Background: In 30% of acute myeloid leukemia (AML) patients, the presence of fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) causes constitutive FLT3 signaling, and these patients relapse rapidly following response to chemotherapy. FLT3 inhibitors have limited and transient efficacy, but their efficacy may be enhanced by combination with other drugs targeting FLT3 signaling. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to its proliferative and antiapoptotic effects and also phosphorylates and stabilizes it in a positive feedback loop. FLT3 activation also inhibits the tumor-suppressor protein phosphatase 2A (PP2A), causing stabilization of Pim-1 protein. FLT3 inhibitors and PP2A-activating drugs (PADs) are known to synergistically induce cytotoxicity in cells with FLT3-ITD, and here we studied mechanisms underlying this effect.
Methods: Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 cells, with FLT3-ITD, were cultured with a FLT3 inhibitor, gilteritinib (ASP2215) or quizartinib (AC220), and the PAD fingolimod (FTY720) at pharmacologically relevant concentrations, or DMSO control. Apoptosis was measured by Annexin V/propidium iodide staining. Drug combination indexes were determined by the Chou-Talalay method with CompuSyn software. Pim-1 kinase, Myc, MycSer62, MycThr58, STAT5, phospho-STAT5, PP2A and phospho-PP2A levels were measured by immunoblotting. Cycloheximide was used to assess protein stability.
Results: Concurrent treatment with ASP2215 or AC220 and FTY720 decreased growth and enhanced apoptosis of cells with FLT3-ITD, relative to single drugs, and produced synergistic cytotoxicity. Concurrent treatment sequentially decreased expression of the 44 kDa Pim-1 isoform (Pim-1L) and then c-Myc, via decreased Pim-1L and c-Myc protein stability. Co-incubation with the proteasome inhibitor MG-132 prevented drug-induced degradation and restored Pim-1L and Myc expression in cells co-treated with FLT3 inhibitor and PAD. Drug-induced Myc destabilization/proteasomal degradation was associated with decreased MycSer62 and increased MycThr58 levels. Treatment with the pan-Pim kinase inhibitor AZD1208 prior to ASP2215 or AC220 and FTY720 treatment markedly increased MycThr58 levels but did not affect MycSer62, consistent with both Pim-dependent and -independent effects of FLT3 inhibition and PP2A activation on c-Myc expression.
Conclusions: Combined FLT3 inhibitor and PAD treatment synergistically induces cytotoxicity in cells with FLT3-ITD by enhancing Pim-1 and Myc proteasomal degradation, and effect on Myc is both Pim-1-dependent and -independent. The data support in vivo testing of FLT3 inhibitor and PAD combinations.
Citation Format: Mario Scarpa, Shivani Kapoor, Sikemi Ibikunle, Andre Rush, Patrick R. Baldwin, Danilo Perrotti, Maria R. Baer. FLT3 inhibitors and PP2A-activating drugs synergistically induce cytotoxicity in AML cells with FLT3-ITD through enhanced Pim-1 and Myc proteasomal degradation [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 834.