Abstract Topic: 3. Acute myeloid leukemia - Biology & Translational Research
Background: Tuspetinib (TUS) is a mutation agnostic, once daily, oral agent that potently inhibits JAK1/2, SYK, RSK1/2, wildtype and mutant forms of FLT3, and mutant forms of KIT kinases, simultaneously suppressing multiple oncogenic signaling pathways that mediate resistance to various drugs. TUS has generated complete remissions in relapsed/refractory (R/R) acute myeloid leukemia (AML) patients with diverse mutations and demonstrated favorable safety in a Phase 1 trial (NCT03850574). This clinical activity led us to investigate alterations in AML cells that may give rise to TUS resistance, and to understand the sensitivity of resistant isolates to other agents used to treat AML. TUS is now in a multinational Phase 1/2 expansion trial (APTIVATE) for R/R AML patients with high unmet need as a monotherapy and as a doublet in combination with venetoclax.
Aims: Investigate mechanisms of acquired resistance to TUS and identify synthetic lethal vulnerabilities that emerge in resistant isolates.
Methods: MOLM-14 cells containing a FLT-ITD mutation were selected for resistance by continuous exposure to progressively higher concentrations of TUS over time. The concentrations of TUS and other drugs that inhibited growth rate by 50% were determined using the CCK8 assay with vehicle controls. Western blot analysis of changes in the phospho-kinome were conducted with antibodies to selected total and phospho- forms of kinases.
Results: MOLM-14 FLT-ITD mutant cells selected for resistance to TUS over a 3-month period developed 61-fold greater resistance (IC50 parental cells 2.3 ± 0.3 nM; resistant cells 140.3 ± 28.7 nM, mean ± SEM). Resistance levels remained stable when tested at 60 days after drug removal. TUS-resistant cells (TUS/R) were 60-fold resistant to gilteritinib (FLT3 inhibitor) but not to quizartinib (FLT3 inhibitor). There was no observed resistance to azacitidine, luxeptinib (lymphoid & myeloid kinase inhibitor), brequinar (DHODH inhibitor), zelavespib (HSP90 inhibitor), or IMP-1088 (NMT1/2 inhibitor), and a small degree of hypersensitivity (<2-fold) of TUS/R cells to luxeptinib, brequinar, and IMP-1088. Unexpectedly, TUS/R cells were ~2,000-fold hypersensitive to venetoclax, indicating that resistance to TUS created a substantial synthetic lethal vulnerability to venetoclax. Initial western blot analysis of parental cells in the absence of TUS and TUS/R cells immediately after removal from 75 nM TUS, demonstrated that pFLT3 was markedly inhibited in the parental and TUS/R cells but that pERK1/2, pSYK and pSTAT5 levels were all elevated relative to the total protein level of each kinase. Since FLT3 activity remained fully inhibited, TUS resistance was associated a FLT3-independent mechanism that altered multiple signaling pathways. TUS/R cells had a large increase in BAX suggesting a basis for the marked hypersensitivity to venetoclax. Additional analysis of pro- and anti-apoptotic proteins will be updated at the meeting.
Summary/Conclusion: Resistance to TUS in MOLM-14 cells required prolonged high-level drug exposure. The fact that FLT3 remained fully inhibited in TUS/R cells growing in 75 nM TUS suggests that resistance is not due to a mutation of FLT3. Drug resistance in TUS/R cells in the absence of TUS over 60 days indicates a stable phenotype, distinct from “persister cell resistance” in which resistance fades during subsequent passages. Strikingly, acquired TUS resistance generated a synthetic lethal vulnerability to venetoclax of unusually high magnitude. This suggests that concurrent administration of TUS and venetoclax may be advantageous clinically as venetoclax could eliminate cells that carry this form of TUS resistance at the start of therapy and discourage the emergence of TUS resistance during treatment.
Keywords: Venetoclax, Kinase inhibitor, flt3 inhibitor, AML