Differential in Vitro Sensitivity of BCR::ABL1 Kinase Domain Mutations to Tyrosine Kinase Inhibitors Depending on the p190 or p210 Background

The p190 BCR::ABL1 fusion protein variant, which is predominant in patients with Ph+ALL, was shown to display signaling properties differing from the less common p210 variant. It was not intuitive therefore to assume that resistance-associated mutations frequently occurring in the kinase domain (KD) of the ABL1 gene display identical response levels to tyrosine kinase inhibitors (TKIs), regardless of the underlying fusion protein variant. Nevertheless, published studies and heatmaps assessing in vitro responses of single and compound mutations in the ABL1 KD were generally restricted to analyses performed in Ba/F3 cell line models in the background of the p210 variant only, which is predominant in patients with CML. The aim of the present study was therefore to determine whether individual KD mutations present in the background of the p190 and p210 BCR::ABL1 fusion protein isoforms can mediate differential TKI responses of potential clinical relevance.

To address this question, wildtype BCR::ABL1 and a large number of single (n=34) and compound (n=33) KD mutations were introduced into full-length BCR::ABL1 gene constructs encoding the p190 and p210 isoforms, respectively. The wildtype and mutant constructs were transduced into Ba/F3 cells by a transposon-mediated approach. Cells carrying only one mutant construct were enriched by flow cytometry and cell sorting according to fluorescence signal intensity, to preclude skewed test results potentially caused by multiple copies of the mutant construct in a relevant proportion of cells. The resulting wildtype and 134 mutant Ba/F3 cell lines generated in this way, i.e. 67 cell lines carrying a large spectrum of single and compound mutations in the p190 and p210 background, respectively, were tested in vitro against different concentrations of eight TKIs including imatinib, nilotinib, dasatinib, bosutinib, ponatinib, vamotinib, asciminib, and flumatinib using the CellTiter-Glo® luminescent cell viability assay (Promega).

The results indicate that individual TKIs mostly show less than 3-fold differences in inhibitory concentration (IC₅₀) values between mutant cell lines expressing the p190 or p210 BCR::ABL1 fusion protein variants, while greater than 3-fold differences in IC₅₀ values were observed in 10-25% of the mutant cell lines, depending on the TKI tested. Some of the differentially increased IC₅₀ values observed for individual mutations either in the p190 or p210 background greatly exceeded the clinically achievable TKI concentrations, suggesting that the expected clinical responses might reveal relevant differences for individual mutations and TKIs. The most prominent shifts were observed for some mutant cell lines upon exposure to the 4^th generation TKI asciminib, where the IC₅₀ values for identical mutations in the p190 versus p210 background differed by more than 100-fold, thus clearly exceeding the clinically achievable drug concentrations in specific settings. Examples of such constellations include the frequently occurring P-loop mutations E255V and Y253H, which revealed high resistance to asciminib, if present in the background of p190, and various (non-T315I) compound mutations, which showed differential responses to this drug, depending on the underlying fusion protein variant. Less pronounced, but potentially clinically relevant differences were also observed for other TKIs in the presence of specific mutations.

The findings of the present study provided the basis for extending and supplementing the existing heatmaps for various mutations and TKIs in the background of p210 by a corresponding heatmap established specifically for mutations occurring in the background of p190, thus filling a clinically important information gap. Once appropriately evaluated in the clinical setting, the comprehensive heatmaps emanating from the present study may provide support for adequate TKI selection (and potentially for selection of the appropriate dose) in the presence of ABL1 KD mutations occurring in the background of the two most common isoforms of the BCR::ABL1 fusion protein in Ph+ALL.

Lion:Angelini: Honoraria; Novartis Pharma: Honoraria, Speakers Bureau; Incyte Inc.: Honoraria, Research Funding, Speakers Bureau; Ascentage Pharma Group Inc.: Honoraria; SymBio Pharmaceuticals Limited: Honoraria, Research Funding.