Abstract

Resistance to FLT3 inhibitors remains a major limitation in the treatment of FLT3 -mutated acute myeloid leukemia (AML). Canonically, ERK is considered the predominant MAPK effector downstream of FLT3 signaling. However, pharmacologic inhibition of MEK/ERK provides limited clinical benefit once resistance develops, suggesting that alternative signaling dependencies may emerge under therapeutic pressure. Using an unbiased kinome-wide CRISPR-Cas9 screen in gilteritinib-resistant AML cells, we identified MAPK14 (encoding p38α), rather than MAPK3 / MAPK1 (encoding ERK1/2), as a prominent context-dependent dependency associated with the resistant state. Genetic impairment or pharmacologic inhibition of p38 enhanced gilteritinib sensitivity and synergized with FLT3 inhibition to suppress leukemic growth. Mechanistically, resistant cells exhibited adaptor-mediated rewiring of FLT3 signaling, in which p46-SHC1 supported an FLT3-associated MKK3/6–p38 signaling module despite FLT3 inhibition, thereby sustaining downstream programs including MYC expression. Consistent with its role as a stress-responsive kinase, p38 supports leukemic cell survival under prolonged drug exposure. Together, these findings define a non-canonical MAPK signaling state associated with FLT3 inhibitor resistance, provide a mechanistic explanation for the limited efficacy of MEK/ERK-directed therapies in the resistant setting, and offer a rationale for combination strategies targeting stress-adaptive pathways to improve the durability of FLT3-directed therapy in AML.