Abstract 3968: AXL is responsible for much of TKI-mediated adaptive resistance in FLT3/ITD AML cells

Abstract

Abstract FMS-like tyrosine kinase-3 (FLT3) is the most frequently mutated receptor tyrosine kinase (RTK) in acute myeloid leukemia (AML). Internal tandem duplication mutations (ITD) confer a poor prognosis on FLT3/ITD patients, making FLT3/ITD AML susceptible to treatment with tyrosine kinase inhibitors (TKI). However, FLT3 TKI success has been hindered by mechanisms of resistance including adaptive resistance, in which downstream signaling pathways are rapidly reactivated after initial inhibition with targeted therapies. Past work has shown that FLT3/ITD cells undergo adaptive resistance through the reactivation of ERK signaling within 24 hours of sustained FLT3 inhibition. We are interested in finding the mechanisms responsible for this ERK reactivation. AXL is a RTK that is implicated in acquired resistance for many cancers, including against targeted therapy in FLT3/ITD AML. However, AXL signaling has not been implicated in early adaptive resistance in the context of FLT3/ITD AML. Thus, we sought to determine if AXL plays a role in the ERK reactivation found in TKI-treated AML cells. To address this, we first treated FLT3/ITD cell lines (Molm14 and MV4;11) with FLT3 TKI for 24 hours and saw an increase in AXL protein and mRNA levels as early as 4 hours after starting treatment. AXL phosphorylation occurred as early as 16 hours, which coincides with the beginning of the ERK phosphorylation (pERK) rebound. Further characterization revealed AXL upregulation, like ERK reactivation, is at least partially serum-dependent and that the ERK reactivation itself is not causing the AXL induction. These results established that TKI-induced AXL activation is associated with the adaptive ERK reactivation. To determine if AXL signaling actually plays a role in ERK reactivation in FLT3/ITD AML, we inhibited AXL activation using both small-molecule inhibitors (TP-0903 and R428) and selective inhibition (shRNA knockdown and ligand trapping with an AXL chimeric protein) concurrently with FLT3 TKI treatment. Each method of AXL inhibition diminished the pERK rebound, decreased cell proliferation and increased apoptosis of FLT3 TKI-treated FLT3/ITD cells. Combined AXL and FLT3 inhibition also suppressed leukemic cell recovery even after brief treatment exposure. Furthermore, 24-hour FLT3 and AXL TKI treatments of both relapsed and newly-diagnosed FLT3/ITD patient leukemic samples yielded the same upregulation of AXL protein and decreased pERK rebound observed in the cell lines. In summation, FLT3 TKI treatment causes rapid AXL upregulation resulting in adaptive ERK reactivation. Concurrent FLT3 and AXL inhibition diminishes pERK rebound and sensitives FLT3/ITD cells to TKI treatment. This data suggests that AXL plays a role in adaptive resistance in FLT3/ITD AML, and the addition of AXL inhibition to FLT3 TKI treatment has the potential to improve FLT3/ITD patient outcomes. Citation Format: Tessa Seale, Li Li, Jaesung Seo, Bao Nguyen, Mark Levis, Christine Pratilas, Donald Small. AXL is responsible for much of TKI-mediated adaptive resistance in FLT3/ITD AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3968.