440105 Bcr-Abl1 V304D Mutation in CML: Friend or Foe?

Wednesday, November 11, 2015
Exhibit Hall 1 (Salt Palace Convention Center)
Sabrina Pricl1, Maurizio Fermeglia1, V. Dal Col2, P. Storici3, B. Giabbai4, M. Semrau5, M. De March6, N. J. Donato7,8 and A. Quintas-Cardama9, (1)Mose-DI3, University of Trieste, Trieste, Italy, (2)1University of Trieste, Trieste, Italy, (3)Structural Biology Laboratory, 2Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy, (4)Structural Biology Laboratory, 2Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy, (5)Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A., Trieste, Italy, (6)Structural Biology Laboratory, Elettra-Sincrotrone Trieste S.C.p.A, Trieste, Italy, (7)University of Trieste, Trieste, Italy, (8)Comprehensive Cancer Center, 3University of Michigan, Ann Arbor, MI, (9)4Cell & Gene Therapies Unit, Novartis Pharma A.G., Basel, Switzerland

Our recent work revealed that some chronic myeloid leukemia (CML) patients failing imatinib therapy carry the BCR-ABL1 V304D mutation, and is associated with poor clinical outcomes. BCR-ABL1 V304D variant has rarely been described in clinical specimens, and its location within the BCR-ABL1 Kinase Domain (KD) appears to be in an area of the protein not involved in the direct binding with tyrosine kinase inhibitors (TKIs) such as Imatinib and Ponatinib. The present study integrates computational, structural, and molecular biology techniques to investigate the role of the BCR-ABL1 V304D mutation in kinase activity and TKI resistance.

Accordingly, in this work we used in silico experiments to probe whether, compared to the wild type (wt), the V304D mutation i) directly affects the kinase domain fold and/or thermodynamic stability and ii) if/how these effects might eventually interfere with BCR-ABL1 binding to the TKIs. Small angle X-ray scattering (SAXS), fluorescence, and circular dichroism spectroscopy are employed for structural characterization of the wt/mutated kinase domain and their interaction with TKIs. Cell proliferation, protein phosphorylation, and kinase activity assays are performed (with/without) the TKIs to assess the effect of this mutation on BCR-ABL1 KD, its enzymatic and transforming activities.

Our preliminary results show that BCR-ABL1V304D does not transform Ba/F3 cells, retains minimal kinase activity, and fails to activate downstream pathways. At the same time, the V304D BCR-ABL1 isoform partially protects cells from cytokine withdrawal and exhibits imatinib resistance. The SAXS-based structural model of the kinase domain mutant in complex with Ponatinib does not reveal major structural perturbations compared to the wild type protein. Molecular simulations on the SH2-linker-SH3-KD construct suggest a possible rationale for the TKI resistance seen in the clinic.

In aggregate, these results indicate that BCR-ABL1V304D results in kinase inactivation, pan-TKI resistance mediated by lower control of protein autoinhibition via perturbation of the SH3 binding domain and very poor prognosis.

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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division