INVESTIGATING ALLOSTERIC REGULATION OF ABL AND BCR-ABL KINASES: IMPLICATIONS FOR SMALL MOLECULE INHIBITORS

BCR-ABL is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of BCR-ABL kinase activity. Despite this clinical success, imatinib is less effective in advanced disease due to the emergence of drug resistant BCR-ABL mutants. Resistant mutations often arise in the drug binding site and include the most recalcitrant gatekeeper mutation, T315I. Other mutations arise outside the active site and allosterically reduce imatinib binding by promoting the active kinase conformation. Recently, a new class of allosteric BCR-ABL inhibitors, of which GNF-2 is the prototype, has been reported that targets the myristate-binding pocket of ABL. These compounds stabilize the inactive conformation of ABL and work in concert with ATP-competitive inhibitors to overcome imatinib resistance. Mounting evidence supports a regulatory influence of the non-catalytic SH3 and SH2 domains on BCR-ABL kinase domain. The major focus of this study was to exploit the intramolecular SH3:linker interaction, to stabilize the downregulated kinase domain conformation of BCR-ABL and sensitize the kinase to both imatinib and GNF-2. To achieve this goal, I engineered High Affinity Linker (HAL) variants of both ABL and BCR-ABL in which SH3:linker interaction was tightened through sequential addition of proline residues to the linker. Enhanced SH3:linker interaction induced long-range suppressive effects on the kinase activity in c-ABL, allosterically stabilized both the active site and the myristate-binding pocket, and sensitized BCR-ABL to small molecule inhibitors. Src family kinases (SFKs) are important mediators of BCR-ABL signal transduction and oncogenesis in CML. SFKs also play important roles in clinical resistance to imatinib in the absence of BCR-ABL mutations. In the second part of my project, I explored the effect of SFK- selective inhibitor, pyrazolopyrimidine A-419259, on myeloid cells transformed with clinically relevant imatinib resistant BCR-ABL mutants. While proliferation of cells expressing BCR-ABL E255V and Y253H was inhibited by A-419259, BCR-ABL T315I cells were not. Surprisingly, cells expressing BCR-ABL-T315I maintained SFK activity in the presence of the inhibitor. This observation suggests that BCR-ABL-T315I induces cross-resistance to drugs that inhibit SFKs in CML through direct phosphorylation of the SFKs

Differential sensitivity of Src-family kinases to activation by SH3 domain displacement

Src-family kinases (SFKs) are non-receptor protein-tyrosine kinases involved in a variety of signaling pathways in virtually every cell type. The SFKs share a common negative regulatory mechanism that involves intramolecular interactions of the SH3 domain with the PPII helix formed by the SH2-kinase linker as well as the SH2 domain with a conserved phosphotyrosine residue in the C-terminal tail. Growing evidence suggests that individual SFKs may exhibit distinct activation mechanisms dictated by the relative strengths of these intramolecular interactions. To elucidate the role of the SH3:linker interaction in the regulation of individual SFKs, we used a synthetic SH3 domain-binding peptide (VSL12) to probe the sensitivity of downregulated c-Src, Hck, Lyn and Fyn to SH3-based activation in a kinetic kinase assay. All four SFKs responded to VSL12 binding with enhanced kinase activity, demonstrating a conserved role for SH3:linker interaction in the control of catalytic function. However, the sensitivity and extent of SH3-based activation varied over a wide range. In addition, autophosphorylation of the activation loops of c-Src and Hck did not override regulatory control by SH3:linker displacement, demonstrating that these modes of activation are independent. Our results show that despite the similarity of their downregulated conformations, individual Src-family members show diverse responses to activation by domain displacement which may reflect their adaptation to specific signaling environments in vivo. © 2014 Moroco et al

The Roles of DNA Demethylases in Triple-Negative Breast Cancer

Triple-negative breast cancers (TNBCs) are very heterogenous, molecularly diverse, and are characterized by a high propensity to relapse or metastasize. Clinically, TNBC remains a diagnosis of exclusion by the lack of hormone receptors (Estrogen Receptor (ER) and Progesterone Receptor (PR)) as well as the absence of overexpression and/or amplification of HER2. DNA methylation plays an important role in breast cancer carcinogenesis and TNBCs have a distinct DNA methylation profile characterized by marked hypomethylation and lower gains of methylations compared to all other subtypes. DNA methylation is regulated by the balance of DNA methylases (DNMTs) and DNA demethylases (TETs). Here, we review the roles of TETs as context-dependent tumor-suppressor genes and/or oncogenes in solid tumors, and we discuss the current understandings of the oncogenic role of TET1 and its therapeutic implications in TNBCs