FLT3-ITD Cell Lines Research Articles

Evaluation of Synergistic Anti-Leukemic Efficacy of PHI-101 in Preclinical Model of FLT3-ITD Acute Myeloid Leukemia

Background: AML is a heterogeneous disease with an overall cure rate of 40-50%. Some types of AML have even poorer prognosis with FLT3 mutant AML patients having only a 20-25% cure rate with chemotherapy alone. In order to improve the outcome for this high-risk AML, a number of tyrosine kinase inhibitors (TKI) have been developed and to date. Given the difficulty of identifying appropriate targets and treatment options for AML, combining drug options for AML treatment may produce clinical benefits. PHI-101 is a selective next-generation type I FLT3 inhibitor that shows potent anti-leukemic activity against AML samples harboring FLT3-ITD and/or TKD mutations. In a clinical trial, PHI-101 demonstrates significant clinical responses and tolerability in refractory or relapsed AML patients as monotherapy. To investigate the optimal way to combine PHI-101 with other therapies, a number of other drugs utilized in treating AML were tested both, in vitro and in vivo to determine synergistic or additive effects that might improve efficacy for relapsed/refractory/elderly FLT3 mutant AML patients. Study Design and Methods: Combination effects of simultaneous versus sequential treatment of PHI-101 with other agents, including daunorubicin, cytarabine, venetoclax (Ven), and azacytidine (Aza) were tested using human leukemia cell lines harboring FLT3-ITD mutations (MV4-11, Molm13, Molm14) or FLT3-wild type as controls. The cell growth inhibition was assessed by CCK-8, MTT and/or CellTiter-Glo assays as well as annexin V-binding by flow cytometry and the Chou-Talalay method was used to calculate the combination index (CI. using Compusyn software. The potential mechanisms of effect on combination therapy were explored by Western blotting of a variety of signal transduction pathways including FLT3 phosphorylation and downstream pathways BCL-2 family expression, and DNA damage. The MV4-11 xenograft mouse model was used to assess in vivo efficacy of PHI-101 combination therapy. Results: Among the substances tested for combination therapy with PHI-101 in FLT3-ITD cell lines MV4-11 and Molm14 cell lines, Aza and Ven treatment both showed synergy with both simultaneous and sequential treatment. Ven was highly synergistic when used simultaneously with PHI-101 in MV4-11 cells. Cell growth inhibition effect (GI 50) of PHI-101, Aza, and Ven used alone was measured first to calculate the CI in combination with increasing concentrations of each drug together of treatment for 72hrs. The GI 50 of PHI-101 in FLT3-mutant cell lines (MV4-11, Molm13, Molm14) were 7.0, 3.8, and 3.4 nM, respectively. The GI 50 of the gilteritinib was 5.7, 10.2, and 10.8 nM, respectively. On the other hand, the GI 50 of Aza were 198.9, 140.4, and 85.4 nM, that of Ven were 38.2, 8.4, and 56.5 nM. PHI-101 was the most efficient antitumor agent compared to other drugs tested in this study. The CI of Aza and PHI-101 was 0.01216 (EC 50), 0.05981 (EC 75), 0.29468 (EC 90) in MV4-11 cells. The observed synergistic effects were confirmed in the other FLT3-ITD cell lines as well. PHI-101 and venetoclax combined therapy in MV4-11 showed synergism (CI<0.1), and the CI was 0.00002 (EC 50), 0.00104 (EC 75), 0.01243 (EC 90). By Western blotting, the combination of Aza or Ven with PHI-101 resulted in a significant induction of p-H2AX and PARP cleavage in the FLT3-ITD cell lines (MV4-11), but not in FLT3-WT cell lines (THP-1). To further validate the synergistic anti-leukemic activity of PHI-101 with these agents, MV4-11-derived mouse xenograft models were generated and treated at various doses. In the MV4-11-xenograft model, PHI-101 alone or combined with Ven or Aza significantly decreased tumor volume. In line with the cell viability data, Ven and Aza combined with PHI-101 induced significant cell-killing in vivo. Conclusion: Treatment of AML cells with the combination of Ven or Aza with PHI-101 in vitro induced rapid induction of apoptosis and inhibition of cell growth that showed significant synergy. In vivo data showed improved efficacy for the combination treatment with these agents. In vitro and in vivo data from the preclinical AML models provides a rationale to evaluate the activity of PHI-101 in combination for patients who are ineligible for more intensive chemotherapeutic induction therapy. Improved anti-leukemic efficacy by combined therapy with PHI-101 represent a promising therapeutic strategy for newly diagnosed or R/R FLT3-mutant AML patients

FLT3-ITD Induces Immune Escape in AML Via up-Regulating CD47 Expression and Decreased Phagocytic Ability of Macrophages

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and confer a poor prognosis. FLT3 inhibitors such as Quizartinib could prolong survival. However, very few relapsed patients are cured. Little attention has been paid to the characteristics of the immune microenvironment in FLT3-ITD AML. Within the bone marrow, Flt3/ITD animals demonstrated significantly increased dendritic cells and T cells levels compared to Flt3/WT mice, which suggested that FLT3-ITD can modulate the immune microenvironment in AML. Here, we found that FLT3-ITD cell lines (MV-4-11 and MOLM-13) induced less LDH release compared to FLT3-WT cell lines (HEL and K562) ( Figure a), indicating less macrophage killing. CD47 gene is an integrin related protein, which acts as an “anti phagocytosis” signal, and it interacts with signal-regulatory protein alpha (SIRPalpha) on macrophages, so that tumor cells expressing CD47 can escape being destroyed by macrophages. Further Flow Cytometry results showed that the CD47 Mean fluorescent intensity (MFI) of the FLT3-ITD group was significantly higher than those of the control group ( Figure b). Therefore, we can ask a hypothesis that FLT3-ITD in tumor cells may leads to decreased phagocytic capacity of macrophages and immune tolerance. To test our hypothesis, the FLT3 wildtype (FLT3-OE, FLT3 over expression) or ITD mutant (FLT3-ITD) stable overexpression HEL/K562 cells were constructed by means of lentiviral transfections. In the FLT3-ITD group, the MFI levels of CD47 was higher than FLT3-OE group and FLT3 NC (FLT3 Normal control) group ( Figure c and d). Green-fluorescent (CFSE-stained) Hel and K562 were then added to cultures of macrophages induced by Peripheral Blood Mononuclear Cell stained with the red dye PKH26 (ratio tumor cell:macrophages, 5:1). Flow Cytometry results showed FLT3-ITD decreased the Phagocytic Capacity of Macrophages ( Figure e and f). The TRANSFAC database was used to predict the transcription factor binding sites of the CD47 promoter. We found that HOXB5 may regulate the expression of CD47 at the transcriptional level. Compared with the FLT3-NC and FLT3-OE group, the relative expression level of the CD47 gene in the FLT3-ITD group was significantly upregulated ( Figure g, p < 0.05). To ascertain the mechanism that HOXB5 regulated CD47, we analysed the location CD47 gene on the chromosome, as well as the sequence of promoter and first non-encoding exon of HOXB5 (−2000 ~ 100 bp) by Jaspar online tool (http://jaspar.genereg.net/). Mechanistically, Dual luciferase reporter gene assay and ChIP experiments suggest HOXB5 directly activate CD47( Figure h and i). Compared to the monotherapy group, Combination therapy with CD47 inhibitor and Quizartinib (AC220) significantly promoted the phagocytic ability of M1-like macrophages against MOLM13 cells ( Figure j and k). The mouse model of AML was constructed by injection of 5x106 FLT3-ITD cells via the tail vein. Mouse models further demonstrate that combination therapy with CD47 inhibitor and Quizartinib significantly reduces the tumor burden and improves survival rate. We first revealed that FLT3-ITD can up-regulates CD47 to promote immune escape of macrophages. These evidences indicates that combination therapy with CD47 inhibitor and FLT3-ITD inhibitor can be a promising therapeutic method for impressive treatment of FLT3-ITD AML patients.

Venetoclax Synergizes with IMGN632, a Novel CD123-Targeting Antibody Conjugated to a DNA Alkylating Payload, By Suppressing DNA Damage Response and Potentiating Apoptosis in Acute Myeloid Leukemia in Vitro Models

CD123, a component of interleukin-3 receptor, is overexpressed on the surface of acute myeloid leukemia (AML) blasts and leukemic stem cells compared with normal hematopoietic progenitors, which makes this antigen an attractive therapeutic target. IMGN632 is a novel antibody-drug-conjugate comprising a high affinity CD123-targeting antibody and DNA alkylating cytotoxic payload. In preclinical studies, IMGN632 demonstrated high potency and selective cytotoxicity against AML leukemic cells vs normal myeloid progenitors, suggesting reduced likelihood of myelosuppression (Kovtun, 2018). Importantly, we previously showed high synergy of IMGN632 combination with BCL-2 inhibitor venetoclax (VEN) and DNA hypomethylating azacytidine (AZA) in AML cell lines and xenograft models (ASH 2020, #617). IMGN632 demonstrated encouraging single agent activity and favorable tolerability in relapsed/refractory AML patients (ASH 2019 #734, ASCO 2020 TPS7563, NCT03386513) and is currently tested in combination with VEN and/ or AZA in relapsed and frontline CD123-positive AML patients (NCT04086264). In this study, we provide new insight into molecular mechanisms underlying the synergistic efficacy of these combinations using AML models in vitro. AML cells with FLT3-ITD mutations expressed higher levels of CD123, as previously reported (Han 2013). FLT3-ITD cell lines were particularly sensitive to IMGN632 with IC 50 values at low ng/ml range. Correspondingly, as evidenced by large scale drug combination screen using BLISS independence model, triple IMGN632/VEN/AZA treatment was synergistic at multiple dosage levels and ratios, suggesting a favorable potency of triple combination in FLT3-mutated subtypes of AML associated with poor prognosis. The efficacy of IMGN632/VEN/AZA combination was however decreased in cells lacking functional TP53. shRNA-mediated knockdown of TP53 in FLT3-ITD MOLM13 cells resulted in increased cell viability, reduced apoptosis and downregulation of cleaved caspase-3 and PARP. While both treated wild-type and TP53 KD cells showed robust overexpression of phosphorylated histone H2AX (p-H2AX), TP53 KD cells expressed higher levels of anti-apoptotic MCL-1 and lower levels of pro-apoptotic BAX, indicative of their intrinsic inability to fully execute apoptosis and resulting in suppressed response to triple combination. Importantly, in sensitive AML cells, IMGN632 induced profound DNA damage, G 1 (MOLM13 and MOLM14 cells) or G 2/M (MV4-11 cells) phase cell cycle arrest and decline in S-phase population, all above augmented by VEN/AZA co-treatment. Observed increase in p-H2AX was accompanied by apoptotic caspase-3 and PARP cleavage and was fully prevented by QVD-OPh caspase inhibitor, suggesting that double-stranded DNA damage and p-H2AX triggered by triple combination were secondary events associated with apoptosis but not the initial cause of cell cycle arrest. Thus, given that cytotoxic payload of IMGN632 is a DNA alkylator, we next tested its impact on mediators of the DNA damage response (DDR). IMGN632 alone induced phosphorylation of p53 at Ser15, a step required for p53 transcriptional activity, and triggered activating phosphorylation of checkpoint kinase Chk1 (Ser345), known to facilitate cell cycle arrest and DNA damage repair, thus preventing treated cells from progressing through S-phase but also increasing the potential of repairing IMGN632-induced DNA damage (Fig. 1A). Upon combined IMGN632+VEN treatment, p53 phosphorylation was sustained, but p-ChK1 decreased, suggesting that VEN may impair protective DDR triggered by IMGN632, resulting in greater cell death. To support this hypothesis, we showed that IMGN632 treatment prior to VEN produced significant synergistic activity (Fig. 1B). Together, these results suggest that VEN, apart from its canonical inhibitory effect on anti-apoptotic BCL-2, exerts previously unrecognized ability to suppress DDR program in AML and augments activity of DNA damaging IMGN632. Failure of cells to sustain DDR in the presence of VEN constitutes a key aspect of high efficacy of IMGN/VEN/AZA combination in AML.

Improved efficacy of quizartinib in combination therapy with PI3K inhibition in primary FLT3-ITD AML cells.

Acute myeloid leukemia is a heterogeneous hematopoietic malignancy, characterized by uncontrolled clonal proliferation of abnormal myeloid progenitor cells, with poor outcomes. The internal tandem duplication (ITD) mutation of the Fms-like receptor tyrosine kinase 3 (FLT3) (FLT3-ITD) represents the most common genetic alteration in AML, detected in approximately 30% of AML patients, and is associated with high leukemic burden and poor prognosis. Therefore, this kinase has been regarded as an attractive druggable target for the treatment of FLT3-ITD AML, and selective small molecule inhibitors, such as quizartinib, have been identified and trialled. However, clinical outcomes have been disappointing so far due to poor remission rates, also because of acquired resistance. A strategy to overcome resistance is to combine FLT3 inhibitors with other targeted therapies. In this study, we investigated the preclinical efficacy of the combination of quizartinib with the pan PI3K inhibitor BAY-806946 in FLT3-ITD cell lines and primary cells from AML patients. We show here that BAY-806946 enhanced quizartinib cytotoxicity and, most importantly, that this combination increases the ability of quizartinib to kill CD34+ CD38-leukemia stem cells, whilst sparing normal hematopoietic stem cells. Because constitutively active FLT3 receptor tyrosine kinase is known to boost aberrant PI3K signaling, the increased sensitivity of primary cells to the above combination can be the mechanistic results of the disruption of signaling by vertical inhibition.

Targeting Extracellular Vesicle Secretion in Combination with Venetoclax Synergistically Induces Apoptosis in FLT3-ITD+ AML

Introduction Intrinsic or acquired resistance to apoptosis is a hallmark of human cancers, including different subtypes of acute myeloid leukemia (AML). Among different mechanisms of achieving resistance, disposal of pro-apoptotic factors via extracellular vesicle (EV) release has been described for several cancer models. In AML, RNA and protein trafficking by leukemia-derived EVs has emerged as a mediator of cell-cell communication and a crucial factor in reprogramming the bone marrow (BM) niche into a leukemia permissive microenvironment. In our study, we aimed to characterize the content of primary AML-derived EVs with a special focus on apoptotic factors, and to evaluate the effect of EV release inhibition with FTY720, a FDA approved oral drug for treatment of multiple sclerosis, on apoptosis in combination with venetoclax (VEN) treatment, an inhibitor of the anti-apoptotic protein Bcl-2. Methods BM-derived EVs were purified from healthy donors (n=3), or patients with newly diagnosed AML (FLT3-WT and -ITD, n=10) after written informed consent and approval by the ethics committee in accordance with the Declaration of Helsinki. EVs were characterized by transmission electron microscopy (TEM), western blotting and nano tracking analysis (NTA). Protein and RNA from EVs were isolated and used for proteomic and RNA analyses. The apoptotic response of FLT3-WT (OCI-AML2, OCI-AML3) or FLT3-ITD (MOLM-13, MV4-11) AML cell lines and primary samples to EV release inhibitor FTY720 was assessed by flow cytometry with AnnexinV/PI. FTY720-induced EV release inhibition and intracellular EV accumulation was determined by Western Blot analysis of EV markers and visualized by immunostaining. Finally, the combination treatment of FTY720 with VEN was tested. Results TEM and immunoblotting confirmed a pure vesicle fraction (30-150 nm) with expression of characteristic markers enriched (CD63, CD81, HSP70) or absent (GAPDH, ß-actin) in EVs. NTA showed no differences in EV numbers released by AML compared to healthy cells. However, AML EVs were significantly larger in size. EV content was significantly different comparing healthy to AML-derived EVs and bioinformatic analysis from RNA sequencing revealed enrichment in pathways involved in regulation of apoptosis specifically in AML FLT3-ITD EVs (Figure 1). Of note, hsa-miR-196b-5p target interaction analysis identified BCL-2 as an exemplary anti-apoptotic target gene (Figure 2). Concordantly, AML cell lines harboring FLT3-ITD showed higher sensitivity to the EV release inhibitor FTY720 compared to FLT3-WT (IC50 = 6.2µM, 6.6µM, 13.7µM and 10.2µM respectively for MOLM-13, MV4-11, OCI-AML2 and OCI-AML3). FTY720 treatment induced intracellular EV accumulation and modulated EV marker expression confirming its effect on the EV machinery. Interestingly, FTY720 co-treatment potentiated apoptosis induced by VEN alone in both FLT3-ITD cell lines while FTL3-WT cells were still resistant to VEN and healthy hematopoiesis was unaffected. Conclusions Taken together, these results suggest that FLT3-ITD AML cells establish a protective mechanism against apoptosis by disposing of positive regulators of apoptosis via EV release. Preventing the disposal of the pro-apoptotic factors in combination with BCL-2 inhibition sensitizes AML FLT3-ITD cells to apoptosis. Additional evaluation in primary patient material and further studies to identify the factors responsible for sensitization to apoptosis upon EV release inhibition are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PIM Inhibition By SEL24/MEN1703 Combines Synergistically with Gilteritinib in FLT3-ITD Preclinical Models of Acute Myeloid Leukemia

BACKGROUND. Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer, derived from an uncontrolled clonal proliferation of malignant myeloid blast cells in the bone marrow, transformed by recurrent genetic alterations (Wu M. et al., J. Hematol Oncol, 2018). SEL24/MEN1703 is a first-in-class, oral, type I dual PIM/FMS-like tyrosine kinase 3 (FLT3) inhibitor investigated in the ongoing Ph2 study in patients with AML (DIAMOND-01, ClinicalTrials.gov identifier: NCT03008187) (Czardybon W. et al., Oncotarget 2018). Gilteritinib is a highly potent and selective oral FLT3 inhibitor, FDA-approved for the treatment of relapsed or refractory AML with FLT3 mutations. Although Gilteritinib showed strong single-agent activity in patients with FLT3+ AML (Levis M. and Perl AE., Blood advances, 2020), the development of Gilteritinib resistance limits durability of the response, indicating that novel combination strategies may be clinically important (Zhang LS. et al., Onco Targets and Therapy, 2022). AIM. The main objective of this study is to assess potential synergistic effects in-vitro and in-vivo of the combination of SEL24/MEN1703 and Gilteritinib in AML cell lines with FLT3-ITD and to elucidate the underlying signaling pathways. METHODS.In-vitro cytotoxicity and apoptosis experiments were carried out in FLT3-ITD AML cell lines (MV4-11, MOLM13, MOLM14), testing SEL24/MEN1703 and Gilteritinib either as single agents or in combination for 72h. The combination index was calculated according to the Chou method (Chou TC., Pharmacol Rev, 2006). In-vitro co-culture experiments were performed with FLT3-ITD cell lines MV4-11 and MOLM14 incubated in the presence of human primary bone marrow stromal cells. In MV4-11 and MOLM13 xenograft models, SEL24/MEN1703 and Gilteritinib were administered per OS every day, for 14 and 22 days, respectively. A signaling pathway analysis (pAKT, pERK, pSTAT5, Mcl-1, c-Myc, Bcl-xL, pS6) was also performed through immuno-capillary electrophoresis on tumor nodules collected from MOLM13 xenograft study. RESULTS. The combination treatment was moderately synergistic/additive in both in-vitro cytotoxicity and apoptosis experiments on MV4-11, MOLM13, and MOLM14, at all concentration levels. However, in co-culture experiments, the presence of primary stromal cells resulted in a significantly increased combination index, in tested AML cell lines. In the in-vivo MV4-11 xenograft model, SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg induced a significant anti-tumor activity as single agent treatment, showing 57.2% and 84.1% of tumor volume inhibition (TVI) respectively, whereas the combination treatment SEL24/MEN1703 plus Gilteritinib induced 99.2% of TVI at day 33 (Figure 1). At day 40, when the Gilteritinib schedule was concluded, SEL24/MEN1703 and Gilteritinib as single agents showed 53.1% and 87.6% of TVI respectively, whereas the combo group induced a complete tumor regression (100% of TVI). Moreover, only the combination of SEL24/MEN1703 and Gilteritinib resulted in a delayed tumor regrowth 23.5 days after discontinuation of the treatment. In the MOLM13 model, at day 29, treatment with SEL24/MEN1703 at 25 mg/kg and Gilteritinib at 3 mg/kg as a monotherapy induced an antitumor activity with 38.8% and 60.1% of TVI respectively, whereas with the combination treatment resulted in a higher TVI (78.3%), however, no complete tumor regression was observed in this model. In the MOLM13 xenograft model, analysis of downstream signaling pathways of FLT3 and PIM kinases showed that the pS6 effector protein is mainly downregulated in the combination treatment group. CONCLUSIONS. Combination therapy with SEL24/MEN1703 and Gilteritinib is moderately synergistic in-vitro, however, results in robust tumor regressions, including complete responses in in-vivo FLT3-ITD models, at tolerable doses. The higher efficacy of this combination in-vivo and compared to monotherapy could be related to a very potent concomitant inhibition of FLT3 and PIM kinases affecting upregulated FLT3 signaling pathway in-vivo as demonstrated by the reduction of phosphorylation of downstream effector proteins, such as pS6. Overall, these experiments have demonstrated the antitumor potential of concomitant inhibition of FLT3 and PIM kinases which could drive a novel therapeutic strategy in AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AD80 INHIBITS FLT3-MEDIATED SIGNALING AND HAS ANTINEOPLASTIC EFFECTS ON ACUTE MYELOID LEUKEMIA CELLS

Acute myeloid leukemia (AML) is one of the most common types of hematological malignancy in adults. Activating mutations in different tyrosine kinases have been identified as important factors in the development and progression of hematological neoplasms. For AML, FLT3-ITD has been described as a key mutation, activating signaling pathways such as PI3K, RAS, and STAT5. AD80 is a multikinase inhibitor able to inhibit the activity of kinases RET, RAF, S6K, and ERK. It is a new compound and, therefore, few studies have been carried out to date verifying its effects on tumor cells. In the present study, the cellular and molecular effects of AD80 in AML cell lines were analyzed. For the study, 6 human (FLT3-ITD: MV4-11 and MOLM13, BCR: ABL1: K562 and KU812, JAK2V617F: SET2 and HEL) and 5 murine (Ba/F3 with activating mutations: BCR::ABL1, BCR:ABL1T315I, JAK2V617F, MPLW515L, CSF3RT618I) cell lines representing different hematological neoplasms were utilized. Cell viability was assessed using MTT assay. Apoptosis, autophagy, and mitochondrial membrane potential were determined by flow cytometry. Colony formation assays were performed to analyze clonogenic ability. Protein expression/activation was assessed by western blot. In all cellular models, AD80 reduced cell viability in a time- and dose-dependent manner (all p < 0.05). MOLM13 and MV4-11 cells were the most sensitive, with IC50 ranging from 0.4 to 1.1 nM. In FLT3-ITD cell lines, AD80 induced apoptosis and autophagy, disrupted mitochondrial membrane potential, as well as decreased clonogenic ability in a dose-dependent manner (all p < 0.05). AD80 markedly decreased the activation of AKT, STAT5, S6RP, and induced PARP1 cleavage and γH2AX. AD80 was more potent in reducing the viability of FLT3-ITD AML cell lines. Indeed, it has been shown that AD80 strongly inhibits wild-type and mutated FLT3 in an in vitro assay (>90% of inhibition; Dar et al. Nature, 486:80–84, 2012). Similarly, AD80 also inhibits, in less extension, wild-type and mutated ABL1 and JAK2, which corroborate our cell viability findings. Previous studies from our group showed that AD80 has effects on AML cell lines without the tyrosine kinase-related mutations, with IC50 values ranging from 1.6 to 27.2 μM. In contrast, AD80 concentrations at 0.64 nM were able to induce apoptosis and disrupt mitochondrial membrane potential at 72 h, and induced autophagy at 24 h of treatment in FLT3-ITD AML cell lines. In the molecular scenario, this compound decreases the expression/activating of proteins related to cell growth and proliferation and increases molecular markers of autophagy and apoptosis. AD80 exhibits antineoplastic effects against FLT3-ITD mutated cell lines at nanomolar concentrations, highlighting a noticeably on-targeted effect, and opening up research possibilities that could potentially evolve to further investigations. FAPESP, CNPq, and CAPES.

AML-326 Novel FLT3-ALK2 Dual Inhibitor TP-0184 Inhibits Leukemia Growth by Targeting Serine Biosynthesis in FLT3-ITD-Positive AML Cells

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) inhibitors have been studied; however, molecular factors contributing to this resistance are unknown. i) To investigate the clinical significance of BMP type 1 receptor ALK2 in AML patients with FLT3-ITD mutations. ii) To elucidate the therapeutic effect of FLT3-ALK2 dual inhibitor TP-0184 on leukemia growth. To determine whether ALK2 is a potential target in FLT3-mutated AML patients, we analyzed OHSU and TCGA datasets. We treated AML cell lines with FLT3-WT or -ITD mutations with ALK2 inhibitors and measured their effect on cell cycle and proliferation. To determine the mechanism of TP-0184, we measured the activation of FLT3 downstream signaling by using western blotting and RNA sequencing. Further, we performed kinase profiling to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, we investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutated PDX and xenograft models. Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (P<0.00001) and predicts poor overall survival (P=0.05). Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD-mutant cell growth at low concentrations (IC50<25 nM), while WT-FLT3 cells were affected only at high concentrations (IC50>100 nM). Interestingly, TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5 and p-ERK, as well as p-PI3K, p-mTOR, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway. Lastly, treatment with TP-0184 significantly prolonged the survival of FLT3-ITD-mutated AML-bearing mice (P<0.0001). Our data indicate that ALK2 is a prognostic marker in FLT3-mutated AML. TP-0184 inhibits cell proliferation by inhibiting FLT3 downstream signaling pathways in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3-ALK2 dual inhibitor. TP-0184 inhibits AML growth in FLT3-mutated PDX and xenograft models.

P472: THE FLT3-ITD RECEPTOR CAN PROMOTE PROTEIN FOLDING IN THE ENDOPLASMIC RETICULUM THROUGH THE OXIDOREDUCTASE ERO1

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy and the most common myeloid tumour in adults. Internal tandem duplications (ITD) in FLT3 receptor appear in 30% of cases and is associated with poor disease outcome. This receptor is synthesized and processed in the endoplasmic reticulum (ER) before being transported to the plasma membrane, where it is activated by ligand binding. Upon activation, it regulates cell differentiation, proliferation, and survival. Mutations in FLT3, such as ITD mutations, not only constitutively activate this receptor, but also prevent its correct processing in the endoplasmic reticulum. Therefore, it is mostly retained in this organelle. Aims: We decided to study if the aberrant accumulation of FLT3-ITD affects the functioning of the ER. Methods: Experiments were performed on wild-type and FLT3-ITD AML cell lines, as well as on stably transfected FLT3 and FLT3-ITD Ba/F3 cell lines. Protein aggregation, reactive oxygen species (ROS) and glutathione (GSH) levels were determined by Thioflavin T, DCFH-DA and Thioltracker Violet fluorescence, respectively. Protein detection was performed by Western Blot. Cell viability and death was analysed by cell count and Trypan Blue exclusion. Results: Protein folding in the ER generates reactive oxygen species (ROS). Antioxidant molecules such as GSH are needed to maintain redox homeostasis. We found that FLT3-ITD cell lines have lower levels of protein aggregates, as well as ROS and GSH. This data could suggest that mutant cell lines have more efficient protein folding mechanisms. In fact, among the ER proteins that are involved in the protein folding process, we found the oxidoreductase ERO1 to be increased in FLT3-ITD cell lines. Supporting our findings, we saw that FLT3-ITD cell lines were less sensitive to protein folding blocking agents, DTT and 2-mercaptoethanol. This was reflected by a slight decrease in cell proliferation compared to wild-type cell lines. We confirmed that the decrease in cell viability in FLT3-ITD cell lines was not accompanied by cell death, as opposed to wild-type cell lines. Under ER stress conditions, such as an accumulation of misfolded proteins in the ER, the unfolded protein response (UPR) is activated. We decided to study if there were differences in the basal levels of UPR by western blot. We found FLT3-ITD cell lines to have lower levels of UPR, reflected by lower protein expression of the active forms of PERK and IRE (p-PERK and p-IRE respectively), together with the protein BiP, the major chaperone found in the ER and binds to misfolded proteins. Altogether, our data suggest that FLT3-ITD cell lines have less ER stress, reflected by lower levels of chaperones and UPR proteins, possibly because they rely on more efficient protein folding mechanisms. To confirm the importance of protein folding process in acute myeloid leukemia, we tested the effect of ERO1 inhibitor (EN460), and found it to have a greater impact in FLT3-ITD cell lines. In order to determine a possible relation between the ITD mutation in the FLT3 receptor and ERO1, we used stably transfected Ba/F3 cell lines carrying the normal or mutated FLT3 receptor. We found ERO1 to be overexpressed on FLT3-ITD Ba/F3 cell lines. Summary/Conclusion: Current AML treatments are mainly based on the administration of FLT3 inhibitors, with limited success. Our data suggest that FLT3-ITD cell lines, with greater resistance to treatments, have more efficient protein folding mechanisms. This process should be studied in more detail to determine its potential as a therapeutic target for AML treatment.

Abstract 5456: Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations

Abstract FMS-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in acute myeloid leukemia (AML), and its activating mutations including internal tandem duplications (ITDs) and missense point mutations in the tyrosine kinase domain (TKD) are found in approximately 30% of AML patients. Although the patients initially well responded to FLT3 inhibitors such as gilteritinib, the most cases relapsed within a few months after the initiation of treatment. For these patients who relapsed after FLT3 inhibitor-based therapy, no effective treatment is available. Mechanisms of resistance are not fully elucidated, but FLT3-ITD-TKD double mutant is considered as one of possible mechanisms. We have discovered a novel FLT3 inhibitor OTS447 that shows the potent and selective inhibition in both FLT3-ITD and FLT3-ITD-TKD double mutants. OTS447 was identified through the screening of our in-house proprietary compound library. It showed potent inhibitory activity against FLT3 with an IC50 value of 0.19 nM. The selectivity of OTS447 was investigated by human kinase profile assay. Among 371 human kinases tested, there were only seven (including FLT3) whose activity were inhibited by 80% or more at 5 nM of OTS447. We then examined the anti-proliferative effects in FLT3-ITD and -wild type (WT) AML cell lines. The proliferation of FLT3-ITD cell lines, MV4-11 and MOLM13, was more strongly suppressed than that of FLT3-WT cell lines. We also investigated the inhibitory effects to FLT3-ITD-TKD double mutants using Ba/F3 cells. FLT3-ITD-D835Y mutant was inhibited as strongly as FLT3-ITD mutant, and FLT3-ITD-F691I mutant was more strongly inhibited than Ba/F3 parental cells. These data suggest that OTS447 has selective inhibitory activity against FLT3 mutants. To confirm that FLT3 inhibition by OTS447 leads to the anti-proliferative effect, FLT3 autophosphorylation and phosphorylation of downstream molecules such as STAT5, ERK and AKT were examined. Both autophosphorylation of FLT3 and phosphorylation of downstream molecules were decreased by OTS447 in dose-dependent manner. Moreover, treatment of OTS447 induced the apoptosis and increased sub-G1 population in MV4-11 cell. Finally, we tested OTS447 anti-tumor effect using MV4-11 mouse xenograft model. OTS447 showed potent growth inhibition against MV4-11 tumor in dose-dependent manner. In summary, we discovered OTS447, a potent and selective FLT3 inhibitor, that can inhibit not only ITD mutation but also ITD-TKD mutations of the FLT3 gene. OTS447 possesses cytotoxic activity induced by inhibition of FLT3 signaling pathway and has anti-tumor activity in mouse xenograft model. We are pursuing further optimization of the inhibitor aiming for the effective treatment of AML patients with several types of FLT3-activating mutations. Citation Format: Yasuhide Okamoto, Naofumi Takamatsu, Takashi Miyamoto, Akiko Taira, Kozue Toyota, Yukiho Imai, Shinobu Fujii, Rumiko Ono, Kyoko Adachi, Yo Matsuo, Suyoun Chung, Jae-Hyun Park. Discovery of OTS447, a highly potent and selective inhibitor of FLT3 for the treatment of AML patients with FLT3-ITD/TKD mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5456.

TP-0184, a Novel FLT3-ALK2 Dual Inhibitor, Targets Amino Acid Transport and Biosynthesis in AML Cells and Sensitizes AML Cells to Chemotherapy and BCL2 Inhibition

Background FMS-like tyrosine kinase 3 (FLT3), a transmembrane receptor tyrosine kinase that is frequently mutated in AML, is associated with poor prognosis. Inhibitors of FLT3 internal tandem duplication (ITD) mutants and wild-type (WT) FLT3 have been studied, but their clinical usefulness is limited owing to treatment resistance. However, the molecular factors contributing to this resistance are unknown. We reported that AML cells induce osteogenic differentiation of bone marrow-derived mesenchymal stromal cells through the bone morphogenetic protein (BMP)-mediated signaling pathway, promoting leukemic growth. However, the effects of targeting BMP signaling in patients with FLT3-mutated AML are unexplored. Here, we hypothesized that the BMP type 1 receptor ALK2 is a key biomarker and a therapeutic target in AML patients with FLT3-ITD mutations and that the FLT3-ALK2 dual inhibitor TP-0184 inhibits leukemia growth.Methods: To determine whether ALK2 is a potential target in AML patients with FLT3-ITD mutations, we analyzed gene expression datasets (OHSU and TCGA). We treated 9 AML cell lines with FLT3-WT or ITD mutations with varying doses of ALK2 inhibitors (LDN-212854 or TP-0184) and measured their effect on cell proliferation and the cell cycle. To determine the mechanism of TP-0184-mediated cell cycle arrest, we measured activation of FLT3 downstream signaling by using Western blotting and RNA sequencing. IncuCyte live-cell imaging was used to determine the apoptotic effects of TP-0184 in combination with chemotherapy or targeted therapy agents. Further, we performed human RTK kinase binding assay to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. Finally, the effect of TP-0184 on AML growth in vivo was investigated using an FLT3-ITD positive AML xenograft model (MOLM13).Results: Analysis of AML datasets showed that ALK2 is significantly upregulated in AML patients with FLT3 mutations compared to those with WT-FLT3 (p < 0.00001) and predicts poor overall survival (p = 0.05). Validating these findings, we found higher ALK2 mRNA expression in AML cell lines with FLT3-ITD mutations than in those with WT-FLT3 (p = 0.039). This suggests that ALK2 could serve as a therapeutic target in AML with FLT3-ITD mutation. Treatment of FLT3-WT and -mutated AML cell lines with the ALK2 inhibitors LDN-212854 and TP-0184 resulted in significant inhibition of FLT3-ITD-mutant cell growth at low concentrations (IC50<25nM), while WT-FLT3 cells were affected only at high concentrations (IC50>100nM). Interestingly, TP-0184 was 10-fold more potent in inhibiting AML cell proliferation than was LDN-212854. TP-0184 induced G1/G0 arrest in AML cell lines with FLT-ITD mutations but had minimal to no effect in FLT3-WT AML cells, suggesting that AML cell lines with FLT3-ITD mutations depend on ALK2 for their survival. Further, we observed that treatment with TP-0184 in AML cell lines significantly inhibited multiple signaling proteins downstream of FLT3, such as p-STAT5, p-MKK3, and p-ERK, as well as p-PI3K, p-AKT, p-mTOR, p-4E-BP1, and p-S6K. Gene expression analysis revealed that treatment with TP-0184 in FLT3-ITD cell lines significantly downregulated the serine biosynthesis pathway, which is essential in these cells (Bjelosevic S. et al., Cancer Discov, 2021). Moreover, molecular docking and kinase-binding studies revealed that TP-0184 is bound to wild-type FLT3 as well as most of the FLT3 mutants with dissociation constants (KD) less than 5nM. These data suggest that TP-0184 inhibits both mutant FLT3 and ALK2 in AML cells. Interestingly, TP-0184 plus chemotherapy showed a synergistic effect only in FLT3-ITD cell lines, whereas TP-0184 plus the BCL2 inhibitor, venetoclax was synergistic in both FLT3-ITD and FLT3-WT cell lines. Lastly, treatment with TP-0184 inhibited AML growth and significantly prolonged survival of FLT3-ITD-mutated AML-bearing mice in a dose-dependent manner (p <0.0001).Conclusion: Our data indicate that ALK2 is a prognostic marker for AML patients with FLT3-ITD mutations. TP-0184 significantly inhibits cell proliferation by inhibiting signaling pathways downstream of FLT3, including serine biosynthesis, in AML cells. Kinase assays confirmed that TP-0184 is a highly specific FLT3 inhibitor as well as an ALK2 inhibitor. TP-0184 sensitizes AML cells to chemotherapeutic agents and targeted therapy and inhibits AML growth in vivo. DisclosuresFoulks: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2. Warner: Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US11040038B2; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: US10752594B2; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: CA3103995A1; Sumitomo Dainippon Pharma Oncology: Patents & Royalties: WO2021102343A1. Battula: Tolero Pharmaceuticals: Research Funding.

FLT3 Inhibition Downregulates EZH2 in AML and Promotes Myeloid Differentiation

Internal tandem duplication mutations in the Fms-like tyrosine kinase 3 (FLT3-ITD) are frequently recurring in AML and confer a poor prognosis. FLT3 inhibitors (FLT3i) such as gilteritinib are efficacious in relapsed AML. Clinical responses to FLT3i include myeloid differentiation of the FLT3-ITD clone in about 50% of patients. How FLT3i induce this response in a subset of patients is unknown.The FLT3i-induced differentiation response seen in clinical trials has not previously been demonstrated in animal models. We modeled FLT3i-induced differentiation in murine Flt3 ITD/ITDDnmt3a -/- AML model (Meyer et al., Cancer Discovery, 2016). Treatment with FLT3i in vitro accelerated differentiation of cKIT+ leukemic splenocytes as assessed by colony morphology in serial re-plating assays. To characterize the differentiation response in vivo, we transplanted CD45.2+ leukemic splenocytes from moribund mice into sub-lethally irradiated healthy congenic CD45.1+ mice. After confirmation of engraftment at 2 weeks post-irradiation, mice were treated with vehicle or gilteritinib for 4 weeks. Animals treated with gilteritinib demonstrated increased neutrophil and decreased stem/progenitor cell populations, recapitulating the clinically observed increase in granulocytic differentiation of the FLT3-ITD clone.We next sought to understand the molecular mechanism of FLT3i-induced differentiation. We used a proteomic-based screen in a human AML cell line treated with FLT3i to identify novel targets of FLT3-ITD that could be potential mediators of the differentiation response. We identified downregulation of Enhancer of Zeste Homolog 2 (EZH2), the catalytic component of the Polycomb Repressive Complex 2 (PRC2). EZH2 and PRC2 were previously shown to be required for leukemic maintenance in mouse models of MLL-AF9 AML. We treated murine Flt3 ITD/ITDDnmt3a -/- cKIT+ leukemic splenocytes with FLT3i or the EZH1/2 inhibitor (EZH1/2i). Both promoted myeloid differentiation to similar degrees as assessed by colony morphology in this model. We hypothesized that FLT3-ITD regulates EZH2 to maintain leukemia cells in a stem/progenitor cell state. We, therefore, characterized the effect of FLT3i on PRC2 in more detail. We confirmed that FLT3i decreases EZH2 protein levels in FLT3-ITD cell lines and primary human AML within 24 hours of treatment as suggested by our proteomic data (Figure 1A-B). We found that the mechanism of EZH2 downregulation is complex with both transcriptional effects and a decrease in EZH2 protein half-life. ChIP-Seq for H3K27me3 demonstrated decreased peaks at the transcription start sites of PRC2 target genes (Figure 1C). RNA-Seq gene expression profiles of FLT3i- and EZH1/2i-treated human AML cells overlapped at 253 differentially expressed genes (Figure 1D). Critically, both FLT3i and EZH1/2i expression profiles enriched in differentiated myeloid cell gene signatures.Overall, we found that EZH2 is a novel, unexpected, and clinically relevant target of FLT3-ITD. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the FLT3i-induced differentiation response seen in patients. These data demonstrate that FLT3-ITD has at least two functions in leukemogenesis, the well described activation of signaling pathways, and second, a previously undefined, regulation of PRC2 to maintain a myeloid stem cell state. Our results may lead to improved approaches to therapy for FLT3 mutated AML. [Display omitted] DisclosuresBernt: Syndax: Research Funding; Merck: Other: Spouse is an employee of Merck.. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.

Modulation of FLT3-ITD Localization and Targeting of Distinct Downstream Signaling Pathways as Potential Strategies to Overcome FLT3-Inhibitor Resistance.

OBJECTIVES: Internal tandem duplications (ITDs) of the Fms-like tyrosine kinase 3 (FLT3) represent the most frequent molecular aberrations in acute myeloid leukemia (AML) and are associated with an inferior prognosis. The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is influenced by the localization of FLT3-ITD depending on its glycosylation status. Different pharmacological approaches can affect FLT3-ITD-driven oncogenic pathways by the modulation of FLT3-ITD localization. AIMS: The objective of this study was to investigate the effects of N-glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) or the histone deacetylase inhibitor valproic acid (VPA) on FLT3-ITD localization and downstream activity. We sought to determine the potential differences between the distinct FLT3-ITD variants, particularly concerning their susceptibility towards combined treatment by addressing either N-glycosylation and the heat shock protein 90 (HSP90) by 17-AAG, or by targeting the PI3K/AKT/mTOR pathway by rapamycin after treatment with VPA. METHODS: Murine Ba/F3 leukemia cell lines were stably transfected with distinct FLT3-ITD variants resulting in IL3-independent growth. These Ba/F3 FLT3-ITD cell lines or FLT3-ITD-expressing human MOLM13 cells were exposed to tunicamycin, 2-deoxy-D-glucose or VPA, and 17-AAG or rapamycin, and characterized in terms of downstream signaling by immunoblotting. FLT3 surface expression, apoptosis, and metabolic activity were analyzed by flow cytometry or an MTS assay. Proteome analysis by liquid chromatography–tandem mass spectrometry was performed to assess differential protein expression. RESULTS: The susceptibility of FLT3-ITD-expressing cells to 17-AAG after pre-treatment with tunicamycin or 2-deoxy-D-glucose was demonstrated. Importantly, in Ba/F3 cells that were stably expressing distinct FLT3-ITD variants that were located either in the juxtamembrane domain (JMD) or in the tyrosine kinase 1 domain (TKD1), response to the sequential treatments with tunicamycin and 17-AAG varied between individual FLT3-ITD motifs without dependence on the localization of the ITD. In all of the FLT3-ITD cell lines that were investigated, incubation with tunicamycin was accompanied by intracellular retention of FLT3-ITD due to the inhibition of glycosylation. In contrast, treatment of Ba/F3-FLT3-ITD cells with VPA was associated with a significant increase of FLT3-ITD surface expression depending on FLT3 protein synthesis. The allocation of FLT3 to different cellular compartments that was induced by tunicamycin, 2-deoxy-D-glucose, or VPA resulted in the activation of distinct downstream signaling pathways. Whole proteome analyses of Ba/F3 FLT3-ITD cells revealed up-regulation of the relevant chaperone proteins (e.g., calreticulin, calnexin, HSP90beta1) that are directly involved in the stabilization of FLT3-ITD or in its retention in the ER compartment. CONCLUSION: The allocation of FLT3-ITD to different cellular compartments and targeting distinct downstream signaling pathways by combined treatment with N-glycosylation and HSP90 inhibitors or VPA and rapamycin might represent new therapeutic strategies to overcome resistance towards tyrosine kinase inhibitors in FLT3-ITD-positive AML. The treatment approaches addressing N-glycosylation of FLT3-ITD appear to depend on patient-specific FLT3-ITD sequences, potentially affecting the efficacy of such pharmacological strategies.

Concurrent Treatment with Pim Kinase Inhibitor Downregulates Alternative Non-Homologous End-Joining Repair and Decreases Genomic Instability in FLT3-ITD Cells Treated with Topoisomerase 2 Inhibitor Chemotherapy Drugs

Internal tandem duplication of fms -like tyrosine like kinase 3 (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML) cases, causing constitutive FLT3 signaling. While FLT3-ITD AML patients generally achieve remission following combination chemotherapy including cytarabine and a topoisomerase (topo) 2 inhibitor, they relapse rapidly, and FLT3-ITD AML cells frequently have new structural chromosome changes at relapse. Genomic instability in FLT3-ITD AML appears to result from increased generation of reactive oxygen species, which cause DNA double-strand breaks (DSBs), and upregulated DNA DSB repair by an error-prone alternative non-homologous end-joining (Alt-NHEJ) pathway.Pim-1 is an oncogenic serine/threonine kinase that is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, creating a positive feedback loop in FLT3-ITD cells. Pim kinase inhibitors are in clinical trials. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances apoptosis induction by topo 2 inhibitors in FLT3-ITD cells. Here, we tested the effect of concurrent Pim kinase inhibitor treatment on Alt-NHEJ repair of topo 2 inhibitor-induced DNA DSBs and on genomic instability.Functional changes in DSB repair activities induced by treatment with the topo 2 inhibitor daunorubicin (DNR) in the absence and presence of the pan-Pim kinase inhibitor AZD1208 were studied in Ba/F3-ITD cells, with FLT3-ITD, using stably integrated green fluorescent protein (GFP)-based DSB repair reporter constructs DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical (C-)NHEJ and Alt-NHEJ, respectively. DNR, which induced DNA DSBs, did not induce HR or C-NHEJ repair activity, but induced a 50% increase in Alt-NHEJ activity at 36 hours. Concurrent treatment with AZD1208, which increased DNR-induced DNA DSBs more than two-fold, completely abrogated the DNR-induced increase in Alt-NHEJ repair activity. Treatment of Ba/F3-ITD cells with the topo 2 inhibitors mitoxantrone (MXR) or etoposide (VP-16) also induced Alt-NHEJ repair activity, which was abrogated by concurrent treatment with AZD1208.We next studied mechanisms underlying Pim kinase inhibitor downregulation of Alt-NHEJ. The effects of DNR, MXR or VP-16 on cellular and nuclear expression of the Alt-NHEJ proteins poly (ADP-ribose) polymerase (PARP) 1, DNA polymerase θ, DNA ligase 3 and co-factor X-ray repair cross-complementing protein 1 (XRCC1) were measured in the FLT3-ITD cell lines Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 in the absence and presence of AZD1208. Cellular and nuclear expression of XRCC1 and DNA ligase 3, but not PARP or DNA polymerase θ, decreased in cells co-treated with AZD1208 and topo 2 inhibitors, compared to topo 2 inhibitors alone. XRCC1 was identified as a potential direct Pim-1 substrate by global profiling using the reverse in-gel kinase assay, and was confirmed as a robust Pim substrate in a reverse in-gel kinase assay. This provides a potential mechanistic link between Pim-1 kinase and ALT NHEJ.Alt-NHEJ repair is associated with genomic instability characterized by increased chromosome deletions, insertions and translocations. We studied induction of chromosome changes by topo 2 inhibitor treatment with and without Pim kinase inhibitor in FLT3-ITD cells. Ba/F3-ITD and 32D-ITD cells were incubated with DNR and/or AZD1208, or DMSO control, for 36 hours, then prepared for cytogenetic analysis.For each treatment, 20 consecutive metaphases were analyzed for induction of chromatid breaks and exchanges. No chromosome changes were seen in either cell line with DMSO or AZD1208 treatment. In Ba/F3-ITD cells, chromosome abnormalities were seen in 17 of 20 metaphases after DNR treatment, but only 8 of 20 after combination treatment (p=0.008). In 32D-ITD cells, chromosome abnormalities were seen in 8 of 20 metaphases with DNR, and 0 metaphases with combination (p=0.003).Thus concurrent treatment with Pim kinase inhibitor abrogates induction of Alt-NHEJ repair by topo 2 inhibitor treatment in FLT3-ITD cells and decreases the genomic instability that may contribute to rapid relapse of FLT3-ITD AML after chemotherapy. The data support further preclinical and clinical testing of Pim kinase inhibitors with chemotherapy in AML with FLT3-ITD. DisclosuresTron:AstraZeneca: Employment, Equity Ownership.

The Novel, Irreversible FLT3 Inhibitor FF-10101 Demonstrates Broad in Vitro Activity Against FLT3 Inhibitor Resistance Mutations

Background: Small-molecule FLT3 tyrosine kinase inhibitors (TKIs) have improved remission rates in relapsed/refractory AML driven by FLT3 internal tandem duplication (ITD) mutations, although nearly all responders eventually relapse. Acquired clinical resistance to type II inhibitors such as quizartinib and sorafenib has largely been due to kinase domain (KD) mutations that destabilize drug binding. Early inhibitors, limited by short half-life and fluctuating plasma levels, were also only able to transiently inhibit FLT3. FF-10101 is a first-in-class, type I inhibitor that covalently binds FLT3 irreversibly, suggesting its activity should be limited by the half-life of FLT3 itself. In a profiling assay of 216 human recombinant kinases FF-10101 showed selectivity for wild type FLT3 and FLT3 D835Y. It also demonstrated potent activity against FLT3-ITD AML cell lines harboring TKI-resistant D835Y and Y842C/H mutations. Here, we report in vitro testing of FF-10101 against 19 additional FLT3 TKI-resistant mutations, as well as six cysteine-to-serine mutations within the KD that could impact covalent drug binding. Profiling of this activity is relevant as FF-10101 enters single-agent phase 1/2a clinical trial in relapsed/refractory FLT3 mutant AML.Results: FF-10101 was profiled in an in vitro viability assay against murine BaF3 and human AML cell lines harboring a spectrum of mutations. FF-10101 inhibited the proliferation of FLT3-driven Molm14 and MV4;11 cells (IC50 4.1 and 1.1 nM, respectively). BaF3 FLT3-ITD cells were comparably inhibited (IC50 3.1 ± 1.6 nM), while growth of BCR-ABL-driven K562 cells were unaffected. To confirm covalent binding of FF-10101, we then analyzed its activity against all cysteine residues in the FLT3 KD. Every C->S substitution except C695S remained sensitive to FF-10101, confirming C695 as the FF-10101 binding site and suggesting C695S may impart clinical resistance to FF-10101. We next assessed 10 BaF3 FLT3-ITD cell lines harboring mutations at D835 and the gatekeeper F691, given discovery of resistance mutations at these sites in patients treated with AC220 and sorafenib. FF-10101 inhibited F691L and all substitutions/deletions at the D835 residue. N676K, a mutation found in a patient at relapse on midostaurin, was also potently inhibited. Notably, F691L previously exhibited relative resistance to crenolanib, a reversible, type I FLT3 inhibitor. Beyond clinically described mutants, we also tested FF-10101 against six mutations previously identified in mutagenesis screening assays. A D698N mutation found in separate crenolanib and PLX3397 mutagenesis assays demonstrated relative resistance to crenolanib but was sensitive to FF-10101 (IC50 8.4 ± 7.8 nM), as were four D839 amino acid substitutions identified in the PLX3397 mutagenesis assay alone. Alternatively, a Y693C mutation found in a crenolanib mutagenesis screen remained relatively insensitive to FF-10101. Finally, two resistance mutations co-occurring in the same KD are well-described in chronic myelogenous leukemia (CML) patients treated with sequential TKI therapy. While not yet found in AML patients treated similarly, we hypothesize that analogous compound mutations could emerge. As such, we tested FF-10101 against BaF3 FLT3-ITD cells containing three compound mutations. Unlike crenolanib, FF-10101 potently inhibited D698N/D835V, but F691L/D835V and Y693C/D835V each exhibited relatively moderate resistance to both drugs. Analysis of additional activation loop (AL) mutations (N831H/K, R845G, G846R, Y842S) and biochemical studies of FLT3 phosphorylation and downstream effector pathways in response to FF10101 is ongoing and will be presented.Conclusions: FF-10101 is a type I, irreversible FLT3 inhibitor that has demonstrated potent activity against all mutations associated with clinical resistance to FLT3 TKIs. Expanded profiling shows anti-proliferative activity against nearly all mutations associated with FLT3 TKI resistance except for C695S (the site of FF-10101 binding) and the crenolanib-resistant Y693C mutation, which has yet to be associated with clinical TKI resistance. These data suggest FF-10101 has potential to be clinically active against the majority of FLT3 TKI resistant KD mutations. FF-10101 is currently beginning phase I/2a clinical assessment as a single agent in patients with relapsed/refractory AML harboring a FLT3 mutation. [Display omitted] DisclosuresSmith:Fujifilm: Research Funding; Astellas: Research Funding.

Inhibition of Bcl-2 Synergistically Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia.

To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) and the underlying molecular mechanism. Using both FLT3-ITD cell lines and primary patient samples, Annexin V-FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot analysis was performed to assess changes in protein expression levels of members of the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of proteins. The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of gilteritinib and venetoclax. Lentiviral overexpression of Mcl-1 was used to confirm its role in cell death induced by midostaurin or gilteritinib with venetoclax. Changes of Mcl-1 transcript levels were assessed by RT-PCR. The combination of midostaurin or gilteritinib with venetoclax potently and synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in "free" Bim, leading to synergistic induction of apoptosis. In vivo results show that gilteritinib in combination with venetoclax has therapeutic potential. Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of midostaurin and gilteritinib in FLT3-mutated AML.See related commentary by Perl, p. 6567.

Homoharringtonine Combined with the Heat Shock Protein 90 Inhibitor IPI504 in the Treatment of FLT3-ITD Acute Myeloid Leukemia

As a heterogeneous group of clonal disorders, acute myeloid leukemia with internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) mutation usually shows an inferior prognosis. In the present study, we found that homoharringtonine (HHT), a protein translation inhibitor of plant alkaloid in China, exhibited potent cytotoxic effect against FLT3-ITD (+) cell lines and primary leukemia cells, and a remarkable synergistic anti-leukemia action was demonstrated in vitro and in vivo in xenograft mouse models when co-treated with the heat shock protein 90 inhibitor IPI504. Mechanistically, HHT combined with IPI504 synergistically inhibited the growth of leukemia cells by inducing apoptosis and G1 phase arrest. This synergistic action resulted in a prominent reduction of total and phosphorylated FLT3 (p-FLT3) as well as inhibition of its downstream signaling molecules such as STAT5, AKT, ERK and 4E-BP1. Furthermore, co-treatment of HHT and IPI504 led to a synergistic or additive effect on 55.56%(10/18) of acute myeloid leukemia cases tested, including three relapsed/refractory patients. In conclusion, our findings indicate that the combination of HHT and HSP90 inhibitor provides an alternative way for the treatment of FLT3-ITD positive acute myeloid leukemia, especially for relapsed/refractory AML.

Abstract 2371: Entospletinib, a potent SYK inhibitor, blocks constitutive and FCgRI activated signaling in FLT3-ITD cell lines

Abstract Spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase, is an important mediator of immune receptor signaling in B cells, mast cells, neutrophils, and macrophages. SYK is expressed in 90% of AML blasts and has been reported to be is constitutively activated (Hahn et al., Cancer Cell 2009). High expression of phosphorylated SYK (pSYK) is associated with unfavorable outcomes, independent of other AML prognostic predictors such as age, cytogenetics, and white blood cell count (Boros et al., Oncotarget 2015). Entospletinib (ENTO) is a potent and selective inhibitor of SYK currently in clinical trials for the treatment of AML as monotherapy and in combination with standard-of-care therapies. We investigated the effects of ENTO on SYK phosphorylation and signaling in FLT3-ITD mutant AML cells. We found that ENTO blocked constitutive phosphorylation of pSYKY352, pSTAT5Y694, and MYC protein expression in FLT3-ITD-driven AML cell lines, MV4-11 and MOLM14. The concentration of ENTO required to inhibit pSYKY352 and pSTAT5Y694 correlated with inhibition of cell viability. Phosphorylation at these sites was inhibited by the selective FLT3 inhibitor, quizartinib; but not inhibited by Src family kinase inhibitor (dasatinib) or JAK inhibitors (ruxolitinib or tofacitinib). Further, a second-generation, highly selective SYK inhibitor, GS-9876, failed to inhibit the constitutive phosphorylation of these sites. Comparison of the biochemical and cellular kinase activities of the SYK and FLT3 inhibitors suggests that the observed inhibition of constitutive phosphorylation of pSYKY352 and pSTAT5Y694 and MYC expression was mediated by the direct activity of ENTO against FLT3-ITD, rather than SYK. Activation of FcγRI has been previously shown to activate SYK (Oellerich et al., Blood 2013). We demonstrate SYK activation at pSYKY348 and pSYKY323 after stimulation of the FCγRI with human IgG in the FLT3-ITD mutant cell line, MV4-11. Downstream signaling molecules pERK1/2T202/Y204, pVAV1Y160, and pLYNY507 were also activated by FCγR stimulation. ENTO inhibited phosphorylation of pSYKY348, pSYKY323 and pVAV1 Y160. The second-generation SYK inhibitor, GS-9876, also inhibited FCγRI-mediated activation of pSYKY348,pSYKY323 and pVAV1 Y160, suggesting that these phosphorylation events were mediated by SYK activity. In conclusion, ENTO inhibited constitutive and FCγRI-activated SYK phosphorylation and signaling pathways in FLT3-ITD mutant cell lines, resulting in inhibition of cell viability. Inhibition of constitutive SYK phosphorylation is mediated by direct activity of ENTO against FLT3-ITD; however, inhibition of FCγRI activated SYK results from direct inhibition of SYK. Citation Format: Stacey L. Tannheimer, Adam Kashishian, Rick Sorensen, Kathleen S. Keegan. Entospletinib, a potent SYK inhibitor, blocks constitutive and FCgRI activated signaling in FLT3-ITD cell lines [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 2371.

Abstract 2363: Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD+ AML

Abstract Activating internal tandem duplication (ITD) mutations in the FMS-like receptor tyrosine kinase 3 (Flt3) are found in approximately 30% of acute myeloid leukemia (AML) patients. Flt3-ITD expression is associated with aggressive, chemotherapy-resistant disease and decreased overall survival. Multiple selective Flt3 kinase inhibitors have been developed as targeted therapy for Flt3-ITD+ AML. However, the clinical utility of these kinase inhibitors has been limited by the rapid recurrence of drug-resistant disease that often results from Flt3 kinase domain mutations that prevent inhibitor action. Recent studies revealed that multiple non-receptor tyrosine kinases are over-expressed in AML, including the myeloid Src-family kinases Hck, Lyn and Fgr, and may cooperate with Flt3 to drive disease. These observations suggest that kinase inhibitors with specificity profiles targeting Flt3 and these AML-associated cytoplasmic kinases may be less prone to acquired resistance. In the present study, we determined the contributions of Hck and Fgr kinase activities to Flt3-ITD+ AML cell growth using a chemical genetics approach. While RNAi-mediated knockdown of each of these kinases reduces proliferation and increases apoptosis in primary AML cells, the impact of pharmacologic inhibition of their kinase activities on AML cell growth is not known. Because no selective Hck or Fgr inhibitors exist, we developed kinase domain mutants that desensitize each kinase to the broad-spectrum SRC-family kinase inhibitor, A-419259. Examination of a previous crystal structure of Hck with A-419259 bound to the active site suggested a role for the ‘gatekeeper’ residue in compound binding. Substitution of the gatekeeper threonine of Hck and Fgr with methionine conferred 3-fold resistance to A-419259 in vitro without diminishing kinase activity. When expressed in human TF-1 myeloid cells transformed with Flt3-ITD, these inhibitor-resistant Hck and Fgr mutants conferred resistance to growth inhibition by A-419259 to a similar extent, while expression of the corresponding wild-type kinases was without effect. The sensitivity of the TF-1/Flt3-ITD cell lines to A-419259 correlated with inhibition of mutant Hck and Fgr kinase activity in the cells. Our findings suggest that suppression of AML cell growth by A-419259 is due in part to inhibition of Hck and Fgr, and identify these AML-associated kinases as therapeutic targets. Ongoing studies are addressing the role of Hck and Fgr as inhibitor targets in Flt3-ITD+ AML cell lines and patient samples as well as the propensity of A-419259 and other multi-targeted inhibitors of AML-associated kinases for acquired drug resistance. Citation Format: Ravi K. Patel, Mark Weir, Sabine Hellwig, Heather Dorman, Thomas E. Smithgall. Targeting Src-family kinases to combat acquired inhibitor resistance in FLT3-ITD+ AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2363. doi:10.1158/1538-7445.AM2017-2363

A Phase I Study of the Multi-Targeted Tyrosine Kinase Inhibitor Cabozantinib in Patients with Acute Myeloid Leukemia

Cabozantinib is a multi-targeted tyrosine kinase inhibitor (TKI) of FLT3, MET, AXL, VEGFR, and KIT, all of which are implicated in the pathogenesis of acute myeloid leukemia (AML). Cabozantinib has been studied in clinical trials of solid tumors, and is FDA approved for use in medullary thyroid and renal cell carcinoma. Despite multiple relevant targets, cabozantinib has not been previously studied in AML. Given up-regulation of these key enzymes and related pathways in AML, we sought to assess the safety and tolerability of cabozantinib in this population.The primary objective of this phase I, 3+3 cohort dose-escalation study was to define the maximum tolerated dose (MTD) of cabozantinib in patients with AML. Eligibility included adults ≥ age 18, with relapsed or refractory (R/R) AML, or those aged ≥ 70, with new diagnosed AML but ineligible for or declining conventional therapy. Study treatment required a white blood cell count (WBC) of < 30,000/mm3, but concurrent treatment with hydroxyurea was allowed to control WBC during the first 15 days of cycle 1. The first dose level was 40MG by mouth daily (QD), a dose previously demonstrated to be well-tolerated in solid tumor malignancies. Cabozantinib was administered in 28 day cycles, and dose escalation occurred via 3 cohorts (40MG QD, 60MG QD, 80MG QD). Escalation to each successive dose level was guided by incidence of dose limiting toxicity (DLT) during cycle 1 of study treatment. Pharmacodynamic evaluation of serial plasma samples obtained from patients, by use of a plasma inhibitory assay (PIA), was used to assess FLT3 inhibitory activity in FLT3-ITDcell lines. Response was determined by bone marrow biopsy after the first cycle of treatment, and those achieving complete or partial remission, or with stable disease and clinical benefit, could continue on study until disease progression, intolerable toxicity, or upon receipt of 12 cycles.18 patients were enrolled on study. The median age was 68 (range 27-85); 12 (67%) were male, and 14 (78%) were Caucasian. FLT3-ITD mutations were detected in 5 (23%) patients, and NPM1 mutations in none. 16 patients (89%) had R/R AML, and 2 patients (11%) had newly diagnosed disease. The majority (75%) of those with R/R AML had been treated with two or more lines of prior treatment. 3 patients were enrolled at the first dose level (40MG QD), of whom one experienced >G3 transaminitis, thought unrelated to cabozantinib, but could not be dosed for this reason and was therefore replaced. No DLTs were detected at the first dose level among the 4 studied patients. However, two of the three patients enrolled at the second dose level (60MG QD) experienced DLT, one with grade 3 pancreatitis and the other with grade 3 transaminitis. Three patients were then enrolled at the next lower level, dose level 1 (40MG QD), of whom two could not complete the DLT period, one because of disease progression and the other due to an infected aortic ulceration unrelated to study treatment. These two patients were replaced, and no additional DLTs were noted. 40MG daily was therefore determined to be the MTD, and 6 patients were subsequently treated in an expansion cohort at this dose. Additional ≥ grade 2 toxicities, possibly or probably related to cabozantinib, each affected 3 or less patients, and included fatigue, nausea, anorexia, constipation, oral mucositis, palmar-plantar erythrodysesthesia, transaminitis, and electrolyte imbalance. Fifteen patients could be assessed for response on study. No patients had a marrow response per formal criteria. Four patients had a reduction in peripheral blasts during treatment, of whom 2 transiently cleared their circulating blasts (one with FLT3-ITD). One patient experienced a reduction in marrow blasts, and one had stable marrow disease. FLT3 inhibitory activity of serially collected patient plasma samples, as determined by the ex vivo PIA, consistently revealed potent and sustained inhibition of FLT3 auto-phosphorylation in FLT3-ITDmutant cell lines by day 8 of cycle 1.Cabozantinib, a multi-targeted TKI, is well-tolerated in patients with AML at the MTD of 40 MG daily. In this molecularly heterogeneous, heavily pre-treated population of AML patients, clinical response to cabozantinib monotherapy was limited. However, we found that cabozantinib inhibits FLT3-ITD tyrosine kinase in a potent and sustained fashion. Additional clinical study of cabozantinib among patients with FLT3 mutant AML is therefore warranted. DisclosuresFathi:Celgene: Consultancy, Research Funding; Merck: Other: Advisory Board participation; Seattle Genetics: Consultancy, Other: Advisory Board participation, Research Funding; Agios Pharmaceuticals: Other: Advisory Board participation; Bexalata: Other: Advisory Board participation. Levis:Millennium: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi-Sankyo: Consultancy, Honoraria; Astellas: Consultancy, Honoraria, Research Funding. Avigan:Astex: Research Funding; DCPrime: Research Funding. Hobbs:Constellation: Other: Advisory Board participation; Incyte: Other: Advisory Board participation.