Alternative polyadenylation (APA) is a gene regulatory process that dictates mRNA 3 ′ -UTR length, resulting in changes in mRNA stability and localization. APA is frequently disrupted in cancer and promotes tumorigenesis through altered expression of oncogenes and tumor suppressors. Pan-cancer analyses have revealed common APA events across the tumor landscape; however, little is known about tumor type-specific alterations that may uncover novel events and vulnerabilities. Here, we integrate RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Atlas (TCGA) to comprehensively analyze APA events in 148 pancreatic ductal adenocarcinomas (PDACs). We report widespread, recurrent, and functionally relevant 3 ′ -UTR alterations associated with gene expression changes of known and newly identified PDAC growth-promoting genes and experimentally validate the effects of these APA events on protein expression. We find enrichment for APA events in genes associated with known PDAC pathways, loss of tumor-suppressive miRNA binding sites, and increased heterogeneity in 3 ′ -UTR forms of metabolic genes. Survival analyses reveal a subset of 3 ′ -UTR alterations that independently characterize a poor prognostic cohort among PDAC patients. Finally, we identify and validate the casein kinase CSNK1A1 (also known as CK1alpha or CK1a) as an APA-regulated therapeutic target in PDAC. Knockdown or pharmacological inhibition of CSNK1A1 attenuates PDAC cell proliferation and clonogenic growth. Our single-cancer analysis reveals APA as an underappreciated driver of protumorigenic gene expression in PDAC via the loss of miRNA regulation.

Alternative polyadenylation (APA) is a gene regulatory process that dictates mRNA 3’-UTR length, resulting in changes in mRNA stability and localization. APA is frequently disrupted in cancer and promotes tumorigenesis through altered expression of oncogenes and tumor suppressors. Pan-cancer analyses have revealed common APA events across the tumor landscape; however, little is known about tumor type-specific alterations that may uncover novel events and vulnerabilities. Here we integrate RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Atlas (TCGA) to comprehensively analyze APA events in 148 pancreatic ductal adenocarcinomas (PDAs). We report widespread, recurrent and functionally relevant 3’-UTR alterations associated with gene expression changes of known and newly identified PDA growth-promoting genes and experimentally validate the effects of these APA events on expression. We find enrichment for APA events in genes associated with known PDA pathways, loss of tumor-suppressive miRNA binding sites, and increased heterogeneity in 3’-UTR forms of metabolic genes. Survival analyses reveal a subset of 3’-UTR alterations that independently characterize a poor prognostic cohort among PDA patients. Finally, we identify and validate the casein kinase CK1α as an APA-regulated therapeutic target in PDA. Knockdown or pharmacological inhibition of CK1α attenuates PDA cell proliferation and clonogenic growth. Our single-cancer analysis reveals APA as an underappreciated driver of pro-tumorigenic gene expression in PDA via the loss of miRNA regulation.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited effective treatment options. This potentiates the importance of uncovering novel drug targets. We have discovered global dysregulation of the gene regulatory process alternative polyadenylation (APA) in PDAC. APA is a pre-mRNA processing mechanism that generates mRNAs with distinct 3’ ends, impacting gene expression and protein function. We revealed that APA dysregulation in PDAC drives oncogenic signatures and predicts poor patient outcome. As APA directs widespread gene expression dysregulation across the PDAC patient population, we hypothesized that inhibition of APA has therapeutic potential. APA is controlled by a complex of proteins, including cleavage and polyadenylation specificity factor 3 (CPSF3). CPSF3 is the endonuclease catalyzing mRNA cleavage, and a potentially druggable target. We now find that CPSF3 is highly expressed and associated with poor prognosis in PDAC patients. CPSF3 knockdown decreases PDAC proliferation and clonogenicity in vitro and tumor growth in vivo. We demonstrate that CPSF3 knockdown induces widespread APA alterations of oncogenes and tumor suppressors, and determine the contribution of one of these events to CPSF3-induced cell proliferation phenotype. Furthermore, we find that PDAC, but not non-transformed pancreatic cells, are sensitive to the CPSF3 small molecule inhibitor JTE-607. Mechanistically, JTE-607 impairs replication-dependent histone processing, disrupting nucleosome assembly and destabilizing chromatin structure. Finally, we determine that JTE-607 attenuates cell proliferation by arresting cells in early S-phase of the cell cycle. Altogether, we identify CPSF3 as a druggable target in PDAC and reveal novel mechanisms by which CPSF3 controls cancer cell growth.SignificanceThis work identifies CPSF3 as a potential drug target in pancreatic ductal adenocarcinoma and reveals new mechanisms by which CPSF3 inhibition attenuates PDAC cell proliferation through modulating alternative polyadenylation and histone processing.

PurposeThis research investigates the association between benzodiazepines (BZDs) and cancer patient survival outcomes. Due to the high prevalence of BZD use in pancreatic cancer patients, we evaluated the effect of commonly prescribed BZDs on the pancreatic cancer tumor microenvironment and cancer-associated fibroblast (CAF) signaling.Experimental DesignMultivariate Cox regression modeling was used to retrospectively measure associations between Roswell Park cancer patient survival outcomes and BZD prescription records. Immunohistochemistry, H&E, Masson’s trichrome,in situhybridization, and RNA sequencing were used to evaluate the impact of lorazepam (LOR) on the PDAC tumor microenvironment, using murine pancreatic cancer models. ELISA and qPCR were used to determine the impact of BZDs on IL-6 expression/secretion by human immortalized pancreatic CAFs. PRESTO-Tango assays, reanalysis of PDAC single cell sequencing/TCGA datasets, and GPR68 CRISPRi knockdown CAF cells were used to mechanistically determine the impact of BZDs on CAF-specific GPR68 signaling.ResultsLOR is associated with worse progression-free survival (PFS) while alprazolam (ALP) is associated with improved PFS, in pancreatic cancer patients receiving chemotherapy. LOR promotes desmoplasia (fibrosis and extracellular matrix protein deposition), inflammatory signaling, IL-6 expression/secretion in CAFs, and ischemic necrosis. LOR promotes inflammatory signaling and IL-6 secretion by CAFs through activation of GPR68. GPR68 is preferentially expressed on human PDAC CAFs, and n-unsubstituted BZDs significantly increase GPR68 activation under acidic conditions. LOR increases IL-6 expression and secretion in CAFs in a pH and GPR68-dependent manner. Conversely, ALP, and other GPR68 non-activator BZDs decrease IL-6 in human CAFs in a pH and GPR68-independent manner. Across many cancer types, LOR is associated with worse survival outcomes relative to ALP and patients not receiving BZDs.ConclusionWe demonstrate that LOR stimulates fibrosis and inflammatory signaling, promotes ischemic necrosis, and is associated with decreased pancreatic cancer patient survival.

Treatment regimens of pancreatic ductal adenocarcinoma (PDAC) are limited and minimally effective which potentiates the importance of discovering novel potential drug targets. We discovered widespread dysregulation of alternative polyadenylation (APA) in PDAC patients. APA is a pre-mRNA processing mechanism that generates mRNAs isoforms with distinct 3’ untranslated regions (3’UTRs). These isoforms contribute to the expression and function of mRNAs and proteins. Our recent study revealed that APA alterations in PDAC drive dysregulation of PDAC-promoting genes and present a reliable prognostic marker. APA is controlled by a suite of genes, including cleavage and polyadenylation specificity factor 3 (CPSF3). Because CPSF3 is the endonuclease that catalyzes mRNA cleavage, we here sought to target CPSF3 in PDAC. We show that CPSF3 expression is significantly upregulated and associated with unfavorable prognosis in PDAC patients. Loss of CPSF3 decreases PDAC cell proliferation, clonogenicity and tumor growth. We show that CPSF3 loss causes APA changes of tumor-associated genes and validate the tumor suppressing activity of one APA-altered gene, FHL1. Using JTE-607, a chemical inhibitor of CPSF3, we find that PDAC cells are preferentially sensitive while non-transformed cell lines are resistant to CPSF3 inhibition. Notably, we show that JTE-607 decreases proliferation-dependent histone expression and disrupts nucleosome assembly. Finally, we find that JTE-607 arrests cells in early to mid S-phase of the cell cycle, resembling histone defect-induced cell cycle arrest. Overall, we reveal alternative polyadenylation and histone processing as two distinct mechanisms underlying CPSF3-induced attenuation of cell proliferation and identify CPSF3 as a potential therapeutic target in PDAC. Citation Format: Abdulrahman A. Alahmari, Aditi Chaubey, Arwen Tisdale, Carla Schwarz, Abigail Cornwell, Kathryn Maraszek, Emily Paterson, Minsuh Kim, Swati Venkat, Eduardo Cortes Gomez, Jianmin Wang, Katerina Gurova, Michael E. Feigin. Inhibition of CPSF3 attenuates pancreatic cancer cell proliferation through disruption of histone processing [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 B042.

The aim of this work was to determine the role of the benzodiazepine (BZD) lorazepam, an anxiolytic drug, in modifying the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment. PDA is a lethal malignancy. Poor survival rates and tumor intrinsic aspects of the disease, such as altered cytokine pools, promote significant levels of anxiety in patients. Approximately 25% of PDA patients will be prescribed BZDs. Epidemiological evidence suggests BZDs increase the risk of cancer development, but no group has determined the impact of BZDs on the PDA tumor microenvironment. Recent research has established that BZDs promote the signaling of GPR68, a proton-sensing G protein-coupled receptor activated by the acidic tumor microenvironment. GPR68 is expressed on PDA cancer-associated fibroblasts (CAFs) and promotes pro-inflammatory and pro-fibrotic signaling. Additionally, we performed covariate adjusted analyses of pancreatic cancer patients who received chemotherapy at Roswell Park from 2004 to 2020. Patients receiving lorazepam (LOR), an n-unsubstituted BZD that strongly activates GPR68, had shorter progression-free survival (PFS) relative to non-users (HR 3.83 (1.53, 9.57). In contrast, patients receiving alprazolam (ALP), an n-substituted BZD and GPR68 non-activator, had improved PFS relative to non-users (HR 0.38 (0.16-0.92). It is essential that we determine the impact of BZDs and GPR68 activation on the PDA tumor microenvironment and therapeutic response. We hypothesize that BZDs which are strong GPR68 activators will stimulate fibrosis and inflammatory signaling, promoting a more desmoplastic tumor microenvironment, decreasing the efficacy of chemotherapy. Using a subcutaneous KPC allograft mouse model, we found that lorazepam, a strong GPR68-activating BZD, modified the tumor microenvironment by increasing α-SMA (smooth muscle actin) expression, collagen deposition, and ischemic necrosis. Similarly, under acidic conditions, lorazepam-treated primary mouse and human CAFs promoted the mRNA expression of α-SMA and the pro-inflammatory cytokine, interleukin-6. Overall, this research indicates that lorazepam treatment significantly impacts the PDA tumor microenvironment. Significance: This research may guide the development of new clinical recommendations for prescribing anxiolytic drugs to PDA patients. The results of this research will likely be applicable to other cancer types which are reliant on CAFs for growth, such as colon cancer. Citation Format: Abigail C. Cornwell, Abdulrahman A. Alahmari, Arwen A. Tisdale, Michael E. Feigin. Lorazepam modifies the pancreatic ductal adenocarcinoma tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-047.

Tumors can co-opt the mRNA processing machinery to dysregulate the expression of oncogenes and tumor suppressors. One such altered process, termed alternative polyadenylation (APA), is responsible for regulating 3’-untranslated region (UTR) length. APA is driven by a core set of factors that recognize a series of highly conserved sequences within the 3’-UTR on the pre-mRNA. The pre-mRNA is then cleaved before addition of the poly(A) tail. As most transcripts contain several polyadenylation sequences, the choice of where to cleave is a critical determinant of 3’-UTR length. The 3’-UTR contains regulatory sequences, including miRNA binding sites, that control mRNA stability, function, and subcellular localization. Therefore, altering the length of a 3’-UTR can have dramatic impacts on gene function and cellular phenotype. APA factor expression is altered in a variety of cancer types, resulting in dysregulated gene expression. For example, loss of the APA factor CFIm25 in glioblastoma increases cell proliferation through 3’-UTR shortening and upregulated expression of cyclin D1. Pan-cancer analyses have revealed that hundreds of transcripts undergo APA. How these changes in 3’-UTR length are mechanistically linked to changes in gene expression and function are complex and only now being functionally addressed. Despite recent efforts to interrogate APA events in human tumors, numerous critical gaps in knowledge remain. Several pan-cancer analyses have revealed common APA events across diverse tumor types. However, no large-scale, single tumor-type analysis has been reported. Furthermore, the role of APA in pancreatic ductal adenocarcinoma (PDA) has been largely unaddressed. An understanding of the gene-regulatory mechanisms driving PDA progression may provide novel targets for therapeutic intervention. We have performed a comprehensive analysis of differential APA events in normal pancreas and PDA tumors, and observe widespread 3’-UTR shortening in PDA, correlating with expression changes in known PDA drivers and alterations in cancer signaling networks. Experimental validation of candidate genes reveals APA as a novel mechanism of regulation for the PDA oncogene ALDOA, and other genes implicated in pancreatic cancer cell growth, migration, and invasion. Hierarchical clustering of APA events allowed the development of a prognostic signature, identifying patients with poor prognosis. We find that 3’-UTR shortening events drive a preferential loss of tumor suppressive miRNA binding sites, and validate these findings through experimental manipulation of 3’-UTRs in cultured cells. Finally, we identify the serine/threonine kinase casein kinase 1 alpha (CK1) as an APA-regulated, targetable factor driving pancreatic cancer cell growth; inhibition of CK1 attenuates cell proliferation, colony formation, and migration. Our study reveals new mechanisms of dysregulation for cancer promoting genes, highlights the prognostic value of APA analysis, and identifies new targetable factors for PDA. Citation Format: Swati Venkat, Arwen A Tisdale, Kevin H. Eng, Michael E. Feigin. Alternative polyadenylation drives oncogenic gene expression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A14.

The goal of this research is to identify the effect of the GPR68-activator benzodiazepine (BZD) lorazepam on the pancreatic ductal adenocarcinoma (PDA) tumor microenvironment (TME). BZDs are commonly prescribed to PDA patients to treat anxiety and anticipatory nausea prior to chemotherapy. Certain types of BZDs are known to promote off-target activation of GPR68 under acidic conditions. We hypothesize that GPR68-activating BZDs will stimulate pro-fibrotic and pro-inflammatory signaling pathways by cancer-associated fibroblasts (CAFs), producing a more desmoplastic TME that will subsequently constrict the tumor vasculature, decreasing chemotherapeutic efficacy, and ultimately decreasing patient survival. Using a subcutaneous KPC allograft mouse model, we found that lorazepam modified the TME by significantly increasing α-SMA (smooth muscle actin) protein expression, collagen deposition, and ischemic necrosis. Preliminary, we found that treating tumor-bearing KPC mice with lorazepam similarly promoted ischemic necrosis. RNA sequencing of the lorazepam-treated allograft tumors indicated that CAF and ECM-related genes such as PDPN, LOX, SERPINB2, and ITGA11 were significantly upregulated. Pathway analysis revealed that lorazepam treatment promoted pathways related to inflammation, EMT, hypoxia, as well as known GPR68 downstream signaling pathways such as TNF-alpha signaling via NF-kB and IL6/JAK/STAT3 signaling. qRT-PCR of immortalized CAFs treated with lorazepam indicated that IL6 expression is increased by lorazepam in a GPR68-dependent manner at acidic pH, supporting that the promotion of IL6 expression we observed in vivo was likely GPR68 and CAF-dependent. To validate the clinical significance of our work, covariate adjusted analyses of pancreatic cancer patients who received chemotherapy at Roswell Park from 2004-2020 was performed. Patients taking GPR68 activator benzodiazepines versus non-activator benzodiazepines had poorer progression-free survival (HR 6.85(2.12,22.06)), suggesting that GPR68 activation by benzodiazepines may negatively impact survival. Overall, these findings suggest that lorazepam significantly modifies the PDA TME by promoting desmoplasia and ischemic necrosis, due in part to activation of GPR68. Future experimental work will determine if lorazepam negatively impacts survival and chemotherapeutic efficacy. Significance: This research may guide the development of new clinical recommendations for prescribing benzodiazepines to PDA patients, which will likely be applicable to other cancer types. Citation Format: Abigail C. Cornwell, Abdulrahman A. Alahmari, Arwen A. Tisdale, Kathryn Maraszek, Swati Venkat, Michael E. Feigin. Lorazepam promotes desmoplasia and ischemic necrosis in murine pancreatic ductal adenocarcinoma [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-100.