Human Positive Coactivator 4 (PC4) is a multifaceted chromatin protein involved in diverse cellular processes including genome organization, transcription regulation, replication, DNA repair and ...autophagy. PC4 exists as a phospho-protein in cells which impinges on its acetylation by p300 and thereby affects its transcriptional co-activator functions via double-stranded DNA binding. Despite the inhibitory effects, the abundance of phosphorylated PC4 in cells intrigued us to investigate its role in chromatin functions in a basal state of the cell. We found that casein kinase-II (CKII)-mediated phosphorylation of PC4 is critical for its interaction with linker histone H1. By employing analytical ultracentrifugation and electron microscopy imaging of in vitro reconstituted nucleosomal array, we observed that phospho-mimic (PM) PC4 displays a superior chromatin condensation potential in conjunction with linker histone H1. ATAC-sequencing further unveiled the role of PC4 phosphorylation to be critical in inducing chromatin compaction of a wide array of coding and non-coding genes in vivo. Concordantly, phospho-PC4 mediated changes in chromatin accessibility led to gene repression and affected global histone modifications. We propose that the abundance of PC4 in its phosphorylated state contributes to genome compaction contrary to its co-activator function in driving several cellular processes like gene transcription and autophagy.
Multifunctional human transcriptional positive co‐activator 4 (PC4) is a bona fide nonhistone component of the chromatin and plays a pivotal role in the process of chromatin compaction and functional ...genome organization. Knockdown of PC4 expression causes a drastic decompaction which leads to open conformation of the chromatin, and thereby altered nuclear architecture, defects in chromosome segregation and changed epigenetic landscape. Interestingly, these defects do not induce cellular death but result in enhanced cellular proliferation, possibly through enhanced autophagic activity. Moreover, PC4 depletion confers significant resistance to gamma irradiation. Exposure to gamma irradiation further induced autophagy in these cells. Inhibition of autophagy by small molecule inhibitors as well as by silencing of a critical autophagy gene drastically reduces the ability of PC4 knockdown cells to survive. On the contrary, complementation with wild‐type PC4 could reverse this phenomenon, confirming the process of autophagy as the key mechanism for radiation resistance in the absence of PC4. These data connect the unexplored role of chromatin architecture in regulating autophagy during stress conditions such as radiation.
Non‐histone chromatin protein positive co‐activator 4 (PC4) plays a key role in chromatin compaction and genome organization. Here, Tapas Kundu and colleagues show that PC4 knockdown cells exhibit an open chromatin structure and irregular nuclear shape. Intriguingly, the resulting nuclear defects do not induce cell death but increase cell proliferation via enhanced autophagy. Depletion of PC4 also confers resistance to gamma radiation, through a further increase in autophagy in these cells. These findings highlight the critical role of autophagy, regulated by changes in chromatin architecture, in the cellular response to stress.
Prostate-specific membrane antigen (PSMA) is an important cell surface target in prostate cancer. There are limited data on the heterogeneity of PSMA tissue expression in metastatic ...castration-resistant prostate cancer (mCRPC). Furthermore, the mechanisms regulating PSMA expression (encoded by the FOLH1 gene) are not well understood. Here, we demonstrate that PSMA expression is heterogeneous across different metastatic sites and molecular subtypes of mCRPC. In a rapid autopsy cohort in which multiple metastatic sites per patient were sampled, we found that 13 of 52 (25%) cases had no detectable PSMA and 23 of 52 (44%) cases showed heterogeneous PSMA expression across individual metastases, with 33 (63%) cases harboring at least 1 PSMA-negative site. PSMA-negative tumors displayed distinct transcriptional profiles with expression of druggable targets such as MUC1. Loss of PSMA was associated with epigenetic changes of the FOLH1 locus, including gain of CpG methylation and loss of histone 3 lysine 27 (H3K27) acetylation. Treatment with histone deacetylase (HDAC) inhibitors reversed this epigenetic repression and restored PSMA expression in vitro and in vivo. Collectively, these data provide insights into the expression patterns and regulation of PSMA in mCRPC and suggest that epigenetic therapies - in particular, HDAC inhibitors - can be used to augment PSMA levels.
Abstract
Background: DNA methylation alterations are a universal feature of cancer. In addition to site specific gain of DNA methylation (hypermethylation), a global loss of methylation ...(hypomethylation) was noted in most cancer genomes. Whereas numerous studies have focused on the role of hypermethylation in disease progression, less is known about the biology of hypomethylation in cancer. Recently, we have identified tumors across all major cancer types that are characterized by severe loss of DNA methylation. Our preliminary data suggest that these DNA hypomethylated cancers (hereafter DHMCs) show distinct alterations influencing gene expression and tumor microenvironment composition.
Objective: We hypothesize that since DNA methylation is critical for genome organization and gene expression. Severe global hypomethylation is likely to result in distinct epigenomic and genomic alterations. We therefore investigate common molecular changes associated with DNA hypomethylation to develop novel therapeutic approaches targeting unique vulnerabilities arising in DHMCs.
Study design: To delineate the biology of DHMCs, we determined methylation changes with validated orthogonal methods in large representative cohorts of patient samples (The Cancer Genome Atlas (TCGA), University of Washington rapid autopsy cohorts), patient derived xenografts and cancer cell lines. We assessed the patterns of common molecular alterations by performing genome wide epigenome mapping experiments and by defining common driver gene events in DHMCs. We further determined tumor cell intrinsic therapeutic vulnerabilities in broad pharmacologic and CRISPR-Cas9 genomic screens.
Results: We observed that DNA hypomethylation is a relatively common feature of solid tumors affecting overall 15-20% of all cancers, with highest rates observed in urothelial, melanoma, hepatocellular and lung and head and neck squamous cell carcinoma. In in vivo tumor models of DHMCs, we observe distinct shifts in epigenetic states upon DNA hypomethylation. Most notably, we found that DHMCs are characterized by unique intrinsic drug sensitivities to drug classes affecting core histone levels.
Impact: This is the first study to show druggable vulnerabilities arising from DNA hypomethylation. Collectively, these studies define a novel epigenetic subtype of cancer, determine its clinical and molecular features, and pave the way for new targeted therapies.
Citation Format: Pallabi Mustafi, Brian Hanratty, Ilsa Coleman, Radhika Patel, Adil Mohamed, Jay Sarthy, Gavin Ha, Michael Haffner. Targeting vulnerabilities arising from global DNA hypomethylation in cancer abstract. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A042.
Abstract
Introduction: Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease which can be classified into clinically relevant subtypes based on the expression of ...transcription factors (TF), such as the androgen receptor (AR) and neuroendocrine markers. Neuroendocrine prostate cancer (NEPC), characterized by gain of stem-like and neuroendocrine features and lack of AR expression is a clinically aggressive variant. Due to the absence of adequate biomarkers, NEPC is usually detected at a very advanced stage. There is mounting evidence that molecular subtype changes seen in NEPC are enforced by widespread epigenetic alterations, in particular DNA methylation changes. In this study, we aim to devise a novel DNA methylation-based assay for molecular subtyping and disease monitoring from cell-free DNA (cfDNA).
Methods: We analyzed genome wide methylation patterns in 60 prostate cancer patient-derived xenograft (PDX) and 133 mCRPC tumors using array- and sequencing-based assays. We integrated DNA methylation with TF cistrome data to determine the landscape of methylation alterations at key lineage TF binding sites (TFBS). A linear regression model was trained on low-pass Enzymatic Methyl-Seq (EM-seq) cfDNA data derived from PDXs to identify molecular subtype specific DNA methylation changes at these TFBS. The model performance was optimized with in silico admixture experiments. This model was then used to discern tumor molecular phenotypes from cfDNA in three independent cohorts of mCRPC patients using low-pass whole genome bisulfite sequencing and EM-seq.
Results: We observed a strong association between TFBS methylation and TF expression. For lineage specific TFs such as AR and ASCL1, we identified core sets of TFBSs whose differential methylation allowed for accurate assay-independent molecular subtype classification in tumor tissues. Applying an optimized quantitative model to mCRPC patients who underwent comprehensive tissue sampling by rapid autopsy we observed perfect subtype prediction from both tissue samples and cfDNA (AUC=1). A similar analytical performance was observed in additional clinical mCRPC cohorts with cfDNA.
Conclusions: We show that methylation patterns at TFBSs can determine TF activity and can be used to classify molecular subtypes from both tumor tissue and cfDNA. For prostate cancer, we demonstrate that this approach can accurately detect NEPC by cost-effective low-pass EM-seq. More broadly, this study provides a novel analysis framework for robustly assessing molecular tumor phenotypes in cfDNA with applications in solid and liquid tumor diagnostics.
Citation Format: Mohamed Adil, Brian Hanratty, Pallabi Mustafi, Ilsa Coleman, Radhika Patel, Anna-Lisa Doebley, Robert Patton, Eden Cruikshank, Patricia Galipeau, Ruth Dumpit, Martine Roudier, Jin-Yih Low, Navonil De Sarkar, Robert Montgomery, Eva Corey, Colm Morrissey, Peter Nelson, Gavin Ha, Michael Haffner. Molecular phenotype classification of metastatic prostate cancer by cell-free DNA methylation analysis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB298.
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