Abstract 4138: CK1δ-BRD4 pathway as novel therapeutic target for SHH subtype of medulloblastoma

Abstract

Abstract Background: Medulloblastoma is the most common malignant pediatric brain tumor with variable prognosis due to its clinical and genomic heterogeneity. Despite decades of treatment advances, nearly 40% of children experience tumour recurrence, and 30% will die from this disease. Thus, new drugs and drug combinations need to be developed that effectively treat medulloblastoma. Casein kinase 1δ (CK1δ) is a serine/threonine kinase that controls cell cycle progression, signal transduction and neurogenesis, and we previously reported high levels of CK1δ in mouse models of medulloblastoma. BRD4 is an epigenetic reader protein that controls expression of several oncogenes. We wanted to determine the mechanism through which CK1δ regulates BRD4 activity in medulloblastoma and assess whether a combination of CK1δ and BRD4 inhibitors could be more effective in decreasing medulloblastoma progression in vivo. Methods: Granule cell progenitors (GCPs), medulloblastoma Ptch-/-P53-/- mouse cells and SUFU-/- mouse embryonic fibroblasts were incubated with the CK1δ inhibitor SR-1277 and the phosphorylation levels of BRD4 were determined by WB and its binding to chromatin by ChIP. In vitro phosphorylation studies with human BRD4 purified from E. coli were performed and whether genetic disruption of CK1δ reduces BRD4 phosphorylation was tested. We combined BRD4 and CK1δ inhibitors (JQ1 and SR-1277, respectively) and analysed Gli1 mRNA expression and EdU incorporation. We deleted CK1δ in GCPs in Ptch-/-;P53-/- mouse models of medulloblastoma by breeding these mice with a Tg(Atoh1-Cre);CK1δfl/fl strain. Furthermore, we intracranially transplanted human SHH medulloblastoma PDX cells expressing luciferase in mice, to validate the combination therapy. Results: CK1δ inhibitor treatment and CK1δ knockout reduced BRD4 phosphorylation, suggesting that CK1δ phosphorylates BRD4 and is required for BRD4 recruitment to chromatin. In vitro phosphorylation studies with purified human BRD4 and CK1δ confirmed CK1δ-mediated BRD4 phosphorylation and CK1δ is required for BRD4 phosphorylation at serine 492 and 494 in vivo. Using loss of function studies in GCPs, both CK1δ and BET inhibition reduced BRD4 association with Gli1 promoter, thereby reducing Gli1 mRNA levels. In addition, SR-1277 suppressed SHH signaling downstream of SUFU of medulloblastoma cells, in vitro and in vivo. We also found synergy by combining these compounds in vitro and in reducing tumor burden in the in vivo model with intracranial allografts. Conclusion: Together, our studies validate the CK1δ-BRD4 pathway as a novel target in medulloblastoma. The significance of our work is underscored by the possibility that simultaneous CK1δ-BRD4 inhibition could overcome the resistance observed with BRD4 inhibitors and enhance therapeutic benefit to patients. We are also the first group to demonstrate that CK1δ acts downstream of SHH signalling in SHH-driven medulloblastoma. Citation Format: Sze Kiat Tan, Clara Penas, Cheng-Ming Chiang, David Robbins, Nagi Ayad. CK1δ-BRD4 pathway as novel therapeutic target for SHH subtype of medulloblastoma [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 4138.