Curcuphenol possesses an unusual histone deacetylase enhancing activity that counters immune escape in metastatic tumours

Curcuphenol, a common component of the culinary spices, naturally found in marine invertebrates and plants, has been identified as a novel candidate for reversing immune escape by restoring expression of the antigen presentation machinery (APM) in invasive cancers, thereby resurrecting the immune recognition of metastatic tumours. Two synthetic curcuphenol analogues, were prepared by informed design that demonstrated consistent induction of APM expression in metastatic prostate and lung carcinoma cells. Both analogues were subsequently found to possess a previously undescribed histone deacetylase (HDAC)-enhancing activity. Remarkably, the H3K27ac ChIPseq analysis of curcuphenol-treated cells reveals that the induced epigenomic marks closely resemble the changes in genome-wide pattern observed with interferon-γ, a cytokine instrumental for orchestrating innate and adaptive immunity. These observations link dietary components to modifying epigenetic programs that modulate gene expression guiding poised immunity

Deciphering non-coding driver mutations in prostate cancer

Androgen receptor (AR) mediated signalling is critical to the growth at all stages of prostate cancer (PCa). Hormone stimulated AR binds to tens of thousands cis-regulatory elements (CRE) to activate transcription of specific set of genes through enhancer activity. Intriguingly, there are 100X more enhancers compared to AR- regulated genes. How this complex network of ARenhancers regulate the AR transcriptome remains poorly understood. We have previously shown that ARBS have significantly higher rate of mutations in PCa compared to other TF- binding sites. Given their critical role, these mutations could alter the transcriptional landscape and influence the cancer growth and progression. However, while we can readily identify these non-coding mutations, they are extremely challenging to characterize due to poor functional annotation and limited understanding of how variants impact enhancer activity. In this thesis we developed an experimental and computational framework to stratify non-coding mutations at AR enhancers. Using massively multi-parallel enhancer assays, we functionally tested thousands of common clinical AR binding regions to create a map of enhancer activity for the first time. Using this functional map, we characterized the genomic features associated with active and inactive types of AR enhancers. Next, we developed a new statistical and computational tool to introduce clinically relevant mutations and measure their impact on enhancer activity. Using this system, we interrogated known PCa risk-associated loci and demonstrated that 35% of them harbour SNPs that significantly altered enhancer activity. We also provided a potential mechanism of action for 20 PCa GWAS risk regions. Lastly, we incorporated the enhancer quantification to single cell settings to better understand heterogenous enhancer usage for the first time. Overall, this work laid the foundation to functionally characterize non-coding ARBS variants in PCa at a nucleotide resolution level.Science, Faculty ofGraduat

Optimized high-throughput screening of non-coding variants identified from genome-wide association studies - code

The scripts used for the analysis of our manuscript "Optimized high-throughput screening of non-coding variants identified from genome-wide association studies". Our original repository is located at here. https://github.com/mortunco/snp-starrseq</p

Systematic characterization of chromatin modifying enzymes identifies KDM3B as a critical regulator in castration resistant prostate cancer

Androgen deprivation therapy (ADT) is the standard care for prostate cancer (PCa) patients who fail surgery or radiotherapy. While initially effective, the cancer almost always recurs as a more aggressive castration resistant prostate cancer (CRPC). Previous studies have demonstrated that chromatin modifying enzymes can play a critical role in the conversion to CRPC. However, only a handful of these potential pharmacological targets have been tested. Therefore, in this study, we conducted a focused shRNA screen of chromatin modifying enzymes previously shown to be involved in cellular differentiation. We found that altering the balance between histone methylation and demethylation impacted growth and proliferation. Of all genes tested, KDM3B, a histone H3K9 demethylase, was found to have the most antiproliferative effect. These results were phenocopied with a KDM3B CRISPR/Cas9 knockout. When tested in several PCa cell lines, the decrease in proliferation was remarkably specific to androgen-independent cells. Genetic rescue experiments showed that only the enzymatically active KDM3B could recover the phenotype. Surprisingly, despite the decreased proliferation of androgen-independent cell no alterations in the cell cycle distribution were observed following KDM3B knockdown. Whole transcriptome analyses revealed changes in the gene expression profile following loss of KDM3B, including downregulation of metabolic enzymes such as ARG2 and RDH11. Metabolomic analysis of KDM3B knockout showed a decrease in several critical amino acids. Overall, our work reveals, for the first time, the specificity and the dependence of KDM3B in CRPC proliferation

Optimized high-throughput screening of non-coding variants identified from genome-wide association studies

The vast majority of disease-associated single nucleotide polymorphisms (SNP) identified from genome-wide association studies (GWAS) are localized in non-coding regions. A significant fraction of these variants impact transcription factors binding to enhancer elements and alter gene expression. To functionally interrogate the activity of such variants we developed snpSTARRseq, a high-throughput experimental method that can interrogate the functional impact of hundreds to thousands of non-coding variants on enhancer activity. snpSTARRseq dramatically improves signal-to-noise by utilizing a novel sequencing and bioinformatic approach that increases both insert size and the number of variants tested per loci. Using this strategy, we interrogated known prostate cancer (PCa) risk-associated loci and demonstrated that 35% of them harbor SNPs that significantly altered enhancer activity. Combining these results with chromosomal looping data we could identify interacting genes and provide a mechanism of action for 20 PCa GWAS risk regions. When benchmarked to orthogonal methods, snpSTARRseq showed a strong correlation with in vivo experimental allelic-imbalance studies whereas there was no correlation with predictive in silico approaches. Overall, snpSTARRseq provides an integrated experimental and computational framework to functionally test non-coding genetic variants

Genetic determinants of chromatin reveal prostate cancer risk mediated by context-dependent gene regulation

Many genetic variants affect disease risk by altering context-dependent gene regulation. Such variants are difficult to study mechanistically using current methods that link genetic variation to steady-state gene expression levels, such as expression quantitative trait loci (eQTLs). To address this challenge, we developed the cistrome-wide association study (CWAS), a framework for identifying genotypic and allele-specific effects on chromatin that are also associated with disease. In prostate cancer, CWAS identified regulatory elements and androgen receptor-binding sites that explained the association at 52 of 98 known prostate cancer risk loci and discovered 17 additional risk loci. CWAS implicated key developmental transcription factors in prostate cancer risk that are overlooked by eQTL-based approaches due to context-dependent gene regulation. We experimentally validated associations and demonstrated the extensibility of CWAS to additional epigenomic datasets and phenotypes, including response to prostate cancer treatment. CWAS is a powerful and biologically interpretable paradigm for studying variants that influence traits by affecting transcriptional regulation

Functional mapping of androgen receptor enhancer activity

Background: Androgen receptor (AR) is critical to the initiation, growth, and progression of prostate cancer. Once activated, the AR binds to cis-regulatory enhancer elements on DNA that drive gene expression. Yet, there are 10–100× more binding sites than differentially expressed genes. It is unclear how or if these excess binding sites impact gene transcription. Results: To characterize the regulatory logic of AR-mediated transcription, we generated a locus-specific map of enhancer activity by functionally testing all common clinical AR binding sites with Self-Transcribing Active Regulatory Regions sequencing (STARRseq). Only 7% of AR binding sites displayed androgen-dependent enhancer activity. Instead, the vast majority of AR binding sites were either inactive or constitutively active enhancers. These annotations strongly correlated with enhancer-associated features of both in vitro cell lines and clinical prostate cancer samples. Evaluating the effect of each enhancer class on transcription, we found that AR-regulated enhancers frequently interact with promoters and form central chromosomal loops that are required for transcription. Somatic mutations of these critical AR-regulated enhancers often impact enhancer activity. Conclusions: Using a functional map of AR enhancer activity, we demonstrated that AR-regulated enhancers act as a regulatory hub that increases interactions with other AR binding sites and gene promoters.Medicine, Faculty ofOther UBCNon UBCUrologic Sciences, Department ofReviewedFacult