kinase involved in myeloproliferative disease (Gautier E F et al, Blood 2012). Transcriptional regulation of CDC25A by STAT5 has not been reported up to now, but the involvement of STAT3 in both negative and positive transcriptional regulation of CDC25A has been described (Barré B et al, JBC 2005). Different studies recognized the importance of STAT5 in FLT3-ITD signaling and leukemic cells transformation (Hayakawa F et al, Mizuki M et al, Spiekermann K et al), and targeting of this pathway constitutes an important axis of therapeutic research (Nelson E A et al). Interestingly, we recently demonstrated that the STAT5/Pim signaling pathway also governs FLT3-ITD positive AML cells resistance and proliferation through direct phosphorylation of CHK1 ser/thr kinase on Ser 280 by Pim (Yuan L L et al, Leukemia 2014). Further experiments are needed to better understand CDC25A regulation by STAT transcription factors in AML cells.
[0159] In this work, we highlighted a central role for CDC25A, a major regulator of cell cycle progression, in the differentiation processes of FLT3-ITD AML subtype. Abnormalities of transcription factors-induced differentiation are observed in one third of AML (PML-RARA, AML1-ETO, CBFβ and C/EBPα). In particular, mutations in the C/EBPα gene are detected in 10% of these pathologies, and C/EBPα plays a key role in normal granulocytic or monocytic differentiation, depending on its dimerization partner (Paz-Priel I et al, Crit Rev Oncog 2011). Differentiation arrest in FLT3-ITD AML was recently described to be dependent on the phosphorylation of C/EBPα on serine 21 by the ERK kinase and/or the mitotic cyclin dependent kinase CDK1/cyclin B1 (Radomska H S et al, JCI 2012). These authors proposed FLT3-ITD-dependent regulation of cyclin B1 protein as a key parameter of CDK1 activity, and consequent C/EBP□□ phosphorylation and differentiation arrest in this model. Our work suggests that CDC25A, in addition to cyclin B1, is another key regulator of CDK1 activity downstream of FLT3-ITD. This would suggest that FLT3-ITD up-regulates CDK1 activity by different ways, both by CDC25A-dependent dephosphorylation of Thr14/Tyr15, and by accumulation of cyclin B1 and its subsequent association with CDK1. The molecular mechanism of cyclin B1 accumulation in this context, and whether CDC25A activity could be involved in this process, remain to be established. CDC25A is involved in different phases of the cell cycle, and to this respect, is considered as regulator of different CDKs. Its role as an activator of CDK2 and the G1/S transition and during DNA replication is well established, and its action on the G1 CDK4/CDK6-cyclin D1 complex has been recently highlighted (Bertero T et al, CDD 2013). Our data do not allow to distinguish which CDKs are regulated by CDC25A and involved in the differentiation process downstream of FLT3-ITD. In consequence, we cannot exclude that CDK2, and/or CDK4/CDK6 are important actors of this process. This hypothesis would be in line with the very recent identification of CDK6 as a critical effector of MLL fusions in leukemogenesis, underscoring that cell cycle regulators may have distinct, non-canonical, and non-redundant functions in different contexts (Placke T et al, Blood 2014). By acting on different CDK/cyclin complexes, CDC25 inhibitors would constitute interesting candidates to increase the therapeutic tools in the AML personalized treatment, and intense research is ongoing to give rise to new potent compounds (Song Y et al, Eur J Med Chem 2014).
[0160] Since a few works reported the possible importance of CDC25C (Caduil J S et al, Leuk. Lymphoma 2008) and CDC25B (Reikvam H et al, J. Hematol 2014) in AML, CDC25 family members inhibition in these pathologies may be of interest in the future (Brenner A K et al, Molecules 2014). From a more general point of view, the ability of cells to escape terminal differentiation is one of the characteristics of cancer (Hanahan D et al, Cell 2000), and AML represents a paradigm for this phenomenon (Tenen D G et al, Nat Rev Cancer 2003). Reinducing leukemic cells differentiation constitutes an important alternative to genotoxic therapeutic agents currently used to treat these pathologies, but up to now, this approach is only routinely used in the case of promyelocytic leukemia with all-transretinoic acid or arsenic trioxide, which transformed the prognosis of this disease. In the non-promyelocytic leukemia, the discovery of key mutations this last decade led to intense efforts to improve therapeutic strategies (Schlenk R F et al, Patel J P et al NEJM 2012, Patel J P, ASH Edu 2012). In parallel, in AML characterized by mutation of the IDH2 gene, preliminary results of a phase I assay of the IDH2 inhibitor AG-221 showed encouraging rates of response, and nice differentiation of treated leukemic cells in vitro and clinically with cases of differentiation syndrome (Agresta S et al, EHA 2014). FLT3 inhibition is one of the major promising targeted therapies in this subset. To date, one of the most potent and clinically advanced molecule is AC220 (quizartinib) (Wander S A et al, Ther Adv Hematol 2014 Cortes J E et al, Blood 2012; Levis M J et al, Blood 2012 and Kampa-Schittenhelm K M et al, Mol Cancer 2013), and relief of differentiation block in vitro and clinically (Sexauer A et al, Blood 2012) is one of the interesting mechanism of action described for this drug. This pro-differentiation effect of FLT3 inhibition seems to be an on-target effect, as the in vitro results were obtained with other FLT3 inhibitors (sorafenib, lestaurtinib and tandutinib) (Sexauer A et al, Blood 2012; Zheng R et al, Blood 2004 and Radomska H S et al, J Exp Med 2006). These recent observations appeared somehow controversial, since transgenic mice models expressing FLT3-ITD developed myeloproliferative neoplasms rather than AML, suggesting that FLT3-ITD expression by itself did not significantly affect the hematopoietic differentiation process in vivo (Li L et al, Blood 2008).
[0161] Better understanding of signaling and cell cycle molecules impacting hematopoietic differentiation downstream of FLT3-ITD will probably give some keys for future treatments of this AML subtype.
TABLE-US-00001 TABLE 1 Biological properties of primary AML samples A summary of the main biological properties of primary AML samples used in FIG. 4d and FIG. 6. French-American-British (FAB) classification, karyotype, as well as FLT3-ITD (with allelic ratio when mutated) and NPM1 mutational status are listed. Sample FAB Karyotype FLT3 NPM1 ITD #1 5 Normal ITD 30% NPM1c ITD #2 NA Normal ITD 27% NPM1c ITD #3 4 Normal ITD 45% WT ITD #4 2 t (6;9) ITD 18% WT ITD #5 2 t (6;9) ITD 69% WT ITD #6 1 Normal ITD 24% WT WT #1 1 del (7p) WT WT WT #2 1 Normal WT NPM1c WT #3 2 Normal WT NPM1c WT #4 2 Normal WT WT WT #5 1 Normal WT NPM1c NA: non available; WT: wild-type; NPM1c: NPM1 cytoplasmic, ie. mutated
TABLE-US-00002 TABLE 2 Table 2: Clonogenic properties of AMI cells according to CDC25A mRN A expression Low CDC25A High CDCZSA median (IQR) median (IQR) p* Total intermediate karyotype (n = 100) 850 (0-7850) 2400 (150-10800) 0.16 Intermediate karyotype with FLT3-ITD (n = 35) 200 (0-1240) 5575 (2200-17850) 0.03 Intermediate karyotype with FLT3-WT (n = 59) 2225 (0-11650) 1100 (100-10800) 0.76 Normal karyotype with FLT3-ITD (n = 31) 650 (0-4650) 5250 (1975-12638) 0.09 Normal karyotype with FLT3-WT (n = 36) 2550 (0-8050) 3800 (250-11850) 0.45 Values are expressed in number of clones per 10.sup.6 cells. *Comparisons between low CDC25A and high CDC25A subgroups were performed using Mann-Whitney U test
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