Abstract 2382: In silico/in vitro studies of novel hot spot mutations in BCR-ABL1: A multidisciplinary approach to shed a new light on TKI resistance

Abstract

Abstract Acquisition of mutations in the BCR-ABL1 kinase domain (KD) is frequently associated with tyrosine kinase inhibitor (TKI) failure in CML. Recently, we revealed a novel mutation “hot- spot” in the BCR-ABL1 KD region (residues 295 - 312), associated with high resistance and poor clinical outcomes [1]. Some of these new BCR-ABL1 variants locate in a protein region, which might not be directly involved in TKI interaction. Therefore, we hypothesized that some of these mutations might be linked to TKI resistance via a direct mechanism while others might exert an indirect effect on drug resistance. The present contribution integrates molecular, computational, and structural biology techniques to understand the eventual role of the newly reported hot-spot mutations of the BCR-ABL1 KD in TKI resistance observed in CML patients using a "two-tier level" investigation: isolated KDs and SH2-linker-SH3-KD BCR- ABL1 constructs. Molecular Modeling will assess in silico whether the clinically observed “hot-spot” BCR-ABL1 mutations might be directly related to a change in the thermodynamic stability and/or protein structure and how these modifications could be involved in the BCR-ABL1 binding profile to specific TKIs (e.g., imatinib, dasatinib, and ponatinib) compared with wild-type (WT) systems and “prototypical” BCR-ABL1 mutations (e.g. T315I). In parallel, in vitro analysis will evaluate the effects exerted by the selected mutations on the intrinsic activity of the kinase and to determine the inhibition activity of the selected drugs on these BCR-ABL1 variants. Specifically, direct drug binding to both KD and SSK constructs will be measured using isothermal titration calorimetry and in vitro kinase assays will monitor the ability of these mutant isoforms to auto-phosphorylate and to phosphorylate a substrate peptide. Finally, structural biology (e.g., SAXS and far-UV CD) evidences of WT and mutant isolated KDs and SH2-linker-SH3-KD (SSK) constructs (per se and in complex with TKIs) will be discussed to support in silico predictions. [1] Gibbons DL, Pricl S et al., Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy. Proc Natl Acad Sci U S A. 2014 Mar 4;111(9):3550-5. doi: 10.1073/pnas.1321173111. Citation Format: Erik Laurini, Suzana Aulic, Natasa Skoko, Sulena Polez, Marco Baralle, Maurizio Romano, Sabrina Pricl. In silico/in vitro studies of novel hot spot mutations in BCR-ABL1: A multidisciplinary approach to shed a new light on TKI resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2382.