Abstract B48: A novel high throughput screening platform identifies statins as inducers of basic Helix-Loop-Helix activity, p21 and growth arrest in pancreatic cancer cell and patient derived xenograft lines

Pancreatic Ductal Adenocarcinoma (PDA) is the most lethal form of cancer with an average survival of merely 6 months, underscoring the need for new therapeutic approaches. A central feature of PDA progression is the reduced expression and activity of basic helix-loop-helix (bHLH) transcription factors. Recently, we showed that restoring bHLH activity in highly aggressive PDA cells, through overexpression of the bHLH factor E47, resulted in growth arrest and reduced tumorigenicity. Cell cycle exit was characterized by reduced expression of Aurora kinase A, cyclin A2 and Topoisomerase 2A and required upregulation of the cell cycle inhibitor p21. In order to translate these findings for clinical utility, here we developed a high throughput screening platform to identify small molecule inducers of E47-like bHLH proteins in PDA. E47 belongs to phylogenic Group A family of bHLH factors. Within Group A are the semi-ubiquitous Class I proteins, e.g. E47, and the tissue restricted Class II bHLH proteins, e.g PTF1a and MIST1. To capture compounds increasing the activity Group A factors we utilized a luciferase reporter driven by a 4x multimer of three unique canonical Group A binding sites. The vector was introduced into human PANC-1 cells and overexpression of E47 was employed as a positive control. Statistical analysis of assay robustness and dynamic range was measured as a z factor, and a promising z factor of 0.58 was achieved, demonstrating both robustness and reproducibility of the screening platform. We therefore performed a screen of 4,375 known drugs, revealing 246 hits which induced bHLH activity up to 40.6 standard deviations from the mean (initial hit rate=5.6%). Each hit was re-assayed in triplicate, resulting in 84 confirmed hits, representing 70 unique inducers of bHLH activity (final hit rate=1.6%). Notably, among the hits were 6 members of the statin class of cholesterol lowering drugs. Statins are currently in clinical trials for multiple cancers, including PDA. Mechanistic studies with pitavastatin in the established PDA cell lines PANC-1 and BxPC3, and in a patient derived cell line, AA0779E, revealed dose responsive inhibition of growth. Concurrently the statins induced dose responsive upregulation of p21 at both the mRNA and protein levels, with dose responsive abrogation of Aurora kinase A, topoisomerase, and cyclin A2. Together, these findings establish the effectiveness of a novel screening platform to identify drugs of interest in pancreatic cancer. Moreover, the studies reveal for the first time that statins regulate bHLH activity and bHLH target genes in PDA. In conclusion, the data underscore the central role of bHLH signaling as an arbiter of cell cycle arrest in PDA and suggest that pharmacologic induction of bHLH signaling for therapeutic benefit may be possible. Citation Format: Reyhaneh Lahmy, Nicholas Villarino, Jaco van Niekerk, Tarek Almaleh, SangWun Kim, Andrew Lowy, Pamela Itkin-Ansari.{Authors}. A novel high throughput screening platform identifies statins as inducers of basic Helix-Loop-Helix activity, p21 and growth arrest in pancreatic cancer cell 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 B48.