Epigenetic Modulators Show Differential Activity On Lung Adenocarcinoma Cells With Loss-Of-Function Mutations Of Swi/Snf Protein Smarca4

SWI/SNF is a multiprotein chromatin remodeler with ATP-dependent activities leading to selective gene expression, DNA repair, recombination and replication. Various sequencing efforts indicated that nearly 20% of cancers bear mutations in at least one subunit of the complex. One of the crucial regulators of the complex is SMARCA4, a member of SWI/SNF family of helicases with ATPase activities, which are thought to regulate transcription of certain genes by altering the chromatin structure. SMARCA4 is mutated in virtually all cases of small cell carcinoma of the ovary and SMARCA4 is fourth the most frequently mutated gene in lung adenocarcinoma. High occurrence of inactivating mutations prompted several screenings projects focused on synthetic lethality interactions with other proteins, which led to the identification of SMARCA2 as an essential gene in SMARCA4 mutated cancers. This vulnerability could be potentially exploited therapeutically and several groups managed to identify potent ligands of SMARCA2 bromodomain. Surprisingly these molecules were inactive in SMARCA4 mutant cells, however additional studies indicated that ATPase rather than bromodomain is a target for novel compounds with anticancer activities. Overall, these results revealed functional complexity of SMARCA2 and SMARCA4 in cancer cells. In order to characterize molecular consequences of SMARCA2 silencing in SMARCA4 mutant lung adenocarcinoma cells, we have carried out a series of gene knockdown experiments, followed by transcriptional profiling by RNAseq and analysis of posttranslational histone modifications. These studies indicated rapid and irreversible loss of viability in SMARCA4 mutant cells after SMARCA2 gene silencing. Interestingly double SMARCA2/SMARCA4 knockdown in SMARCA4 WT cells has not resulted in lowered viability. Transcriptional profiling of SMARCA2 knockdown in SMARCA4 mutated cells revealed broad, predominantly repressory effects on gene expression levels. Gene set enrichment analysis showed significant inhibitory effects of SMARCA2 knockdown, particularly on transcripts which could be induced after reintroduction of SMARCA4. Furthermore MS/MS analysis indicated global rearrangements in histone epigenetic marks. These results prompted us to test activity of small-molecule epigenetic modulators, including DNA Methyltransferases (DNMTs), Histone Acetyltransferases (HATs), Histone Deacetylases (HDACs), Histone Methyltransferases (HMTs), Histone Demethylases (HDMs) and a distinct set of chromatin readers, bromodomains. Differential activity of these compounds provides a strong basis for novel strategies for the treatment of cancers with loss-of-function mutations of SMARCA4. Citation Format: Tomasz Rzymski, Anna Wrobel, Michal Mikula, Karolina Pyziak, Anna Bartosik, Agnieszka Sroka, Agnieszka Paziewska, Aleksandra Grochowska, Malgorzata Statkiewicz, Katarzyna Paczkowska, Michalina Dabrowska, Jerzy Ostrowski, Krzysztof Brzozka. Epigenetic modulators show differential activity on lung adenocarcinoma cells with loss-of-function mutations of SWI/SNF protein SMARCA4 [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 5063. doi:10.1158/1538-7445.AM2017-5063