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In normal pancreatic acinar cells, acquisition of activating mutations in Kras leads to protective oncogene-induced-senescence (OIS) (Collado et al, Nature 2005). This barrier to malignant transformation is lost upon subsequent mutation and LOH of p53, DNA-methylation based silencing of the p16 locus, and pancreatitis (Guerra et al, Cancer Cell 2011). We have previously shown that pancreatic ductal adenocarcinoma (PDA) tumors express high levels of ID3, an inhibitor of bHLH transcription factor activity. Moreover, overexpression of ID3 alone in human exocrine cells was sufficient to induce cell cycle entry suggesting that the bHLH/ID3 axis is a powerful arbiter of cell cycle status in vitro and in vivo (Lee et al, Molecular Cancer Research 2011). Here we report that restoring bHLH activity in aggressive PDA cells reinstates senescence in the absence of wild-type p53 or p16 in both established PDA lines and patient-derived xenograft lines. Previously we showed that overexpression of the bHLH protein, E47, in established cell lines - Panc-1, Bx-PC3 and MiaPaCa-2 - induced growth arrest through a mechanism that involved upregulation of p21 expression (Kim et al, Pancreas 2015). Here we report that two patient-derived pancreatic cancer cell lines - AA0779E from a primary human PDA, and AA1334E, a liver metastasis from a primary human PDA, also stopped proliferating within 48 hours in response to high levels of E47. We used RNA-sequencing to characterize E47 induced genome-wide changes in the transcriptomes of all five cell lines and found that the most significant and consistent alterations occurred in expression of a large cohort of genes that code for regulators of the cell cycle. In all lines, p21 was among the most highly upregulated. In addition, the RNA-sequencing data revealed that E47 restored normal expression of genes that are specifically linked to senescence, including laminB1, Dec1/bHLHe40 and CENP-A. Moreover, all of the cell lines underwent extensive senescence-associated changes in cell size and morphology concomitant with induction of senescence-associated beta-galactosidase activity (SA-beta-gal). In conclusion, we have shown that overexpressed E47 initiates a senescence program that is independent of wild-type p53 or p16 in all PDA cell lines examined including those derived from a liver metastasis of PDA. Interestingly, Panc-1 and AA0779E cells remained stably arrested for weeks in culture while Bx-PC3, MiaPaCa-2, and AA1334E cells died within 96 hours. This outcome is consistent with a model in which genetic variability among lines determines whether the cells fully execute the cellular senescence program or succumb to cell death after initiation. Importantly, since most patients present with disseminated disease, the data suggest that a therapy aimed at restoring bHLH activity could be effective in halting growth of both primary tumors and metastases. Citation Format: Kathleen M. Scully, Reyhaneh Lahmy, Heejung Kim, Andrew Lowy, Pamela Itkin-Ansari.{Authors}. Overexpression of the basic helix-loop-helix transcription factor, E47, promotes p16-independent senescence in established and patient-derived xenograft lines. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A55.