Functional Role of DREAM and DYRK1A in High-Grade Serous Ovarian Cancer Cell Dormancy

High-grade serous ovarian cancer (HGSOC) is the most common form of ovarian cancer. The majority of women are disproportionately diagnosed at an advanced stage (stage III-IV) of the disease when tumours have progressed beyond the ovaries or fallopian tubes and into the peritoneal cavity. Survival rates at late-stage are as low as 25% and chemoresistant disease recurrence is common, affecting up to 90% of patients. Multicellular clusters called spheroids contribute to dormancy, chemoresistance, and metastases and are a major challenge to treatment of HGSOC. Spheroid cells undergo reversible quiescence to evade chemotherapy in a process mediated by the mammalian DREAM complex and its initiating kinase, DYRK1A. Depletion of DYRK1A reduces spheroid cell survival and increases sensitivity to chemotherapy, highlighting it as an attractive therapeutic target. Herein we demonstrate the long-term consequences of DREAM loss in adult mice. DREAM deficient mice do not have proliferative control defects but develop systemic amyloidosis as a result of overexpression of apolipoproteins Apoa1 and Apoa4. Overexpression of Apoa1 and Apo4 were marked with increased B-MYB-MuvB (MMB) and decreased H2AZ deposition within gene bodies. The prolonged latency before developing amyloidosis suggests depriving cells of quiescence is tolerable for short periods of time. To broadly identify genetic vulnerabilities in spheroid cells, we employed an integrated strategy in which we investigated the transcriptional programming and also performed a loss-of-function genome-wide CRISPR screen in HGSOC spheroid cells. Towards this aim, we developed novel bioinformatic tools and methodology to facilitate high-throughput discovery of essential genes and pathways and anticipate these tools will have broad usability in transcriptional and loss-of-function studies. Using these tools, we identified the netrin signaling pathway as an essential mediator of HGSOC spheroid cell survival. Specifically, components of netrin signaling are upregulated in spheroid cells and depletion of netrin ligands or receptors was sufficient to reduce spheroid cell viability. Our work highlights netrin signaling as a potential target for new metastatic ovarian cancer therapies. Taken together, the work presented herein provide more insight into the roles of DREAM and DYRK1A in HGSOC spheroid survival as well as implications of therapeutically targeting this pathway. HGSOC is a very deadly disease and there is an urgent need to develop new therapeutic strategies that can specifically target dormant chemoresistant spheroids in patients to treat or prevent relapse

BEAVR: A browser-based tool for the exploration and visualization of RNA-seq data

Background: The use of RNA-sequencing (RNA-seq) in molecular biology research and clinical settings has increased significantly over the past decade. Despite its widespread adoption, there is a lack of simple and interactive tools to analyze and explore RNA-seq data. Many established tools require programming or Unix/Bash knowledge to analyze and visualize results. This requirement presents a significant barrier for many researchers to efficiently analyze and present RNA-seq data. Results: Here we present BEAVR, a Browser-based tool for the Exploration And Visualization of RNA-seq data. BEAVR is an easy-to-use tool that facilitates interactive analysis and exploration of RNA-seq data. BEAVR is developed in R and uses DESeq2 as its engine for differential gene expression (DGE) analysis, but assumes users have no prior knowledge of R or DESeq2. BEAVR allows researchers to easily obtain a table of differentially-expressed genes with statistical testing and then visualize the results in a series of graphs, plots and heatmaps. Users are able to customize many parameters for statistical testing, dealing with variance, clustering methods and pathway analysis to generate high quality figures. Conclusion: BEAVR simplifies analysis for novice users but also streamlines the RNA-seq analysis process for experts by automating several steps. BEAVR and its documentation can be found on GitHub at https://github.com/developerpiru/BEAVR. BEAVR is available as a Docker container at https://hub.docker.com/r/pirunthan/beavr

Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM\u27s absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes