Activin-like Kinase 2 (ALK2/ACVR1) Is a Resistance Factor and Therapeutic Vulnerability to FLT3 Inhibition in Acute Myeloid Leukemia (2022)

Background: FMS-like tyrosine kinase-3 (FLT3), a transmembrane receptor, is frequently mutated in acute myeloid leukemia (AML), and this is associated with poor prognosis. Although FLT3 inhibitors (FLT3i) have significantly improved overall survival, their clinical effectiveness is limited due to treatment resistance, and the molecular mechanism remains unknown. We previously reported that the bone morphogenetic protein (BMP) signaling pathway supports AML cell growth via osteogenic differentiation in mesenchymal stromal cells. However, the role of BMP in FLT3i resistance has not been studied. We hypothesized that BMP type-1 receptor (ALK2/ACVR1) is a resistance factor for FLT3i, and therefore targeting FLT3 and ACVR1 using a novel dual inhibitor, TP-0184, would overcome FLT3i resistance and sensitize AML cells to standard therapy. 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.