(627b) Structural, Thermodynamics and Kinetics Role of Novel Hot-Spot Mutations of BCR-ABL1 in Resistance Towards "Ibs" Inhibitors

Authors: 
Pricl, S., University of Trieste
Laurini, E., University of Trieste
Fermeglia, M., University of Trieste
Aulic, S., University of Trieste
Marson, D., University of Trieste
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.A question then arise: “Do the majority of the BCR-ABL1 KD mutations determined so far, studied and characterized by considering just the BCR-ABL KD domain, exert their resistance mechanism by directly interfering with TKI binding, as often claimed?”The present contribution integrates structural, computational, and molecular 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-liker-SH3-KD BCR-ABL1 constructs.

Specifically, in silicoexperiments will disclose the effects exerted by the “hot-spot” BCR-ABL1 mutations on the protein structure, thermodynamic stability, and ability to interfere with BCR-ABL1 binding to specific TKIs (e.g., imatinib, dasatinib, and ponatinib).

Structural biology evidences of wild type and mutant isolated KDs and SH2-linker-SH3-KD (SSK) constructs (per seand in complex with TKIs) will be discussed to support in silico predictions.

Direct drug binding to both KD and SSK constructs will be measured using isothermal titration calorimetry and surface plasmon resonance.

In vitrokinase assays will monitor the ability of these mutant isoforms to auto-phosphorylate and to phosphorylate a substrate peptide.

“Prototypical” BCR-ABL1 KD mutations will be analyzed in parallel for comparison and to confirm/dispute the current, classical view of TKI resistance.

[1] Gibbons DL, Pricl S, Posocco P, 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.