Abstract PO-001: ARNTL2 is a hypoxia-responsive master regulator of PDAC malignancy
Authors: Curiel-Garcia Alvaro , Maurer Carlo H. , Laise Pasquale , Sagalovskiy Irina , Chabot John A. , Manji Gulam A. , Iuga Alina , Johnson Kristen , Califano Andrea , Olive Kenneth P. 
Published: November 2021

Abstract
Large-scale sequencing studies have identified a limited number of genetic drivers of PDAC malignancy, with only four genes found mutated a high penetrance across human pancreatic tumors. This suggests that the cell-autonomous ability to progress, dedifferentiate and metastasize is determined by non-genetic alterations to transcriptional regulation. Regulatory network analysis is a computational means of identifying changes in the activity of transcription factors on a genome-wide scale. Briefly, the approach calculates the activity of each regulatory protein (i.e. transcription factors, co-factors, and other proteins that regulate RNA transcript abundance) based on the expression of its positive and negative transcriptional targets (which are inferred de novo in a context-specific manner using the ARACNe algorithm). In this way, it is possible to identify the most hyper-activated and hyper-repressed regulatory proteins between groups of samples that represent different phenotypes – i.e. the “master regulators” (MRs) of those phenotypes. We applied regulatory network analysis to identify MRs of PDAC malignancy by comparing groups of samples representing several phenotypes. First, we performed laser capture microdissection and RNA sequencing (LCM-RNAseq) on 199 human PDAC samples, 26 low-grade PanIN, and 19 low-grade IPMN, isolating just the epithelial cells for transcriptional profiling. We then identified MRs of four different phenotypes among these samples: 1) precursor versus PDAC; 2) low-grade versus high-grade PDAC (based on histopathology); 3) low versus high Kras signaling; and long versus short overall survival. Integrating these analyses, the most highly activated regulatory protein in the genome across these four phenotypes was ARNTL2, an understudied member of the ARNT (HIF1β) superfamily. Notably, this protein was only modestly differentially expressed at the RNA level, indicating that its activation occurs primarily at a post-translational level. Computational analysis of the function of ARNTL2 indicated an enrichment of pathways involved in hypoxia responsiveness. Experimental analysis found that ARNTL2 had little impact in proliferation or survival under normoxic conditions, but drove increased invasiveness under hypoxic conditions. The functional associations of ARNTL2 in cultured cells expanded substantially under hypoxic conditions. Together, these findings suggest that ARNTL2 may serve as a Kras-associated, hypoxia-responsive driver of PDAC malignancy.
Citation Format: Alvaro Curiel-Garcia, Carlo H. Maurer, Pasquale Laise, Irina Sagalovskiy, John A. Chabot, Gulam A. Manji, Alina Iuga, Kristen Johnson, Andrea Califano, Kenneth P. Olive. ARNTL2 is a hypoxia-responsive master regulator of PDAC malignancy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-001.