Abstract The Hippo pathway is an evolutionarily conserved signaling pathway that regulates multiple biological functions, including organ size, cell proliferation, contact inhibition, and is also significantly implicated in cancer. In multiple cancer types, dysregulation of the Hippo pathway leads to heightened activity of the oncoproteins YAP and TAZ that contributes to oncogenesis. In mammalian cells, YAP is regulated by a phosphorylated cascade mediated by the kinases MST1/2 and LATS1/2. This cascade, known as the canonical Hippo pathway, receives upstream cues such as cell density, mechanical stress and other factors and ultimately results in phosphorylation of YAP/TAZ and its cytosolic degradation. Pertubation of the Hippo pathway leads to YAP intranuclear translocation and downstream activation of target genes, inducing proliferative signals and thus initiating oncogenesis. YAP/TAZ upregulation have been described in cancer, and YAP driven animal cancer models have been established. Much effort has been towards studying downstream effects of YAP activation in cancer, but the role of upstream epigenetic regulation of YAP is unclear. We asked whether if histone deacetylation has a role in YAP expression. Histone deacetylases (HDACs) are essential in controlling transcription, and HDAC inhibitors are approved in several cancer types. We discovered that HDAC inhibitors decrease YAP expression significantly without inhibiting TAZ levels, in multiple cancer cell lines. Interestingly, the downregulation occurs at the transcriptional level, suggesting that this inhibition is independent of canonical Hippo pathway. HDAC inhibition mediated downregulation of YAP leads to decreased nuclear translocation of YAP. We then show that the transcription inhibition of YAP expression is mediated by HDAC1, a member of the class I HDACs. This is of particular interest since HDAC1, normally involved in transcriptional silencing, conversely maintains YAP expression in human cancers. To clarify the exact mechanism, transcriptional profiling by RNA-seq and miR-seq were then performed on samples from cells treated with HDAC inhibitors or siRNA targeting HDAC1. Overlap of the consensus differentially expressed genes identified transcription factors that play a role in this pathway. Analysis of human RNA-seq data in cancers with HDAC inhibition validated our findings in human gene expression databases.In summary, we have discovered a novel pathway showing that inhibition of HDAC1 downregulates YAP expression, further inhibiting its intranuclear translocation and related downstream signaling. Interestingly, inhibition of HDAC1 conversely suppresses YAP expression. Our study uncovers a novel link between HDAC1 and the Hippo pathway and paves rationale for HDAC inhibitors, of which several drugs are approved, for a potential role of YAP driven cancers. Citation Format: Nai-Kuan Wang, Ming-Yuan Chao, Yu-Hsuan Huang, Jiun-I Lai. Mechanism of the novel HDAC1-YAP epigenetic regulatory mechanism in Hippo pathway of human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5303.