Elucidating the mechanism of anti-GSK3 adjuvant therapy for Myc-driven lymphomas

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PROJECT SUMMARY Many cancers harbor translocations, amplifications, or activating mutations of the c-Myc proto- oncogene. Myc regulates many programs in the cell including growth and proliferation, and apoptosis as well, predominantly through the activation of p53. However, Myc deregulation and p53 inactivation frequently co-occur in B-cell lymphomas such as Burkitt's lymphoma (BL); thus, chemotherapies for patients are not always curative. Studies have shown that very high levels of oncogenic Myc are required to engage apoptosis, thus one strategy to improve current therapies would be to transiently increase Myc expression and stability. GSK3β regulates Myc protein stability by phosphorylating Myc at the Thr58 residue, marking it for ubiquitylation and degradation. GSK3β can be targeted pharmacologically with specific inhibitors, making it an attractive target to modulate Myc levels. Preliminary data show that transient up-regulation of Myc by inhibiting GSK3β with CHIR99021 enhances apoptosis in B-cell lymphomas when combined with chemotherapeutic drugs and suggest that the extrinsic apoptotic pathway is important for this enhanced apoptosis. Extrinsic apoptosis is activated by binding of ligands (FasL, TNF, TRAIL) to death receptors on the cell surface (Fas, TNFR1, DR4, DR5). The goal of this proposal will be to determine the mechanism by which anti- GSK3β adjuvant therapy enhances apoptosis in B-cell lymphomas and to investigate both Myc- dependent and independent mechanisms. My first aim will be to examine the role of Myc and other GSK3β targets in anti-GSK3β adjuvant therapy. To assess the role of Myc, I will compare induction of apoptosis in BL cells with WT vs. mutant Thr58 Myc, where Myc cannot be stabilized upon GSK3β inhibition. I will investigate PTEN as another GSK3β target and validate the effect of GSK3β inhibition on PTEN protein level, stability, Thr366 phosphorylation (the site targeted by GSK3β) and activity. I will then compare the response of BL cells engineered with WT vs. Thr366 mutant PTEN to anti- GSK3β adjuvant therapy. My second aim will investigate the contribution of extrinsic apoptosis by using CRISPR/Cas9 to delete death receptors on BL cells complemented by modulation of FLIP-long expression, the negative regulator of the extrinsic pathway; I will test the response of these modified cells to anti-GSK3β adjuvant therapy. Finally I will investigate the role of the extrinsic pathway in vivo by comparing the response of subcutaneous xenografts of BL cells with intact vs. perturbed extrinsic apoptotic signaling to anti-GSK3β adjuvant therapy as well as GSK3β inhibition + the extrinsic ligand TRAIL. These proposed experiments will provide rationale and mechanism for GSK3β inhibition as adjuvant therapy for Myc-driven B-cell lymphomas.