Abstract B002: ARNTL2 is a Kras-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma
Authors: Maurer H. Carlo , Curiel-Garcia Alvaro , Laise Pasquale , Palermo Carmine F. , Sastra Stephen A. , Manji Gulam A. , Iuga Alina , Johnson Kristen , Genkinger Jeanine , Lyssiotis Costas A. , Califano Andrea , Olive Kenneth P. 
Published: November 2022

Abstract
We employed regulatory network analysis to identify master transcriptional regulators whose activity drives key pancreatic cancer phenotypes. Regulatory network analysis is a systems biology approach that computationally infers the activity of transcriptional regulators based on the integrated expression of their positive and negative target genes. To focus on epithelial cell biology, we performed laser capture microdissection and RNA sequencing on 200 human PDAC samples and 45 low-grade precursors (PanIN and IPMN), generating a rich dataset with clinical, epidemiological, and histopathological annotation. Using a transcriptional regulatory network derived from this dataset, we performed master regulator analysis to identify master regulators of four phenotypes: precursors vs. PDAC (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Changes in the activity of multiple established PDAC drivers and suppressors were associated with tumor initiation and progression, providing a strong validation from existing biology. Integrating across these phenotypes, the top master regulator of PDAC malignancy was found to be ARNTL2, a member of the PAS family of bHLH transcription factors. The canonical function of ARNTL2 is as the heterodimeric binding partner of the circadian rhythm protein CLOCK. However, functional analysis ARNTL2 inferred transcriptional targets highlighted a potential role in hypoxia response and prior studies have demonstrated the ability of ARNTL2 to heterodimerize with HIF1A. Indeed, ARNTL2 was inferred to directly or indirectly regulate every single hypoxia-responsive protein in a previously published hypoxia signature. We therefore studied the role of ARNTL2 in both normoxic and hypoxic conditions and found that its knockdown in PDAC cells led to a selective decrease in viability and invasion under hypoxic conditions. ARNTL2 activity was induced in response to hypoxia and inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its computationally-ingerred association with RAS activity. Strikingly, we found that ARNTL2 was required in PDAC cells for the stabilization of HIF1A in response to hypoxia. Conversely, HIF2A was further stabilized in response to hypoxia upon ARNTL2 deletion, suggesting that ARNTL2 serves to control the balance between HIF1A- and HIF2A dependent metabolic programs in PDAC. Consistent with this, loss of ARNTL2 had sizeable impacts on profiles in PDAC cells and led to a significant reduction of lactate dehydrogenase A under hypoxic conditions. Together these data demonstrate that ARNTL2 is a previously underappreciated master regulator of hypoxia response in pancreatic ductal adenocarcinoma.
Citation Format: H. Carlo Maurer, Alvaro Curiel-Garcia, Pasquale Laise, Carmine F. Palermo, Stephen A. Sastra, Gulam A. Manji, Alina Iuga, Kristen Johnson, Jeanine Genkinger, Costas A. Lyssiotis, Andrea Califano, Kenneth P. Olive. ARNTL2 is a Kras-dependent master regulator of hypoxic response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B002.