Mechanistic studies of asciminib resistance mechanisms for M244V and assessment of sensitivity to combined asciminib/imatinib treatment. (A) Representative of 3 cellular thermal shift assays performed in Ba/F3 cells of BCR::ABL1 isoforms indicated. (B) Isothermal titration calorimetry (ITC) measurements of recombinant ABL1 SH3-SH2-kinase domain (SH3-SH2-KD) unit native (left panel) and M244V mutant (right panel) (10 μM, respectively) with asciminib (100 μM) at 25°C. Each panel shows the raw heat signal of an ITC experiment (top) and the integrated calorimetric data of the area of each peak (bottom). The continuous line represents the best fit of the data based on a 1:1 binding model computed from the MicroCal software. A representative measurement of 2 independent experiments is shown with Kd value, stoichiometry (N), and enthalpy (ΔH) calculated from the fit. (C) Assessment of sensitivity of Ba/F3 cells harboring native BCR::ABL1 or BCR::ABL1/M244V to imatinib, asciminib, or the combination. Biological and technical triplicates were performed. (D) Proposed model of BCR::ABL1/M244V-mediated disruption of asciminib-induced allosteric modulation. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
