Fms-like Tyrosine Kinase 3 Activation Research Articles

PU.1 promotes development of rheumatoid arthritis via repressing FLT3 in macrophages and fibroblast-like synoviocytes

ObjectivesTo uncover the function and underlying mechanism of an essential transcriptional factor, PU.1, in the development of rheumatoid arthritis (RA).MethodsThe expression and localisation of PU.1 and its potential target, FMS-like...

Development of PLM-102, a novel dual inhibitor of FLT3 and RET as a new therapeutic option for acute myeloid leukemia.

e15103 Background: Mutations of the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 30% of all acute myeloid leukemia (AML) cases, with the internal tandem duplication (ITD) representing the most common type of FLT3 mutation (FLT3-ITD; approximately 25% of all AML cases). Although several FLT3 inhibitors have been developed, occurrence of secondary TKD mutations of FLT3 such as FLT3/D835Y and FLT3/F691L causes the acquired resistance to the current FLT3 inhibitors and eventually become a key area of unmet medical needs. Here, we have revealed that PLM-102, a novel, orally active FLT3 and RET dual inhibitor, has a potential to overcome the acquired resistance to current FLT3 inhibitors. Methods: 1. Kinase assay- Biochemical assays for FLT3 (WT and D835Y) and RET (WT and Mutants) were performed according to ADP-Glo kinase assay protocol(Promega). 2. Cell proliferation and Apoptosis- Human leukemia cell line MV4-11 and MOLM-14 were purchased from ATCC and DSMZ. Cells were seeded at a density of 2 X 103 cells per well and treated with the indicated concentrations of inhibitors for 72 hours at 37°C. Cell viability was determined by an Alamar Blue assay (Bio-Rad). Caspase-3/7 activity was measured by using the Caspase-Glo 3/7 assay (Promega). 3. Western blot analysis- Immunoblotting using MOLM-14 cells was performed using anti-phospho-FLT3 (Cell Signaling Technology #3461) and anti-FLT3 antibody (Cell Signaling Technology #3462). 4. In vivo mouse models- The MV4-11 and MOLM-14 cells are implanted into the subcutaneous space of the left flank of the mice. Resulting tumors are monitored by calipering twice weekly. Treatment started after randomization when tumor volumes had reached a size of approximately 100-150 mm3. For statistical analysis, analysis of variance (ANOVA) was performed using Prism 9.0 to examine statistical differences. Results: In compared to FDA-approved Gilteritinib, PLM-102 showed stronger sub-nanomolar IC50 values in FLT3 kinases regardless of wild-type, ITD, TKD and ITD/TKD mutants in both kinase- and cell-based assays. PLM-102 inhibited phosphorylation of FLT3 and its downstream signaling pathways, and induced apoptosis as evidenced by PARP-cleavage and caspase-3 activation. Moreover, PLM-102 showed an excellent anti-tumor activity in mouse xenograft models implanted with MV4-11 and MOLM-14 AML cells. Conclusions: Taken together, PLM-102 showing potent anti-cancer activity against various in vitro and in vivo AML models could be developed as a valuable agent overcoming the acquired resistance to the current FLT3 inhibitor.

Relation between FMS-like tyrosine kinase 3 factor and hematological parameter in acute lymphoblastic leukemia patients by flow cytometry

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a heterogeneous disorder that is caused by the clonal expansion of immature lymphoid cells with a high rate among children more than adults. FMS-like tyrosine kinase 3 (FLT3) is a cellular receptor belongs to the Class III receptor tyrosine kinase family. The main expression of FLT3 on bone marrow (BM) cells especially CD34+ hematopoietic stem cells, early progenitor cells, dendritic progenitor cells, and other cells of organs (brain, placenta, and testis). Activation of FLT3 results in increased cell proliferation, decreased cell apoptosis, and inhibition of differentiation of cells. This study aims to detect the expression of the FLT3 cluster of differentiation antigen 135 (CD135) in childhood B-ALL patients. Moreover, to correlate this expression with hematological parameters include a complete blood count and BM examination findings and clinical parameters. PATIENTS, MATERIALS AND METHODS: This study was conducted on 30 newly diagnosed pediatric ALL patients. Diagnosis of the disease was based on the blood film, BM examination findings, cytochemistry, and flowcytometry of peripheral blood (PB) and/or BM sample, 1 ml of PB and/or BM sample was collected in EDTA tubes for flowcytometry for detection of CD135. RESULTS: This study found that male patients were more than females with a male-to-female ratio (1.14:1) and a median age of 5 years. Most of the patients had a positive expression of the FLT3 receptor and according to NCI risk groups, 60% of patients fall in the standard risk and 40% in the high-risk group. There was a significant correlation between the level of FLT3 (CD135) and age but no significant correlation with hemoglobin, white blood count, platelets, and peripheral or BM blast percentage. CONCLUSION: In this study, the patients with positive FLT3 blast cells (which is a bad prognostic factor) were associated with good prognostic factors. This proves that FLT3 is an independent prognostic factor.

Hematopoietic cell transplantation for FLT3-mutated acute myeloid leukemia

FMS-like tyrosine kinase 3 (FLT3) mutations are observed in 25-30% of patients with acute myeloid leukemia (AML). Due to poor prognosis associated with FLT3-mutated AML, allogeneic hematopoietic transplantation is commonly performed to induce remission. With the availability of active FLT3 inhibitors and improvement in transplants techniques, the outcomes of AML have improved drastically. The results of many clinical trials have shown that FLT3 inhibitors in combination with chemotherapy and post-transplant maintenance therapy are most effective for AML. Furthermore, these developments have opened up the possibility of transplantation for elderly patients in whom transplantation is difficult. Here, we discuss the optimal approach for treating FLT3-mutated AML using FLT3 inhibitors, allogenic transplantation, and post-transplant maintenance therapy.

Abstract 1442: Targeting ATM kinase and mTOR signaling reverses bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy

Abstract Internal tandem duplication (ITD) mutations in FMS-like tyrosine kinase 3 (FLT3) are among the most common mutations in acute myeloid leukemia (AML) and are associated with poor prognosis. FLT3-ITD causes constitutive activation of FLT3, and the strong evidence that activated FLT3 drives leukemogenesis has led to the development of several FLT3-targeted inhibitors. However, clinical studies of FLT3-targeted inhibitors have demonstrated much more effective clearing of leukemic burden in the periphery than in the bone marrow, implicating the bone marrow microenvironment as a potential contributor to drug resistance. As previously reported, the conditioned media of human bone marrow stromal cells (CM-BMSC) protects human FLT3-ITD AML cells from the killing effect of FLT3-targeted therapy despite complete inhibition of FLT3. We further show that genetic knockdown of Ataxia Telangiectasia Mutated (ATM) in combination with FLT3 inhibition substantially reverses the protection from cell death mediated by CM-BMSC. While autophosphorylation activity of ATM is downregulated upon FLT3 inhibition in regular media, CM-BMSC prevented the loss of ATM activity. Interestingly, genetic knockdown of ATM in human FLT3-ITD AML cells impaired the activity of mTOR Complex 1, and unbiased analyses of gene expression and signaling pathways by RNA-seq analysis and Reverse Phase Protein Arrays (RPPA) showed that mTOR signaling is downregulated with FLT3 inhibition in regular media, coinciding with marked inhibition of the protein translation machinery. Both mTOR signaling and protein translation were maintained during FLT3 inhibition in the presence of CM-BMSC. Furthermore, human FLT3-ITD AML cells treated with mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib reversed the protection mediated by CM-BMSC, and combination therapies are currently being tested in mouse models. These data suggest that ATM kinase and mTOR signaling play a key role in bone marrow stoma-mediated protection against FLT3 targeted therapy. Findings from this research provide new insights into the mechanism of bone marrow stroma-mediated protection of FLT3-ITD AML from FLT3-targeted therapy, identifying additional candidates for combinatorial therapies designed to overcome the protective effects of bone marrow stromal cells and improve patient outcomes. Citation Format: Hae J. Park, Mark A. Gregory, Vadym Zaberezhnyy, James DeGregori. Targeting ATM kinase and mTOR signaling reverses bone marrow stromal cell-mediated protection of FLT3-ITD AML from FLT3-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1442.

A Phase II Study of Midostaurin and 5-Azacitidine for Elderly Patients with Acute Myeloid Leukemia

A Phase II Study of Midostaurin and 5-Azacitidine for Elderly Patients with Acute Myeloid Leukemia

Investigation of Selected Flavonoid Derivatives as Potent FLT3 Inhibitors for the Potential Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis and a high degree of relapse seen in patients. Overexpression of FMS-like tyrosine kinase 3 (FLT3) is associated with up to 70% of AML patients. Wild-type FLT3 induces proliferation and inhibits apoptosis in AML cells, while uncontrolled proliferation of FLT3 kinase activity is also associated with FLT3 mutations. Therefore, inhibiting FLT3 activity is a promising AML therapy. Flavonoids are a group of phytochemicals that can target protein kinases, suggesting their potential antitumor activities. In this study, several plant-derived flavonoids have been identified with FLT3 inhibitory activity. Among these compounds, compound 40 (5,7,4'-trihydroxy-6-methoxyflavone) exhibited the most potent inhibition against not only FLT3 (IC50 = 0.44 μM) but also FLT3-D835Y and FLT3-ITD mutants (IC50 = 0.23 and 0.39 μM, respectively). The critical interactions between the FLT3 binding site and the compounds were identified by performing a structure-activity relationship analysis. Furthermore, the results of cellular assays revealed that compounds 28, 31, 32, and 40 exhibited significant cytotoxicity against two human AML cell lines (MOLM-13 and MV-4-11), and compounds 31, 32, and 40 resulted in cell apoptosis and G0/G1 cell cycle arrest. Collectively, these flavonoids have the potential to be further optimized as FLT3 inhibitors and provide valuable chemical information for the development of new AML drugs.

Discovery of Oxazol-2-amine Derivatives as Potent Novel FLT3 Inhibitors.

Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) is the most common mutation in patients with acute myeloid leukemia (AML). FLT3-ITD+ induces constitutive activation of FLT3, causing an abnormally rapid proliferation of cancer cells. In this study, we identified novel FLT3 inhibitors and investigated 5-(4-fluorophenyl)-N-phenyloxazol-2-amine (compound 7; 7c) as candidates for the treatment of AML. The results showed that 7c inhibited the activities of FLT3 and mutated FLT3 in a cell-free kinase assay and Molm-13 and MV4-11 cells, as well as the proliferation of FLT3-ITD+ AML cells, increasing apoptosis. The anti-leukemic activity of 7c was confirmed by in vivo tumor growth inhibition in MV4-11 xenograft mice. Besides, 7c suppressed the expression of DNA damage repair genes. Combination treatment with 7c and olaparib (a poly (ADP-ribose) polymerase [PARP] inhibitor) synergistically inhibited cell proliferation in Molm-13 and MV4-11 cells. Our findings demonstrated that 7c is a therapeutic candidate targeting FLT3 for AML treatment and suggested that combination treatment with 7c and a PARP inhibitor may be an effective therapy regimen for FLT3-mutated AML.

Novel Approaches to Target Mutant FLT3 Leukaemia.

Simple SummaryAcute myeloid leukemia (AML) is a haematologic disease in which oncogenic mutations in the receptor tyrosine kinase FLT3 frequently lead to leukaemic development. Potent treatment of AML patients is still hampered by inefficient targeting of leukemic stem cells expressing constitutive active FLT3 mutants. This review summarizes the current knowledge about the regulation of FLT3 activity at cellular level and discusses therapeutical options to affect the tumor cells and the microenvironment to impair the haematological aberrations. Fms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinases (RTK) and is involved in cell survival, proliferation, and differentiation of haematopoietic progenitors of lymphoid and myeloid lineages. Oncogenic mutations in the FLT3 gene resulting in constitutively active FLT3 variants are frequently found in acute myeloid leukaemia (AML) patients and correlate with patient’s poor survival. Targeting FLT3 mutant leukaemic stem cells (LSC) is a key to efficient treatment of patients with relapsed/refractory AML. It is therefore essential to understand how LSC escape current therapies in order to develop novel therapeutic strategies. Here, we summarize the current knowledge on mechanisms of FLT3 activity regulation and its cellular consequences. Furthermore, we discuss how aberrant FLT3 signalling cooperates with other oncogenic lesions and the microenvironment to drive haematopoietic malignancies and how this can be harnessed for therapeutical purposes.

Small-Molecule Fms-like Tyrosine Kinase 3 Inhibitors: An Attractive and Efficient Method for the Treatment of Acute Myeloid Leukemia

Fms-like tyrosine kinase 3 (FLT3) is an important member of the class III receptor tyrosine kinase (RTK) family, which is involved in the proliferation of hematopoietic cells and lymphocytes. In recent years, increasing evidence have demonstrated that the activation and mutation of FLT3 is closely implicated in the occurrence and development of acute myeloid leukemia (AML). The exploration of small-molecule inhibitors targeting FLT3 has aroused wide interest of pharmaceutical chemists and is expected to bring new hope for AML therapy. In this review, we specifically highlighted FLT3 mediated JAK/STAT, RAS/MAPK, and PI3K/AKT/mTOR signaling. The structural properties and biological activities of representative FLT3 inhibitors reported from 2014 to the present were also summarized. In addition, the major challenges in the current advance of novel FLT3 inhibitors were further analyzed, with the aim to guide future drug discovery.

Deficiency of core fucosylation activates cellular signaling dependent on FLT3 expression in a Ba/F3 cell system.

Fms-like tyrosine kinase 3 (FLT3) is a glycoprotein, that is a member of the class III receptor tyrosine kinase family. Approximately one-third of acute myeloid leukemia (AML) patients have mutations of this gene, and activation of the FLT3 downstream pathway plays an important role in both normal and malignant hematopoiesis. However, the role of N-glycosylation for FLT3 activation remains unclear. In this study, we showed that the N-glycan structures on wild type (WT), internal tandem duplication (ITD), and tyrosine kinase domain (TKD) mutants of FLT3 were different. Interestingly, expression of either WT or mutant FLT3 in Ba/F3 cells, an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell, greatly induced core fucosylation. To elucidate the function of core fucosylation in FLT3-mediated signaling, we used a CRISPR/Cas9 system to establish α1,6-fucosyltransferase (Fut8) knockout (KO) cells. Surprisingly, the Fut8KO resulted in cell proliferation in an IL-3-independent manner in FLT3-WT cells, which was not observed in the parental cells, and suggested that this proliferation is dependent on FLT3 expression. Fut8KO greatly increased cellular tyrosine phosphorylation levels, together with an activation of STAT5, AKT, and ERK signaling, which could be completely neutralized by restoration with Fut8 in the KO cells. Consistently, a tyrosine kinase inhibitor efficiently inhibited cell proliferation induced by Fut8KO or specific fucosylation inhibitor. Additionally, immunostaining with FLT3 showed that the proteins were mainly expressed on the cell surface in the KO cells, which is similar to FLT3-WT cells, but different from the ITD mutant. Finally, we found that Fut8KO could induce dimer-formation in FLT3 without ligand-stimulation. Taken together, the present study clearly defines the regulatory function of core fucosylation in FLT3, which could provide a valuable direction for development of drugs could be effective in the treatment of AML.

Abstract 926: Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib, in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) remains a highly fatal disease due to the development of drug resistance. Mutational activation of kinases is a frequent event in leukemia, driving malignant cell growth. The most commonly mutated kinase in AML is the FMS-like tyrosine kinase 3 (FLT3) receptor. Internal tandem duplication (ITD) events in the juxtamembrane domain of FLT3 have been reported in ~ 20% of patients and lead to constitutive activation of FLT3. Previous work from our laboratory has shown that fibroblast growth factor 2 (FGF2) secreted by mesenchymal stromal cells protects FLT3-ITD AML cells from quizartinib, a FLT3 inhibitor (Cancer Research, 2016). FGF2 binds the FGFR1 receptor and activates MAPK signaling, enabling leukemia cells to become drug resistant. Over months of treatment, FGF2-protected leukemia cells acquired FLT3 resistance mutations. This two-step mechanism of resistance in vitro mirrors what happens in patients treated with quizartinib. Newer FLT3 inhibitors, such as gilteritinib, have been developed that have activity against both ITD and kinase domain mutations that impart resistance to quizartinib. We used a similar approach and treated the FLT3-ITD+ AML cell line, MOLM-14, with 100 nM of gilteritinib in media alone (n=4), or supplemented with 10 ng/ml FGF2 (n=4) or FLT3 ligand (FL, n=4). After 7 weeks, all cultures supplemented with FGF2 or FL eventually resumed growth. In contrast, no MOLM-14 cells cultured in gilteritinib alone resumed growth even after 16 weeks, suggesting the importance of extrinsic factors in facilitating early drug resistance. Selective pressure was then put on the ligand-dependent cultures by removal of FGF2 and FL. This transiently restored sensitivity to gilteritinib, however within a month the cultures resumed exponential growth. To further explore the mechanism of resistance, we performed immunoblots of kinase signaling pathways on MOLM-14 resistant cultures pre- and post-withdrawal of ligands. We saw robust activation of FLT3 receptor in resistant cultures with FL. FGF2 activated the MAPK pathway, circumventing inhibition by gilteritinib. After removal of extrinsic FGF2 and FL, there was re-activation of MAPK signaling but FLT3 itself remained inhibited. Whole Exome Sequencing of resistant cells identified NRAS mutations as the source of MAPK signaling after ligand withdrawal. Two of the four FGF2-supplemented cultures also developed novel FLT3 point mutations, although there was no clear re-activation of FLT3 signaling in these cultures. Our in vitro results suggest that extrinsic signals from the microenvironment can re-activate MAPK pathways enough to keep cells alive but continued selective pressure eventually leads to direct activation of MAPK through RAS mutations. As such, there is a need to consider rational inhibitor combinations early in disease therapy to prevent the emergence of resistance. Citation Format: Sunil K. Joshi, Stephen Christy, Renata Scopim Ribeiro, Shannon McWeeney, Jeffrey W. Tyner, Cristina E. Tognon, Brian J. Druker, Elie Traer. Extrinsic and intrinsic activation of RAS/MAPK signaling enables resistance to FLT3 inhibitor, gilteritinib, in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 926.

FMS-like Tyrosine Kinase 3/FLT3: From Basic Science to Clinical Implications.

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is expressed almost exclusively in the hematopoietic compartment. Its ligand, FLT3 ligand (FL), induces dimerization and activation of its intrinsic tyrosine kinase activity. Activation of FLT3 leads to its autophosphorylation and initiation of several signal transduction cascades. Signaling is initiated by the recruitment of signal transduction molecules to activated FLT3 through binding to specific phosphorylated tyrosine residues in the intracellular region of FLT3. Activation of FLT3 mediates cell survival, cell proliferation, and differentiation of hematopoietic progenitor cells. It acts in synergy with several other cytokines to promote its biological effects. Deregulated FLT3 activity has been implicated in several diseases, most prominently in acute myeloid leukemia where around one-third of patients carry an activating mutant of FLT3 which drives the disease and is correlated with poor prognosis. Overactivity of FLT3 has also been implicated in autoimmune diseases, such as rheumatoid arthritis. The observation that gain-of-function mutations of FLT3 can promote leukemogenesis has stimulated the development of inhibitors that target this receptor. Many of these are in clinical trials, and some have been approved for clinical use. However, problems with acquired resistance to these inhibitors are common and, furthermore, only a fraction of patients respond to these selective treatments. This review provides a summary of our current knowledge regarding structural and functional aspects of FLT3 signaling, both under normal and pathological conditions, and discusses challenges for the future regarding the use of targeted inhibition of these pathways for the treatment of patients.

Abstract 2192A: Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML

Abstract Acute myeloid leukemia (AML) has a 5-year overall survival rate under 26% in adults and current cytotoxic chemotherapies are toxic, can cause long-term side effects and are often contraindicated for elderly patients. Targeted therapies may have reduced toxicity compared to chemotherapy; however, resistance to single-agents often develops and combination therapies may provide more durable responses. MERTK is aberrantly expressed in >80% of AML patient samples and is a promising therapeutic target. MRX-2843 is a novel small molecule dual MERTK and FMS-like tyrosine kinase 3 (FLT3) inhibitor that is currently in clinical trials. Here, we report synergistic anti-leukemia activity mediated by MRX-2843 and rho-associated, coiled-coil-containing protein kinases 1 and 2 (ROCK1/2) inhibitors. Both MERTK and ROCK1/2 have been implicated in actin/microtubule dynamics and cell cycle progression and previous publications demonstrated induction of apoptosis in AML cells in response to RNAi-mediated MERTK or ROCK1 inhibition. ROCK1/2 inhibitors RKI-1447 or GSK269962A synergized with sub-therapeutic doses of MRX-2843 to reduce cell density as indicated by decreased Presto Blue staining in cultures of 4 of 6 AML cell lines tested and had an additive effect in the remaining 2 cell lines. Similar interactions were observed between RKI-1447 and UNC3997, a MERTK-selective TKI with 15-fold weaker FLT3 activity. Flow cytometric analysis of cells stained with popro-1-iodide and propidium iodide (PI) dyes revealed synergistic induction of cell death in the KG-1, OCI-AML5, and NB4 cell lines and an additive effect in Kasumi-1 cultures. In addition, cells that survived treatment exhibited subsequent defects in expansion, even when they were cultured without inhibitor(s), and these effects were significantly more pronounced in cells treated with the combination therapy compared to single agents. Flow cytometric analysis of PI-stained permeabilized cells revealed an increased fraction of cells in G2/M phase in cultures treated with MRX-2843. This effect was more pronounced in cultures treated with the combination therapy and was accompanied by accumulation of a population with slightly reduced DNA content relative to untreated G2 cells, suggestive of a defect in late S-phase. Thus, the combination therapy mediates anti-leukemia activity by multiple mechanisms, including abrogation of cell cycle progression and induction of cell death. In addition, a preliminary analysis of The Cancer Genome Atlas database revealed significantly poorer overall survival in patients with higher levels of ROCK1 expression (p=0.00147, n=172). Together, these data suggest that combination therapies targeting MERTK and ROCK1/2 may be particularly effective for treatment of AML and support further studies to test the effects of this novel strategy in animal models. Citation Format: Dawn E. Barnes, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2192A.

Emerging treatment paradigms with FLT3 inhibitors in acute myeloid leukemia.

Mutations in the fms-like tyrosine kinase 3 (FLT3) gene are detected in approximately one-third of patients with newly diagnosed acute myeloid leukemia (AML). These consist of the more common FLT3-internal tandem duplication (ITD) in approximately 20–25% of AML cases, and point mutations in the tyrosine kinase domain (TKD) in approximately 5–10%. FLT3 mutations, especially FLT3-ITD, are associated with proliferative disease, increased risk of relapse, and inferior overall survival when treated with conventional regimens. However, the recent development of well tolerated and active FLT3 inhibitors has significantly improved the outcomes of this aggressive subtype of AML. The multikinase inhibitor midostaurin was approved by the United States Food and Drug Administration (US FDA) in April 2017 for the frontline treatment of patients with FLT3-mutated (either ITD or TKD) AML in combination with induction chemotherapy, representing the first new drug approval in AML in nearly two decades. In November 2018, the US FDA also approved the second-generation FLT3 inhibitor gilteritinib as a single agent for patients with relapsed or refractory FLT3-mutated AML. Promising phase I and II efficacy data for quizartinib is likely to lead to a third regulatory approval in relapsed/refractory AML in the near future. However, despite the significant progress made in managing FLT3-mutated AML, many questions remain regarding the best approach to integrate these inhibitors into combination regimens, and also the optimal sequencing of different FLT3 inhibitors in various clinical settings. This review comprehensively examines the FLT3 inhibitors currently in clinical development, with an emphasis on their spectra of activity against different FLT3 mutations and other kinases, clinical safety and efficacy data, and their current and future roles in the management of AML. The mechanisms of resistance to FLT3 inhibitors and potential combination strategies to overcome such resistance pathways are also discussed.

Fms-like tyrosine kinase 3 is a key factor of male fertility

Fms-like tyrosine kinase 3 (FLT3) is a type III kinase that is highly expressed in seminal plasma of infertile men. FLT3 activation can be blocked by inhibition of its phosphorylation using the nontoxic and selective inhibitor, quizartinib. We investigated the function of FLT3 and the corresponding effects of quizartinib in mouse spermatozoa. Spermatozoa were treated with different concentrations (0.1, 1, 10, 20, and 30 μM) of quizartinib for 90 min at 37 °C in 5% CO2 in air. FLT3 was detected in capacitated and non-capacitated spermatozoa. While the level of FLT3 was unaffected, the levels of phospho-FLT3 were significantly altered in spermatozoa by quizartinib. Exposure of spermatozoa to higher concentrations of quizartinib significantly altered sperm viability, motility, motion kinematics, levels of intracellular ATP, and capacitation status. Fertilization and early embryonic development were suppressed by quizartinib. This may have occurred as a consequence of decreased protein kinase A (PKA) activity and tyrosine phosphorylation. The inhibition of FLT3 by quizartinib may affect the fertilization and embryonic development by reducing tyrosine phosphorylation through a PKA-dependent pathway. Our data implicate FLT3 as a biomarker for diagnosis and prognosis of male fertility. In addition, quizartinib has potential for development as a new contraceptive agent.

Novel Therapy for FLT3-ITD Acute Myeloid Leukemia Utilizing the Combination of CUDC-907 and Gilteritinib

Introduction:FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase, and is mutated in approximately one third of acute myeloid leukemia (AML) patients; this mutation confers a poor prognosis. Two FLT3 mutations are commonly seen in AML: internal tandem duplications (ITD) in the juxtamembrane domain (~25% of AML), and point mutations in the receptor tyrosine kinase at codon 835 (D835) (~7% of AML). Both mutations result in constitutive FLT3 activation, causing downstream activation of multiple pathways, in particular, those involved in cell survival including the RAS-RAF-MEK-ERK, JAK-STAT5, and PI3K/AKT pathways. PI3K-AKT may also be activated by AXL, also a tyrosine kinase, via its targets PLC, Grb2, and PI3K. Logically, then, inhibition of FLT3 is a promising pharmacological approach for treating this subtype of AML. Gilteritinib (ASP-2215) is a novel dual inhibitor of FLT3 and AXL, exposure to which results in upregulation of FLT3 as a resistance mechanism. Previously, we found that the novel dual PI3K/histone deacetylase (HDAC) inhibitor CUDC-907 downregulates FLT3 expression in AML cells (Figure 1A). Additionally, inhibition of FLT3 and AXL by gilteritinib may not result in robust inactivation of both the PI3K-Akt and MEK/ERK pathways due to crosstalk between the two pathways. Thus, our hypothesis was that CUDC-907 would sensitize AML cells to gilteritinib, resulting in concurrent inhibition of all the downstream signaling pathways of FLT3 and AXL, leading to synergistic antileukemic activities again FLT3-mutated AML (Figure 1B).Methods:FLT3-ITD AML cell lines (MV4-11 and MOLM-13) and primary patient samples were treated with CUDC-907, gilteritinib, both, or neither for 24 hours, at clinically achievable concentrations. Annexin V/Propidium Iodide (PI) staining and flow cytometry analyses was performed, and combination indexes (CI) calculated; CI<1, CI=1, and CI>1 indicating synergistic, additive, or antagonistic effects, respectively. Western blots were performed after treatment for 0-24 hours to determine protein expression of relevant targets.Results:CUDC-907 and gilteritinib demonstrated potent synergistic antileukemic effects in FLT3-ITD AML cell lines and FLT3-ITD patient samples (AML#171, AML#180), the combination exceeding either in isolation (Figure 1C). These findings were confirmed via western blot, which showed accentuated upregulation of cleaved caspase3 with combination therapy, in both cell lines and one patient sample, demonstrating drug-induced apoptosis. We confirmed that CUDC-907 abolishes gilteritinib-induced expression of FLT3 in a time-dependent fashion in cell lines MV4-11 and MOLM-13 (Figure 1D). Gilteritinib treatment decreased p-AKT, p-S6, and p-STAT5, while inhibition of the ERK pathway, as assessed by p-ERK expression, varied amongst the samples (Figure 1E). CUDC-907 treatment decreased both p-AKT and p-ERK. MOLM-13 cells showed increased p-ERK following gilteritinib treatment and increased p-STAT5 after CUDC-907 treatment. In all samples, combination of gilteritinib with CUDC-907 resulted in decrease of p-STAT5 and p-S6, similar to gilteritinib treatment alone, and further reduction of p-AKT and p-ERK compared to single drug treatments. Gilteritinib treatment also reduced expression of anti-apoptotic protein Mcl-1, which was further decreased in combination treated cells. Subsequently, time-course analysis was performed in both cell lines; findings were consistent with prior observations, and confirmed that protein expression changed over time, in relation to gilteritinib/CUDC-907/combined treatment exposure.Conclusion:We confirmed that CUDC-907 and Gilteritinib synergistically induce apoptosis in both cell lines and primary patient samples derived from patients with FLT3-ITD AML, and that CUDC-907 abolishes Gilteritinib-induced FLT3 expression. Additionally, the combination cooperatively inhibits the PI3K-AKT, JAK-STAT, and RAS-RAF pathways, while preventing escape via alternative pathways. Our results provide a strong foundation for subsequent in vivo murine studies, and eventual clinical evaluation of the combination of gilteritinib and CUDC-907 for the treatment of AML. [Display omitted] DisclosuresGe:MEI Pharma: Research Funding.

Induction of cytotoxicity and apoptosis in FLT3 mutant expressing cells using novel pyrimido cyanoacrylates and quinoline derivatives.

Aberrant activation of FMS-like tyrosine kinase 3 (FLT3) is associated with acute myeloid leukemia (AML). Leukemic cells expressing constitutively active FLT3 mutants are resistance to the current cancer therapies (radiotherapy and chemotherapy); hence, there is an increased interest to identify new agents for the treatment of AML. The main aim of this study was evaluating cytotoxic effects of novel pyrimidocyanoacrylates and quinoline derivatives on FLT3 overexpressing cells. Five novel pyrimidocyanoacrylates & 2-chloro 3-carbaldehyde quinolone derivative compounds, E1QAC1, E1QAC2, E1QAC3, E1QAC4, and E1QAC5 were designed and synthesized at the Department of Chemistry, Faculty of Sciences, Ferdowsi University, Mashhad, Iran. FDC-P1 cells expressing human wild-type FLT3 (FD-FLT3-WT) and internal tandem duplication (ITD) mutants (FD-FLT3-ITD) used in this study. The cells maintained in DMEM medium supplemented with 10% fetal calf serum (FCS) and murine granulocyte-macrophage colony stimulating factor (mGM-CSF). Potency for induction of cytotoxicity (IC50 value) and apoptosis was determined after treating the cells with concentration of the compounds by resazurin assay. Bax and Bcl2 activation status was also investigated by Western blot analysis. All the compounds had concentration-dependent effects on inhibition of cell proliferation and induction of apoptosis in both cell lines. E1QAC4 was the most potent compound for inhibition of cell proliferation (with IC50 value of 19 μM) and apoptosis induction in the FLT3-WT cells. However, FD-FLT3-ITD cells were nearly five-times more resistant to all the compounds (except than E1QAC2) that the FLT3-WT expressing cells. Western blotting results also showed that FD-FLT3-ITD cells had lower levels of Bax and higher levels of Bcl2 than the FD-FLT3-WT cells. The five novel heterocyclic compounds (E1QAC1-5) had cytotoxic effects and induced apoptosis in FD-FLT3 cells. Therefore, it is worthwhile to consider them as potential lead compound for development of new therapeutic agents for AML patients.

Tyrosine kinase inhibitor–induced defects in DNA repair sensitize FLT3(ITD)-positive leukemia cells to PARP1 inhibitors

AbstractMutations in FMS-like tyrosine kinase 3 (FLT3), such as internal tandem duplications (ITDs), can be found in up to 23% of patients with acute myeloid leukemia (AML) and confer a poor prognosis. Current treatment options for FLT3(ITD)-positive AMLs include genotoxic therapy and FLT3 inhibitors (FLT3i's), which are rarely curative. PARP1 inhibitors (PARP1i's) have been successfully applied to induce synthetic lethality in tumors harboring BRCA1/2 mutations and displaying homologous recombination (HR) deficiency. We show here that inhibition of FLT3(ITD) activity by the FLT3i AC220 caused downregulation of DNA repair proteins BRCA1, BRCA2, PALB2, RAD51, and LIG4, resulting in inhibition of 2 major DNA double-strand break (DSB) repair pathways, HR, and nonhomologous end-joining. PARP1i, olaparib, and BMN673 caused accumulation of lethal DSBs and cell death in AC220-treated FLT3(ITD)-positive leukemia cells, thus mimicking synthetic lethality. Moreover, the combination of FLT3i and PARP1i eliminated FLT3(ITD)-positive quiescent and proliferating leukemia stem cells, as well as leukemic progenitors, from human and mouse leukemia samples. Notably, the combination of AC220 and BMN673 significantly delayed disease onset and effectively reduced leukemia-initiating cells in an FLT3(ITD)-positive primary AML xenograft mouse model. In conclusion, we postulate that FLT3i-induced deficiencies in DSB repair pathways sensitize FLT3(ITD)-positive AML cells to synthetic lethality triggered by PARP1i's. Therefore, FLT3(ITD) could be used as a precision medicine marker for identifying AML patients that may benefit from a therapeutic regimen combining FLT3 and PARP1i's.

Abstract 4821: A targeted differentiation therapy for the treatment of acute myeloid leukemia

Abstract Objectives: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse genetic abnormalities present in all ages, but mainly prevalent in an elderly population with an average age of above 60 years. The treatment of elderly AML remains a formidable challenge as the long-term outcomes of elderly AML patients have not improved in the last three decades, with ≤5% overall survival recorded at 5 years, calling for novel treatment options. Epigenetic therapy has a significant impact on the management of hematologic malignancies. Recent findings show that using very low dosages of decitabine depletes DNA methyl transferase 1 (DNMT1) without cytotoxicity. Decitabine-mediated epigenetic therapy does not induce FLT3 (fms-like tyrosine kinase 3) ligand, which hinders the effectiveness of FLT3 inhibitors. The inhibition of FLT3 and DNMT1 is associated with terminal myeloid differentiation of AML cells. Therefore, combining the inhibitors of FLT3 and DNMT1 may be an effective therapeutic approach for the treatment of poor-risk AML. Ponatinib is a third-generation receptor tyrosine kinase inhibitor. Various in vitro and in vivo preclinical studies demonstrated antileukemic activities of ponatinib against AML cells bearing FLT3-ITD mutations. Methods: Apoptosis and CD11b were measured by flow cytometry. FLT3 signaling and DNMT1 levels were analyzed by immunoblotting. Results: FLT3-ITD expressing AML cell lines MV4-11, MOLM-13, and MOLM-14 were used in the study. Co-treatment with decitabine (10-100 nM) and ponatinib (2 nM) in AML cells induced apoptosis in association with PARP cleavage. Increased levels of pro-apoptotic protein BAD and decreased levels of anti-apoptotic protein MCL1 were observed in these treated cells. The combination of decitabine and ponatinib also showed similar effects in primary AML cells expressing FLT3-ITD. The Western blot analysis demonstrated that treatment of decitabine decreased levels of DNMT1 and ponatinib inhibited FLT3 signaling and activation of downstream effectors STAT5, AKT, and ERK1/2 with induction of PU.1 a key regulator of myeloid differentiation. Co-treatment with ponatinib and decitabine using low concentrations in MV4-11 and MOLM14 cells induced myeloid differentiation. The percentage of differentiated cells was measured by increased surface expression of CD11b analyzed by flow cytometry and granulocytic/monocytic morphology examined by Wright-Giemsa staining. Conclusions: Mechanistically, the hypomethylating agent initiates the differentiation process and FLT3 inhibition augments differentiation leading to apoptosis of AML cells. Altogether, these preclinical findings of downregulation of DNMT1 and induction of PU.1 are a novel differentiation approach to induce apoptosis in AML cells and warrant future therapeutic potential for the treatment of AML patients expressing FLT3 mutations. Citation Format: Sudhakiranmayi Kuravi, Myles Taylor, Tara L. Lin, Jensen Roy, Joseph McGuirk, Ramesh Balusu. A targeted differentiation therapy for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4821.