Numerous clinically-detected asciminib-resistant BCR::ABL1 mutations disrupt allostery and confer cross-resistance to the novel clinically-active allosteric TKI tgrx-678 Free

Objectives: Asciminib is a first-in-class potent and selective allosteric inhibitor of BCR::ABL1 that has demonstrated substantial clinical efficacy and excellent tolerability in patients with Philadelphia chromosome positive chronic myeloid leukemia in chronic phase (Ph+CML-CP) who exhibit resistance or intolerance to prior TKI therapies that target the ATP binding site, and in newly diagnosed patients. Asciminib has received accelerated approval by the US FDA as a treatment option for Ph+CML-CP in any line of therapy. Asciminib inhibits BCR::ABL1 kinase activity via a unique mechanism of action that involves binding to the allosteric regulatory myristoyl binding pocket (MBP) and inducing an assembled, autoinhibited confirmation. This aspect of asciminib enables a reduced propensity for off-target inhibition in patients, thereby potentially enhancing its therapeutic index. However, an increasing number of asciminib-resistant mutations are emerging clinically and have been identified both within the MBP and elsewhere in the kinase domain. We previously demonstrated that mutations in the kinase N-lobe unexpectedly confer in vitro and clinical asciminib resistance despite retaining binding affinity for asciminib and provided indirect evidence for disruption of allostery as a candidate mechanism for one mutation (Leyte-Vidal et al, Blood 2024). We now present a comprehensive analysis of 22 asciminib-resistant mutations recently identified in clinical isolates, including in regions outside of the kinase domain.

Methods: BCR::ABL1 mutants were created in MSCVpuro by site-directed mutagenesis and transduced into Ba/F3 cells, which were selected for growth factor independence and tested for sensitivity to asciminib and a second clinically active allosteric TKI, TGRX-678. Drug binding assessments were conducted utilizing an in cellulo nanoBRET assay with full length BCR::ABL1. Limited proteolysis experiments were performed to assess for evidence of global alteration of purified kinase protein in vitro. Mutants were cloned into MSCVpuro ABL1b, transduced into Ba/F3 cells, and assessed for growth factor independence/kinase activation.

Results: Twenty-two BCR::ABL1 mutations identified in asciminib-resistant patients were assessed. These substitutions are located in or near the MBP (n=8), in the kinase C-lobe/hinge region but removed from the MBP (n=4), in the kinase activation loop (n=4), in the kinase N-lobe (n=4), in the SH2-kinase linker region (n=1), and in the SH2 domain (n=1). All assessed mutants conferred resistance of varying degrees to asciminib in vitro. Relative resistance to asciminib highly correlated with in vitro resistance to the novel clinically-active allosteric TKI TGRX-678. Only a minority of the 22 mutations, primarily those in the MBP, substantially altered the binding affinity of asciminib. Partial proteolysis studies of several mutants demonstrated evidence of global structural alterations. Most mutations activated the kinase activity of ABL1b in cellulo, as reflected by their ability to confer growth factor independence in Ba/F3 cells associated with elevated levels of phosphotyrosine.

Conclusion: Asciminib is clinically susceptible to a substantially larger number of drug-resistant mutants than 2G or 3G TKIs. All 22 BCR::ABL1 mutations tested conferred varying degrees of resistance to asciminib, with a majority retaining asciminib binding affinity. Several mutants displayed activation of kinase activity and alteration of kinase structure, providing the first direct evidence for disruption of allostery as the most common resistance mechanism of asciminib resistant-mutations. Notably, this study also marks the first report of a clinically-detected asciminib-resistant SH2 domain mutation, further expanding the spectrum of resistance mutations and demonstrating the need to assess regions of BCR::ABL1 outside of the kinase domain in patients with asciminib resistance. The high degree of cross-resistance observed between asciminib and TGRX-678 suggests a class-like effect of mutations that confer resistance to allosteric TKIs, primarily through disruption of allostery. These findings underscore the need for continued investigation into resistance mechanisms to inform the development of next generation therapies and treatment strategies for CML patients.