Behavioral and transcriptomic analyses of mecp2 function in zebrafish.

Rett syndrome (RTT), a human neurodevelopmental disorder characterized by severe cognitive and motor impairments, is caused by dysfunction of the conserved transcriptional regulator Methyl-CpG-binding protein 2 (MECP2). Genetic analyses in mouse Mecp2 mutants, which exhibit key features of human RTT, have been essential for deciphering the mechanisms of MeCP2 function; nonetheless, our understanding of these complex mechanisms is incomplete. Zebrafish mecp2 mutants exhibit mild behavioral deficits but have not been analyzed in depth. Here, we combine transcriptomic and behavioral assays to assess baseline and stimulus-evoked motor responses and sensory filtering in zebrafish mecp2 mutants from 5 to 7 days post-fertilization (dpf). We show that zebrafish mecp2 function is required for normal thigmotaxis but is dispensable for gross movement, acoustic startle response, and sensory filtering (habituation and sensorimotor gating), and reveal a previously unknown role for mecp2 in behavioral responses to visual stimuli. RNA-seq analysis identified a large gene set that requires mecp2 function for correct transcription at 4 dpf, and pathway analysis revealed several pathways that require MeCP2 function in both zebrafish and mammals. These findings show that MeCP2's function as a transcriptional regulator is conserved across vertebrates and supports using zebrafish to complement mouse modeling in elucidating these conserved mechanisms.

A clinical-grade liquid biomarker detects neuroendocrine differentiation in prostate cancer.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).

Reversible epigenetic alterations regulate class I HLA loss in prostate cancer.

Downregulation of HLA class I (HLA-I) impairs immune recognition and surveillance in prostate cancer and may underlie the ineffectiveness of checkpoint blockade. However, the molecular mechanisms regulating HLA-I loss in prostate cancer have not been fully explored. Here, we conducted a comprehensive analysis of HLA-I genomic, epigenomic and gene expression alterations in primary and metastatic human prostate cancer. Loss of HLA-I gene expression was associated with repressive chromatin states including DNA methylation, histone H3 tri-methylation at lysine 27, and reduced chromatin accessibility. Pharmacological DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibition decreased DNA methylation and increased H3 lysine 27 acetylation and resulted in re-expression of HLA-I on the surface of tumor cells. Re-expression of HLA-I on LNCaP cells by DNMT and HDAC inhibition increased activation of co-cultured prostate specific membrane antigen (PSMA)27-38-specific CD8+ T-cells. HLA-I expression is epigenetically regulated by functionally reversible DNA methylation and chromatin modifications in human prostate cancer. Methylated HLA-I was detected in HLA-Ilow circulating tumor cells (CTCs), which may serve as a minimally invasive biomarker for identifying patients who would benefit from epigenetic targeted therapies.

SEEMLIS: a flexible semi-automated method for enrichment of methylated DNA from low-input samples.

BACKGROUND: DNA methylation alterations have emerged as hallmarks of cancer and have been proposed as screening, prognostic, and predictive biomarkers. Traditional approaches for methylation analysis have relied on bisulfite conversion of DNA, which can damage DNA and is not suitable for targeted gene analysis in low-input samples. Here, we have adapted methyl-CpG-binding domain protein 2 (MBD2)-based DNA enrichment for use on a semi-automated exclusion-based sample preparation (ESP) platform for robust and scalable enrichment of methylated DNA from low-input samples, called SEEMLIS. RESULTS: We show that combining methylation-sensitive enzyme digestion with ESP-based MBD2 enrichment allows for single gene analysis with high sensitivity for GSTP1 in highly impure, heterogenous samples. We also show that ESP-based MBD2 enrichment coupled with targeted pre-amplification allows for analysis of multiple genes with sensitivities approaching the single cell level in pure samples for GSTP1 and RASSF1 and sensitivity down to 14 cells for these genes in highly impure samples. Finally, we demonstrate the potential clinical utility of SEEMLIS by successful detection of methylated gene signatures in circulating tumor cells (CTCs) from patients with prostate cancer with varying CTC number and sample purity. CONCLUSIONS: SEEMLIS is a robust assay for targeted DNA methylation analysis in low-input samples, with flexibility at multiple steps. We demonstrate the feasibility of this assay to analyze DNA methylation in prostate cancer cells using CTCs from patients with prostate cancer as a real-world example of a low-input analyte of clinical importance. In summary, this novel assay provides a platform for determining methylation signatures in rare cell populations with broad implications for research as well as clinical applications.

Prospective Evaluation of Clinical Outcomes Using a Multiplex Liquid Biopsy Targeting Diverse Resistance Mechanisms in Metastatic Prostate Cancer.

PURPOSE: Nearly all men with prostate cancer treated with androgen receptor (AR) signaling inhibitors (ARSIs) develop resistance via diverse mechanisms including constitutive activation of the AR pathway, driven by AR genomic structural alterations, expression of AR splice variants (AR-Vs), or loss of AR dependence and lineage plasticity termed neuroendocrine prostate cancer. Understanding these de novo acquired ARSI resistance mechanisms is critical for optimizing therapy. MATERIALS AND METHODS: A novel liquid biopsy technology was used to collect mRNA from circulating tumor cells (CTCs) to measure expression of AR-Vs, AR targets, and neuroendocrine prostate cancer markers. An institutional review board-approved prospective cohort (N = 99) was used to identify patterns of gene expression. Two prospective multicenter phase II clinical trials of ARSIs for men with castration-resistant prostate cancer (ClinicalTrials.gov: NCT01942837 [enzalutamide, N = 21] and NCT02025010 [abiraterone, N = 27]) were used to further validate these findings. RESULTS: Hierarchical clustering of CTC transcripts identified two distinct clusters. Cluster 2 (C2) exhibited increased expression of AR-regulated genes and was associated with worse overall survival (median 8.6 v 22.4 months; P < .01; hazard ratio [HR] = 3.45 [1.9 to 6.14]). In multivariable analysis, C2 was prognostic independent of other clinicopathologic variables. AR-V status was not significant when accounting for C2. Upon further validation in pooled multicenter phase II trials, C2 was associated with worse overall survival (15.2 months v not reached; P < .01; HR = 8.43 [2.74 to 25.92]), prostate-specific antigen progression-free survival (3.6 v 12 months; P < .01; HR = 4.64 [1.53 to 14.11]), and radiographic progression-free survival (2.7 v 40.6 months; P < .01; HR = 4.64 [1.82 to 17.41]). CONCLUSION: We demonstrate that a transcriptional profile detectable in CTCs obtained from liquid biopsies can serve as an independent prognostic marker beyond AR-V7 in patients with metastatic prostate cancer and can be used to identify the emergence of multiple ARSI resistance mechanisms. This is currently being investigated in additional prospective trials.