Abstract 5013: Effects of PP2A-activating drugs on FLT3 inhibitor resistance mediated by diverse mechanisms in acute myeloid leukemia with FLT3-ITD
Authors: Ali Moaath Mustafa , Chatterjee Aditi , Lee Jonelle K. , Scarpa Mario , Baer Maria R. 
Published: April 2023

Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase is present in AML cells in 25% of patients, causing constitutive activation and aberrant signaling. FLT3-ITD+ AML patients have a high relapse rate following chemotherapy and short disease-free and overall survival. Incorporation of FLT3 inhibitors (FLT3is) into therapy for FLT3-ITD+ AML has improved outcomes, but FLT3i efficacy is limited by onset of resistance, which occurs by diverse mechanisms. The tumor suppressor protein phosphatase 2A (PP2A) is inactivated in FLT3-ITD+ AML, and we previously showed that PP2A-activating drugs (PADs), including DT-061 and FTY720, enhance FLT3i efficacy through enhanced proteasomal degradation of c-Myc and Pim-1, mediated by activation of the serine/threonine kinase GSK-3β (Mol Cancer Ther 20:676, 2021). Here we sought to determine whether concurrent treatment with PADs overcomes resistance to FLT3is. We studied MOLM-14 human AML cells, with heterozygous FLT3-ITD, and FLT3i-resistant MOLM-14 cells with D835Y tyrosine kinase domain and F691L gatekeeper FLT3 mutations and M14(R)701 MOLM-14 cells (from Dr. Donald Small, Johns Hopkins) with a G12D NRAS activating mutation. IC50s, determined by WST-1 cytotoxicity assays, of Type 1 FLT3i gilteritinib in MOLM-14, D835Y, F691L and M14(R)701 cells were 9.2, 18.6, 56 and 57.5 nM, and the Type II FLT3i quizartinib, 2.8, 211, 659 and &gt;4,000 nM. Both DT-061 and FTY720 sensitized MOLM-14 D835Y cells to quizartinib (IC50s &lt;10 nM) but not to gilteritinib in cytotoxicity assays and showed synergy with quizartinib (combination indexes 0.6, 0.5), but not with gilteritinib in drug combination studies using Chou-Talalay analysis. In contrast, DT-061 and FTY720 did not sensitize to or synergize with quizartinib or gilteritinib in MOLM-14 F691L cells. Finally, FTY720 markedly sensitized M14(R)701 cells to both quizartinib and gilteritinib and showed substantial synergy (combination indexes &lt;0.1), while DT-061 did not. In mechanistic studies, quizartinib alone did not decrease phospho-FLT3 expression by western blot analysis in MOLM-14 D835Y cells, but DT-061 or FTY720 and quizartinib co-treatment markedly decreased phospho-FLT3, but not FLT3, expression. Thus PADs resensitize cells with D835Y to quizartinib by inactivating FLT3-ITD, thereby enabling the binding of quizartinib to FLT3-ITD. Additionally, while FTY720, but not DT-061, markedly sensitized M14(R)701 cells to quizartinib and gilteritinib, FTY720 and DT-061 increased phosphatase activity similarly in a PP2A immunoprecipitation phosphatase assay, indicating that sensitization by FTY720 likely occurs by a mechanism other than PP2A activation. Potential induction of ceramide accumulation by FTY720, a sphingolipid analog, in M14(R)701 cells is being studied.

Citation Format: Moaath Mustafa Ali, Aditi Chatterjee, Jonelle K. Lee, Mario Scarpa, Maria R. Baer. Effects of PP2A-activating drugs on FLT3 inhibitor resistance mediated by diverse mechanisms in acute myeloid leukemia with FLT3-ITD. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5013.