Select P-Loop Mutants in BCR::ABL1 Confer Moderate to High Degrees of Resistance to Asciminib

Abstract

Introduction: NCCN CML Guidelines describe BCR::ABL1 mutants that are contraindicated for select ATP-competitive BCR:ABL1 TKIs due to their association with in vitro and clinical resistance. The allosteric ABL1 kinase inhibitor asciminib was recently approved in the US at a "low” dose of 40 mg twice daily (or 80 mg once daily) in patients with resistance or intolerance to two prior TKIs, and a "high” dose of 200 mg twice daily in CML patients with BCR::ABL1/T315I. Prior work demonstrated ~10x reduced sensitivity of BCR::ABL1/T315I to asciminib compared with native BCR::ABL1 in cellular proliferation studies. Based on its unique mode of binding to a myristate binding pocket, it has been hypothesized that asciminib may complement all approved ATP-competitive TKIs due to presumed non-overlapping mechanisms of resistance with respect to kinase domain mutation. Although several ATP-competitive kinase domain mutants (Q252H, V299L, E355G, F359C/I/V) confer a degree of in vitro resistance similar to T315I (Wylie et al, Nature 2017; Eide et al, Cancer Cell 2019), at the present time, treatment with low dose asciminib is indicated in cases that harbor these mutations. There are no formal guidelines for dose escalation in the event of poor response. To date, documentation of clinical activity of asciminib in patients with pre-existing BCR::ABL1 kinase domain mutations has been limited, and several kinase domain mutants have yet to be assessed for sensitivity to asciminib in vitro. NCCN CML Guidelines list mutations at two positions for which asciminib is contraindicated. These amino acid positions map to the asciminib binding pocket (A337, P465). Mutations at these residues retain sensitivity to ATP-competitive TKIs. Results: We recently treated two chronic phase CML patients whose disease harbored the imatinib-resistant BCR::ABL1/M244V mutation with asciminib 80 mg once daily. Both patients demonstrated failure to respond. In both cases, despite dose escalation (to 120 mg twice daily and 200 mg twice daily), asciminib was discontinued due to rising blood counts or BCR::ABL1 transcript level. One patient was subsequently switched to ponatinib 15 mg daily and has responded well; the other patient has maintained disease control on dasatinib. The in vitro activity of asciminib against BCR::ABL1/M244V has not been previously reported. We therefore assessed the sensitivity of Ba/F3 cells transduced with BCR::ABL1/M244V. BCR::ABL1/M244V confers a surprisingly high degree of resistance to asciminib (IC50 increased > 10-fold relative to BCR::ABL1/T315I). BCR::ABL1/M244V confers a surprisingly high degree of resistance to asciminib (IC50 increased > 10-fold relative to BCR::ABL1/T315I), albeit less than that conferred by the previously described asciminib-specific mutant BCR::ABL1/A337V. Interestingly, M244 resides at the base of the P-loop, at considerable distance from the asciminib binding pocket. We therefore tested three other resistance-conferring mutants in proximity to M244V that have not been previously profiled (L248R/V, Y253F), and found that two of these mutants (L248V and Y253F) indeed exhibit moderate resistance relative to native BCR::ABL1, similar to that observed with BCR::ABL1/T315I. These findings suggest that select alterations in the kinase P-loop can lead to conformational changes in myristate binding pocket. Conclusions: BCR::ABL1/M244V confers a high degree of in vitro resistance and is associated with clinical resistance to even high doses of asciminib. Asciminib should therefore be considered contraindicated in patients with the BCR::ABL1/M244V mutation. Furthermore, several additional commonly encountered BCR::ABL1 kinase domain P-loop mutations (L248V, Y253F, F359C/I/V) that confer clinical resistance to ATP-competitive TKIs confer substantial cross-resistance to asciminib in vitro. While assessment of clinical response in patients with these mutations to asciminib is required, it appears unlikely that the currently approved dose of asciminib will be active in CML cases that harbor these mutants, and they may lead to acquired resistance. These mutations represent critical vulnerabilities for ongoing investigational efforts to combine asciminib with imatinib in an attempt to suppress the outgrowth of drug-resistant BCR::ABL1 mutants.