FMS-like Tyrosine Kinase 3 Expression Research Articles

Modulation of FLT3 through decitabine-activated C/EBPa-PU.1 signal pathway in FLT3-ITD positive cells

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) which occurs in approximately 30% of all AML patients still has a poor prognosis. This study aimed to examine the effect of decitabine (DAC) on FLT3-ITD positive AML. In our study, we found that expression of FLT3 and its downstream targets was decreased in FLT3-ITD mutant cell lines treated with DAC. DAC treatment could increase the percentage of apoptotic cells and CD11b positive cells tested by flow cytometry and upregulate the expression of cleaved caspase3, cleaved PARP, C/EBPa and PU.1 detected by western blot. To explore the effect of increased expression of PU.1 on FLT3 protein, we transiently transfected MOLM13 and MV4-11 cells with siRNA against PU.1 and a siRNA control. In both FLT3-ITD positive cells, the effect of DAC on downregulation of FLT3 was diminished in PU.1-konckdown MOLM13 and MV4-11 cells and there was a decrease of CD11b expression after PU.1 knockdown. Furthermore, the percentage of apoptotic cells was also decreased in PU.1-konckdown cells compared with siRNA control-expressing cells with the same dose of DAC. These findings indicated that DAC upregulated PU.1 to induce downregulation of FLT3 to trigger apoptosis. DAC was also found efficacious in mouse xenograft models of FLT3-ITD AML in our study. These findings may provide a novel theoretical basis for treatment of FLT3-ITD positive AML patients.

PF217 TARGETING FLT3 IN AML: MODULATION OF FLT3‐BITE® ACTIVITY THROUGH COMBINATION WITH VARIOUS TKI

Background:T cell recruiting antibody constructs represent a novel platform for immunotherapy of acute myeloid leukemia (AML). We previously characterized FLT3 (CD135; FMS‐like tyrosine kinase 3) as a suitable target antigen for treatment of AML based on significantly higher expression on primary AML (pAML) cells in comparison to healthy hematopoietic subpopulations (Lindl ASH 2017).Aims:Here, we evaluated T cell‐redirected target cell lysis mediated by a FLT3 BiTE® antibody construct (FLT3 BiTE®) against AML cells in an ex vivo model for pAML cells, an in vivo NOD‐SCID mouse admixture model and in non human primates (NHP). We hypothesized that combining FLT3 BiTE® with a tyrosine kinase inhibitor (TKI) would enhance target cell lysis. Accordingly, FLT3 expression, mutational status, and TKI‐mediated T‐cell dysfunction were analyzed as relevant factors for FLT3 BiTE®‐mediated cytotoxicity.Methods:FLT3 BiTE®‐mediated cytotoxicity was evaluated by coculture of various FLT3+ AML cell lines with healthy donor (HD) T cells. Cytotoxicity against pAML cells and T‐cell proliferation were tested ex vivo using a long‐term culture system (Krupka Blood 2014) and assessed by flow cytometry. The Effector:Target cell ratio was based on residual T cells within the sample.In vivo, a NOD‐SCID mouse admixture model with human CD3+ T cells and MOLM‐13 cells was used. In NHP, FLT3 BiTE® was administered with step‐dosing at 5, 15, 45, and 100 μg/kg/d in a 16‐day continuous intravenous study. Pharmacodynamics were determined by measuring blood (PB) and bone marrow (BM) FLT3 transcript and soluble FLT3 ligand.TKI‐mediated modulation of T‐cell function was assessed by CD3/CD28‐bead stimulation of CFSE‐labeled HD T cells. Cytotoxicity of 3 TKIs against AML cell lines with different FLT3‐ITD mutational status was evaluated.Last, the combination of TKI with FLT3 BiTE® was tested in cytotoxicity assays using HD T cells with FLT3‐ITD+ AML cell lines or pAML cells.Results:FLT3 BiTE® exhibited cytotoxicity against FLT3+ AML cell lines at picomolar concentrations irrespective of FLT3 mutational status (EC50:1.9 ± 0.7pM, ± SD, n = 6). No cytotoxicity was observed against FLT3− cell lines. pAML cells were lysed (%specific lysis day 9: 43 ± 9%, ± SEM, n = 14) even at low E:T ratios (1:3–1:74) accompanied by strong T‐cell proliferation (fold change CD2+ day 9: 5.9 ± 1.8, ± SEM) independent of FLT3 mutational status. In mice treated intraperitoneally with 200 μg/kg/d FLT3 BiTE®, reduction in tumor volume was observed (tumor volume d15 FLT3 BiTE® vs control BiTE®: 221 vs 1431 mm3, p≤0.0001, n = 10 per group). Furthermore, a decrease in FLT3 transcript levels (d17: 92.1 ± 0.1%, ± SD, n = 3) as well as a time‐dependent increase in circulating soluble FLT3 ligand levels were observed in PB and BM samples from NHP, supporting a FLT3 BiTE®‐mediated decrease in FLT3+cells.Pharmacologically relevant concentrations of TKIs induced cytotoxicity against FLT3‐ITD+ AML cell lines (Table, top) without altering T‐cell proliferation. Higher concentrations of TKI led to a decrease in T‐cell proliferation (Table, middle).Next, we combined TKIs with FLT3 BiTE®, resulting in increased cytotoxicity against FLT3‐ITD+ AML cell lines (p = 0.03, n = 6) as well as pAML cells in 2 FLT3‐ITD+ patients (Table, bottom).Summary/Conclusion:The FLT3 BiTE® antibody construct showed strong cytotoxicity against FLT3+ cells in vitro and ex vivo, mediated antitumor activity and depleted FLT3+ cells in vivo. The addition of clinically relevant concentrations of TKI enhanced FLT3‐BiTE®‐mediated cytotoxicity in FLT3‐ITD+ AML while higher TKI concentrations compromised T‐cell activity.image

Nonclinical safety assessment of AMG 553, an investigational anti-FLT3 CAR-T therapy.

7032 Background: FMS-like tyrosine kinase 3 (FLT3) is a tyrosine-protein kinase involved in hematopoiesis. In acute myeloid leukemia (AML), higher FLT3 transcript levels, independent of the presence of FLT3 mutations, correlate with higher leukocyte counts and higher degrees of bone marrow infiltration by leukemic cells. FLT3 protein is detectable on the cell surface of more than 80% of leukemia isolates from adult AML patients whereas FLT3 mutations are present only in approximately one third of patients. AMG 553 is a novel, investigational, adoptive cellular immunotherapy for the treatment of relapsed refractory AML, consisting of autologous T cells genetically modified ex vivo to express a transmembrane chimeric antigen receptor (CAR) to target FLT3 protein on the surface of AML cells irrespective of FLT3 mutational status. Methods: The nonclinical safety evaluation of AMG 553 includes assessment of FLT3 expression in normal tissues; in vitro assessment of cytotoxicity against human cells with or without FLT3 expression; toxicology studies in cynomolgus monkeys administered autologous T-cells transduced with an anti-FLT3 CAR or administered anti-FLT3 bi-specific T-cell engager (BiTE) antibodies. Results: Assessment of FLT3 expression in normal tissues indicates that FLT3 protein is detected on the cell surface of a subpopulation of human bone marrow hematopoietic stem and progenitor cells; expression in other tissues is cytoplasmic and not anticipated to be accessible to AMG 553. AMG 553-induced cytotoxicity was only observed in a percentage of primary bone marrow CD34+ cells. There was no evidence of CAR-T cell-mediated toxicity, expansion, or persistence in cynomolgus monkeys likely due to restricted cell surface FLT3 protein expression in healthy animals which demonstrates the limited value of such studies for this target. All findings in toxicology studies with anti-FLT3 BiTEs were consistent with the targeted killing of cells expressing FLT3 on the plasma membrane. Conclusions: The nonclinical safety data support AMG 553 as a novel therapeutic to target FLT3 protein on AML cells irrespective of FLT3 mutational status, while only affecting a percentage of normal hematopoietic stem and progenitor cells.

Angiogenic and FLT3 Receptors Expression in Acute Lymphoblastic Leukemia in Pediatric Age Group

Angiogenesis has an important role in pathophysiology of cancer. FMS-like tyrosine kinase 3 (FLT3) is implicated in hematopoietic malignancies. Their role in childhood acute lymphoblastic leukemia (ALL) pathogenesis needs more enlightenment. Expression of vascular endothelial growth factor receptor-1 and -2 (VEGFR-1 and -2), as well as FLT3 were assessed by flow cytometry in bone marrow (BM) blasts of 55 newly diagnosed children with ALL. Patients included B cell ALL (B-ALL) group (n = 41) and T cell ALL (T-ALL) group (n = 14). Comparison between groups revealed a significant increase in blasts percent (%) expressing FLT3 and FLT3 intensity detected in B-ALL group (p = 0.004 and p = 0.02, respectively). In B-ALL patients, a significant positive correlation was seen between blasts % expressing FLT3 and blasts percentage infiltrating BM (r = 0.405; p = 0.009), also positive correlation was seen between % of blasts expressing VEGFR-1 and VEGFR-2 (r = 0.704; p < 0.001). In T-ALL group, blast % expressing FLT3 revealed significant positive correlations with blast % expressing VEGFR-1, and those expressing VEGFR-2 (r = 0.627; p = 0.016, and r = 0.654; p = 0.011, respectively). In addition, significant correlation was seen in blasts % expressing all; FLT3, VEGFR-1 and -2, with blasts % expressing stem cell marker CD34 (r = 0.826; p = 0.001, r = 0.596; p = 0.041, and r = 0.798; p = 0.002, respectively). Conclusion: Expression of VEGFR-1, VEGFR-2 and FLT3 were demonstrated and linked on leukemic blasts of ALL which highlights their role in pathogenesis. FLT3 expression plays a role in facilitating blasts proliferation in BM in B-ALL. FLT3, VEGFR-1 and -2 could be used in future profiling of CD34+ leukemic stem cell pool in T-ALL.

FLT3 Inhibitor Treatment Increases FLT3 Expression That Exposes FLT3-ITD+ AML Blasts to Elimination By FLT3 CAR-T Cells

Background: FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein uniformly expressed on leukemic blasts in acute myeloid leukemia (AML), and driver of leukemia-genesis in FLT3-ITD+ (Internal tandem duplication) AML. There is an increasing body of pre-clinical and clinical data suggesting that FLT3-ITD+ AML blasts respond to FLT3 inhibitor treatment by augmenting FLT3 expression in order to sustain the survival signal provided by this mutation. Here, we analyzed FLT3 expression on FLT3 wild type and FLT3-ITD+ AML cells after treatment with the FLT3 inhibitors midostaurin, quizartinib and crenolanib, and determined the antileukemia efficacy of combination treatment with FLT3 inhibitors and FLT3 CAR T cells in vitro and in vivo.Methods: MOLM-13 and MV4;11 AML cells (both FLT3-ITD+) were cultured in the presence of IC50 doses of midostaurin, quizartinib and crenolanib, respectively to induce resistance (MOLM-13R/MV4;11R). A FLT3-CAR comprised of BV10 scFv binding domain, CD28-CD3ζ signal module and EGFRt marker was encoded in a lentiviral vector and expressed in CD8+ and CD4+ T cells of healthy donors and patients (n=6). T cell mediated cytolytic activity was evaluated in luminescence-based assay, cytokine production analyzed by ELISA and proliferation assessed by CFSE dye dilution. NSG mice (n= 4-6 per group) were engrafted with MOLM-13/ffLuc AML cells and treated with 5x106 CAR T cells alone or in combination with FLT3 inhibitors.Results: We detected a significant increase in FLT3 expression on both MV4;11 and MOLM-13 AML cells after treatment with each of the inhibitors as assessed by mean fluorescence intensity (quizartinib > crenolanib > midostaurin). The increase in FLT3 expression occurred specifically on these FLT3-ITD+ AML cell lines and was not observed on FLT3 wt AML (THP-1), acute lymphoblastic leukemia (NALM-16), mixed lineage leukemia (KOPN-8 and SEM) cell lines and normal hematopoietic stem cells. We applied single molecule sensitive super-resolution microscopy to demonstrate that the average number of FLT3 molecules (per micrometer sq.) on MV4;11 AML cells had increased from 0.80 (untreated) to 10.7 (quizartinib), 4.7 (crenolanib), and 3.3 (midostaurin) (p<.05). Of interest, midostaurin induced clustering of FLT3, while FLT3 was still present as monomers after quizartinib and crenolanib treatment. Intriguingly, the higher FLT3 density after FLT3 inhibitor treatment translated into superior antileukemia reactivity of FLT3 CAR T cells against AML cell lines and primary AML cells in vitro and in vivo. We observed the strongest increase in cytolytic activity, cytokine production and proliferation by CD8+ and CD4+ FLT3 CAR T cells after treatment with crenolanib and quizartinib, followed by midostaurin (p<.05).We confirmed that upregulation of FLT3 occurred on MOLM-13 cells during FLT3 inhibitor therapy in NSG mice in vivo, and observed synergistic antileukemia efficacy of FLT3 CAR T cells in combination with each of the compounds. The mean frequency of FLT3 CAR T cells in mice that received FLT3 CAR T cells and an FLT3 inhibitor was 2-4 fold higher compared to mice had received FLT3 CAR T cells alone (p<.05) and was the highest in the cohort of mice that had received FLT3 CAR T cells in combination with crenolanib. FLT3 CAR T cells alone and each of the combination treatments of FLT3 CAR T & FLT3 inhibitor achieved 100% response rate which compares favorably to untreated or FLT3 inhibitor alone (0%). However, the mean fold reduction in leukemia burden (b/w day 7 and 10) was greater in all three combination treatment compare to only CAR treatment (p<.05). The most potent combination was FLT3 CAR T cells & crenolanib that accomplished the strongest reduction in leukemia burden as assessed by bioluminescence imaging and flow cytometry.Conclusion: Collectively, the data show that FLT3 inhibitors augment cell surface expression of FLT3 in FLT3-ITD+ AML cells which leads to enhanced recognition and elimination by FLT3 CAR T cells. This is, to our knowledge, the first demonstration that small molecule inhibitors and CAR T cell immunotherapy can be used synergistically to treat a hematologic malignancy. We confirmed this principle with each of the FLT3 inhibitors in our panel, and observed the strongest antileukemia activity of FLT3 CAR T cells in combination with crenolanib. Our data encourage the clinical evaluation of this combination treatment in high risk patients with FLT3-ITD+ AML. DisclosuresJetani:University hospital wuerzburg: Employment, Patents & Royalties: H.J. and M.H are co-inventors on a patent related to the use of FLT3-CAR T-cells to treat AML filed by the University of Wuerzburg, Wuerzburg, Germany. Bonig:Kiadis Pharma: Consultancy.

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.

PRMT1 Mediated FLT3 Methylation As a Therapeutic Vulnerability in FLT3-ITD+ AML

The current view is that treatment failures of AML patients are due to persistence of leukemia stem cells (LSCs). The presence of FMS-like tyrosine kinase-3 (FLT3) Internal tandem duplication (ITD) is associated with poor prognosis. But, FLT3 tyrosine kinase inhibitors (TKI) demonstrate transient clinical activity in FLT3-ITD+ AML patients. Persistent FLT3-ITD+ AML LSC represent a source of relapse. There is a pressing need to target LSC and improve outcomes for FLT3-ITD+ AML patients.PRMT1, the predominant arginine methyltransferase, has been implicated in pathogenesis of AML rare subtype (i.e., acute megakaryoblastic leukemia). Herein, we show that PRMT1 protein expression is significantly increased in LSC-enriched AML CD34+CD38- cells relative to the counterparts of normal peripheral blood stem cells (PBSC) (AML n=9, normal n=8, p=0.0004,). Following PRMT1-knockdown (KD), AML CD34+ cells (n=15) demonstrated varying degrees of apoptosis while the survival of normal cells were not affected. Interestingly, we observed significant apoptosis-induction in a subset of samples bearing FLT3-ITD mutation (6 out of total 15) upon PRMT1-KD (ShCtrl 13.9±3.6%, ShPRMT1 32.5±4.6%, p<0.001). PRMT1-KD induced apoptosis was also more evident in cord blood (CB) CD34+ cells expressing FLT3-ITD (ShCtrl 19.4±1.3%, ShPRMT1 45.2±2.5%, p<0.001) relative to that of FLT3-WT (ShCtrl 27.47±1.8%, ShPRMT1 36.9±1.6%, p=0.0167). In 293T cells ectopically overexpressing FLT3-WT or FLT3-ITD, co-immunoprecipitation (co-IP) indicated greater interaction between PRMT1 and FLT3-ITD. Through RNA-Seq profiling two AML lines (MV4-11, OCI-AML3) plus one FLT3-ITD transduced CB CD34+ cells with PRMT1-KD, we obtained a differentially-regulated gene set as a “PRMT1 signature”. This signature was enriched in FLT3-ITD+ AML relative to FLT3-WT AML according to ssGSEA analysis using two AML datasets (GSE14468, GSE10358), indicating PRMT1 may cooperate with FLT3-ITD regulating AML maintenance.Given that PRMT1 directly interacts with FLT3-ITD, we next asked whether PRMT1 catalyzes FLT3-ITD protein methylation. Through mass-spectrometry analysis of a FLT3-ITD+ AML specimen and in vitro methylation assay, we identified that PRMT1 catalyzes FLT3-ITD arginine (R) methylation (Me) at two conserved residues, 972 and 973. Using in-house R972/973 Me antibody, we validated the expression of FLT3 R-Me in FLT3-ITD AML speciemens (7 out of 7). To test R-Me function, we transduced MLL-AF9 (MA9) overexpressing murine c-Kit+ cells with methylation-deficient FLT3-ITD (R972/973K, arginine [R] to lysine [K]) construct, and found that MA9 cells expressing R972/973K underwent more apoptosis than that of WT FLT3-ITD (WT FLT3-ITD 9.7±1.1%, R972/973K 23.7±2.1%, p=0.003). The double transformed cells were further transplanted into recipients for leukemia development. Mice receiving MA9 cells expressing R972/973K exhibited longer survival (median survival: WT FLT3-ITD 36 days, R972/973K 50 days, p=0.002, n=6). Mechanistically, expression of R972/973K did not affect the total tyrosine phosphorylation level of FLT3-ITD. Additionally, FLT3-ITD R-Me expression persisted after a TKI (AC220) treatment. These facts indicated that FLT3-ITD R-Me function is independent of FLT3-ITD kinase activity. We then used a FLT3-ITD+ patient derived xenograft (PDX) model to assess the effects of TKI and PRMT1 inhibition. Following engraftment >1% in peripheral blood, we divided mice (n=24) into 4 groups and treated each with vehicle, MS023 (a type I PRMT inhibitor, ACS Chem Biol. 2016;11:772-781) (160 mg/kg/i.p), AC220 (10 mg/kg/i.g) or combination for 4 weeks. MS023 treatment downregulating FLT3 R-Me levels enhanced elimination of FLT3-ITD AML cells by AC220 treatment (AC220 24.6±13.4% vs combination 7.6±6.5%, p=0.02, n=6). At 16 weeks post-secondary BMT, significant AML burden in single drug treated transplants was observed, but less AML cells were detected in combination-treated transplants (AC220 52.3%, vs combination 25.4%, p<0.001, n=6). MS023 had little effect on long-term in vivo engraftment of CD34+ from a human CB specimen (vehicle 76.3±5.9%, MS023 72.2±3.4%, p=0.21, n=5).In summary, our study demonstrated PRMT1 overexpression contributes to AML stem/progenitor cell survival possibly through FLT3-ITD methylation, supporting further exploration into how PRMT1-mediated FLT3 methylation governs LSC survival. DisclosuresKhaled:Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy; Juno: Other: Travel Funding.

The Cellular p53 Inhibitor MDM2 and the Growth Factor Receptor FLT3 as Biomarkers for Treatment Responses to the MDM2-Inhibitor Idasanutlin and the MEK1 Inhibitor Cobimetinib in Acute Myeloid Leukemia.

The tumor suppressor protein p53 is inactivated in a large variety of cancer cells. Cellular p53 inhibitors like the mouse double minute 2 homolog (MDM2) commonly suppress the p53 function in acute myeloid leukemia (AML). Moreover, fms like tyrosine kinase 3 (FLT3) growth factor signaling pathways including the mitogen-activated kinase (MAPK) cascade (RAS-RAF-MEK-ERK) are highly active in AML cells. Consequently, the combined administration of MDM2 and MEK inhibitors may present a promising anti-leukemic treatment strategy. Here we assessed the MDM2 antagonist idasanutlin and the MEK1 inhibitor cobimetinib as single agents and in combination in a variety of AML cell lines and primary AML blast cells for their ability to induce apoptosis and cell death. AML cell lines and blast cells comprised all major AML subtypes based on the mutational status of TP53, FLT3 and NPM1 genes. We observed a considerably varying anti-leukemic efficacy of idasanutlin and cobimetinib. AML cells with high sensitivity to the single compounds as well as to the combined treatment emerged with normal karyotype, wild-type TP53 and elevated FLT3 and MDM2 protein levels. Our data indicate that AML cells with normal karyotype (NK) and wild-type status of TP53 with elevated FLT3 and MDM2 expression emerge to be most sensitive to the combined treatment with cobimetinib and idasanutlin. FLT3 and MDM2 are biomarkers for treatment response to idasanutlin and cobimetinib in AML.

CAR T-cells targeting FLT3 have potent activity against FLT3-ITD+ AML and act synergistically with the FLT3-inhibitor crenolanib.

FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein expressed on normal hematopoietic stem and progenitor cells (HSC) and retained on malignant blasts in acute myeloid leukemia (AML). We engineered CD8+ and CD4+ T-cells expressing a FLT3-specific chimeric antigen receptor (CAR) and demonstrate they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3 with internal tandem duplication (FLT3-ITD). We also show that treatment with the FLT3-inhibitor crenolanib leads to increased surface expression of FLT3 specifically on FLT3-ITD+ AML cells and consecutively, enhanced recognition by FLT3-CAR T-cells in vitro and in vivo. As anticipated, we found that FLT3-CAR T-cells recognize normal HSCs in vitro and in vivo, and disrupt normal hematopoiesis in colony-formation assays, suggesting that adoptive therapy with FLT3-CAR T-cells will require subsequent CAR T-cell depletion and allogeneic HSC transplantation to reconstitute the hematopoietic system. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD+ AML. Further, our data provide the first proof-of-concept that CAR T-cell immunotherapy and small molecule inhibition can be used synergistically, as exemplified by our data showing superior antileukemia efficacy of FLT3-CAR T-cells in combination with crenolanib.

High expression of FLT3 is a risk factor in leukemia.

Several studies have shown that internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) can result in the failure of leukemia treatment and contribute to a poor prognosis. However, the role of the overexpression of FLT3 in leukemia remains to be fully elucidated. By mining public database, the present study first identified that the expression of FLT3 in leukemia was markedly higher, compared with that in other types of tumor and cell lines, indicating that FLT3 is important in leukemia. In leukemia, FLT3 was found to be significantly upregulated in acute myeloid leukemia and acute lymphoblastic leukemia, and a high expression of FLT3 contributed to reduced survival rates. By analyzing Gene Expression Omnibus and The Cancer Genome Atlas data, it was found that genetic alterations and modification of DNA methylation increased the expression of FLT3 in leukemia. FLT3-ITD and FLT3 tyrosine kinase domain point mutations increased the expression of FLT3 in four independent datasets. In addition, the status of FLT3 gene methylation was negatively correlated with the expression of FLT3, and haploinsufficiency of DNA methyltransferase 1 increased the expression of Flt3 in mouse leukemia cells. By analyzing the enrichment of differentially-expressed genes in chemical and genetic perturbation datasets, it was found that genes, which were upregulated in the FLT3 high expression group had myeloid lymphoid leukemia- and nucleophosmin 1-like signatures, indicating that the overexpression of FLT3 may use the same mechanism to promote leukemia. Collectively, the results of the present study showed that the overexpression of FLT3 is a potential risk factor in leukemia.

High Expression of AHSP, EPB42, GYPC and HEMGN Predicts Favorable Prognosis in FLT3-ITD-Negative Acute Myeloid Leukemia

Background/Aims: Acute myeloid leukemia (AML) is a heterogeneous clonal disease and patients with AML who harbor an FMS-like tyrosine kinase 3 (FLT3) mutation present several dilemmas for the clinician. This study aims to identify novel targets for explaining the dilemmas. Methods: We analyzed four microarray gene expression profiles to investigate changes in whole genome expression associated with FLT3-ITD mutation. Results: We identified 22 differentially expressed genes which are commonly expressed among all four profiles. Kaplan-Meier analysis of the dataset GSE12417 revealed that low expression of AHSP, EPB42, GYPC and HEMGN predicted poor prognosis (AHSP: P=0.0317, HR=1.894; EPB42: P=0.0382, HR=1.859; GYPC: P=0.0015, HR=2.051; HEMGN: P=0.0418, HR=1.838 in GSE12417 test cohort; AHSP: P=0.0279, HR=1.548; EPB42: P=0.0398, HR=1.505; GYPC: P=0.0408, HR=1.501; HEMGN: P=0.0143, HR=1.630 in GSE12417 validation cohort). When patients were FLT3-ITD positive, the expression of FLT3 was significantly increased (all P<0.05 in four profiles), and correleation analysis of four profiles revealed that the expression of the four candidate genes negatively correlated with FLT3 expression. Conclusions: Our findings suggest that AHSP, EPB42, GYPC and HEMGN may be suitable biomarkers for diagnostic or therapeutic strategies for FLT3-ITD-positive AML patients.

Preclinical Development of FLT3-Redirected Chimeric Antigen Receptor T Cell Immunotherapy for Acute Myeloid Leukemia

Background: Outcomes for adults and children with acute myeloid leukemia (AML) are dismal with 20-40% and 60% 5-year event-free survival, respectively. Alternative therapeutic strategies for AML are thus needed to improve outcomes. Chimeric antigen receptor (CAR) T cell immunotherapy has induced remarkable clinical responses in multiple phase 1 clinical trials for patients with relapsed or chemorefractory B cell leukemias, encouraging great interest in developing similar approaches for AML. Prior studies have demonstrated efficacy of CD33 or CD123-redirected CAR T cells in AML models, although the genetic heterogeneity of AML will likely require identification of additional therapeutic targets. In the current studies, we report preliminary in vitro and in vivo efficacy of new CAR T cells targeting the FMS-like tyrosine kinase 3 (FLT3) in human AML. FLT3 mutations via internal tandem duplication or kinase domain point mutations occur in approximately 25% of AML and result in FLT3 surface protein overexpression, suggesting potential efficacy of FLT3-targeting therapies. Both types of FLT3 alterations induce ligand-independent activation of FLT3 signaling, further demonstrating a critical role of FLT3 in AML pathogenesis.Hypothesis: FLT3 is a promising target for CAR T cell immunotherapy based treatment of AML.Results: Quantitative flow cytometric analysis of human AML cell lines demonstrated FLT3 surface expression ranging from 1338 (MOLM-13), 2594 (MOLM-14), and 2710 (MV4;11) receptors/cell versus 623 receptors/cell on negative control U937 cells. We first generated FLT3-redirected CAR construct consisting of a single chain variable fragment (scFv) derived from a well-characterized anti-human FLT3 antibody coupled to T cell 4-1BB (CD137) costimulatory and CD3-zeta activation domains. CD33 CAR T cells based on Gemtuzumab created by identical methodologies were also used as AML CAR T cell controls. In vitro studies verified that human T cells transduced with the FLT3 CAR construct induced interferon-gamma and interleukin-2 production after co-culture with AML cell lines MOLM-13, MOLM-14, and MV4;11. One dose of FLT3 CAR T cells inhibited leukemia proliferation in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with FLT3-mutant MOLM-13 or MOLM14 cell lines. These first data demonstrate potent preclinical activity of FLT3 CAR T cells and warrant further study in additional AML models.However, on target/off tumor toxicities can occur with AML antigen-targeted immunotherapies, as previously reported in studies of CD33 and CD123 CAR T cells. Normal expression of FLT3 has been mainly described on CD34+ hematopoietic progenitor stem cell populations, and FLT3-targeted therapies have potential to induce aplastic anemia. To address this question of hematologic toxicity of FLT3 CAR T cells, we created normal human hematopoiesis xenograft models in NOD scid gamma Il3-GM-SF (NSGS) mice engrafted with CD34+ cord blood cells for treatment with anti-AML CAR T cells. No difference in human granulocyte numbers was observed in marrows of engrafted mice treated with FLT3 CAR T cells, CD33 CAR T cells, or non-transduced T cells. A significant reduction in monocytes was observed in FLT3 CAR T cell-treated animals, however (p<0.05 by t test). To determine potential for increased hematologic toxicity in the presence of greater target antigen levels, we injected MOLM-14 into CD34+ cell-engrafted mice, then treated animals with control or anti-AML CAR T cells. We surprisingly found no decrement in defined hematopoietic stem cell (HSC) or granulocyte macrophage progenitor (GMP) populations, but did observe increased multipotent and common myeloid progenitor (MPP, CMP) cell numbers and an increase in total human cell engraftment 5 days after FLT3 CAR treatment in comparison to non-transduced T cell-treated animals. Relative to CD33 CAR T cells, FLT3 CAR T cells induced less toxicity to HSCs and MPPs and equivalent toxicity to CMPs and GMPs, indicating lower hematologic toxicity with FLT3 targeting.Conclusions: Taken together, these initial data demonstrate potent in vitro and in vivo anti-AML activity with limited hematopoietic toxicity of FLT3 CAR T cell immunotherapy. Future studies are focused on testing the effectiveness on other AML cell lines with varying expression of FLT3. DisclosuresNo relevant conflicts of interest to declare.

FLT3/ITD Cooperates with Rac1/DOCK2 to Modulate the Sensitivity of Leukemic Cells to Chemotherapeutic Agents Via Regulation of DNA Repair Pathways

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm characterized by clonal expansion of myeloid blasts. The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in AML, and patients who harbor a FLT3/ITD mutation have a relatively poor prognosis. FLT3/ITD activates Rac1 GTPase, and both FLT3/ITD and Rac1 play key roles in the signaling network that activates signal transducer and activator of transcription 5 (STAT5) and promotes leukemogenesis.Utilizing a co-immunoprecipitation/mass spectrometry analysis, we found that FLT3/ITD interacts with Dedicator of Cytokinesis 2 (DOCK2), which is a guanine nucleotide exchange factor for Rac GTPases. Expression of DOCK2 is limited to hematopoietic cells, and it is expressed in leukemic blasts of patients with FLT3/ITD AML. Knockdown (KD) of DOCK2 results in reduced Rac1 activity and leads to decreased survival of leukemic cells with elevated FLT3 activity, both alone and in combination with cytarabine (Ara-C) treatment in both in vitro studies and a mouse xenograft model.We further investigated the mechanisms by which Rac1/DOCK2 activity affects cell survival and chemotherapeutic response in FLT3/ITD leukemic cells. Exogenous expression of FLT3/ITD in TF-1 cells resulted in elevated Rac1 activity, an increase in the level of reactive oxygen species, and increased activation of STAT5. Western and qPCR studies in these cells demonstrated a concomitant statistically significant increase in DNA damage response (DDR) factors, including Chk1, Rad51, Wee1, and PIM-1. Furthermore, expression of FLT3/ITD led to increased expression of AP-1 and its downstream targets, the mismatch repair (MMR) factors MSH2, MLH1 and MSH6. These FLT3/ITD-expressing TF-1 cells exhibited markedly increased resistance to Ara-C treatment, and the increase in IC50 positively correlates with the expression level of FLT3/ITD. These effects on DDR factors appear to be dependent on FLT3/ITD activity, as they were not observed in TF-1 cells expressing even relatively high levels of wild-type (WT) FLT3. These findings suggest that enhanced DDR may provide a mechanism of resistance to Ara-C treatment in FLT3/ITD cells.We then utilized DOCK2 KD in patient-derived leukemia cell lines that express FLT3/ITD (MV4;11 and Molm 14) to evaluate whether decreased Rac1 activity would restore sensitivity to Ara-C. DOCK2 KD via shRNA resulted in significantly reduced expression of Meis1 and c-Myb, which are known regulators of FLT3 expression. Accordingly, the expression level and activity of FLT3 in cells with DOCK2 KD are markedly decreased. The DOCK2 KD cells showed reduced STAT5 activity and decreased expression of Chk1, Rad51, Wee1 and PIM-1, as well as lower expression levels of AP1, MSH2, MSH6 and MLH1. Consistent with the reduction in activity of DDR factors, DOCK2 KD cells exhibited significantly increased sensitivity to Chk1 inhibitor MK8776, Wee1 inhibitor MK1775 and Rad51 inhibitor B02. Moreover, synergistic effects between these DDR inhibitors and Ara-C, as indicated by increased apoptosis and reduction in cell proliferation, were observed at markedly lower concentrations in FLT3/ITD cells with DOCK2 KD. In contrast, DOCK2 KD in a leukemia cell line expressing WT FLT3 (REH) did not lead to down-regulation of the activity of FLT3 or DDR factors, and no enhancement in sensitivity toward DDR inhibitors was observed.These findings suggest that FLT3/ITD and Rac1 activity cooperatively modulate DDR and MMR activity in leukemic cells. The increased DNA damage repair activity of FLT3/ITD leukemia cells may play a role in the relatively poor response of FLT3/ITD AML to standard AML chemotherapeutic regimens. Therefore, the addition of DDR inhibitors to conventional chemotherapy may be useful in the treatment of FLT3/ITD AML, and pharmacologic inhibition of the Rac signaling pathways via DOCK2 may provide a novel and promising therapeutic target for FLT3/ITD AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High fms-like tyrosine kinase-3 (FLT3) receptor surface expression predicts poor outcome in FLT3 internal tandem duplication (ITD) negative patients in adult acute myeloid leukaemia: A prospective pilot study from India.

Background & objectives:Mutations in fms-like tyrosine kinase 3 (FLT3) receptor have significant role in assessing outcome in patients with acute myeloid leukaemia (AML). Data for FLT3 surface expression in relation to FLT3 internal tandem duplication (ITD) status and outcome are not available from India. The objective of the current study was to investigate adult patients with AML for FLT3 expression and FLT3 ITD mutation, and their association with long-term outcome.Methods:Total 51 consecutive de novo AML patients aged 18-60 yr were enrolled in the study. FLT3 ITD was detected by polymerase chain reaction (PCR); flowcytometry and qPCR (Taqman probe chemistry) were used for assessment of FLT3 protein and transcript, respectively. Kaplan Meier curves were obtained for survival analysis followed by log rank test.Results:FLT3 ITD was present in eight (16%) patients. Complete remission was achieved in 33 (64.6%) patients. At 57.3 months, event free survival (EFS) was 26.9±6.3 per cent, disease free survival (DFS) 52.0±9.2 per cent, and overall survival event (OS) 34.5±7.4 per cent. FLT3 surface expression was positive (>20%) by flow-cytometry in 38 (88%) of the 51 patients. FLT3 surface expression and transcripts were not associated with FLT3 ITD status. FLT3 expression was significantly associated with inferior EFS (P=0.026) and OS (P=0.018) in those who were negative for FLT3 ITD.Interpretation & conclusions:This study evaluated FLT3 ITD mutation along with FLT3 expression in AML patients, and associated with survival. Negative impact of FLT3 surface expression on survival was observed in AML patients who were FLT3 ITD negative.

AGS62P1, a Novel Anti-FLT3 Antibody Drug Conjugate, Employing Site Specific Conjugation, Demonstrates Preclinical Anti-Tumor Efficacy in AML Tumor and Patient Derived Xenografts

FLT3 (FMS-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase family, which is highly expressed in the blasts of both AML and ALL patients. In addition to FL ligand stimulation, FLT3 can also be activated by two distinct clusters of mutations: internal tandem duplications (FLT3/ITDs) in 20% to 25% patients and point mutations at position D835 in the tyrosine-kinase domain (FLT3/TKD) in 7% to 10% patients. FLT3 tyrosine kinase inhibitors (TKI) are mainly active against FLT3 mutant AML. An antibody drug conjugate (ADC), directed against the extracellular domain of FLT3 may only require FLT3 cell surface expression independent of mutation status. The restricted cellular distribution of FLT3 receptor and a higher expression in AML than in normal bone marrow makes FLT3 a favorable ADC target. Therefore, this ADC based strategy may offer a therapeutic alternative for AML patients independent of FLT3 status.Here, we report the preclinical assessment of a novel FLT3 targeting ADC, AGS62P1. AGS62P1 consists of a human anti-FLT3 monoclonal antibody, site specifically conjugated to a potent cytotoxic payload. FLT3 expression is confirmed in a large panel of AML and ALL tumor cells as well as in AML patient specimens via flow cytometry. The anti-leukemic activity of AGS62P1 was evaluated against AML and ALL tumor cell lines, in vitro and in vivo.AGS62P1 demonstrated strong binding affinity (0.1-0.5 nM) and potent cytotoxic activity in FLT3/ITD and Non-ITD tumor models, in vitro. Cytotoxic IC50 potency for AGS62P1 was 0.5-13 nM in FLT3/ITD and 0.2-12 nM in FLT3 non-ITD models. A fluorescence based assay confirmed that AGS62P1 is rapidly internalized in AML tumor cell lines. AGS62P1 is highly efficacious in FLT3/ITD and non-ITD tumor xenografts, leading to significant tumor growth inhibition or complete tumor regression. In primary AML patient xenograft drug treatment studies, the engraftment and outgrowth of 5/6 samples were significantly reduced when treated with AGS62P1.Taken together our data demonstrate that AGS62P1 exhibits potent antitumor activity against a broad panel of AML tumor models and primary AML samples, regardless of FLT3 status. We believe AGS62P1 may be an effective and alternative therapeutic for AML patients, which can bypass the TKI mediated resistance and deliver target specific effect through a different mode of action. DisclosuresJin:Agensys: Research Funding. Anand:Agensys: Employment. Dick:Agensys: Research Funding.

Targeted Treatment of FLT3 -Overexpressing Acute Myeloid Leukemia with MiR-150 Nanoparticles Guided By Conjugated FLT3 Ligand Peptides

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies. With standard chemotherapies, only 30-50% of younger (aged <60) and 5-10% of older patients with AML survive longer than 5 years. Aberrancy of FMS-like tyrosine kinase 3 (FLT3) occurs in the majority cases of AML. Two major classes of constitutively activating mutations of FLT3, i.e. internal-tandem duplications (ITDs) and tyrosine kinase domain (TKD) point mutations are found in more than 30% of AML cases and usually predict poor prognosis. Overexpression of FLT3 has also been reported in more than 70% of AML cases with a variety of AML subtypes, e.g. MLL (Mixed Lineage Leukemia)-rearranged or FLT3 -ITD AML, and may be associated with poor survival in AML patients. Given the disappointing results with FLT3 tyrosine kinase inhibitors (TKIs) in clinical trials in the past decade, decreasing the overall abundance of FLT3 at the RNA and protein levels would be an alternative strategy to treat AMLs with FLT3 overexpression and/or FLT3 -ITD/TKD mutations.MicroRNAs (miRNA) are a class of small, non-coding RNAs that play important roles in post-transcriptional gene regulation. We recently reported that miR-150 functions as a pivotal tumor-suppressor gatekeeper in MLL-rearranged and other subtypes of AML, through targeting FLT3 and MYB directly, and the MYC/LIN28/HOXA9/MEIS1 pathway indirectly. Our data showed that MLL-fusion proteins up-regulate FLT3 level through inhibiting the maturation of miR-150. Therefore, our findings strongly suggest a significant clinical potential of restoration of miR-150 expression/function in treating FLT3 -overexpressing AML.In the present study, we first analyzed FLT3 expression patterns and prognostic impact in a large cohort of AML patients (n=562). We found that FLT3 is aberrantly highly expressed in FAB M1/M2/M5 AML or AML with t(11q23)/MLL -rearrangements, FLT3 -ITD or NPM1 mutations, and that increased expression of FLT3 is an independent predictor of poor prognosis in patients with FLT3 -overexpressing AML.To treat FLT3 -overexpressing AML, we developed a novel targeted nanoparticle system consisting of FLT3 ligand (FLT3L)-conjugated G7 poly(amidoamine) (PAMAM) dendriplexes encapsulating miR-150 oligos (see Figure 1A). In FLT3 -overexpressing cell lines, the uptake ratios of the G7-FLT3L dendrimers were much higher (50.3~97.1%) than the G7-histone 2B (H2B) control nanoparticles (4.3~33.2%). And the uptake only took minutes. By integrating the miR-150 oligo with G7-FLT3L dendrimers, we constructed the G7-FLT3L-miR-150 dendriplexes, which significantly reduced the viability and increased the apoptosis of MONOMAC-6 cells carrying t(9;11) in a dose-dependent manner. To increase the stability of miR-150 oligos, we incorporated a 2'-o -methyl (2'-O Me) modification into the miRNA oligos. Indeed, the G7-FLT3L nanoparticles carrying 2'-O Me modified miR-150 exhibited a more sustained inhibition on cell growth.In order to further investigate the in vivo therapeutic effects of the miR-150 nanoparticles, we used a MLL -rearranged leukemia model. We transplanted wild-type recipient mice with primary mouse leukemic cells bearing the MLL-AF9 fusion. After the onset of leukemia, the mice were treated with G7-Flt3L or G7-NH2 control nanoparticles complexed with 2'-O Me-modified miR-150 oligos. In these treated animals, G7-Flt3L-miR-150 nanoparticles tended to be enriched in the bone marrow. The G7-Flt3L-miR-150 nanoparticles showed the best therapeutic effect (with median survival of 86 days), as compared with the control group (Ctrl; PBS treated; with median survival of 54 days) or the G7-NH2-miR-150 treated group (with median survival of 63 days). Nanoparticles carrying miR-150 mutant oligos showed no anti-leukemia effect at all. Notably, the G7-Flt3L-miR-150 treatment almost completely blocked MLL-AF9 -induced leukemia in 20% of the mice (Fig. 1B). Furthermore, the G7-Flt3L-miR-150 nanoparticles showed a synergistic effect with JQ1, a small-molecule inhibitor of the MYC pathway, in treating AML in vivo (Fig. 1C).Collectively, we have developed a novel targeted therapeutic strategy to treat FLT3-overexpressing AML, such as MLL-rearranged leukemias, which are resistant to currently available therapies, with both high specificity and efficacy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Combinatorial Targeting of XPO1 and FLT3-ITD Exerts Synergistic Anti-Tumor Effects in FLT3-Mutated Acute Myeloid Leukemias

Internal tandem duplication (ITD) mutations of the fms-like tyrosine kinase-3 (FLT3) gene are common in patients with acute myeloid leukemia (AML) and associated with poor disease prognosis (Kottaridis et al., 2003). FLT3-targeted therapies using novel small-molecule inhibitors such as sorafenib are under development with some notable effects (Adnane et al., 2005). However, acquired resistance to these agents is frequent following long-term therapy. One of the main resistance mechanisms is the acquisition of point mutations in the tyrosine kinase (TK) domains (TKD) ofFLT3, in addition to ITD mutations (Smith et al., 2012; Zhang et al., 2014). Another novel target for AML therapy is exportin-1 (XPO1), one of the major transporters of proteins and mRNAs from the nucleus. High expression of XPO1 is associated with decreased overall survival in AML patients (Kojima et al., 2013). and its levels were increased in patients with FLT3-ITD mutations. Interestingly, targeting CRM1 with the small molecule inhibitor KPT-185 down-regulated FLT3 expression (Ranganathan et al., 2012). The XPO1 inhibitor selinexor (PKT-330) has been developed recently and has shown encouraging anti-tumor effects against AML-initiating cells in primary human AML-engrafted NSG mice (Etchin et al., 2015) and in ongoing clinical trials.In this study, we investigated the anti-tumor activity of combined treatment with sorafenib and selinexor in FLT3-mutant AML cells, including those with ITD and TKD point mutations, (Smith et al., 2012; Zhang et al., 2014). Selinexor alone exerted marked cell killing in human and murineFLT3-mutant AML cells, including those harboring D835Y or ITD with Y842C or F691L mutations. Interestingly, FLT3 and MAPK or FLT3 and AKT signaling pathways were activated by the selinexor treatment for 24 h in murine FLT3 -mutant AML cells. Combination treatment with selinexor and sorafenib demonstrated synergistic or additive pro-apoptotic effects in TKD point- and FLT3-ITD-mutanted AML cells, respectively. Mechanistically, the combination treatment triggered significant suppression of phospho-ERK and phospho-AKT compared to selinexor treatment alone in all examined AML cell lines. Of note, the combination treatment promoted the retention of ERK, AKT, NFκB, and FOXO3a the in nucleus, which could explain the low levels of phospho-ERK and phospho-AKT observed in the cytosol. In addition, a 5-day in vitro combination treatment with extremely low 5 to 10 nM concentrations of each agent promoted myeloid differentiation of FLT3-ITD mutant MOLM14 cells. No cell killing was observed using this treatment scenario. Furthermore, the combination treatment demonstrated profound anti-tumor efficacy in a human FLT3-mutated leukemia model in NOG mice. The median survival was doubled from 16 days in the vehicle-treated mice to 31 or 23 days with selinexor or sorafenib treatments, respectively, and this effect was increased to 51 days in the mice that received the combination treatment (p = 0.0001).Our findings provide a pre-clinical basis for a combinatorial treatment strategy targeting XPO1 and FLT3-ITD in FLT3 -mutated AML patients, especially in those patients who have acquired resistance to FLT3-targeted therapy. DisclosuresZhang:Karyopharm: Research Funding. Ishizawa:Karyopharm: Research Funding. Daver:ImmunoGen: Other: clinical trial, Research Funding.

DOCK2 Interacts with FLT3 and Modulates the Survival of FLT3-Expressing Leukemia Cells

The FMS-like tyrosine kinase-3 (FLT3) receptor gene is the most commonly mutated gene in acute myeloid leukemia (AML), and patients carrying FLT3/ITD mutations have a poor prognosis. Despite continuing progress in the development of more effective FLT3 inhibitors, long-term success in inhibition of FLT3 activity in AML patients is still elusive. In order to achieve a better understanding of FLT3 biology and more effective strategies for the inhibition of FLT3 activity, a screen was performed on leukemia cell lines to search for FLT3-interacting proteins. One of the proteins identified in the screen was dedicator of cytokinesis 2 (DOCK2).The DOCK family of proteins acts as guanine nucleotide exchange factors (GEFs) for Rho family of GTPases, which includes Rac GTPases. DOCK2 expression is limited to hematopoietic cells, and is known to regulate several crucial processes, including lymphocyte migration, activation and differentiation of T cells, and cell-cell adhesion and bone marrow homing of various immune cells.We first verified that DOCK2 is expressed in primary AML samples from patients, and co-immunoprecipitation experiments showed that DOCK2 interacts with both wild-type FLT3 and FTL3/ITD in these cells. Co-immunoprecipitation experiments using leukemia cell lines demonstrated that DOCK2 interacts with FLT3, FLT3/ITD, FLT3/D835Y and FLT3/D835H, and that it predominantly interacts with the unphosphorylated form of FLT3. Knock-down of DOCK2 by shRNA did not significantly affect the growth of cell lines that lack expression of FLT3, but greatly reduced growth of cell lines expressing amplified wild type FLT3 (Sem K2), FLT3/D835H (HB11;19) and FLT3/ITD (MV4;11). Accordingly, colony formation assays revealed that cell lines with elevated expression of wild type or mutant FLT3 produced fewer, smaller and more compact colonies when the expression of DOCK2 was decreased, while colonies from cell lines lacking FLT3 expression showed no significant difference in response to the knock-down of DOCK2. Furthermore, an Annexin V binding assay indicated that reduction in DOCK2 expression level greatly sensitized cells with elevated FLT3 activity (MV4;11 and Sem K2) to cytarabine, resulting in increased apoptosis, but no significant sensitization was observed in cell lines that lack FLT3 expression.These findings demonstrate that DOCK2 interacts with FLT3 in leukemia cell lines, and suggest that this interaction has important roles in regulating the survival of leukemia cells with elevated FLT3 activity, both alone and in combination with conventional anti-leukemic agents. Therefore, DOCK2 is a potential therapeutic target for AML treatment, and better understanding of the interaction between DOCK2 and FLT3 may contribute to the development of novel strategies to effectively inhibit FLT3 activity in AML patients. DisclosuresNo relevant conflicts of interest to declare.

Responsiveness of FLT3-ITD+ AML to Kinase Inhibition Correlates with High Allele Ratio and Relapsed Disease but Not to Inhibition of Canonical Targets

Fms-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase and is the most commonly mutated gene in acute myeloid leukemia (AML). Second generation FLT3 inhibitors such as quizartinib (AC220) are clinically active in relapsed FLT3-ITD+ patients. However, not all patients respond and to date primary resistance has not been characterized. A previous study proposed that AML cells from patients with relapsed FLT3 mutant AML and samples with high allelic burden for FLT3 ITD are more sensitive to FLT3 inhibitor cytotoxicity (Pratz KW, et al Blood 2010). We performed studies to test this hypothesis and determine if cells with homodimeric FLT3 ITD are more heavily dependent on FLT3 ITD for growth and survival than cells expressing heterodimeric FLT3 WT:FLT3 ITD. 16 primary AML samples that contain FLT3 ITD mutations were incubated in increasing concentrations of the second generation FLT3 inhibitor crenolanib and assayed for survival in short term liquid culture assays. Only 6 samples demonstrated greater than 30% inhibition of survival in this culture system whereas 10 samples showed little or no cytotoxic response. Consistent with previous results (Pratz, et. al, Blood 2010), responding samples tended to be from relapsed patients and to have higher FLT3 ITD allelic ratios. We then analyzed FLT3 expression and phosphorylation levels as well as inhibition of and crenolanib inhibition of FLT3 phosphorylation, as well as the canonical downstream signal transduction pathways STAT5, ras/MAPK, and PI3K/AKT/mTOR in 13 FLT3-ITD+ primary AML samples. For 11/13 samples, crenolanib strongly inhibited phosphorylation of FLT3 kinase. However, neither FLT3 protein expression nor baseline phosphorylation level correlated with cytotoxic response in liquid culture assays. Crenolanib inhibited phosphorylation of STAT5, ribosomal S6 and ERK to varying degrees and inhibition of none of these pathways consistently correlated with cytotoxicity. Overall, these results are consistent with the hypothesis that FLT3 ITD mutant AML with high allele burden or relapsed samples are more addicted to FLT3 ITD. To further examine this topic, we studied AML cell lines that express only wild type FLT3 (THP1), both FLT3-ITD and WT FLT3 (MOLM14) or FLT3-ITD but not WT-FLT3 (Mv4;11, TF1-ITD). Growth of all three cell lines expressing FLT3-ITD but not THP1 cells was inhibited by crenolanib. Crenolanib inhibited tyrosine phosphorylation and activation of downstream signaling pathways in all three FLT3-ITD+ cell lines. Importantly, crenolanib was active at 10 nM in TF1-ITD cells but higher concentrations were required to inhibit signaling in Mv4;11 cells. This suggests that allele ratio alone does not determine sensitivity to FLT3 inhibitors. Interestingly, FLT3 ligand (FL) impairs inhibition of FLT3 by kinase inhibitor and, again, mutant cell lines are similarly responsive to FL in the presence of kinase inhibitor. In summary, these data demonstrate that AML cells have variable dependence on mutant FLT3 for survival. Samples with high allele ratio and relapsed samples behave in a manner consistent with oncogene addiction and are likely to show cytotoxicity to FLT3 inhibition. However, the basis for oncogene addiction is unclear and does not depend on activation of the canonical signal transduction pathways known to be downstream of FLT3. Interestingly, a recent unbiased screen of phosphorylated proteins in FLT3-ITD+ AML to predict clinical response to AC220 did not identify tyrosine phosphorylation of canonical FLT3 targets, but correlated clinical response with serine phosphorylation on EEPD1-S160, BCL11A-S630, and RANBP3-S333 (Schaab C, et al. Leukemia 2014). Analysis of the effects of FLT3 inhibitors upon these proteins in FLT3 mutant primary samples and cell lines is ongoing. DisclosuresPerl:Arog pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.