Investigating the Role of the Lysine-Specific Demethylase 4C in Pancreatic Ductal Adenocarcinoma

MennatAllah Shaheen, MS (Advisor: Anirban Maitra, MBBS)

           Pancreatic cancer is the third leading cause of cancer deaths in the United States, and the 10th most diagnosed. Pancreatic ductal adenocarcinoma is the most common type of pancreatic cancer representing over 90% of patient cases. Late diagnosis, the complex nature of this disease and the relative lack of druggable mutations have been greatly limiting progress in treating pancreatic cancer patients. Extensive research efforts in pancreatic cancer across the years have characterized the genetic mutations driving cancer initiation, and progression, stratified patients into clinically relevant subtypes, and studied the pancreatic tumor microenvironment to identify therapeutic targets and to develop more effective chemotherapy agents. Indeed, survival rates of pancreatic cancer has increased from 7% to 13% over the past decade, raising hopes for more effective treatment options in the future. For a long time, mutant KRAS, the main oncogenic driver in pancreatic cancer, was considered undruggable, although multiple KRAS inhibitor molecules are currently being tested in the clinic.  On the other hand, recent research studies have demonstrated the role of epigenetic aberrations in driving tumor progression and maintenance. The deregulation of proteins involved in chromatin regulation is common in pancreatic ductal adenocarcinoma (PDAC) and has remained a largely untapped area of research. Lysine demethylase 4C (KDM4C) is one of the chromatin-modifying proteins frequently overexpressed across multiple solid cancers. KDM4C overexpression is linked to chromatin instability, increased cell proliferation, and enhanced stem cell-like behavior. We observed the upregulation of KDM4C protein in a panel of human and murine PDAC cell lines, as well as in PDAC patient samples compared to non-neoplastic controls. CRISPR/Cas9-mediated deletion of KDM4C in human and murine PDAC cells resulted in reduced proliferation and clonogenicity and increased survival of orthotopically implanted murine PDAC allografts. Notably, in multiple KDM4C-null PDAC clones analyzed by reverse phase protein arrays (RPPA), we observed a downregulation of activated mitogen-activated protein kinase (MAP kinase) pathway components (including phospho-MEK and phospho-ERK), which are key effector pathways downstream of mutant KRAS. In vitro propagation of KDM4C-null PDAC lines eventually led to adaptation and restitution of MAP kinase signaling via compensatory upregulation of other KDM4 family members (KDM4A and KDM4B). In order to bypass genetic compensation by KDM4 family members, we utilized a preclinical grade pan-KDM4 inhibitor (TACH107, Tachyon Therapeutics) in PDAC cell lines, which confirmed profound reduction in proliferation and colony formation; and increased survival of in vivo xenograft and allograft mice treated with TACH107. To characterize signaling pathways perturbed by KDM4C deregulation, we investigated the transcriptional changes in PDAC cells with and without KDM4C deletion by RNA-seq and integrated that with alterations in H3K9me3 and H3K36me3 histone marks using ChIP-Seq. Importantly, gene set enrichment analysis showed downregulation in RAS signaling signature and given our data on MAP kinase signaling, we prioritized candidates implicated in regulation of Ras and Ras effector signaling. We found that kinase suppressor of ras 2 (KSR2) a scaffolding protein and positive regulator of MAPK cascade, is transcriptionally downregulated and has increased H3K9me3 marks near the promoter region in KDM4C depleted cells, suggesting that KDM4C epigenetically activates KSR2 thus activating MAPK signaling. To further dissect the mechanism of action of KDM4C we sought to identify direct protein binding partners of KDM4C through proximity labeling. We validated the histone deacetylase SIRT1 as a novel binding partner of KDM4C. We also found that there are increased H3K36me3 and H3K27Ac marks at the DUSP2 promoter suggesting that KDM4C and SIRT1 might be working in synergy to repress the expression of specific downstream targets, like DUSP2, to maintain active MAPK signaling. Further, we created KDM4C catalytic domain deletion construct, and reader domain deletion constructs that we used to identify that Tudor domain is responsible for SIRT1 binding. Our data suggests that KDM4C is an important regulator of MAPK signaling, which is the main effector pathway downstream of mutant Ras. To the best of our knowledge, this is the first demonstration linking the requirement of sustained activity of the KDM4 family of lysine demethylases to MAPK signaling in cancer and presents an opportunity to leverage this oncogenic pathway for therapeutic purposes.

Advisory Committee:

• Anirban Maitra, MBBS, Chair
• Chanda Joya, PhD
• Kunal Rai, PhD
• Subrata Sen, PhD
• Haoqiang Ying, PhD

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