Overcoming asciminib resistance by targeting compound BCR::ABL1 mutations in chronic myeloid leukemia Free

Introduction:

Chronic myeloid leukemia (CML) is driven by the BCR::ABL1 fusion gene, which encodes a constitutively active tyrosine kinase that promotes uncontrolled proliferation of myeloid cells. Although the introduction of ABL1 tyrosine kinase inhibitors (TKIs) has significantly improved outcomes, the emergence of resistance due to compound mutations within the BCR::ABL1 kinase domain remains a major therapeutic challenge. Asciminib, a first-in-class allosteric inhibitor targeting the myristoyl pocket of ABL1, has shown clinical efficacy in patients who have failed at least two prior TKIs or harbor the T315I mutation. However, the molecular mechanisms underlying resistance to asciminib are not fully understood. In this study, we established and characterized a CML cell line with acquired resistance to asciminib, identified associated BCR::ABL1 mutations. We investigated therapeutic strategies using both conventional agents and the novel third-generation TKI, olverembatinib to overcome resistance in murine and human CML cells.

Materials and Methods:

Asciminib-resistant Ba/F3 cells (Ba/F3 asc-R) were established to model resistance. BCR::ABL1 mutations associated with resistance were identified through direct sequencing. To discover potential therapeutic agents, a high-throughput screen of 1,700 clinically approved compounds was conducted. Drug responses were evaluated using standardized assays for viability, cytotoxicity, and apoptosis. Selected candidate compounds demonstrating efficacy were validated across a panel of CML models, including Ba/F3 cells expressing wild-type BCR::ABL1, the T315I mutant (Ba/F3 T315I), and compound mutations (Ba/F3 PR: Y253H, E255K, and T315I), as well as K562 cells and their drug-resistant derivatives, K562 imatinib-resistant (K562 IR) and K562 ponatinib-resistant (K562 PR).

Results:

Ba/F3 asc-R cells harboring the T315I and Y139D mutations exhibited marked resistance to asciminib. High-throughput screening identified ponatinib (a third-generation tyrosine kinase inhibitor), bortezomib (a proteasome inhibitor), and selinexor (a selective inhibitor of nuclear export, XPO1 inhibitor) as candidate therapeutic agents. While Ba/F3 asc-R cells remained sensitive to ponatinib, Ba/F3 PR cells displayed resistance to both asciminib and ponatinib. In contrast, K562 PR cells retained sensitivity to asciminib, whereas K562 IR cells exhibited partial resistance. Co-treatment with ponatinib and asciminib reduced cell viability in Ba/F3 asc-R and Ba/F3 T315I cells but was ineffective in Ba/F3 PR cells. Selinexor monotherapy effectively suppressed proliferation and induced apoptosis across all CML models tested, including Ba/F3 PR, K562 IR, and K562 PR cells. Bortezomib also demonstrated dose-dependent anti-proliferative and pro-apoptotic activity; however, its combination with ponatinib showed only limited synergistic effects. Olverembatinib exhibited potent cytotoxic activity against Ba/F3 asc-R and Ba/F3 T315I cells, and partial efficacy at higher concentrations in Ba/F3 PR cells. Combination treatment with olverembatinib and selinexor induced synergistic cytotoxicity and robust apoptosis, significantly suppressing proliferation in asciminib- and ponatinib-resistant cells. This effect was accompanied by a marked increase in caspase 3/7 activity, indicating enhanced activation of apoptotic pathways. Furthermore, the combination therapy significantly reduced colony formation in K562 cells, highlighting its potential to impair leukemic clonogenicity.

Conclusion:

CML cells with compound BCR::ABL1 mutations exhibited resistance to asciminib and, in part, to ponatinib, while bortezomib and selinexor retained efficacy in these resistant cells. Olverembatinib effectively suppressed proliferation and induced apoptosis in ABL TKI-resistant CML cells. The combination of olverembatinib and selinexor synergistically induced apoptosis and suppressed proliferation, offering a promising approach for overcoming TKI-resistant CML with compound mutations.