Epigenomic signatures of sarcomatoid differentiation to guide the treatment of renal cell carcinoma.

Renal cell carcinoma with sarcomatoid differentiation (sRCC) is associated with poor survival and a heightened response to immune checkpoint inhibitors (ICIs). Two major barriers to improving outcomes for sRCC are the limited understanding of its gene regulatory programs and the low diagnostic yield of tumor biopsies due to spatial heterogeneity. Herein, we characterized the epigenomic landscape of sRCC by profiling 107 epigenomic libraries from tissue and plasma samples from 50 patients with RCC and healthy volunteers. By profiling histone modifications and DNA methylation, we identified highly recurrent epigenomic reprogramming enriched in sRCC. Furthermore, CRISPRa experiments implicated the transcription factor FOSL1 in activating sRCC-associated gene regulatory programs, and FOSL1 expression was associated with the response to ICIs in RCC in two randomized clinical trials. Finally, we established a blood-based diagnostic approach using detectable sRCC epigenomic signatures in patient plasma, providing a framework for discovering epigenomic correlates of tumor histology via liquid biopsy.

Detecting small cell transformation in patients with advanced EGFR mutant lung adenocarcinoma through epigenomic cfDNA profiling.

PURPOSE: Histologic transformation to small cell lung cancer (SCLC) is a mechanism of treatment resistance in patients with advanced oncogene-driven lung adenocarcinoma (LUAD) that currently requires histologic review for diagnosis. Herein, we sought to develop an epigenomic cell-free (cf)DNA-based approach to non-invasively detect small cell transformation in patients with EGFR mutant (EGFRm) LUAD. EXPERIMENTAL DESIGN: To characterize the epigenomic landscape of transformed (t)SCLC relative to LUAD and de novo SCLC, we performed chromatin immunoprecipitation sequencing (ChIP-seq) to profile the histone modifications H3K27ac, H3K4me3, and H3K27me3, methylated DNA immunoprecipitation sequencing (MeDIP-seq), assay for transposase-accessible chromatin sequencing (ATAC-seq), and RNA sequencing on 26 lung cancer patient-derived xenograft (PDX) tumors. We then generated and analyzed H3K27ac ChIP-seq, MeDIP-seq, and whole genome sequencing cfDNA data from 1 ml aliquots of plasma from patients with EGFRm LUAD with or without tSCLC. RESULTS: Analysis of 126 epigenomic libraries from the lung cancer PDXs revealed widespread epigenomic reprogramming between LUAD and tSCLC, with a large number of differential H3K27ac (n=24,424), DNA methylation (n=3,298), and chromatin accessibility (n=16,352) sites between the two histologies. Tumor-informed analysis of each of these three epigenomic features in cfDNA resulted in accurate non-invasive discrimination between patients with EGFRm LUAD versus tSCLC (AUROC=0.82-0.87). A multi-analyte cfDNA-based classifier integrating these three epigenomic features discriminated between EGFRm LUAD versus tSCLC with an AUROC of 0.94. CONCLUSIONS: These data demonstrate the feasibility of detecting small cell transformation in patients with EGFRm LUAD through epigenomic cfDNA profiling of 1 ml of patient plasma.

Plasma Kidney Injury Molecule-1 for Preoperative Prediction of Renal Cell Carcinoma Versus Benign Renal Masses, and Association With Clinical Outcomes.

PURPOSE: Both clear cell and papillary renal cell carcinomas (RCCs) overexpress kidney injury molecule-1 (KIM-1). We investigated whether plasma KIM-1 (pKIM-1) may be a useful risk stratification tool among patients with suspicious renal masses. METHODS: Prenephrectomy pKIM-1 was measured in two independent cohorts of patients with renal masses. Cohort 1, from the prospective K2 trial, included 162 patients found to have clear cell RCC (cases) and 162 patients with benign renal masses (controls). Cohort 2 included 247 patients with small (cT1a) renal masses from an academic biorepository, of whom 184 had RCC. We assessed the relationship between pKIM-1, surgical pathology, and clinical outcomes. RESULTS: In Cohort 1, pKIM-1 distinguished RCC versus benign masses with area under the receiver operating curve (AUC-ROC, 0.81 [95% CI, 0.76 to 0.86]). In Cohort 2 (cT1a only), pKIM-1 distinguished RCC versus benign masses (AUC-ROC, 0.74 [95% CI, 0.67 to 0.80]) and the addition of pKIM-1 to an established nomogram for predicting malignancy improved the model AUC-ROC (0.65 [95% CI, 0.57 to 0.74] v 0.78 [95% CI, 0.72 to 0.85]). A pKIM-1 cutpoint identified using Cohort 2 demonstrated sensitivity of 92.5% and specificity of 60% for identifying RCC in Cohort 1. In long-term follow-up of RCC cases (Cohort 1), higher prenephrectomy pKIM-1 was associated with worse metastasis-free survival (multivariable MFS hazard ratio [HR] 1.29 per unit increase in log pKIM-1, 95% CI, 1.10 to 1.53) and overall survival (multivariable OS HR 1.31 per unit increase in log pKIM-1, 95% CI, 1.10 to 1.54). In long-term follow-up of Cohort 2, no metastatic events occurred, consistent with the favorable prognosis of resected cT1a RCC. CONCLUSION: Among patients with renal masses, pKIM-1 is associated with malignant pathology, worse MFS, and risk of death. pKIM-1 may be useful for selecting patients with renal masses for intervention versus surveillance.

Increased frequency of CHD1 deletions in prostate cancers of African American men is associated with rapid disease progression without inducing homologous recombination deficiency.

We analyzed genomic data derived from the prostate cancer of African and European American men in order to identify differences that may contribute to racial disparity of outcome and that could also define novel therapeutic strategies. In addition to analyzing patient derived next generation sequencing data, we performed FISH based confirmatory studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CRISPR edited, CHD1 deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. We found that subclonal deletion of CHD1 is nearly three times as frequent in prostate tumors of African American men than in men of European ancestry and it associates with rapid disease progression. We further showed that CHD1 deletion is not associated with homologous recombination deficiency associated mutational signatures in prostate cancer. In prostate cancer cell line models CHD1 deletion did not induce HR deficiency as detected by RAD51 foci formation assay or mutational signatures, which was consistent with the moderate increase of olaparib sensitivity. CHD1 deficient prostate cancer cells, however, showed higher sensitivity to talazoparib. CHD1 loss may contribute to worse outcome of prostate cancer in African American men. A deeper understanding of the interaction between CHD1 loss and PARP inhibitor sensitivity will be needed to determine the optimal use of targeted agents such as talazoparib in the context of castration resistant prostate cancer.

Decoding the Epigenetics and Chromatin Loop Dynamics of Androgen Receptor-Mediated Transcription.

Androgen receptor (AR)-mediated transcription plays a critical role in normal prostate development and prostate cancer growth. AR drives gene expression by binding to thousands of cis-regulatory elements (CRE) that loop to hundreds of target promoters. With multiple CREs interacting with a single promoter, it remains unclear how individual AR bound CREs contribute to gene expression. To characterize the involvement of these CREs, we investigated the AR-driven epigenetic and chromosomal chromatin looping changes. We collected a kinetic multi-omic dataset comprised of steady-state mRNA, chromatin accessibility, transcription factor binding, histone modifications, chromatin looping, and nascent RNA. Using an integrated regulatory network, we found that AR binding induces sequential changes in the epigenetic features at CREs, independent of gene expression. Further, we showed that binding of AR does not result in a substantial rewiring of chromatin loops, but instead increases the contact frequency of pre-existing loops to target promoters. Our results show that gene expression strongly correlates to the changes in contact frequency. We then proposed and experimentally validated an unbalanced multi-enhancer model where the impact on gene expression of AR-bound enhancers is heterogeneous, and is proportional to their contact frequency with target gene promoters. Overall, these findings provide new insight into AR-mediated gene expression upon acute androgen simulation and develop a mechanistic framework to investigate nuclear receptor mediated perturbations.

Increased chromatin accessibility mediated by nuclear factor I drives transition to androgen receptor splice variant dependence in castration-resistant prostate cancer.

Androgen receptor (AR) splice variants, of which ARv7 is the most common, are increased in prostate cancer (PC) that develops resistance to androgen signaling inhibitor drugs, but the extent to which these variants drive AR activity, and whether they have novel functions or dependencies, remain to be determined. We generated a subline of VCaP PC cells (VCaP16) that is resistant to the AR inhibitor enzalutamide (ENZ) and found that AR activity was independent of the full-length AR (ARfl), despite its continued high-level expression, and was instead driven by ARv7. The ARv7 cistrome and transcriptome in VCaP16 cells mirrored that of the ARfl in VCaP cells, although ARv7 chromatin binding was weaker, and strong ARv7 binding sites correlated with higher affinity ARfl binding sites across multiple models and clinical samples. Notably, although ARv7 expression in VCaP cells increased rapidly in response to ENZ, there was a long lag before it gained chromatin binding and transcriptional activity. This lag was associated with an increase in chromatin accessibility, with the AR and nuclear factor I (NFI) motifs being most enriched at these more accessible sites. Moreover, the transcriptional effects of combined NFIB and NFIX knockdown versus ARv7 knockdown were highly correlated. These findings indicate that ARv7 can drive the AR program, but that its activity is dependent on adaptations that increase chromatin accessibility to enhance its intrinsically weak chromatin binding.

The PENGUIN approach to reconstruct protein interactions at enhancer-promoter regions and its application to prostate cancer.

We introduce Promoter-Enhancer-Guided Interaction Networks (PENGUIN), a method for studying protein-protein interaction (PPI) networks within enhancer-promoter interactions. PENGUIN integrates H3K27ac-HiChIP data with tissue-specific PPIs to define enhancer-promoter PPI networks (EPINs). We validated PENGUIN using cancer (LNCaP) and benign (LHSAR) prostate cell lines. Our analysis detected EPIN clusters enriched with the architectural protein CTCF, a regulator of enhancer-promoter interactions. CTCF presence was coupled with the prevalence of prostate cancer (PrCa) single nucleotide polymorphisms (SNPs) within the same EPIN clusters, suggesting functional implications in PrCa. Within the EPINs displaying enrichments in both CTCF and PrCa SNPs, we also show enrichment in oncogenes. We substantiated our identified SNPs through CRISPR/Cas9 knockout and RNAi screens experiments. Here we show that PENGUIN provides insights into the intricate interplay between enhancer-promoter interactions and PPI networks, which are crucial for identifying key genes and potential intervention targets. A dedicated server is available at https://penguin.life.bsc.es/ .

Liquid biopsy epigenomic profiling for cancer subtyping.

Although circulating tumor DNA (ctDNA) assays are increasingly used to inform clinical decisions in cancer care, they have limited ability to identify the transcriptional programs that govern cancer phenotypes and their dynamic changes during the course of disease. To address these limitations, we developed a method for comprehensive epigenomic profiling of cancer from 1 ml of patient plasma. Using an immunoprecipitation-based approach targeting histone modifications and DNA methylation, we measured 1,268 epigenomic profiles in plasma from 433 individuals with one of 15 cancers. Our assay provided a robust proxy for transcriptional activity, allowing us to infer the expression levels of diagnostic markers and drug targets, measure the activity of therapeutically targetable transcription factors and detect epigenetic mechanisms of resistance. This proof-of-concept study in advanced cancers shows how plasma epigenomic profiling has the potential to unlock clinically actionable information that is currently accessible only via direct tissue sampling.

Characteristics and Outcomes of Patients Requiring Repeat Intensive Care Unit Consults.

Objective: To better understand the mortality and notable characteristics of patients initially denied intensive care unit (ICU) admission that are later admitted on reconsultation. Patients and Methods: We collected data regarding all adult inpatients (n=3725) who received one or more ICU consults at an academic tertiary care hospital medical center between January 1, 2018 and October 1, 2021. We compared patients who were initially denied ICU admission and later admitted on reconsultation (C2A1, n=144) with those who were admitted after the first consultation (C1A1, n=2286) and those denied at first consult and never later admitted (C1A0, n=1295). Results: Ten percent of patients initially rejected by the ICU were later admitted on reconsultation. There was no significant difference in the adjusted hospital death odds ratios between C1A1 and C2A1 (0.67; 95% CI 0.43-1.01; P=.11). Assessing subgroups of the C2A1 population, we found that 8.2% (n=100) of full code patients were later admitted to the ICU on reconsultation vs 23.2% (n=40) of do not attempt resuscitation patients (P<.001); 7.6% (n=77) of patients initially consulted from the emergency department were later admitted to the ICU on reconsultation vs 15.1% (n=52) of patients initially consulted from an inpatient setting (P<.001). Conclusion: In this cohort, we demonstrated that patients admitted on repeat ICU consultation have no significant difference in mortality compared with equivalent patients admitted after the first consultation. Understanding and further exploring the consequences of these ICU reconsultations is vital to developing optimal critical care triaging practices.

A biallelic multiple nucleotide length polymorphism explains functional causality at 5p15.33 prostate cancer risk locus.

To date, single-nucleotide polymorphisms (SNPs) have been the most intensively investigated class of polymorphisms in genome wide associations studies (GWAS), however, other classes such as insertion-deletion or multiple nucleotide length polymorphism (MNLPs) may also confer disease risk. Multiple reports have shown that the 5p15.33 prostate cancer risk region is a particularly strong expression quantitative trait locus (eQTL) for Iroquois Homeobox 4 (IRX4) transcripts. Here, we demonstrate using epigenome and genome editing that a biallelic (21 and 47 base pairs (bp)) MNLP is the causal variant regulating IRX4 transcript levels. In LNCaP prostate cancer cells (homozygous for the 21 bp short allele), a single copy knock-in of the 47 bp long allele potently alters the chromatin state, enabling de novo functional binding of the androgen receptor (AR) associated with increased chromatin accessibility, Histone 3 lysine 27 acetylation (H3K27ac), and ~3-fold upregulation of IRX4 expression. We further show that an MNLP is amongst the strongest candidate susceptibility variants at two additional prostate cancer risk loci. We estimated that at least 5% of prostate cancer risk loci could be explained by functional non-SNP causal variants, which may have broader implications for other cancers GWAS. More generally, our results underscore the importance of investigating other classes of inherited variation as causal mediators of human traits.

Grade Group 1 Prostate Cancers Exhibit Tumor-defining Androgen Receptor-driven Programs.

Grade group 1 (GG1) primary prostate cancers with a pathologic Gleason score of 6 are considered indolent and generally not associated with fatal outcomes, so treatment is not indicated for most cases. These low-grade cancers have an overall negligible risk of locoregional progression and metastasis to distant organs, which is why there is an ongoing debate about whether these lesions should be reclassified as "noncancerous". However, the underlying molecular activity of key disease drivers, such as the androgen receptor (AR), have thus far not been thoroughly characterized in low-grade tumors. Therefore, we set out to delineate the AR chromatin-binding landscape in low-grade GG1 prostate cancers to gain insights into whether these AR-driven programs are actually tumor-specific or are normal prostate epithelium-like. These analyses showed that GG1 tumors do not harbor a distinct AR cistrome and, similar to higher-grade cancers, AR preferentially binds to tumor-defining cis-regulatory elements. Furthermore, the enhancer activity of these regions and the expression of their respective target genes were not significantly different in GG1 tumors. From an epigenetic perspective, this finding supports the cancer designation currently given to these low-grade tumors and clearly distinguishes them from noncancerous benign tissue. PATIENT SUMMARY: We characterized the molecular activity of the androgen receptor protein, which drives prostate cancer disease, in low-grade tumors. Our results show that these tumors are true cancers and are clearly separate from benign prostate tissue despite their low clinical aggressiveness.

Patients Who Decompensate and Trigger Rapid Response Immediately Upon Hospital Admission Have Higher Mortality Than Equivalent Patients Without Rapid Responses.

BACKGROUND: Rapid response teams (RRTs) have impacted the management of decompensating patients, potentially improving mortality. Few studies address the significance of RRT timing relative to hospital admission. We aimed to identify outcomes of adult patients who trigger immediate RRT activation, defined as within 4 hours of admission and compare with RRT later in admission or do not require RRT activation, and identify risk factors that predispose toward immediate RRT activation. METHODS: A retrospective case-control study was performed using an RRT activation database, comprising 201,783 adult inpatients at an urban, academic, tertiary care hospital. This group was subdivided by timing of RRT activation regarding admission: within the first 4 hours (immediate RRT), between 4 and 24 hours (early RRT), and after 24 hours (late RRT). The primary outcome was 28-day all-cause mortality. Individuals triggering an immediate RRT were compared with demographically matched controls. Mortality was adjusted for age, Quick Systemic Organ Failure Assessment score, intensive care unit admission, and Elixhauser Comorbidity Index. RESULTS: Patients with immediate RRT had adjusted 28-day all-cause mortality of 7.1% (95% confidence interval [CI], 5.6%-8.5%) and death odds ratio of 3.27 (95% CI, 2.5-4.3) compared with those who did not (mortality, 2.9%; 95%CI, 2.8%-2.9%; P < 0.0001). Patients triggering an immediate RRT were more likely to be Black, be older, and have higher Quick Systemic Organ Failure Assessment scores than those who did not trigger RRT activation. CONCLUSIONS: In this cohort, patients who require immediate RRT experienced higher 28-day all-cause mortality, potentially because of evolving or unrecognized critical illness. Further exploring this phenomenon may create opportunities for improved patient safety.

Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development.

The transcription factors MECOM, PAX8, SOX17 and WT1 are candidate master regulators of high-grade serous 'ovarian' cancer (HGSC), yet their cooperative role in the hypothesized tissue of origin, the fallopian tube secretory epithelium (FTSEC) is unknown. We generated 26 epigenome (CUT&TAG, CUT&RUN, ATAC-seq and HiC) data sets and 24 profiles of RNA-seq transcription factor knock-down followed by RNA sequencing in FTSEC and HGSC models to define binding sites and gene sets regulated by these factors in cis and trans. This revealed that MECOM, PAX8, SOX17 and WT1 are lineage-enriched, super-enhancer associated master regulators whose cooperative DNA-binding patterns and target genes are re-wired during tumor development. All four TFs were indispensable for HGSC clonogenicity and survival but only depletion of PAX8 and WT1 impaired FTSEC cell survival. These four TFs were pharmacologically inhibited by transcriptional inhibitors only in HGSCs but not in FTSECs. Collectively, our data highlights that tumor-specific epigenetic remodeling is tightly related to MECOM, PAX8, SOX17 and WT1 activity and these transcription factors are targetable in a tumor-specific manner through transcriptional inhibitors.

Landscape of prostate-specific membrane antigen heterogeneity and regulation in AR-positive and AR-negative metastatic prostate cancer.

Tumor expression of prostate-specific membrane antigen (PSMA) is lost in 15-20% of men with castration-resistant prostate cancer (CRPC), yet the underlying mechanisms remain poorly defined. In androgen receptor (AR)-positive CRPC, we observed lower PSMA expression in liver lesions versus other sites, suggesting a role of the microenvironment in modulating PSMA. PSMA suppression was associated with promoter histone 3 lysine 27 methylation and higher levels of neutral amino acid transporters, correlating with 18F-fluciclovine uptake on positron emission tomography imaging. While PSMA is regulated by AR, we identified a subset of AR-negative CRPC with high PSMA. HOXB13 and AR co-occupancy at the PSMA enhancer and knockout models point to HOXB13 as an upstream regulator of PSMA in AR-positive and AR-negative prostate cancer. These data demonstrate how PSMA expression is differentially regulated across metastatic lesions and in the context of the AR, which may inform selection for PSMA-targeted therapies and development of complementary biomarkers.

Enhancer profiling identifies epigenetic markers of endocrine resistance and reveals therapeutic options for metastatic castration-resistant prostate cancer patients.

Androgen Receptor (AR) signaling inhibitors, including enzalutamide, are treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC), but resistance inevitably develops. Using metastatic samples from a prospective phase II clinical trial, we epigenetically profiled enhancer/promoter activities with H3K27ac chromatin immunoprecipitation followed by sequencing, before and after AR-targeted therapy. We identified a distinct subset of H3K27ac-differentially marked regions that associated with treatment responsiveness. These data were successfully validated in mCRPC patient-derived xenograft models (PDX). In silico analyses revealed HDAC3 as a critical factor that can drive resistance to hormonal interventions, which we validated in vitro . Using cell lines and mCRPC PDX tumors in vitro , we identified drug-drug synergy between enzalutamide and the pan-HDAC inhibitor vorinostat, providing therapeutic proof-of-concept. These findings demonstrate rationale for new therapeutic strategies using a combination of AR and HDAC inhibitors to improve patient outcome in advanced stages of mCRPC.

Analysis of evolutionary dynamics and clonal architecture in prostate cancer.

The extent to which clinical and genomic characteristics associate with prostate cancer clonal architecture, tumor evolution, and therapeutic response remains unclear. Here, we reconstructed the clonal architecture and evolutionary trajectories of 845 prostate cancer tumors with harmonized clinical and molecular data. We observed that tumors from patients who self-reported as Black had more linear and monoclonal architectures, despite these men having higher rates of biochemical recurrence. This finding contrasts with prior observations relating polyclonal architecture to adverse clinical outcomes. Additionally, we utilized a novel approach to mutational signature analysis that leverages clonal architecture to uncover additional cases of homologous recombination and mismatch repair deficiency in primary and metastatic tumors and link the origin of mutational signatures to specific subclones. Broadly, prostate cancer clonal architecture analysis reveals novel biological insights that may be immediately clinically actionable and provide multiple opportunities for subsequent investigation. Statement of significance: Tumors from patients who self-reported as Black demonstrate linear and monoclonal evolutionary trajectories yet experience higher rates of biochemical recurrence. In addition, analysis of clonal and subclonal mutational signatures identifies additional tumors with potentially actionable alterations such as deficiencies in mismatch repair and homologous recombination.

Epigenomic charting and functional annotation of risk loci in renal cell carcinoma.

While the mutational and transcriptional landscapes of renal cell carcinoma (RCC) are well-known, the epigenome is poorly understood. We characterize the epigenome of clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC) by using ChIP-seq, ATAC-Seq, RNA-seq, and SNP arrays. We integrate 153 individual data sets from 42 patients and nominate 50 histology-specific master transcription factors (MTF) to define RCC histologic subtypes, including EPAS1 and ETS-1 in ccRCC, HNF1B in pRCC, and FOXI1 in chRCC. We confirm histology-specific MTFs via immunohistochemistry including a ccRCC-specific TF, BHLHE41. FOXI1 overexpression with knock-down of EPAS1 in the 786-O ccRCC cell line induces transcriptional upregulation of chRCC-specific genes, TFCP2L1, ATP6V0D2, KIT, and INSRR, implicating FOXI1 as a MTF for chRCC. Integrating RCC GWAS risk SNPs with H3K27ac ChIP-seq and ATAC-seq data reveals that risk-variants are significantly enriched in allelically-imbalanced peaks. This epigenomic atlas in primary human samples provides a resource for future investigation.

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.