CPSF3 inhibition blocks pancreatic cancer cell proliferation through disruption of core histone mRNA processing.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited effective treatment options, potentiating the importance of uncovering novel drug targets. Here, we target cleavage and polyadenylation specificity factor 3 (CPSF3), the 3' endonuclease that catalyzes mRNA cleavage during polyadenylation and histone mRNA processing. We find that CPSF3 is highly expressed in PDAC and is associated with poor prognosis. CPSF3 knockdown blocks PDAC cell proliferation and colony formation in vitro and tumor growth in vivo. Chemical inhibition of CPSF3 by the small molecule JTE-607 also attenuates PDAC cell proliferation and colony formation, while it has no effect on cell proliferation of nontransformed immortalized control pancreatic cells. Mechanistically, JTE-607 induces transcriptional readthrough in replication-dependent histones, reduces core histone expression, destabilizes chromatin structure, and arrests cells in the S-phase of the cell cycle. Therefore, CPSF3 represents a potential therapeutic target for the treatment of PDAC.

Biopsy and blood-based molecular biomarker of inflammation in IBD.

OBJECTIVE: IBD therapies and treatments are evolving to deeper levels of remission. Molecular measures of disease may augment current endpoints including the potential for less invasive assessments. DESIGN: Transcriptome analysis on 712 endoscopically defined inflamed (Inf) and 1778 non-inflamed (Non-Inf) intestinal biopsies (n=498 Crohn's disease, n=421 UC and 243 controls) in the Mount Sinai Crohn's and Colitis Registry were used to identify genes differentially expressed between Inf and Non-Inf biopsies and to generate a molecular inflammation score (bMIS) via gene set variance analysis. A circulating MIS (cirMIS) score, reflecting intestinal molecular inflammation, was generated using blood transcriptome data. bMIS/cirMIS was validated as indicators of intestinal inflammation in four independent IBD cohorts. RESULTS: bMIS/cirMIS was strongly associated with clinical, endoscopic and histological disease activity indices. Patients with the same histologic score of inflammation had variable bMIS scores, indicating that bMIS describes a deeper range of inflammation. In available clinical trial data sets, both scores were responsive to IBD treatment. Despite similar baseline endoscopic and histologic activity, UC patients with lower baseline bMIS levels were more likely treatment responders compared with those with higher levels. Finally, among patients with UC in endoscopic and histologic remission, those with lower bMIS levels were less likely to have a disease flare over time. CONCLUSION: Transcriptionally based scores provide an alternative objective and deeper quantification of intestinal inflammation, which could augment current clinical assessments used for disease monitoring and have potential for predicting therapeutic response and patients at higher risk of disease flares.

Alternative polyadenylation drives oncogenic gene expression in pancreatic ductal adenocarcinoma.

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 Characterizes Epithelial and Fibroblast Phenotypic Heterogeneity in Pancreatic Ductal Adenocarcinoma.

Human tumors are characterized by extensive intratumoral transcriptional variability within the cancer cell and stromal compartments. This variation drives phenotypic heterogeneity, producing cell states with differential pro- and anti-tumorigenic properties. While bulk RNA sequencing cannot achieve cell-type-specific transcriptional granularity, single-cell sequencing has permitted an unprecedented view of these cell states. Despite this knowledge, we lack an understanding of the mechanistic drivers of this transcriptional and phenotypic heterogeneity. 3' untranslated region alternative polyadenylation (3' UTR-APA) drives gene expression alterations through regulation of 3' UTR length. These 3' UTR alterations modulate mRNA stability, protein expression and protein localization, resulting in cellular phenotypes including differentiation, cell proliferation, and migration. Therefore, we sought to determine whether 3' UTR-APA events could characterize phenotypic heterogeneity of tumor cell states. Here, we analyze the largest single-cell human pancreatic ductal adenocarcinoma (PDAC) dataset and resolve 3' UTR-APA patterns across PDAC cell states. We find that increased proximal 3' UTR-APA is associated with PDAC progression and characterizes a metastatic ductal epithelial subpopulation and an inflammatory fibroblast population. Furthermore, we find significant 3' UTR shortening events in cell-state-specific marker genes associated with increased expression. Therefore, we propose that 3' UTR-APA drives phenotypic heterogeneity in cancer.

Lorazepam Stimulates IL6 Production and Is Associated with Poor Survival Outcomes in Pancreatic Cancer.

PURPOSE: This research investigates the association between benzodiazepines (BZD) and cancer patient survival outcomes, the pancreatic cancer tumor microenvironment, and cancer-associated fibroblast (CAF) signaling. EXPERIMENTAL DESIGN: Multivariate Cox regression modeling was used to retrospectively measure associations between Roswell Park cancer patient survival outcomes and BZD prescription records. IHC, H&E, Masson's trichrome, RNAscope, and RNA sequencing were used to evaluate the impact of lorazepam (LOR) on the murine PDAC tumor microenvironment. ELISA and qPCR were used to determine the impact of BZDs on IL6 expression or secretion by human-immortalized pancreatic CAFs. PRESTO-Tango assays, reanalysis of PDAC single-cell sequencing/TCGA data sets, and GPR68 CRISPRi knockdown CAFs were used to determine the impact of BZDs on GPR68 signaling. RESULTS: LOR is associated with worse progression-free survival (PFS), whereas alprazolam (ALP) is associated with improved PFS, in pancreatic cancer patients receiving chemotherapy. LOR promotes desmoplasia (fibrosis and extracellular matrix protein deposition), inflammatory signaling, and ischemic necrosis. GPR68 is preferentially expressed on human PDAC CAFs, and n-unsubstituted BZDs, such as LOR, significantly increase IL6 expression and secretion in CAFs in a pH and GPR68-dependent manner. Conversely, ALP and other GPR68 n-substituted BZDs decrease IL6 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. CONCLUSIONS: We demonstrate that LOR stimulates fibrosis and inflammatory signaling, promotes desmoplasia and ischemic necrosis, and is associated with decreased pancreatic cancer patient survival.

Regulation of Cyclin D1 Degradation by Ubiquitin-Specific Protease 27X Is Critical for Cancer Cell Proliferation and Tumor Growth.

Cyclin D1 (CCND1) is a critical regulator of cell proliferation and its overexpression has been linked to the development and progression of several malignancies. CCND1 overexpression is recognized as a major mechanism of therapy resistance in several cancers; tumors that rely on CCND1 overexpression to evade cancer therapy are extremely sensitive to its ablation. Therefore, targeting CCND1 is a promising strategy for preventing tumor progression and combating therapy resistance in cancer patients. Although CCND1 itself is not a druggable target, it can be targeted indirectly by inhibiting its regulators. CCND1 steady-state levels are tightly regulated by ubiquitin-mediated degradation, and defects in CCND1 ubiquitination are associated with increased CCND1 protein levels in cancer. Here, we uncover a novel function of ubiquitin-specific protease 27X (USP27X), a deubiquitinating enzyme (DUB), in regulating CCND1 degradation in cancer. USP27X binds to and stabilizes CCND1 in a catalytically dependent manner by negatively regulating its ubiquitination. USP27X expression levels correlate with the levels of CCND1 in several HER2 therapy-resistant breast cancer cell lines, and its ablation leads to a severe reduction of CCND1 protein levels, inhibition of tumor growth, and resensitization to targeted therapy. Together, the results presented in our study are the first to expose USP27X as a major CCND1 deubiquitinase and provide a mechanistic explanation for how this DUB fosters tumor growth. IMPLICATIONS: As a deubiquitinating enzyme, USP27X is a druggable target. Our study illuminates new avenues for therapeutic intervention in CCND1-driven cancers.

Endogenous DOPA inhibits melanoma through suppression of CHRM1 signaling.

Melanoma risk is 30 times higher in people with lightly pigmented skin versus darkly pigmented skin. Using primary human melanocytes representing the full human skin pigment continuum and preclinical melanoma models, we show that cell-intrinsic differences between dark and light melanocytes regulate melanocyte proliferative capacity and susceptibility to malignant transformation, independent of melanin and ultraviolet exposure. These differences result from dihydroxyphenylalanine (DOPA), a melanin precursor synthesized at higher levels in melanocytes from darkly pigmented skin. We used both high-throughput pharmacologic and genetic in vivo CRISPR screens to determine that DOPA limits melanocyte and melanoma cell proliferation by inhibiting the muscarinic acetylcholine receptor M1 (CHRM1) signaling. Pharmacologic CHRM1 antagonism in melanoma leads to depletion of c-Myc and FOXM1, both of which are proliferation drivers associated with aggressive melanoma. In preclinical mouse melanoma models, pharmacologic inhibition of CHRM1 or FOXM1 inhibited tumor growth. CHRM1 and FOXM1 may be new therapeutic targets for melanoma.

Drivers of Gene Expression Dysregulation in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease with a poor prognosis. The functional consequences of common genetic aberrations and their roles in treatment strategies have been extensively reviewed. In addition to these genomic aberrations, consideration of non-genetic drivers of altered oncogene expression is essential to account for the diversity in PDAC phenotypes. In this review we seek to assess our current understanding of mechanisms of gene expression dysregulation. We focus on four drivers of gene expression dysregulation, including mutations, transcription factors, epigenetic regulators, and RNA stability/isoform regulation, in the context of PDAC pathogenesis. Recent studies provide much-needed insight into the role of gene expression dysregulation in dissecting tumor heterogeneity and stratifying patients for the development of personalized treatment strategies.

Manganese-induced trafficking and turnover of GPP130 is mediated by sortilin.

Elevated, nontoxic doses of manganese (Mn) protect against Shiga toxin-1-induced cell death via down-regulation of GPP130, a cycling Golgi membrane protein that serves as an endosome-to-Golgi trafficking receptor for the toxin. Mn binds to GPP130 in the Golgi and causes GPP130 to oligomerize/aggregate, and the complexes are diverted to lysosomes. In fact, based on experiments using the self-interacting FM domain, it appears generally true that aggregation of a Golgi protein leads to its lysosomal degradation. How such oligomers are selectively sorted out of the Golgi is unknown. Here we provide evidence that Mn-induced exit of GPP130 from the trans-Golgi network (TGN) toward lysosomes is mediated by the sorting receptor sortilin interacting with the lumenal stem domain of GPP130. In contrast, FM-induced lysosomal trafficking of the Golgi protein galactosyltransferase was sortilin independent and occurred even in the absence of its native lumenal domain. Thus sortilin-dependent as well as sortilin-independent sorting mechanisms target aggregated Golgi membrane proteins for lysosomal degradation.