Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster I
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, apoptosis, Translational Research, Combination therapy, Diseases, Therapies, metabolism, Myeloid Malignancies, Biological Processes, molecular biology, Study Population, Animal model
Methods: We assessed the association of ACVR1 expression with molecular mutations in AML by analyzing gene expression datasets (OHSU and TCGA). We also measured ACVR1 expression using qRT-PCR in FLT3-mutant AML cell lines (molm-13, molm-14, and MV4-11) treated with different doses of FLT3i (gilteritinib or midostaurin), as well as in peripheral blood mononuclear cells isolated from patients with FLT3-mutant AML treated with FLT3i. To determine the role of ACVR1 in FLT3i resistance, we knocked down ACVR1 in FLT3-mutant cells, treated them with various doses of FLT3i, and measured apoptosis using IncuCyte. We also measured the effect of TP-0184 on cell proliferation and the cell cycle in FLT3–wild-type (WT) and FLT3-mutant cells. To determine the mechanism of TP-0184–mediated cell cycle arrest, we measured the effect of TP-0184 on cell signaling pathways using Western blot analysis and RNA sequencing. We also performed human RTK kinase binding assay to understand the binding specificity of TP-0184 with 11 different FLT3 mutants. MTT assay was used to determine the synergistic effect of TP-0184 with cytarabine or venetoclax (BCL2 inhibitor). We investigated the effect of TP-0184 on AML growth in vivo using FLT3-mutant and FLT3-WT AML xenograft and patient-derived xenograft models.
Results: Analysis of AML datasets showed that ACVR1 expression is upregulated in FLT3-mutant compared with FLT3-WT AML patient samples (p<0.01) and predicts poor overall survival (p=0.05). ACVR1 expression was also higher in FLT3-mutant than in FLT3-WT cell lines (p=0.039). Interestingly, ACVR1 expression was upregulated in patient samples of FLT3-mutant AML treated with gilteritinib (p=0.02) and FLT3-mutant cell lines treated with FLT3i in a dose- and time-dependent manner. Moreover, ACVR1 knockdown in FLT3-mutant cell lines sensitized AML cells to FLT3i 2- to 3-fold compared with scrambled control cells, suggesting that ACVR1 is a resistance factor in AML cells treated with FLT3i, and that targeting ACVR1 could sensitize AML cells to FLT3i. In FLT3-WT and FLT3-mutant AML cell lines treated with TP-0184, cell growth was significantly reduced in FLT3-mutant cells (IC50<25nM) but not in FLT3-WT cells. TP-0184 induced G1/G0 arrest in FLT3-mutant cells but had minimal or no effect in FLT3-WT cells, suggesting that TP-0184 overcomes ACVR1-mediated resistance in FLT3-mutant cells. Treatment with TP-0184 significantly inhibited multiple proteins downstream of FLT3, including STAT-5, ERK, PI3K, and mTOR pathways, and decreased in pSmad1/5 proteins downstream of ACVR1 in AML cells. These findings confirmed that TP-0184 inhibits both mutant FLT3 and ACVR1 in AML cells. Gene expression analysis revealed that TP-0184 significantly downregulated the serine biosynthesis pathway in FLT3-mutant cell lines. Moreover, molecular docking and kinase-binding studies confirmed that TP-0184 binds to FLT3 mutants with high affinity (KD=<5nM). Interestingly,TP-0184 with venetoclax or cytarabine showed a synergistic effect in FLT3-mutant cells (combination index 0.01; p<0.01). In vivo data also showed that TP-0184 inhibited AML growth and significantly prolonged survival in FLT3-mutant AML xenograft and patient-derived xenograft models (p<0.01).
Conclusion: Our data indicate that ACVR1 causes resistance to FLT3 inhibitors. TP-0184, a dual inhibitor, targets mutant FLT3 and ACVR1 in AML cell lines. TP-0184 sensitizes AML cells with chemotherapy and venetoclax and inhibits AML growth in vivo.
Disclosures: Borthakur: Pacylex, Novartis, Cytomx, Bio Ascend: Membership on an entity's Board of Directors or advisory committees; Astex Pharmaceuticals, Ryvu, PTC Therapeutics: Research Funding; Catamaran Bio, Abbvie, PPD Development, Protagonist Therapeutics, Janssen: Consultancy. Foulks: Sumitomo Pharma Oncology, Inc.: Current Employment, Patents & Royalties: WO2021102343A1; CA3103995A1; US11040038B2. Warner: Sumitomo Dainippon Pharma Oncology: Current Employment, Patents & Royalties: WO2021102343A1; CA3103995A1; US11040038B2; US10752594B2. Battula: Tolero Pharmaceuticals, Inc.: Research Funding; Nektar Therapeutics: Research Funding; Daiichi Sankyo: Research Funding; Y-mAbs Therapeutics: Research Funding.
See more of: Oral and Poster Abstracts