Mechanistically distinct classes of BCR::ABL1 mutations confer resistance to asciminib

The first-in-class allosteric BCR::ABL1 inhibitor asciminib has expanded the therapeutic landscape of chronic myeloid leukemia (CML). However, the molecular mechanisms underlying secondary resistance remain incompletely understood. We investigated clinically observed asciminib resistance mutations by integrating structural modeling, hydrogen-deuterium exchange mass spectrometry (HDX-MS), quantitative biochemical assays and molecular dynamics simulations. Resistance mutations were grouped into two mechanistically distinct classes: Mutations within the BCR::ABL1 myristate pocket (class I) and mutations located up to 40 Å from the asciminib binding site in the kinase N-lobe, SH2-kinase domain (KD) linker and SH3 domain (class II). HDX-MS demonstrated that asciminib induces conformational stabilization across the KD extending beyond the myristate pocket, establishing a long-range allosteric network. We show that class I mutations reduced asciminib affinity up to 90-fold, consistent with steric interference at the binding site. In contrast, class II mutations retained wildtype binding affinity despite conferring clinical resistance. Instead, the class II mutations F359I and M244V increased intrinsic kinase activity and enhanced BCR::ABL1 autophosphorylation, indicating overactivation. Molecular dynamics simulations revealed allosteric communication linking the myristate and ATP-binding pockets, which is disrupted by class II mutations. Together, these findings establish that asciminib resistance arises through two distinct mechanisms: direct impairment of drug binding or disruption of the asciminib binding-induced autoinhibited conformation. Our study provides a mechanistic framework for understanding resistance to allosteric BCR::ABL1 inhibitors and has important implications for resistance monitoring, combination therapies and therapeutic sequencing.