Novel BCR::ABL1 fusion alongside T315I reveals mechanism for dual ponatinib/asciminib resistance and proves sensitive to addition of axitinib

Abstract Despite significant progress in targeting the BCR::ABL1 kinase in chronic myeloid leukemia (CML), the emergence of therapeutic resistance remains a major clinical challenge. Even the most potent ABL1 inhibitors are vulnerable to selection for compound mutations that confer cross-resistance to all currently approved ABL1 inhibitors. While a combination of ponatinib and asciminib can effectively suppress most compound mutations through enforced stabilization of the kinase inactive state, patients can develop resistance via mutations of the gatekeeper residue (T315I or T315M) alongside mutations that destabilize the autoinhibited, inactive conformation of BCR::ABL1. We studied a highly informative patient who relapsed on dual ponatinib- asciminib therapy, and identified a novel BCR::ABL1 fusion (e6a3) together with T315I (BCR::ABL1e6a3/T315I) that displayed resistance to all FDA approved ABL1 inhibitors. Our structural modeling suggests that the novel e6a3 fusion acts by deleting a critical motif in the SH3 domain that mediates kinase autoinhibition, indicating that combined treatment with ponatinib and asciminib can select for novel mutational resistance mechanisms that markedly activate the kinase. In a screen of drug combinations, we identified axitinib as a potent agent when combined with ponatinib, nilotinib, asciminib, or ribastinib, ABL1 inhibitors that target the inactive conformation. Given that axitinib binds both active and inactive conformations of the kinase, we reason that the three-drug combination enforces a shift in the conformational dynamics of a BCR::ABL1 oligomer, stabilizing the inactive state for even this highly activated BCR::ABL1e6a3/T315I kinase. Using primary human cord blood–derived CD34⁺ hematopoietic progenitor cells, we demonstrate that expression of the highly active BCR::ABL1e6a3/T315I kinase is sufficient to induce transformation into B-cell acute lymphoblastic leukemia (B-ALL), further strengthening the correlation of high kinase activity and predisposition towards B-lymphoid disease. In conclusion, we have identified and characterized a novel BCR::ABL1 fusion (e6a3) that together with T315I represents a compound mutation that is pan-resistant to all currently approved ABL1 inhibitors, but which remains fully sensitive to a triple combination therapy comprising axitinib, ponatinib, and asciminib. These preclinical findings demonstrate the remarkable mutability of the BCR::ABL1 kinase when under selection by even the most potent of ABL1 kinase inhbitors, and provides a strong rationale for the clinical assessment of this triple combination as a strategy to overcome resistance to dual ponatinib and asciminib therapy.