Abstract 6266: Group 3 medulloblastoma transcriptional networks are selectively sensitive to EP300 and CBP bromodomain inhibition

Abstract

Abstract EP300/CBP are paralogous, multidomain histone acetyltransferases (HATs) that regulate gene expression by establishing enhancer and promoter marks on histone proteins. While EP300 and CBP proteins bind and regulate genes broadly across the genome, they also function to promote the expression of tumor-selective oncogenes such as the transcription factor c-MYC. This activity makes these proteins attractive targets for preclinical therapeutic development. EP300/CBP proteins contain highly homologous domains, including catalytic HAT domains and bromodomains (BRDs) that mediate physical binding of EP300/CBP to acetylated proteins. Importantly, however, the relative contribution of these distinct domains to tumor cell proliferation remains unresolved. To identify the relative contribution of the EP300/CBP BRD and HAT domains, we used a chemical strategy. We performed a cell viability-based high-throughput screen of the potent EP300/CBP BRD- or HAT-specific inhibitor, CCS1477 or A-485 on 454 barcoded cancer cell lines representing 31 distinct cancer types. These data revealed that the proliferation of most tumor cell lines regardless of the type of cancer were equally affected by targeting either domain of EP300/CBP. Intriguingly, Group 3 medulloblastoma (G3MB) cell lines were the most sensitive to EP300/CBP BRD inhibition, compared to HAT inhibition. Since G3MB cells are highly sensitive to BRD4 inhibition, we conducted a bromoscan analysis of the phase 1 compound CCS1477, which revealed high selectivity for EP300/CBP (Kd~4-5nM), with limited off-target effects on BRD4 (Kd~250nM) or other bromodomain-containing proteins. To validate that the effects on G3MB were primarily due to inhibition of EP300/CBP, we performed biotinylated CCS1477 pulldown assays in G3MB neurosphere cultures using in vitro relevant dosing of CCS1477, which demonstrated selective pulldown of EP300 and CBP, but not BRD4. Further, crystallographic studies of the EP300, CBP and BRD4 bromodomains in complex with CCS1477 demonstrated that the selectivity for the EP300/CBP BRD was critically dependent on interactions between the difluorophenyl group of CCS1477 and amino acid side chains in the BRD of EP300/CBP. In contrast, the analogous sidechains of BRD4 were unable to coordinate efficient binding of CCS1477. Mechanistically, treatment of MB cells with CCS1477, but not A-485 or the BRD4 inhibitor JQ1, caused rapid early loss of mRNA expression of specific G3MB dependency networks including the driver oncogene c-MYC. These studies identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB cells and provide new chemical approaches to disrupting malignant transcription in Group 3 medulloblastoma. Citation Format: Noha Ahmed Mohammed Ahmed Shendy. Group 3 medulloblastoma transcriptional networks are selectively sensitive to EP300 and CBP bromodomain inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6266.