Abstract
Abstract Despite advances in targeted agents and immunotherapy, non-small cell lung cancer (NSCLC) remains the number one cause of cancer-related death. To identify new therapeutic targets in NSCLC, we performed an siRNA screen directed against genes involved in chromatin remodeling. This screen showed that RUVBL1 and RUVBL2 (herein collectively referred to as RUVBL1/2) were universally but differentially required for the viability of 24 NSCLC cell lines, which was an on-target effect. Various independent gene expression datasets/platforms show that NSCLC patient tumors have increased levels of RUVBL1 and RUVBL2 mRNAs, in comparison to normal lung, and that patients with high levels of RUVBL1 or RUVBL2 have a poorer prognosis, suggesting that RUVBL1/2 may play an important role in NSCLC tumors. To confirm this at the protein level, we validated an antibody against RUVBL1 for immunohistochemistry, stained clinically annotated NSCLC tissue microarrays for RUVBL1, and found that patients with higher levels of RUVBL1 protein also have a poorer prognosis. To better understand the role of RUVBL1/2 in NSCLC at a molecular level, we measured the distribution of cells in the cell cycle following RUVBL1/2 KD, determined RUVBL1/2 interacting proteins by immunoprecipitation followed by tandem mass-spec (IP-MS/MS), and measured gene expression changes following RUVBL1/2 KD by RNA-seq. Depletion of RUVBL1/2 arrested cells in S-phase and promoted pan-γH2AX positivity, IP-MS/MS showed an over-representation of proteins involved in DNA repair and replication, and gene set enrichment analysis of the RNA-seq data displayed a downregulation of transcripts involved in DNA replication and repair, strongly implicating RUVBL1/2 in these processes. To further probe the effects of RUVBL1/2 loss, we performed low-level knock down (KD) of RUVBL1/2, such that viability is largely unaffected, and then measured the viability of cells in response to various drugs (n=35). These drugs target a wide variety of biological processes; however, the only drugs with increased efficacy in the presence of RUVBL1/2 KD were those that damage DNA, target ATR or its downstream partner CHEK1, or target some mitotic proteins, further implicating RUVBL1/2 in DNA replication and/or repair. Due to their roles in replication, we reasoned that RUVBL1/2 KD may enhance the effects of ionizing radiation, a treatment frequently given to NSCLC patients. Low level KD of RUVBL1/2 decreased the clonogenic potential of multiple NSCLC cell lines, which can be phenocopied by depleting independent subunits of RUVBL1/2-containing chromatin remodeling complexes. Finally, we show that RUVBL1/2 depend upon their ATPase activity to support NSCLC viability, suggesting that small molecule inhibitors of this protein may be efficacious in the treatment of NSCLC, especially when combined with radiation therapy. Citation Format: Paul Yenerall, Rahul Kollipara, Amit Das, Pamela Villalobos, Long Shan Li, Brenda Timmons, Luc Girard, Jaime Rodriguez-Canales, Ignacio Wistuba, John Minna, Ralf Kittler. The chromatin remodelers RUVBL1 and RUVBL2 are prognostic factors and therapeutic targets in non-small cell lung cancer due to their roles in DNA replication, repair, and radiosensitization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1111. doi:10.1158/1538-7445.AM2017-1111
Published Version
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