Abstract 1467: RUVBL1 controls protein synthesis and tumor progression via MYC-dependent EEF1A1 expression

Abstract Dysregulation of MYC family proto-oncogene is a hallmark of tumorigenesis in various cancers (e.g., in Ewing sarcoma [EwS]) so identifying additional MYC mechanisms and dependencies that can be targeted is of critical therapeutic value. Particularly, the epigenetic regulations that dictate the oncogenic MYC signaling and its downstream mechanisms remain unclear. In this study, we conducted a CRISPR interference (CRISPRi) library screen of 3,669 sgRNAs that targeted the transcription start sites of 728 epigenetic-related genes, and identified the requirement of the NuA4 (Nucleosome acetyltransferase of histone H4) complex in EwS. Subsequently, we developed a custom CRISPR-knockout (KO) library targeting all 18 members of the NuA4 complex at 25 sgRNA/gene. These orthogonal CRISPR screens revealed an essential role of RUVBL1 (an ATPase component of NuA4) in EwS. Suppression of RUVBL1 led to the loss of histone H4 acetylation (mass spectrometry), attenuated in vivo tumor growth (xenograft modeling), and ablated MYC signaling (RNA-seq). While the expression of MYC protein was unaffected by RUVBL1-KO, we observed a drastic reduction of the chromatin-associated MYC (ChIP-seq) in the sgRUVBL1 transduced cells, highlighting the pivotal role of RUVBL1 in MYC chromatin binding and target gene transactivation. Mechanistically, we discovered a RUVBL1/MYC co-regulated gene EEF1A1 (a translation elongation factor) to be essential for the survival and protein synthesis of EwS cells. We also found that RUVBL1 interacts with MYC and recruits KAT5 (a histone acetyltransferase in NuA4) to modify the histone H4K8 and H4K12 for acetylation, which are required for the expression of EEF1A1. Furthermore, we conducted a high-density CRISPR gene body scan of 194 sgRNAs that tiled the coding exons of RUVBL1 at ~7.1 bp/sgRNA (i.e., ~2.4 aa/sgRNA). Using this unbiased approach and combined with 3D structural modeling, we uncovered the critical role of a previously unrecognized lysine residue K108 in RUVBL1, which locates at the center of the RUVBL1 hexameric ring structure, to mediate the MYC interaction and MYC target gene expression. Based on these observations, we rationalized a combinational approach to block the oncogenic MYC signaling via simultaneously targeting RUVBL1 (by CB-6644; inhibits RUVBL1’s ATPase activity) and MYC (by JQ1; suppresses MYC expression), and revealed an improved therapeutic index in both established EwS cell lines and the patient-derived EwS samples. In conclusion, our results identified RUVBL1 as a novel vulnerability in EwS, which controls the amplitude of MYC signaling via tuning the chromatin binding of MYC. This notion can be exploited to synergize with other MYC-targeting therapeutics for an improved EwS therapy. We also expect this mechanism to be broadly applicable to other MYC-driven cancers. Citation Format: Mingli Li, Lu Yang, Sheela Pokharel, Anthony Chan, Nicole Mattson, Qiao Liu, Xiaobao Xu, Chun-Wei Chen. RUVBL1 controls protein synthesis and tumor progression via MYC-dependent EEF1A1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1467.