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1.  Pacritinib (SB1518), a JAK2/FLT3 inhibitor for the treatment of acute myeloid leukemia 
Blood Cancer Journal  2011;1(11):e44-.
FMS-like tyrosine kinase 3 (FLT3) is the most commonly mutated gene found in acute myeloid leukemia (AML) patients and its activating mutations have been proven to be a negative prognostic marker for clinical outcome. Pacritinib (SB1518) is a tyrosine kinase inhibitor (TKI) with equipotent activity against FLT3 (IC50=22 n) and Janus kinase 2 (JAK2, IC50=23 n). Pacritinib inhibits FLT3 phosphorylation and downstream STAT, MAPK and PI3 K signaling in FLT3-internal-tandem duplication (ITD), FLT3-wt cells and primary AML blast cells. Oral administration of pacritinib in murine models of FLT3-ITD-driven AML led to significant inhibition of primary tumor growth and lung metastasis. Upregulation of JAK2 in FLT3-TKI-resistant AML cells was identified as a potential mechanism of resistance to selective FLT3 inhibition. This resistance could be overcome by the combined FLT3 and JAK2 activities of pacritinib in this cellular model. Our findings provide a rationale for the clinical evaluation of pacritinib in AML including patients resistant to FLT3-TKI therapy.
doi:10.1038/bcj.2011.43
PMCID: PMC3256753  PMID: 22829080
Pacritinib; SB1518; FLT3; JAK2; AML
2.  A potential therapeutic target for FLT3-ITD AML: PIM1 Kinase 
Leukemia Research  2011;36(2):224-231.
Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) mutation have a poor prognosis, and FLT3 inhibitors are now under clinical investigation. PIM1, a serine/threonine kinase, is up-regulated in FLT3-ITD AML and may be involved in FLT3-mediated leukemogenesis. We employed a PIM1 inhibitor, AR00459339 (Array Biopharma Inc.), to investigate the effect of PIM1 inhibition in FLT3-mutant AML. Like FLT3 inhibitors, AR00459339 was preferentially cytotoxic to FLT3-ITD cells, as demonstrated in the MV4-11, Molm-14, and TF/ITD cell lines, as well as 12 FLT3-ITD primary samples. Unlike FLT3 inhibitors, AR00459339 did not suppress phosphorylation of FLT3, but did promote the de-phosphorylation of downstream FLT3 targets, STAT5, AKT, and BAD. Combining AR00459339 with a FLT3 inhibitor resulted in additive to mildly synergistic cytotoxic effects. AR00459339 was cytotoxic to FLT3-ITD samples from patients with secondary resistance to FLT3 inhibitors, suggesting a novel benefit to combining these agents. We conclude that PIM1 appears to be closely associated with FLT3 signaling, and that inhibition of PIM1 may hold therapeutic promise, either as monotherapy, or by overcoming resistance to FLT3 inhibitors.
doi:10.1016/j.leukres.2011.07.011
PMCID: PMC3380375  PMID: 21802138
3.  The Flt3 Internal Tandem Duplication Alters Chemotherapy Response In Vitro and In Vivo in a p53-Dependent Manner 
Experimental hematology  2011;39(4):473-485.e4.
Objective
The FLT3 internal tandem duplication (Flt3-ITD) confers a worse prognosis for patients with acute myeloid leukemia (AML); however, the mechanisms involved are unknown. As AML is treated with cytarabine (Ara-C) and an anthracycline we sought to determine the effects of the Flt3-ITD on response to these agents.
Methods
A genetically defined mouse model of AML was used to examine the effects of the Flt3-ITD on response to cytarabine and doxorubicin in vitro and in vivo.
Results
In vitro, the Flt3-ITD conferred resistance to doxorubicin and doxorubicin plus Ara-C, but sensitivity to Ara-C alone. This resistance was reversible by the Flt3-ITD inhibitor sorafenib. The Flt3-ITD did not affect DNA damage levels following treatment but was associated with increased levels of p53. The p53 response was critical to the observed changes as the Flt3-ITD had no effect on chemotherapy response in the setting of p53 null AML. In vivo, the Flt3-ITD accelerated engraftment that was partially reversible by Ara-C but not doxorubicin. Additionally, Ara-C provided a significant reduction in disease burden and a survival advantage that was not increased by the addition of doxorubicin. Doxorubicin alone lead to only minimal disease reduction and no survival benefit.
Conclusions
These data demonstrate that the Flt3-ITD confers sensitivity to cytarabine, but resistance to doxorubicin in a manner that depends on p53. Thus, patients with Flt3-ITD positive AML may not benefit from treatment with an anthracycline.
doi:10.1016/j.exphem.2011.01.009
PMCID: PMC3062750  PMID: 21288478
4.  Functional Characterization of FLT3 Receptor Signaling Deregulation in Acute Myeloid Leukemia by Single Cell Network Profiling (SCNP) 
PLoS ONE  2010;5(10):e13543.
Background
Molecular characterization of the FMS-like tyrosine kinase 3 receptor (FLT3) in cytogenetically normal acute myeloid leukemia (AML) has recently been incorporated into clinical guidelines based on correlations between FLT3 internal tandem duplications (FLT3-ITD) and decreased disease-free and overall survival. These mutations result in constitutive activation of FLT3, and FLT3 inhibitors are currently undergoing trials in AML patients selected on FLT3 molecular status. However, the transient and partial responses observed suggest that FLT3 mutational status alone does not provide complete information on FLT3 biological activity at the individual patient level. Examination of variation in cellular responsiveness to signaling modulation may be more informative.
Methodology/Principal Findings
Using single cell network profiling (SCNP), cells were treated with extracellular modulators and their functional responses were quantified by multiparametric flow cytometry. Intracellular signaling responses were compared between healthy bone marrow myeloblasts (BMMb) and AML leukemic blasts characterized as FLT3 wild type (FLT3-WT) or FLT3-ITD. Compared to healthy BMMb, FLT3-WT leukemic blasts demonstrated a wide range of signaling responses to FLT3 ligand (FLT3L), including elevated and sustained PI3K and Ras/Raf/Erk signaling. Distinct signaling and apoptosis profiles were observed in FLT3-WT and FLT3-ITD AML samples, with more uniform signaling observed in FLT3-ITD AML samples. Specifically, increased basal p-Stat5 levels, decreased FLT3L induced activation of the PI3K and Ras/Raf/Erk pathways, decreased IL-27 induced activation of the Jak/Stat pathway, and heightened apoptotic responses to agents inducing DNA damage were observed in FLT3-ITD AML samples. Preliminary analysis correlating these findings with clinical outcomes suggests that classification of patient samples based on signaling profiles may more accurately reflect FLT3 signaling deregulation and provide additional information for disease characterization and management.
Conclusions/Significance
These studies show the feasibility of SCNP to assess modulated intracellular signaling pathways and characterize the biology of individual AML samples in the context of genetic alterations.
doi:10.1371/journal.pone.0013543
PMCID: PMC2965086  PMID: 21048955
5.  The multi-targeted receptor tyrosine kinase inhibitor, Linifanib (ABT-869), induces apoptosis through an AKT and Glycogen Synthase Kinase 3β-dependent pathway 
Molecular cancer therapeutics  2011;10(6):949-959.
The FMS-like receptor tyrosine kinase 3 (FLT3) plays an important role in controlling differentiation and proliferation of hematopoietic cells. Activating mutations in FLT3 occur in patients with acute myeloid leukemia (15-35%) resulting in abnormal cell proliferation. Furthermore, both adult and pediatric patients with acute myeloid leukemia (AML) harboring the FLT3 internal tandem duplication (ITD) mutation have a poor prognosis. Several inhibitors have been developed to target mutant FLT3 for the treatment of AML, yet the molecular pathways affected by drug inhibition of the mutated FLT3 receptor alone have not yet been characterized. Linifanib (ABT-869) is a multi-targeted tyrosine kinase receptor inhibitor that suppresses FLT3 signaling. In this paper, we demonstrate that treatment with Linifanib inhibits proliferation and induces apoptosis in ITD mutant cells in vitro and in vivo. We show that treatment with Linifanib reduces phosphorylation of AKT and glycogen synthase kinase 3β (GSK3β). In addition, we show that inhibition of GSK3β decreases Linifanib-induced apoptosis. This study demonstrates the importance of GSK3 as a potential target for AML therapy, particularly in patients with FLT3 ITD mutations.
doi:10.1158/1535-7163.MCT-10-0904
PMCID: PMC3112478  PMID: 21471285
AML; FLT3 Inhibitor
6.  BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor–induced apoptosis of human leukemic cells and transduced hematopoietic progenitors carrying oncogenic FLT3 
Blood  2008;113(10):2302-2311.
Constitutively activating internal tandem duplications (ITD) of FLT3 (FMS-like tyrosine kinase 3) are the most common mutations in acute myeloid leukemia (AML) and correlate with poor prognosis. Receptor tyrosine kinase inhibitors targeting FLT3 have developed as attractive treatment options. Because relapses occur after initial responses, identification of FLT3-ITD–mediated signaling events are important to facilitate novel therapeutic interventions. Here, we have determined the growth-inhibitory and proapototic mechanisms of 2 small molecule inhibitors of FLT3, AG1295 or PKC412, in hematopoietic progenitor cells, human leukemic cell lines, and primary AML cells expressing FLT3-ITD. Inactivation of the PI3-kinase pathway, but not of Ras–mitogen-activated protein (MAP) kinase signaling, was essential to elicit cytotoxic responses. Both compounds induced up-regulation of proapoptotic BH3-only proteins Bim and Puma, and subsequent cell death. However, only silencing of Bim, or its direct transcriptional activator FOXO3a, abrogated apoptosis efficiently. Similar findings were made in bone marrow cells from gene-targeted mice lacking Bim and/or Puma infected with FLT3-ITD and treated with inhibitor, where loss of Puma only provided transient protection from apoptosis, but loss of Bim preserved clonal survival upon FLT3-ITD inhibition.
doi:10.1182/blood-2008-07-167023
PMCID: PMC3272395  PMID: 19064725
7.  Pim-1 Kinase Phosphorylates and Stabilizes 130 kDa FLT3 and Promotes Aberrant STAT5 Signaling in Acute Myeloid Leukemia with FLT3 Internal Tandem Duplication 
PLoS ONE  2013;8(9):e74653.
The type III receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) is expressed on both normal hematopoietic stem cells and acute myeloid leukemia (AML) cells and regulates their proliferation. Internal tandem duplication (ITD) mutation of FLT3 is present in a third of AML cases, results in constitutive activation and aberrant signaling of FLT3, and is associated with adverse treatment outcomes. While wild-type (WT) FLT3 is predominantly a 150 kDa complex glycosylated cell surface protein, FLT3-ITD is partially retained in the endoplasmic reticulum as a 130 kDa underglycosylated species associated with the chaperones calnexin and heat shock protein (HSP) 90, and mediates aberrant STAT5 signaling, which upregulates the oncogenic serine/threonine kinase Pim-1. FLT3 contains a Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that it might be a Pim-1 substrate. Pim-1 was indeed found to directly interact with and serine-phosphorylate FLT3. Pim-1 inhibition decreased the expression and half-life of 130 kDa FLT3, with partial abrogation by proteasome inhibition, in association with decreased FLT3 binding to calnexin and HSP90, and increased 150 kDa FLT3 expression and half-life, with abrogation by inhibition of glycosylation. These findings were consistent with Pim-1 stabilizing FLT3-ITD as a 130 kDa species associated with calnexin and HSP90 and inhibiting its glycosylation to form the 150 kDa species. Pim-1 knockdown effects were similar. Pim-1 inhibition also decreased phosphorylation of FLT3 at tyrosine 591 and of STAT5, and expression of Pim-1 itself, consistent with inhibition of the FLT3-ITD-STAT5 signaling pathway. Finally, Pim-1 inhibition synergized with FLT3 inhibition in inducing apoptosis of FLT3-ITD cells. This is, to our knowledge, the first demonstration of a role of Pim-1 in a positive feedback loop promoting aberrant signaling in malignant cells.
doi:10.1371/journal.pone.0074653
PMCID: PMC3764066  PMID: 24040307
8.  Evaluation of the Antitumor Effects of BPR1J-340, a Potent and Selective FLT3 Inhibitor, Alone or in Combination with an HDAC Inhibitor, Vorinostat, in AML Cancer 
PLoS ONE  2014;9(1):e83160.
Overexpression or/and activating mutation of FLT3 kinase play a major driving role in the pathogenesis of acute myeloid leukemia (AML). Hence, pharmacologic inhibitors of FLT3 are of therapeutic potential for AML treatment. In this study, BPR1J-340 was identified as a novel potent FLT3 inhibitor by biochemical kinase activity (IC50 approximately 25 nM) and cellular proliferation (GC50 approximately 5 nM) assays. BPR1J-340 inhibited the phosphorylation of FLT3 and STAT5 and triggered apoptosis in FLT3-ITD+ AML cells. The pharmacokinetic parameters of BPR1J-340 in rats were determined. BPR1J-340 also demonstrated pronounced tumor growth inhibition and regression in FLT3-ITD+ AML murine xenograft models. The combination treatment of the HDAC inhibitor vorinostat (SAHA) with BPR1J-340 synergistically induced apoptosis via Mcl-1 down-regulation in MOLM-13 AML cells, indicating that the combination of selective FLT3 kinase inhibitors and HDAC inhibitors could exhibit clinical benefit in AML therapy. Our results suggest that BPR1J-340 may be further developed in the preclinical and clinical studies as therapeutics in AML treatments.
doi:10.1371/journal.pone.0083160
PMCID: PMC3885398  PMID: 24416160
9.  Treatment of FLT3-ITD acute myeloid leukemia 
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy which is cured in a minority of patients. A FLT3-internal tandem duplication (ITD) mutation, found in approximately a quarter of patients with de novo AML, imparts a particularly poor prognosis. Patients with FLT3-ITD AML often present with more aggressive disease and have a significantly higher propensity for relapse after remission. The therapeutic approach for these patients has traditionally included intensive induction chemotherapy, followed by consolidative chemotherapy or hematopoietic cell transplantation (HCT). In recent years, multiple small molecule inhibitors of the FLT3 tyrosine kinase have been studied preclinically and in clinical trials. The earlier generation of these agents, often non-specific and impacting a variety of tyrosine kinases, produced at best transient peripheral blood responses in early clinical trials. Additionally, the combination of FLT3 inhibitors with cytotoxic regimens has not, as of yet, demonstrated an improvement in overall survival. Nevertheless, multiple current trials, including those with sorafenib, lestaurtinib, and midostaurin, continue to study the combination of FLT3 inhibitors with standard chemotherapy. Factors such as sustained FLT3 inhibition, protein binding, pharmacokinetics, and the presence of elevated FLT3-ligand levels appear to significantly impact the potency of these agents in vivo. In recent years, the development of more specific and potent agents has generated hope that FLT3 inhibitors may play a more prominent role in the treatment of FLT3-ITD AML in the near future. Nevertheless, questions remain regarding the optimal timing and schedule for incorporation of FLT3 inhibitors. The suitability, type, and timing of allogeneic HCT in the therapeutic approach for these patients are also issues which require further study and definition. Recent retrospective data appears to support the efficacy of allogeneic HCT in first complete remission, possibly due to a graft versus leukemia effect. However, larger prospective studies are necessary to further elucidate the role of HCT and its potential combination with FLT3 inhibitor therapy. We are hopeful that current clinical investigation will lead to an optimization and improvement of outcomes for these patients.
PMCID: PMC3301423  PMID: 22432079
10.  Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns 
Leukemia  2012;26(7):1462-1470.
Acquired resistance to selective FLT3 inhibitors, is an emerging clinical problem in the treatment of FLT3-ITD+ acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has limited investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD+ human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD+ allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD+ patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13- RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild type allele and duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.
doi:10.1038/leu.2012.52
PMCID: PMC3523391  PMID: 22354205
FLT3; Aurora; Kinase; AML; Resistance; Inhibitor
11.  PRL-3, a Metastasis Associated Tyrosine Phosphatase, Is Involved in FLT3-ITD Signaling and Implicated in Anti-AML Therapy 
PLoS ONE  2011;6(5):e19798.
Combination with other small molecule drugs represents a promising strategy to improve therapeutic efficacy of FLT3 inhibitors in the clinic. We demonstrated that combining ABT-869, a FLT3 inhibitor, with SAHA, a HDAC inhibitor, led to synergistic killing of the AML cells with FLT3 mutations and suppression of colony formation. We identified a core gene signature that is uniquely induced by the combination treatment in 2 different leukemia cell lines. Among these, we showed that downregulation of PTP4A3 (PRL-3) played a role in this synergism. PRL-3 is downstream of FLT3 signaling and ectopic expression of PRL-3 conferred therapeutic resistance through upregulation of STAT (signal transducers and activators of transcription) pathway activity and anti-apoptotic Mcl-1 protein. PRL-3 interacts with HDAC4 and SAHA downregulates PRL-3 via a proteasome dependent pathway. In addition, PRL-3 protein was identified in 47% of AML cases, but was absent in myeloid cells in normal bone marrows. Our results suggest such combination therapies may significantly improve the therapeutic efficacy of FLT3 inhibitors. PRL-3 plays a potential pathological role in AML and it might be a useful therapeutic target in AML, and warrant clinical investigation.
doi:10.1371/journal.pone.0019798
PMCID: PMC3093398  PMID: 21589872
12.  Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia 
EMBO Molecular Medicine  2013;5(9):1351-1366.
FLT3-ITD mutations are prevalent mutations in acute myeloid leukaemia (AML). PRL-3, a metastasis-associated phosphatase, is a downstream target of FLT3-ITD. This study investigates the regulation and function of PRL-3 in leukaemia cell lines and AML patients associated with FLT3-ITD mutations. PRL-3 expression is upregulated by the FLT3-STAT5 signalling pathway in leukaemia cells, leading an activation of AP-1 transcription factors via ERK and JNK pathways. PRL-3-depleted AML cells showed a significant decrease in cell growth. Clinically, high PRL-3 mRNA expression was associated with FLT3-ITD mutations in four independent AML datasets with 1158 patients. Multivariable Cox-regression analysis on our Cohort 1 with 221 patients identified PRL-3 as a novel prognostic marker independent of other clinical parameters. Kaplan–Meier analysis showed high PRL-3 mRNA expression was significantly associated with poorer survival among 491 patients with normal karyotype. Targeting PRL-3 reversed the oncogenic effects in FLT3-ITD AML models in vitro and in vivo. Herein, we suggest that PRL-3 could serve as a prognostic marker to predict poorer survival and as a promising novel therapeutic target for AML patients.
doi:10.1002/emmm.201202183
PMCID: PMC3799491  PMID: 23929599
acute myeloid leukaemia; antibody therapy; FLT3-ITD mutation; PRL-3; prognostic marker
13.  Targeting FLT3 for treatment of leukemia 
Seminars in hematology  2008;45(3 Suppl 2):S17-S21.
FLT3 is a receptor tyrosine kinase with important roles in hematopoietic stem/progenitor cell survival and proliferation. It is frequently overexpressed in acute leukemias and is frequently mutated in acute myeloid leukemia (AML). FLT3 internal tandem duplication (ITD) mutations in AML portend poor prognosis in both adult and pediatric patients. A number of small molecule tyrosine kinase inhibitors (TKIs) with activity against FLT3 have been discovered. Many of these are still in preclinical development, but several have entered clinical phase 1 and 2 trials as monotherapy in patients with relapsed AML. These trials have resulted in frequent but short-lived responses of peripheral blasts and less frequent responses of bone marrow blasts. This led to clinical testing of FLT3 TKIs in combination with conventional chemotherapy. Several combination trials are ongoing or planned in both relapsed and newly diagnosed FLT3-mutant AML patients. Anti-FLT3 antibodies may also prove to be an excellent way of targeting FLT3 in AML and acute lymphocytic leukemia (ALL) by inhibiting signaling and through antibody-dependent cell-mediated cytotoxicity.
doi:10.1053/j.seminhematol.2008.07.007
PMCID: PMC2597087  PMID: 18760705
14.  SU11652 Inhibits tyrosine kinase activity of FLT3 and growth of MV-4-11 cells 
Background
FLT3-ITD and FLT3-TKD mutations are frequently found in acute myeloid leukemia (AML). This makes tyrosine kinase FLT3 a highly attractive target for therapeutic drug development. However, effective drugs have not yet emerged. This study is intended to identify and to characterize new FLT3 inhibitors.
Methods
By using the protein substrate GST-FLT3S to analyze kinase activity of recombinant proteins carrying the catalytic domain of wild type and mutant forms of FLT3, we screened a chemical library containing 80 known protein kinase inhibitors. We identified SU11652 as a potent FLT3 inhibitor and further employed FLT3-ITD-positive MV- 4–11 cells to study its effects on cell growth, apoptosis, cell cycles, and cell signaling.
Results
SU11652 strongly inhibited the activity of wild type, D835Y, and D835H mutant forms of FLT3 with IC50 values of 1.5, 16, and 32 nM, respectively. It effectively blocked the growth of FLT3-ITD -positive MV-4-11 cells at nanomolar concentrations but exhibited much less effects on several other cells which do not carry mutations of FLT3. SU11652 inhibited growth of MV-4-11 cells by inducing apoptosis, causing cell cycle arrest, and blocking activation of the ERK, Akt, and STAT signaling pathways.
Conclusion
SU11652 is a potent FLT3 inhibitor which selectively targets FLT3-ITD-positive cells. It should serve as a good candidate for development of therapeutic drugs to treat AML.
doi:10.1186/1756-8722-5-72
PMCID: PMC3524753  PMID: 23216927
Tyrosine kinase; FLT3; Inhibitor screening; SU11652; Acute myeloid leukemia
15.  Targeting MCL-1 sensitizes FLT3-ITD-positive leukemias to cytotoxic therapies 
Blood Cancer Journal  2012;2(3):e60-.
Patients suffering from acute myeloid leukemias (AML) bearing FMS-like tyrosine kinase-3-internal tandem duplications (FLT3-ITD) have poor outcomes following cytarabine- and anthracyclin-based induction therapy. To a major part this is attributed to drug resistance of FLT3-ITD-positive leukemic cells. Against this background, we have devised an antibody array approach to identify proteins, which are differentially expressed by hematopoietic cells in relation to activated FLT3 signaling. Selective upregulation of antiapoptotic myeloid cell leukemia-1 (MCL-1) was found in FLT3-ITD-positive cell lines and primary mononuclear cells from AML patients as compared with FLT3-wild-type controls. Upregulation of MCL-1 was dependent on FLT3 signaling as confirmed by its reversion upon pharmacological inhibition of FLT3 activity by the kinase inhibitor PKC412 as well as siRNA-mediated suppression of FLT3. Heterologously expressed MCL-1 substituted for FLT3 signaling by conferring resistance of hematopoietic cells to antileukemia drugs such as cytarabine and daunorubicin, and to the proapoptotic BH3 mimetic ABT-737. Conversely, suppression of endogenous MCL-1 by siRNA or by flavopiridol treatment sensitized FLT3-ITD-expressing hematopoietic cells to cytotoxic and targeted therapeutics. In conclusion, MCL-1 is an essential effector of FLT3-ITD-mediated drug resistance. Therapeutic targeting of MCL-1 is a promising strategy to overcome drug resistance in FLT3-ITD-positive AML.
doi:10.1038/bcj.2012.5
PMCID: PMC3317524  PMID: 22829255
AML; MCL-1; FLT3-ITD; flavopiridol; resistance; kinase inhibitors
16.  The STAT5 Inhibitor Pimozide Displays Efficacy in Models of Acute Myelogenous Leukemia Driven by FLT3 Mutations 
Genes & Cancer  2012;3(7-8):503-511.
Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myelogenous leukemia (AML) containing the FLT3 internal tandem duplication (ITD) mutation. FLT3 ITD is a constitutively active tyrosine kinase that drives the activation of STAT5, leading to the growth and survival of AML cells. Although there has been some success in identifying tyrosine kinase inhibitors that block the function of FLT3 ITD, there remains a continued need for effective treatment of this disease. We have identified the psychotropic drug pimozide as an effective inhibitor of STAT5 function. Pimozide inhibits the tyrosine phosphorylation of STAT5, leading to the death of AML cells through the induction of apoptosis. Pimozide shows a combinatorial effect with the tyrosine kinase inhibitors midostaurin (PKC412) and sunitinib in the inhibition of STAT5 tyrosine phosphorylation and the induction of apoptosis. Significantly, pimozide reduces the tumor burden in a mouse model of FLT3-driven AML. Therefore, identifying STAT5 inhibitors may provide a new avenue for the treatment of AML, and these may be effective alone or in combination with tyrosine kinase inhibitors.
doi:10.1177/1947601912466555
PMCID: PMC3527989  PMID: 23264850
STAT5; pimozide; FLT3; AML
17.  Inhibition of the PI3K/Akt/GSK3 Pathway Downstream of BCR/ABL, Jak2-V617F, or FLT3-ITD Downregulates DNA Damage-Induced Chk1 Activation as Well as G2/M Arrest and Prominently Enhances Induction of Apoptosis 
PLoS ONE  2013;8(11):e79478.
Constitutively-activated tyrosine kinase mutants, such as BCR/ABL, FLT3-ITD, and Jak2-V617F, play important roles in pathogenesis of hematopoietic malignancies and in acquisition of therapy resistance. We previously found that hematopoietic cytokines enhance activation of the checkpoint kinase Chk1 in DNA-damaged hematopoietic cells by inactivating GSK3 through the PI3K/Akt signaling pathway to inhibit apoptosis. Here we examine the possibility that the kinase mutants may also protect DNA-damaged cells by enhancing Chk1 activation. In cells expressing BCR/ABL, FLT3-ITD, or Jak2-V617F, etoposide induced a sustained activation of Chk1, thus leading to the G2/M arrest of cells. Inhibition of these kinases by their inhibitors, imatinib, sorafenib, or JakI-1, significantly abbreviated Chk1 activation, and drastically enhanced apoptosis induced by etoposide. The PI3K inhibitor GD-0941 or the Akt inhibitor MK-2206 showed similar effects with imatinib on etoposide-treated BCR/ABL-expressing cells, including those expressing the imatinib-resistant T315I mutant, while expression of the constitutively activated Akt1-myr mutant conferred resistance to the combined treatment of etoposide and imatinib. GSK3 inhibitors, including LiCl and SB216763, restored the sustained Chk1 activation and mitigated apoptosis in cells treated with etoposide and the inhibitors for aberrant kinases, PI3K, or Akt. These observations raise a possilibity that the aberrant kinases BCR/ABL, FLT3-ITD, and Jak2-V617F may prevent apoptosis induced by DNA-damaging chemotherapeutics, at least partly through enhancement of the Chk1-mediated G2/M checkpoint activation, by inactivating GSK3 through the PI3K/Akt signaling pathway. These results shed light on the molecular mechanisms for chemoresistance of hematological malignancies and provide a rationale for the combined treatment with chemotherapy and the tyrosine kinase or PI3K/Akt pathway inhibitors against these diseases.
doi:10.1371/journal.pone.0079478
PMCID: PMC3832535  PMID: 24260231
18.  Frequency and Prognostic Relevance of FLT3 Mutations in Saudi Acute Myeloid Leukemia Patients 
Advances in Hematology  2014;2014:141360.
The Fms-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase that plays a key role in cell survival, proliferation, and differentiation of hematopoietic stem cells. Mutations of FLT3 were first described in 1997 and account for the most frequent molecular mutations in acute myeloid leukemia (AML). AML patients with FLT3 internal tandem duplication (ITD) mutations have poor cure rates the prognostic significance of point mutations; tyrosine kinase domain (TKD) is still unclear. We analyzed the frequency of FLT3 mutations (ITD and D835) in patients with AML at diagnosis; no sufficient data currently exist regarding FLT3 mutations in Saudi AML patients. This study was aimed at evaluating the frequency of FLT3 mutations in patients with AML and its significance for prognosis. The frequency of FLT3 mutations in our study (18.56%) was lower than many of the reported studies, FLT3-ITD mutations were observed in 14.4%, and FLT3-TKD in 4.1%, of 97 newly diagnosed AML patients (82 adult and 15 pediatric). Our data show significant increase of FLT3 mutations in male more than female (13 male, 5 female). Our results support the view that FLT3-ITD mutation has strong prognostic factor in AML patients and is associated with high rate of relapse, and high leucocytes and blast count at diagnosis and relapse.
doi:10.1155/2014/141360
PMCID: PMC3950551
19.  FLT3-regulated antigens as targets for leukemia-reactive cytotoxic T lymphocytes 
Blood Cancer Journal  2011;1(3):e11-.
The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD+ myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. Antigens, such as PR-3, RHAMM, Survivin, WT-1 and PRAME, were upregulated by constitutively active FLT3-ITD as well as FLT3-WT activated by FLT3 ligand (FL). Cytotoxic T-cell (CTL) clones against PR-3, RHAMM, Survivin and an AML-directed CTL clone recognized AML cell lines and primary AML blasts expressing FLT3-ITD, as well as FLT3-WT+ myeloid dendritic cells in the presence of FL. Downregulation of FLT3 led to the abolishment of CTL recognition. Comparing our findings concerning LAA upregulation by the FLT3 kinase with those already made for the Bcr-Abl kinase, we found analogies in the LAA expression pattern. Antigens upregulated by both FLT3 and Bcr-Abl may be promising targets for the development of immunotherapeutical approaches against myeloid leukemia of different origin.
doi:10.1038/bcj.2011.12
PMCID: PMC3255276  PMID: 22829124
acute myeloid leukemia; FLT3 kinase; leukemia-associated antigens; T-cell clones; immunotherapy
20.  Inhibition of Histone Deacetylases 1 and 6 Enhances Cytarabine-Induced Apoptosis in Pediatric Acute Myeloid Leukemia Cells 
PLoS ONE  2011;6(2):e17138.
Background
Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.
Methodology
Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.
Results
Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.
Conclusion
Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.
doi:10.1371/journal.pone.0017138
PMCID: PMC3040224  PMID: 21359182
21.  Activating FLT3 Mutants Show Distinct Gain-of-Function Phenotypes In Vitro and a Characteristic Signaling Pathway Profile Associated with Prognosis in Acute Myeloid Leukemia 
PLoS ONE  2014;9(3):e89560.
About 30% of patients with acute myeloid leukemia (AML) harbour mutations of the receptor tyrosine kinase FLT3, mostly internal tandem duplications (ITD) and point mutations of the second tyrosine kinase domain (TKD). It was the aim of this study to comprehensively analyze clinical and functional properties of various FLT3 mutants.
In 672 normal karyotype AML patients FLT3-ITD, but not FLT3-TKD mutations were associated with a worse relapse free and overall survival in multivariate analysis. In paired diagnosis-relapse samples FLT3-ITD showed higher stability (70%) compared to FLT3-TKD (30%). In vitro, FLT3-ITD induced a strong activating phenotype in Ba/F3 cells. In contrast, FLT3-TKD mutations and other point mutations – including two novel mutations – showed a weaker but clear gain-of-function phenotype with gradual increase in proliferation and protection from apoptosis. The pro-proliferative capacity of the investigated FLT3 mutants was associated with cell surface expression and tyrosine 591 phosphorylation of the FLT3 receptor. Western blot experiments revealed STAT5 activation only in FLT3-ITD positive cell lines, in contrast to FLT3-non-ITD mutants, which displayed an enhanced signal of AKT and MAPK activation. Gene expression analysis revealed distinct difference between FLT3-ITD and FLT3-TKD for STAT5 target gene expression as well as deregulation of SOCS2, ENPP2, PRUNE2 and ART3.
FLT3-ITD and FLT3 point mutations show a gain-of-function phenotype with distinct signalling properties in vitro. Although poor prognosis in AML is only associated with FLT3-ITD, all activating FLT3 mutations can contribute to leukemogenesis and are thus potential targets for therapeutic interventions.
doi:10.1371/journal.pone.0089560
PMCID: PMC3946485  PMID: 24608088
22.  Silvestrol exhibits significant in vivo and in vitro antileukemic activities and inhibits FLT3 and miR-155 expressions in acute myeloid leukemia 
Background
Activating mutations [internal tandem duplication (ITD)] or overexpression of the FMS-like tyrosine kinase receptor-3 (FLT3) gene are associated with poor outcome in acute myeloid leukemia (AML) patients, underscoring the need for novel therapeutic approaches. The natural product silvestrol has potent antitumor activity in several malignancies, but its therapeutic impact on distinct molecular high-risk AML subsets remains to be fully investigated. We examined here the preclinical activity of silvestrol in FLT3-ITD and FLT3 wild-type (wt) AML.
Methods
Silvestrol in vitro anti-leukemic activity was examined by colorimetric cell viability assay, colony-forming and flow cytometry assays assessing growth inhibition and apoptosis, respectively. Pharmacological activity of silvestrol on FLT3 mRNA translation, mRNA and protein expression was determined by RNA-immunoprecipitation, qRT-PCR and immunoblot analyses, respectively. Silvestrol in vivo efficacy was investigated using MV4-11 leukemia-engrafted mice.
Results
Silvestrol shows antileukemia activity at nanomolar concentrations both in FLT3-wt overexpressing (THP-1) and FLT3-ITD (MV4-11) expressing AML cell lines (IC50 = 3.8 and 2.7 nM, respectively) and patients’ primary blasts [IC50 = ~12 nM (FLT3-wt) and ~5 nM (FLT3-ITD)]. Silvestrol increased apoptosis (~4fold, P = 0.0001), and inhibited colony-formation (100%, P < 0.0001) in primary blasts. Silvestrol efficiently inhibited FLT3 translation reducing FLT3 protein expression by 80–90% and decreased miR-155 levels (~60%), a frequently co-regulated onco-miR in FLT3-ITD-positive AML. The median survival of silvestrol-treated vs vehicle-treated mice was 63 vs 29 days post-engraftment, respectively (P < 0.0001).
Conclusions
Silvestrol exhibits significant in vivo and in vitro antileukemic activities in AML through a novel mechanism resulting in inhibition of FLT3 and miR-155 expression. These encouraging results warrant a rapid translation of silvestrol for clinical testing in AML.
doi:10.1186/1756-8722-6-21
PMCID: PMC3623627  PMID: 23497456
23.  H2O2 Production Downstream of FLT3 Is Mediated by p22phox in the Endoplasmic Reticulum and Is Required for STAT5 Signalling 
PLoS ONE  2012;7(7):e34050.
The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H2O2-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H2O2 after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H2O2 in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H2O2 production in AML cells is mediated by p22phox and is critical for STAT5 signalling.
doi:10.1371/journal.pone.0034050
PMCID: PMC3396659  PMID: 22807997
24.  Combining the FLT3 Inhibitor PKC412 and the Triterpenoid CDDO-Me Synergistically Induces Apoptosis in Acute Myeloid Leukemia with ITD mutation 
Molecular cancer research : MCR  2010;8(7):986-993.
Mutations of the FLT3 receptor tyrosine kinase consisting of internal tandem duplications (ITD) have been detected in blasts from 20–30% of patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. FLT3/ITD results in constitutive auto-phosphorylation of the receptor and factor-independent survival in leukemia cell lines. The C-28 methyl ester of the oleane triterpenoid (CDDO-Me) is a multifunctional molecule that induces apoptosis of human myeloid leukemia cells. Here we report that CDDO-Me blocks targeting of NFκB to the nucleus by inhibiting IKKβ-mediated phosphorylation of IκBα. Moreover, CDDO-Me blocked constitutive activation of signal transducer and activator of transcription 3 (STAT3). We report the potent and selective anti-proliferative effects of CDDO-Me on FLT3/ITD-positive myeloid leukemia cell lines and primary AML cells. The present studies demonstrate that CDDO-Me treatment results in caspase-3-mediated induction of apoptosis of FLT3/ITD expressing cells and its anti-proliferative effects are synergistic with PKC412, a FLT3-tyrosine kinase inhibitor currently in clinical trials. Taken together, our studies indicate that CDDO-Me greatly enhanced the efficacy of the FLT3 inhibitor PKC412, suggesting that combining two separate pathway inhibitors may be a viable therapeutic strategy for AML associated with a FLT3/ITD mutation.
doi:10.1158/1541-7786.MCR-10-0154
PMCID: PMC2905481  PMID: 20571062
FLT3-ITD; AML; STAT3; apoptosis
25.  Incorporating FLT3 inhibitors into acute myeloid leukemia treatment regimens 
Leukemia & lymphoma  2008;49(5):852-863.
FMS-Like-Tyrosine kinase-3 (FLT3) mutations are found in about 30% of cases of acute myeloid leukemia and confer an increased relapse rate and reduced overall survival. Targeting of this tyrosine kinase by direction inhibition is the focus of both preclinical and clinical research in AML. Several molecules in clinical development inhibit FLT3 with varying degrees of specificity. Preclinical models suggest that these compounds enhance the cytotoxicity of conventional chemotherapeutics against FLT3 mutant leukemia cells. The pharmacodynamic interactions between FLT3 inhibitors and chemotherapy appear to be sequence dependent. When the FLT3 inhibitor is used prior to chemotherapy, antagonism is displayed, while if FLT3 inhibition is instituted after to exposure to chemotherapy, synergistic cytotoxicity is seen. The combination of FLT3 inhibitors with chemotherapy is also complicated by potential pharmacokinetic obstacles, such as plasma protein binding and p-glycoprotein interactions. Ongoing and future studies are aimed at incorporating FLT3 inhibitors into conventional induction and consolidation therapy specifically for patients with FLT3 mutant AML.
doi:10.1080/10428190801895352
PMCID: PMC3031857  PMID: 18452067
Pharmacotherapeutics; signal transduction; signalling therapies; myeloid leukemias and dysplasias

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