Strategies targeting FLT3 beyond the kinase inhibitors.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by clonal expansion and differentiation arrest of the myeloid progenitor cells, which leads to the accumulation of immature cells called blasts in the bone marrow and peripheral blood. Mutations in the receptor tyrosine kinase FLT3 occur in 30% of normal karyotype patients with AML and are associated with a higher incidence of relapse and worse survival. Targeted therapies against FLT3 mutations using small-molecule FLT3 tyrosine kinase inhibitors (TKIs) have long been investigated, with some showing favorable clinical outcomes. However, major setbacks such as limited clinical efficacy and the high risk of acquired resistance remain unresolved. FLT3 signaling, mutations, and FLT3 inhibitors are topics that have been extensively reviewed in recent years. Strategies to target FLT3 beyond the small molecule kinase inhibitors are expanding, nevertheless they are not receiving enough attention. These modalities include antibody-based FLT3 targeted therapies, immune cells mediated targeting strategies, and approaches targeting downstream signaling pathways and FLT3 translation. Here, we review the most recent advances and the challenges associated with the development of therapeutic modalities targeting FLT3 beyond the kinase inhibitors.

Comparison of effects of midostaurin, crenolanib, quizartinib, gilteritinib, sorafenib and BLU-285 on oncogenic mutants of KIT, CBL and FLT3 in haematological malignancies.

Mutations in two type-3 receptor tyrosine kinases (RTKs), KIT and FLT3, are common in both acute myeloid leukaemia (AML) and systemic mastocytosis (SM) and lead to hyperactivation of key signalling pathways. A large number of tyrosine kinase inhibitors (TKIs) have been developed that target either FLT3 or KIT and significant clinical benefit has been demonstrated in multiple clinical trials. Given the structural similarity of FLT3 and KIT, it is not surprising that some of these TKIs inhibit both of these receptors. This is typified by midostaurin, which has been approved by the US Food and Drug Administration for mutant FLT3-positive AML and for KIT D816V-positive SM. Here, we compare the in vitro activities of the clinically available FLT3 and KIT inhibitors with those of midostaurin against a panel of cells expressing a variety of oncogenic FLT3 or KIT receptors, including wild-type (wt) FLT3, FLT3-internal tandem duplication (ITD), FLT3 D835Y, the resistance mutant FLT3-ITD+ F691L, KIT D816V, and KIT N822K. We also examined the effects of these inhibitors in vitro and in vivo on cells expressing mutations in c-CBL found in AML that result in hypersensitization of RTKs, such as FLT3 and KIT. The results show a wide spectrum of activity of these various mutations to these clinically available TKIs.

The molecular mechanism behind resistance of the kinase FLT3 to the inhibitor quizartinib.

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is a drug target for leukemias. Several potent inhibitors of FLT3 exist, and bind to the inactive form of the enzyme. Unfortunately, resistance due to mutations in the kinase domain of FLT3 limits the therapeutic effects of these inhibitors. As in many other cases, it is not straightforward to explain why certain mutations lead to drug resistance. Extensive fully atomistic molecular dynamics (MD) simulations of FLT3 were carried out with an inhibited form (FLT-quizartinib complex), a free (apo) form, and an active conformation. In all cases, both the wild type (wt) proteins and two resistant mutants (D835F and Y842H) were studied. Analysis of the simulations revealed that impairment of protein-drug interactions cannot explain the resistance mutations in question. Rather, it appears that the active state of the mutant forms is perturbed by the mutations. It is therefore likely that perturbation of deactivation of the protein (which is necessary for drug binding) is responsible for the reduced affinity of the drug to the mutants. Importantly, this study suggests that it is possible to explain the source of resistance by mutations in FLT3 by an analysis of unbiased MD simulations.

Arsenic trioxide and all-trans-retinoic acid selectively exert synergistic cytotoxicity against FLT3-ITD AML cells via co-inhibition of FLT3 signaling pathways.

FLT3-ITD mutations occur in approximately 30% of acute myeloid leukemia (AML) and are associated with a poor outcome. Currently available FLT3 inhibitors have in vitro but limited clinical activity in FLT3-ITD AML. Reports have shown that an arsenic trioxide (ATO)/all-trans-retinoic acid (ATRA) combination improves prognosis in acute promyelocytic leukemia, especially with FLT3-ITD, and ATO or ATRA alone enhances apoptosis in FLT3-ITD AML cells treated with FLT3 inhibitors, providing a rationale to investigate the role of ATO/ATRA in FLT3-ITD AML. Here, we demonstrate that an ATO/ATRA combination selectively exerts synergistic cytotoxicity against FLT3-ITD AML cell lines (MV4;11/MOLM-13). The signaling pathways affected by ATO/ATRA include FLT3/STAT5/MYC, FLT3/STAT5/E2F1, FLT3/ERK/ATF5 and FLT3/AKT/ATF5.ATF5 may function as an oncogene in FLT3-ITD AML. Our findings provide experimental evidence that supports further exploration of ATO/ATRA in FLT3-ITD AML in vivo and warrants a clinical evaluation of regimens comprising an ATO/ATRA combination.

Pacritinib versus best available therapy for the treatment of myelofibrosis irrespective of baseline cytopenias (PERSIST-1): an international, randomised, phase 3 trial.

BACKGROUND: Available therapies for myelofibrosis can exacerbate cytopenias and are not indicated for patients with severe thrombocytopenia. Pacritinib, which inhibits both JAK2 and FLT3, induced spleen responses with limited myelosuppression in phase 1/2 trials. We aimed to assess the efficacy and safety of pacritinib versus best available therapy in patients with myelofibrosis irrespective of baseline cytopenias. METHODS: This international, multicentre, randomised, phase 3 trial (PERSIST-1) was done at 67 sites in 12 countries. Patients with higher-risk myelofibrosis (with no exclusions for baseline anaemia or thrombocytopenia) were randomly assigned (2:1) to receive oral pacritinib 400 mg once daily or best available therapy (BAT) excluding JAK2 inhibitors until disease progression or unacceptable toxicity. Randomisation was stratified by risk category, platelet count, and region. Treatment assignments were known to investigators, site personnel, patients, clinical monitors, and pharmacovigilance personnel. The primary endpoint was spleen volume reduction (SVR) of 35% or more from baseline to week 24 in the intention-to-treat population as assessed by blinded, centrally reviewed MRI or CT. We did safety analyses in all randomised patients who received either treatment. Here we present the final data. This trial is registered with ClinicalTrials.gov, number NCT01773187. FINDINGS: Between Jan 8, 2013, and Aug 1, 2014, 327 patients were randomly assigned to pacritinib (n=220) or BAT (n=107). Median follow-up was 23·2 months (IQR 14·8-28·7). At week 24, the primary endpoint of SVR of 35% or more was achieved by 42 (19%) patients in the pacritinib group versus five (5%) patients in the BAT group (p=0·0003). 90 patients in the BAT group crossed over to receive pacritinib at a median of 6·3 months (IQR 5·8-6·7). The most common grade 3-4 adverse events through week 24 were anaemia (n=37 [17%]), thrombocytopenia (n=26 [12%]), and diarrhoea (n=11 [5%]) in the pacritinib group, and anaemia (n=16 [15%]), thrombocytopenia (n=12 [11%]), dyspnoea (n=3 [3%]), and hypotension (n=3 [3%]) in the BAT group. The most common serious adverse events that occurred through week 24 were anaemia (10 [5%]), cardiac failure (5 [2%]), pyrexia (4 [2%]), and pneumonia (4 [2%]) with pacritinib, and anaemia (5 [5%]), sepsis (2 [2%]), and dyspnoea (2 [2%]) with BAT. Deaths due to adverse events were observed in 27 (12%) patients in the pacritinib group and 14 (13%) patients in the BAT group throughout the duration of the study. INTERPRETATION: Pacritinib therapy was well tolerated and induced significant and sustained SVR and symptom reduction, even in patients with severe baseline cytopenias. Pacritinib could be a treatment option for patients with myelofibrosis, including those with baseline cytopenias for whom options are particularly limited. FUNDING: CTI BioPharma Corp.

[The efficacy of sorafenib to prevent relapse in patients with FLT3-ITD mutation positive acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation].

To study the efficacy of sorafenib to prevent relapse in patients with FLT3-ITD mutation positive acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 7 cases with FLT3-ITD positive AML have received allo-HSCT in our department from May 2013 to January 2015. Six cases were administrated with sorafenib after hematopoietic reconstruction. Another patient relapsed on day 192 past allo-HSCT, then she started to use sorafenib after remission of re-induction regimens. Five patients survived. The median progression free survival was 280(126-366)day. This study suggests that sorafenib might prevent relapse past allo-HSCT and improve survival in patients with FLT3-ITD positive AML.

Flt3 is a target of coumestrol in protecting against UVB-induced skin photoaging.

While skin aging is a naturally occurring process by senescence, exposure to ultraviolet (UV) radiation accelerates wrinkle formation and sagging of skin. UV induces skin aging by degrading collagen via activating matrix metalloproteinases (MMPs). In this study, we show that coumestrol, a metabolite of the soybean isoflavone daidzein, has a preventive effect on skin photoaging in three-dimensional human skin equivalent model. Coumestrol inhibited UVB-induced MMP-1 expression and activity. Whole human kinase profiling assay identified FLT3 kinase as a novel target protein of coumestrol in UVB-induced signaling pathway in skin. Coumestrol suppresses FLT3 kinase activity, and subsequently, Ras/MEK/ERK and Akt/p70 ribosomal S6 kinase pathway. This suppresses AP-1 activity and in turn, diminishes MMP-1 gene transcription. Using X-ray crystallography, the binding of coumestrol to FLT3 was defined and implied ATP-competitive inhibition. Residues Lys644 and Phe830 showed local changes to accommodate coumestrol in the ATP-binding pocket. 4-APIA, a pharmacological inhibitor of FLT3, inhibited MMP-1 expression and induced signal transduction changes similar to coumestrol. Taken together, coumestrol inhibits UVB-induced MMP-1 expression by suppressing FLT3 kinase activity. These findings suggest that coumestrol is a novel dietary compound with potential application in preventing and improving UVB-associated skin aging.

Overcoming myelosuppression due to synthetic lethal toxicity for FLT3-targeted acute myeloid leukemia therapy.

Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.

Activity of sunitinib in extraskeletal myxoid chondrosarcoma.

BACKGROUND: Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue sarcoma, marked by NR4A3 rearrangement. Herein we report on the activity of sunitinib in a series of 10 patients, strengthening what initially observed in two cases. PATIENTS AND METHODS: From July 2011, 10 patients with progressive metastatic translocated EMC have been consecutively treated with sunitinib 37.5mg/day, on a named-use basis. In an attempt to interpret the activity of sunitinib in EMC, genotype/phenotype correlations were carried out by fluorescence in situ hybridization (FISH) analyses. Moreover, transcriptome, immunohistochemical and biochemical analyses of a limited set of samples were performed focusing on some putative targets of sunitinib. RESULTS: Eight of 10 patients are still on therapy. Six patients had a Response Evaluation Criteria in Solid Tumours (RECIST) partial response (PR), two were stable, two progressed. Positron emission tomography (PET) was consistent in 6/6 evaluable cases. One patient underwent surgery after sunitinib, with evidence of a pathologic response. At a median follow-up of 8.5 months (range 2-28), no secondary resistance was detected. Median progression free survival (PFS) has not been reached. Interestingly, all responsive cases turned out to express the typical EWSR1-NR4A3 fusion, while refractory cases carried the alternative TAF15-NR4A3 fusion. Among putative sunitinib targets, only RET was expressed and activated in analysed samples. CONCLUSIONS: This report confirms the therapeutic activity of sunitinib in EMC. Genotype/phenotype analyses support a correlation between response and EWSR1-NR4A3 fusion. Involvement of RET deserves further investigation.

The role of FLT3 kinase as an AML therapy target.

FMS-like tyrosine kinase-3 (FLT3) is a tyrosine kinase receptor involved in the survival and expansion of hematopoietic stem progenitors. A constitutively activated, mutated form of FLT3, is expressed in approximately 30% of de novo acute myeloid leukemia (AML) and about 6% of acute lymphoblastic leukemia (ALL) cases. Since mutant FLT3 has emerged as an attractive therapeutic target, there are several FLT3 inhibitors currently undergoing evaluation in different phases of clinical trials. However, although many aspects of the intracellular signaling mediated by oncogenic FLT3 have been revealed, what is the best strategy to inhibit FLT3 and how FLT3 inhibitors should be developed for AML treatment is poorly defined. Despite promising in vitro studies, where most FLT3 inhibitors show potent efficacy at nanomolar concentrations, clinical responses in AML patients are moderate and temporary. Furthermore, under prolonged therapy, FLT3 mutation-positive leukemic cells rapidly develop resistance to FLT3 inhibitors when used as monotherapy. Considering that there is no uniform mechanism of resistance triggered by FLT3 inhibitors, it will be necessary to develop new agents that target FLT3, and that can be used consecutively or in combination with conventional cytotoxic therapeutics. On the other hand, given that overexpression of FLT3 ligand (FL), occurring after myelosuppressive therapy, reduces the efficacy of FLT3 inhibitors, targeting both FL and FLT3 kinase, might be more effective approach in AML treatment. Here, we summarize up-to-date studies on FLT3 structure, its mutation status and role in malignant signal trafficking. We also review why FLT3 targeted therapies have not revolutionized AML treatment.

Ligand-induced Flt3-downregulation modulates cell death associated proteins and enhances chemosensitivity to idarubicin in THP-1 acute myeloid leukemia cells.

Sustained ligand stimulation of the receptor tyrosine kinase Flt3 resulted in its downregulation and a refractory signaling phase in primary acute myeloid leukemia (AML) cells and in the AML cell line THP-1. Stable isotope amino acid labeling in cell culture and mass spectrometry were used to compare protein expression patterns in THP-1 before and after Flt3-downregulation. 375 distinct proteins were identified where ATP-dependent RNA helicase DDX3, HNRPU, Matrin-3, Importin-7 and Bax were among the 25 most upregulated proteins and Hausp/UBP7, UBE2N and ERp29 among the 17 most downregulated. THP-1 cells with receptor downregulation were sensitized to idarubicin-induced apoptosis but not cytarabine. We hypothesize that FL-induced receptor modulation may chemosensitize selected AML subsets.

PKC 412 small-molecule tyrosine kinase inhibitor: single-compound therapy for pancreatic cancer.

BACKGROUND: PKC412 is a kinase inhibitor that blocks protein kinase C (PKC), vascular endothelial growth factor receptors, platelet-derived growth factor receptor FLT3, and other class III receptor tyrosine kinases. The enthusiasm for this compound is based on its inhibitory effect even in the case of FLT3 mutations. The aim of this study was to analyze the role of FLT3 in pancreatic cancer and to study the biological activity of combined inhibition of neovascularization and mitogenesis in this disease. METHODS: FLT3 expression was analyzed in 18 pancreatic cancer specimens by real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Sixteen pancreatic cancer cell lines were screened for ITD and D835 point mutations of the FLT3 gene. MTT assays and anchorage-independent growth assays were used to study cell growth. Flow cytometry was used for cell cycle analysis and apoptosis quantification. In vivo AsPC-1 and HPAF-II cells were used for orthotopic tumor modeling. Immunohistochemistry was used to quantify tumor angiogenesis. RESULTS: FLT3 expression is down-regulated in pancreatic cancer. Activating FLT3 mutations (ITD, D835) were not detectable in any of the pancreatic cancer cell lines. Cell growth was significantly inhibited as cell-cycle progression was reduced and programmed cell death increased. In vivo PKC412 therapy resulted in a significant inhibition of orthotopic tumor growth with abrogation of tumor angiogenesis. CONCLUSIONS: These data highlight that PKC412 may be a new compound in target therapy of inoperable pancreatic cancer patients and suggest a potential role for the combined use of broad spectrum kinase inhibitors in the management of these patients.

FLT3 as a therapeutic target in childhood acute leukemia.

During the past few years, a major focus of leukemia research has centered on tyrosine kinases as potential therapeutic targets. This is due in large part to the success of imatinib mesylate (STI571, Gleevec), which has proven effective as a therapy for chronic myeloid leukemias bearing the t(9;22) encoding the BCR-ABL kinase. It has become increasingly evident that mutations producing constitutively active tyrosine kinases play a role in leukemogenesis. Another kinase that has drawn significant attention is the FMS-like tyrosine kinase 3 (FLT3). FLT3 is expressed in most childhood acute leukemias. Select genetic subgroups possess particularly high-level expression, with a significant percentage therein harboring activating mutations. In this review we will discuss FLT3 as a potential therapeutic target in childhood acute leukemias. We will highlight the role of FLT3 in hematopoiesis, and how when activated, it may play a role in the development of acute myeloid or acute lymphoblastic leukemia. We will examine the successes in elucidating FLT3 function in acute leukemias, highlight current FLT3 targeted therapeutics, and discuss how FLT3 inhibitors might be used in combination therapies in the future.

Synergism between etoposide and 17-AAG in leukemia cells: critical roles for Hsp90, FLT3, topoisomerase II, Chk1, and Rad51.

PURPOSE: DNA-damaging agents, such as etoposide, while clinically useful in leukemia therapy, are limited by DNA repair pathways that are not well understood. 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90 (Hsp90), inhibits growth and induces apoptosis in FLT3(+) leukemia cells. In this study, we evaluated the effects of etoposide and 17-AAG in leukemia cells and the roles of Hsp90, FMS-like tyrosine kinase 3 (FLT3), checkpoint kinase 1 (Chk1), Rad51, and topoisomerase II in this inhibition. EXPERIMENTAL DESIGN: The single and combined effects of 17-AAG and etoposide and the mechanism of these effects were evaluated. FLT3 and the DNA repair-related proteins, Chk1 and Rad51, were studied in small interfering RNA (siRNA)-induced cell growth inhibition experiments in human leukemia cells with wild-type or mutated FLT3. RESULTS: We found that etoposide and the Hsp90/FLT3 inhibitor 17-AAG, had synergistic inhibitory effects on FLT3(+) MLL-fusion gene leukemia cells. Cells with an internal tandem duplication (ITD) FLT3 (Molm13 and MV4;11) were more sensitive to etoposide/17-AAG than leukemias with wild-type FLT3 (HPB-Null and RS4;11). A critical role for FLT3 was shown in experiments with FLT3 ligand and siRNA targeted to FLT3. An important role for topoisomerase II and the DNA repair-related proteins, Chk1 and Rad51, in the synergistic effects was suggested from the results. CONCLUSIONS: The repair of potentially lethal DNA damage by etoposide in leukemia cells is dependent on intact and functioning FLT3 especially leukemias with ITD-FLT3. These data suggest a rational therapeutic strategy for FLT3(+) leukemias that combines etoposide or other DNA-damaging agents with Hsp90/FLT3 inhibitors such as 17-AAG.