Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance in Myeloid Diseases: Poster III
Methods. BaF3 cells harboring various FLT3 mutations, Molm13 and MV4-11 cells with FLT3-ITD mutation, as well as FLT3-wild type BaF3 and OCI-AML3 cells were treated with C82 or TKI (sorafenib or quizartinib) alone, or in combination. Apoptosis and cell cycle distribution were determined by flow cytometry, synergistic cytotoxicity (CI) of the combination treatment was calculated by isobologram analysis, and cellular localization of β-catenin was determined by confocal imaging. Protein expression was measured by western blotting and RNA expression by qRT-PCR.
Results. Combination of C82 with sorafenib synergistically induced apoptosis in BaF3 FLT3-ITD, BaF3 FLT3-D835G, BaF3 FLT3-ITD plus 676, Molm13, and MV4-11 cells, and in sorafenib resistant BaF3 FLT3-ITD plus 842 cells, even when they were co-cultured with human bone marrow-derived mesenchymal stem cells (MSCs) to mimic the AML microenvironment (CI < 1). Similar results were obtained with Molm13 and MV4-11 cells treated with C82 plus quizartinib. Cell cycle analysis showed that the combination of C82 and sorafenib suppressed cell cycle progression and increased the proportion of cells in subG1 and G1 phases. Furthermore, the combination of C82 and sorafenib inhibited nuclear localization of β-catenin and reduced CD44 levels. Pathway PCR array analysis revealed that C82 and sorafenib, either alone or in combination, suppressed the expression of survivin, cyclin D1, and c-Myc, all downstream targets of β-catenin and several frizzled family receptors/co-receptor and ligands, such as FZD1, FZD3, FZD5, FZD6, FZD7, low-density lipoprotein receptor, WNT10B, and WNT3 (≥2 fold). Western blot analysis confirmed that C82 and TKI (sorafenib or quizatinib) either alone or in combination suppressed c-Myc, survivin, and CD44. Selective inhibition of FLT3-ITD downstream signaling indicated that TKI-induced c-Myc inhibition is primarily mediated through the FLT3-ITD/STAT5/c-Myc signaling cascade.
Conclusion: These results demonstrate that combined inhibition of β-catenin and FLT3 tyrosine kinase exerts synergistic cytotoxicity in AML cells with FLT3 mutations. These findings provide a rationale for developing clinical protocols employing β-catenin/CBP modulators and TKIs to overcome FLT3 inhibitor resistance and to determine whether treatment outcomes in patients with FLT3 mutant AML can be improved.
Disclosures: Zhang: Karyopharm: Research Funding . Cortes: BMS: Consultancy , Research Funding ; Teva: Consultancy , Research Funding ; Pfizer: Consultancy , Research Funding ; Novartis: Consultancy , Research Funding ; ARIAD Pharmaceuticals Inc.: Consultancy , Research Funding . Andreeff: Oncoceutics, Inc.: Membership on an entity’s Board of Directors or advisory committees . Carter: PrismBiolab: Research Funding .
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