G9a Responds to KRAS‐Mediated Replication Stress by Increased Origin Licensing

Abstract

Pancreatic Ductal Adenocarcinoma (PDAC) remains as one of the leading causes of cancer death in the US. KRAS mutations are recognized as the leading oncogenic driver of PDAC growth. Activation of KRAS causes increased proliferation that requires a level of adaptation during processes, such as DNA replication. This rapid duplication of the genome and epigenome generates havoc in the timing and coordination of replication origin firing, triggering a response known as replication stress (RS). The epigenomic regulator G9a is responsible for catalyzing histone H3 lysine 9 mono‐ and di‐methylation (H3K9me1 and H3K9me2). G9a is abundantly present in many forms of cancer, including PDAC, and because of its interaction with replication proteins, such as RPA and PCNA, it is known to have an important role during DNA replication. DNA replication happens at the S phase of the cell cycle, which requires that replication origins are recognized at late G1 phase by the origin recognition complex (ORC) followed by assembly of the pre‐replication complex (pre‐RC), comprised of CDC6, cdt1 and mini chromosome maintenance (MCM) proteins, to allow origin licensing and subsequent firing. We have shown that the G9a complex increases in response to KRAS activation and is critical to the growth‐promoting effects of this oncogene in PDAC. Though it is known that KRAS reduces distances between replication origins, there has been a paucity of knowledge regarding the role of G9a during replication origin licensing and whether it is directly involved with the pre‐RC. In the current study, we aim to better understand whether G9a interacts with the replication origins and the pre‐RC to increase origin licensing under conditions of KRAS‐mediated RS. To simulate the early events in oncogene‐induced RS to study compensatory mechanisms, we generated an inducible KRAS model (iKRAS‐HPNE) in an hTERT‐immortalized human non‐cancerous pancreatic ductal cell line, hTERT‐HPNE E6/E7. We found that KRAS induction for 48h in this iKRAS‐HPNE model activated the ATR RS‐response pathway, indicated by increased levels of P‐T1989‐ATR, P‐S345‐CHK1 and P‐S33‐RPA32 by western blot and immunofluorescence‐based microscopy. Concurrently, members of the G9a epigenomic complex (G9a, GLP and WIZ) significantly increased along with two subunits of the ORC (ORC1 and ORC2) and several other pre‐RC proteins (Cdt1, MCM2, MCM5 and MCM7). Co‐immunoprecipitation of G9a in the iKRAS‐HPNE cells revealed that G9a interacts with ORC2 protein under conditions of oncogene‐induced RS. Pharmacological inhibition of G9a with UNC0642 during KRAS‐induced RS resulted in a decrease of chromatin‐bound ORC1 and ORC2 as well as pre‐RC proteins in subcellular fractionation experiments, suggesting that functional G9a is critical for enhanced origin licensing. In summary, our results demonstrate that G9a directly interacts with replication origins and the pre‐RC during origin licensing under conditions of RS. Our study reveals one potential mechanism by which cancerous cells with KRAS mutation meet the demands for rapid cellular proliferation during RS and reinforces the role of G9a as a promising therapeutic target for PDAC.