The DREAM complex has emerged as a central repressor of DNA repair, raising questions as to whether such repression exerts long-term effects on human health. Here we establish that DREAM-associated activity significantly impacts lifetime somatic mutation burden, and that such effects are linked to altered lifespan and age-related disease pathology. First, joint profiling of DREAM-associated activity (quantified from the expression of genes transcriptionally repressed by DREAM) and somatic mutations across a single-cell atlas of 21 mouse tissues shows that cellular niches with lower DREAM-associated activity have decreased mutation rates. Second, DREAM-associated activity predicts the varied lifespans observed across 92 mammals, with low activity marking longer-lived species. Third, reduced DREAM-associated activity in individuals with Alzheimer's disease predicts late disease onset and decreased risk for severe neuropathology. Finally, DREAM knockout in mice protects against mutation accumulation, reducing single-base substitutions by 4.2% and insertion/deletions by 19.6% in the brain. These findings position DREAM as a key regulator of aging.

Abstract Cancer cell dormancy and the resultant resistance to conventional therapies present significant challenges for the successful treatment of high-grade serous ovarian cancer (HGSC). We used genome wide, and specialized sgRNA, libraries in CRISPR-based screens to identify critical cell survival mechanisms in dormancy and metastasis. Our findings demonstrate that low expression Wnt ligands WNT8B and WNT9B are essential for sustaining cell survival during prolonged dormant spheroid culture conditions. These Wnt ligands utilize non-canonical signaling to activate expression of stem cell genes such as ALDH1A1 , CD44 and others during spheroid dormancy. The loss of WNT8B and WNT9B reduced survival of xenografted ovarian cancer cells during early dissemination of disease that extended survival. Furthermore, treatment of WNT8B/9B deficient xenografts with carboplatin demonstrated increased sensitivity that further reduced dissemination and extended survival. These findings reveal that rare Wnt ligands can possess outsized functions in cancer pathogenesis and offer new avenues for improving treatment outcomes for HGSC through their inhibition.

The DREAM complex has emerged as a central repressor of DNA repair, raising questions as to whether such repression exerts long-term effects on human health. Here we establish that DREAM activity significantly impacts lifetime somatic mutation burden, and that such effects are linked to altered lifespan and age-related disease pathology. First, joint profiling of DREAM activity and somatic mutations across a single-cell atlas of 21 mouse tissues shows that cellular niches with lower DREAM activity have decreased mutation rates. Second, DREAM activity predicts the varied lifespans observed across 92 mammals, with low activity marking longer-lived species. Third, reduced DREAM activity in Alzheimer's patients predicts late disease onset and decreased risk for severe neuropathology. Finally, we show DREAM knockout protects against mutation accumulation in vivo, reducing single-base substitutions by 4.2% and insertion/deletions by 19.6% in brains of mice. These findings position DREAM as a key regulator of aging.

Background/Objectives: The lack of canonical biomarkers and strategies to target radioresistance contribute to poor patient outcomes in triple-negative breast cancer (TNBC). Identifying and targeting novel radioresistance genes will benefit in enhancing radiotherapy response and treatment outcomes in TNBC patients. Methods: A genome-wide CRISPR screen was performed to identify radioresistance genes in the TNBC cell line. An in vitro clonogenic assay was used to assess the antiproliferative effects of Artemis knockout or pharmacologic inhibition of Artemis, either alone or in combination with RT. Tumor doubling time and animal survival were assessed using an in vivo xenograft model. RNA-seq analysis was performed to identify genes and pathways deregulated under Artemis knockout conditions, both alone and in combination with RT. Cellular senescence was evaluated using a β-galactosidase assay. Results: Our CRISPR screen identified Artemis as a top hit in RT-treated TNBC cells, whose depletion led to radiosensitization in TNBC. Artemis knockout significantly reduced cell proliferation and enhanced the antiproliferative effects of RT in vitro. Compared to mice-bearing control MDA-MB-231 xenografts, Artemis knockout exhibited prolonged survival that was further enhanced with RT. Bulk RNA-sequencing indicated that the antiproliferative and radiosensitization effects of Artemis depletion were mediated by the activation of cellular senescence which was confirmed with a β-galactosidase assay. Conclusions: Taken together, our results highlight the critical role of Artemis in TNBC cell proliferation and response to radiation. Our findings identify Artemis as a potential biomarker indicative of sensitivity to radiation and a putative target that could be inhibited to enhance the efficacy of RT in TNBC.

Androgen receptor inhibitors are commonly used for prostate cancer treatment, but acquired resistance is a significant problem. Co-deletion of RB and p53 is common in castration resistant prostate cancers, however they are difficult to target pharmacologically. To comprehensively identify gene loss events that contribute to enzalutamide response, we performed a genome-wide CRISPR knockout screen in LNCaP prostate cancer cells. This revealed novel genes implicated in resistance that are largely unstudied. Gene loss events that confer enzalutamide sensitivity are enriched for GSEA categories related to stem cell and epigenetic regulation. We investigated the myeloid lineage stem cell factor HOXA9 as a candidate gene whose loss promotes sensitivity to enzalutamide. Cancer genomic data reveals that HOXA9 overexpression correlates with poor prognosis and characteristics of advanced prostate cancer. In cell culture, HOXA9 depletion sensitizes cells to enzalutamide, whereas overexpression drives enzalutamide resistance. Combination of the HOXA9 inhibitor DB818 with enzalutamide demonstrates synergy. This demonstrates the utility of our CRISPR screen data in discovering new approaches for treating enzalutamide resistant prostate cancer.

Dormancy in cancer is a clinical state in which residual disease remains undetectable for a prolonged duration. At a cellular level, rare cancer cells cease proliferation and survive chemotherapy and disseminate disease. We created a suspension culture model of high-grade serous ovarian cancer (HGSOC) dormancy and devised a novel CRISPR screening approach to identify survival genes in this context. In combination with RNA-seq, we discovered the Netrin signaling pathway as critical to dormant HGSOC cell survival. We demonstrate that Netrin-1, –3, and its receptors are essential for low level ERK activation to promote survival, and that Netrin activation of ERK is unable to induce proliferation. Deletion of all UNC5 family receptors blocks Netrin signaling in HGSOC cells and compromises viability during the dormancy step of dissemination in xenograft assays. Furthermore, we demonstrate that Netrin-1 and –3 overexpression in HGSOC correlates with poor outcome. Specifically, our experiments reveal that Netrin overexpression elevates cell survival in dormant culture conditions and contributes to greater spread of disease in a xenograft model of abdominal dissemination. This study highlights Netrin signaling as a key mediator HGSOC cancer cell dormancy and metastasis.

Fibrosing interstitial lung diseases (ILDs) encompass a diverse range of scarring disorders that lead to progressive lung failure. Previous gene expression profiling studies focused on idiopathic pulmonary fibrosis (IPF) and bulk tissue samples. We employed digital spatial profiling to gain new insights into the spatial resolution of gene expression across distinct lung microenvironments (LMEs) in IPF, chronic hypersensitivity pneumonitis (CHP) and non-specific interstitial pneumonia (NSIP). We identified differentially expressed genes between LMEs within each condition, and across histologically similar regions between conditions. Uninvolved regions in IPF and CHP were distinct from normal controls, and displayed potential therapeutic targets. Hallmark LMEs of each condition retained distinct gene signatures, but these could not be reproduced in matched lung tissue samples. Based on these profiles and unsupervised clustering, we grouped previously unclassified ILD cases into NSIP or CHP. Overall, our work uniquely dissects gene expression profiles between LMEs within and across different types of fibrosing ILDs.

Genome-wide CRISPR screens are an effective discovery tool for genes that underlie diverse cellular mechanisms that can be scored through cell fitness. Loss-of-function screens are particularly challenging compared to gain-of-function because of the limited dynamic range of decreased sgRNA sequence detection. Here we describe Guide-Only control CRISPR (GO-CRISPR), an improved loss-of-function screening workflow, and its companion software package, Toolset for the Ranked Analysis of GO-CRISPR Screens (TRACS). We demonstrate a typical GO-CRISPR workflow in a non-proliferative 3D spheroid model of dormant high grade serous ovarian cancer and demonstrate superior performance to standard screening methods. The unique integration of the pooled sgRNA library quality and guide-only controls allows TRACS to identify novel molecular pathways that were previously unidentified in tumor dormancy and undetectable to analysis packages that lack the guide only controls. Together, GO-CRISPR and TRACS can robustly improve the discovery of essential genes in challenging biological scenarios such as growth arrested cells.

PDF fie 148K, Description of additional genotypes of cells analyzed for gammaH2AX distribution and detailed instability phenotypes of heterozygous cells

In mammalian cells expression of repetitive genomic sequences is repressed by heterochromatin, underscoring the potential threat of repeat expression to cellular homeostasis. However, the specific consequences of ectopic repeat expression remains unclear. Here we demonstrate that EZH2 inhibitors stimulate repeat misexpression and cell death in resting splenic B cells. We show that B cells are uniquely sensitive to these agents because of high levels of H3K27me3 at repeats and correspondingly low DNA methylation. We generated a pattern recognition receptor loss-of-function mouse model called RIC with mutations in Rigi , Ifih1 (MDA5), and Cgas to specifically block the consequences of repeat misexpression. In both WT and RIC mutant B cells, EZH2 inhibition caused focused loss of H3K27me3 at repetitive elements and upregulated their expression. However, expression of inflammatory chemokines and cell death were interrupted by the RIC mutations. Furthermore, the chemokine expression patterns induced by EZH2 inhibitors resemble the B cell response to Epstein-Barr virus infection. This study demonstrates a viral mimicry effect induced by pharmacological activation of repeat expression that induces inflammation and B cell death.

PDF file 125K, Detailed methods for ChIP, microscopy, and computational analysis. Lists of cell lines and antibodies used are included

S1. Cumulative viability score of siRNA knockdowns. S2. Spheroid morphology of control and gene knock down cells. S3. Reattachment of control and gene knockdown spheroid cells after 72 hours. S4. Relative protein expression levels of Dyrk1A and Dyrk1B in cell lines used in this study. S5. Expression and function of CIP/KIPproteins in non-adherent culture. S6. Flow cytometry analysis of p130 depleted cells.

Mammalian cells repress expression of repetitive genomic sequences by forming heterochromatin. However, the consequences of ectopic repeat expression remain unclear. Here we demonstrate that inhibitors of EZH2, the catalytic subunit of the Polycomb repressive complex 2 (PRC2), stimulate repeat misexpression and cell death in resting splenic B cells. B cells are uniquely sensitive to these agents because they exhibit high levels of histone H3 lysine 27 trimethylation (H3K27me3) and correspondingly low DNA methylation at repeat elements. We generated a pattern recognition receptor loss‐of‐function mouse model, called RIC, with mutations in Rigi (encoding for RIG‐I), Ifih1 (MDA5), and Cgas . In both wildtype and RIC mutant B cells, EZH2 inhibition caused loss of H3K27me3 at repetitive elements and upregulated their expression. However, NF‐κB‐dependent expression of inflammatory chemokines and subsequent cell death was suppressed by the RIC mutations. We further show that inhibition of EZH2 in cancer cells requires the same pattern recognition receptors to activate an interferon response. Together, the results reveal chemokine expression induced by EZH2 inhibitors in B cells as a novel inflammatory response to genomic repeat expression. Given the overlap of genes induced by EZH2 inhibitors and Epstein–Barr virus infection, this response can be described as a form of viral mimicry.

Introduction: Castration resistant prostate cancer (CRPC) cells can acquire resistance to the androgen receptor (AR) inhibitor enzalutamide (EZ). These cells can switch lineages from an adenocarcinoma to a neuroendocrine (NE) cell type that proliferate independently of the AR signaling pathway. Cancer genomic and molecular studies identified that co-deletion of the retinoblastoma (RB1) and TP53 genes can promote the acquisition of EZ resistance and neuroendocrine features. However, RB and p53 are both tumour suppressor and are therefore difficult to target pharmacologically. The purpose of this study is to identify an actionable molecular factor downstream of RB and p53 that drives EZ resistance in CRPC. Methods and Results: To characterize functional and molecular features of RB and p53 loss, CRISPR-Cas9 was used to generate a double knockout (DKO) line in LNCaP prostate cancer cells. Compared with LNCaP wild type (WT) cells, only DKOs formed distinct colonies over a 4-week colony forming assay under EZ treatment. RNA sequencing of DKO and WT cells, followed by gene ontology (GO) analysis, revealed NE and stemness genes, including HOXA9, were significantly upregulated in DKO cells. To categorize gene loss events in EZ-treated LNCaP cells, a genome-wide CRISPR knockout screen was performed. Pools of KO cells were treated with either EZ or DMSO and then analyzed by next generation sequencing to identify gene mutations that confer increased resistance or sensitivity to EZ. GO analysis of de-enriched genes following EZ treatment identified stemness genes, including HOXA9, highlighting the potential importance of a stem-like phenotype for acquiring EZ resistance EZ. To further investigate the functional significance of HOXA9 we analyzed EZ-resistant prostate tumour genomic data. HOXA9 is mutated or mis-expressed in 10% of cases. Importantly, HOXA9 is either amplified or overexpressed in virtually all these cases, and is associated with poorer prognosis, suggesting an oncogenic role for HOXA9 in CRPC. HOXA9 transcript levels were positively correlated with neuroendocrine features and negatively correlated with RB1expression in these tumour samples. LNCaP WT and DKO cells we then engineered to overexpress HOXA9 displayed increased IC50 values following a 6-day EZ treatment, compared with either parental line. DKO cells that overexpress HOXA9 also formed significantly more colonies following a 4-week EZ treatment, compared with parentals. In contrast, shRNA knockdown of HOXA9 caused a reduction in IC50 values and formed fewer drug resistant colonies compared with control cells. Finally, DKO and WT cells were co-treated with varying concentrations of the HOXA9 inhibitor DB818 and EZ. DKO cells were more sensitive to DB818 alone at high concentrations and displayed higher synergy as measured by a ZIP synergy score when co-treated with EZ. Conclusions: Overall, these results suggest that HOXA9 regulates EZ resistance in prostate cancer. Furthermore, HOXA9 inhibition may be of therapeutic benefit for treating EZ-resistant CRPC. Citation Format: Michael V. Roes, Fred A. Dick. HOXA9 promotes enzalutamide resistance in RB-p53 deficient prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B015.

PDF file 3766K, Analysis of gammaH2AX distribution, chromosomal instability phenotypes in heterozygous cells, and verification of genotypes in human cells

The Farnesoid X Receptor (FXR) belongs to the nuclear receptor superfamily and is an essential bile acid (BA) receptor that regulates the expression of genes involved in the metabolism of BAs. FXR protects the liver from BA overload, which is a major etiology of hepatocellular carcinoma. Herein, we investigated the changes in gene expression and chromatin accessibility in hepatocytes by performing RNA-seq in combination with the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) using a novel FXR knockout mouse model (Fxrex5Δ: Nr1h4ex5Δ/ex5Δ) generated through CRISPR/Cas9. Consistent with previous Fxr knockout models, we found that Fxrex5Δ mice develop late-onset HCC associated with increased serum and hepatic BAs. FXR deletion was associated with a dramatic loss of chromatin accessibility, primarily at promoter-associated transcription factor binding sites. Importantly, several genes involved in BA biosynthesis and circadian rhythm were downregulated following loss of FXR, also displayed reduced chromatin accessibility at their promoter regions. Altogether, these findings suggest that FXR helps to maintain a transcriptionally active state by regulating chromatin accessibility through its binding and recruitment of transcription factors and coactivators.

In cancer, dormancy refers to a clinical state in which microscopic residual disease becomes non-proliferative and is largely refractory to chemotherapy. Dormancy was first described in breast cancer where disease can remain undetected for decades, ultimately leading to relapse and clinical presentation of the original malignancy. A long latency period can be explained by withdrawal from cell proliferation (cellular dormancy), or a balance between proliferation and cell death that retains low levels of residual disease (tumor mass dormancy). Research into cellular dormancy has revealed features that define this state. They include arrest of cell proliferation, altered cellular metabolism, and unique cell dependencies and interactions with the microenvironment. These characteristics can be shared by dormant cells derived from disparate primary disease sites, suggesting common features exist between them. High-grade serous ovarian cancer (HGSOC) disseminates to locations throughout the abdominal cavity by means of cellular aggregates called spheroids. These growth-arrested and therapy-resistant cells are a strong contributor to disease relapse. In this review, we discuss the similarities and differences between ovarian cancer cells in spheroids and dormant properties reported for other cancer disease sites. This reveals that elements of dormancy, such as cell cycle control mechanisms and changes to metabolism, may be similar across most forms of cellular dormancy. However, HGSOC-specific aspects of spheroid biology, including the extracellular matrix organization and microenvironment, are obligatorily disease site specific. Collectively, our critical review of current literature highlights places where HGSOC cell dormancy may offer a more tractable experimental approach to understand broad principles of cellular dormancy in cancer.

Clear evidence supports a causal link between Merkel cell polyomavirus (MCPyV) and the highly aggressive human skin cancer called Merkel cell carcinoma (MCC). Integration of viral DNA into the human genome facilitates continued expression of the MCPyV small tumor (ST) and large tumor (LT) antigens in virus-positive MCCs. In MCC tumors, MCPyV LT is truncated in a manner that renders the virus unable to replicate yet preserves the LXCXE motif that facilitates its binding to and inactivation of the retinoblastoma tumor suppressor protein (pRb). We previously developed a MCPyV transgenic mouse model in which MCC tumor-derived ST and truncated LT expression were targeted to the stratified epithelium of the skin, causing epithelial hyperplasia, increased proliferation, and spontaneous tumorigenesis. We sought to determine if any of these phenotypes required the association between the truncated MCPyV LT and pRb. Mice were generated in which K14-driven MCPyV ST/LT were expressed in the context of a homozygous RbΔLXCXE knock-in allele that attenuates LT-pRb interactions through LT's LXCXE motif. We found that many of the phenotypes including tumorigenesis that develop in the K14-driven MCPyV transgenic mice were dependent upon LT's LXCXE-dependent interaction with pRb. These findings highlight the importance of the MCPyV LT-pRb interaction in an in vivo model for MCPyV-induced tumorigenesis.