Neuroendocrine (NE) prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (PCa) arising either de novo or from transdifferentiated prostate adenocarcinoma following ...androgen deprivation therapy (ADT). Extensive computational analysis has identified a high degree of association between the long noncoding RNA (lncRNA) H19 and NEPC, with the longest isoform highly expressed in NEPC. H19 regulates PCa lineage plasticity by driving a bidirectional cell identity of NE phenotype (H19 overexpression) or luminal phenotype (H19 knockdown). It contributes to treatment resistance, with the knockdown of H19 re-sensitizing PCa to ADT. It is also essential for the proliferation and invasion of NEPC. H19 levels are negatively regulated by androgen signaling via androgen receptor (AR). When androgen is absent SOX2 levels increase, driving H19 transcription and facilitating transdifferentiation. H19 facilitates the PRC2 complex in regulating methylation changes at H3K27me3/H3K4me3 histone sites of AR-driven and NEPC-related genes. Additionally, this lncRNA induces alterations in genome-wide DNA methylation on CpG sites, further regulating genes associated with the NEPC phenotype. Our clinical data identify H19 as a candidate diagnostic marker and predictive marker of NEPC with elevated H19 levels associated with an increased probability of biochemical recurrence and metastatic disease in patients receiving ADT. Here we report H19 as an early upstream regulator of cell fate, plasticity, and treatment resistance in NEPC that can reverse/transform cells to a treatable form of PCa once therapeutically deactivated.
In osteoarthritis (OA), articular chondrocytes display phenotypic and functional changes associated with epigenomic alterations. These changes contribute to the disease progression, which is ...characterized by dysregulated reparative processes and abnormal extracellular matrix remodeling leading to cartilage degradation. Recent studies using a murine model of posttraumatic OA highlighted the contribution of changes in DNA hydroxymethylation (5hmC) to OA progression. Here, we integrated transcriptomic and epigenomic analyses in cartilage after induction of OA to show that the structural progression of OA is accompanied by early transcriptomic and pronounced DNA methylation (5mC) changes in chondrocytes. These changes accumulate over time and are associated with recapitulation of developmental processes, including cartilage development, chondrocyte hypertrophy, and ossification. Our integrative analyses also uncovered that Lrrc15 is differentially methylated and expressed in OA cartilage, and that it may contribute to the functional and phenotypic alterations of chondrocytes, likely coordinating stress responses and dysregulated extracellular matrix remodeling.
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Childhood posterior fossa (PF) ependymomas cause substantial morbidity and mortality. These tumors lack recurrent genetic mutations, but a subset of these ependymomas exhibits CpG island (CpGi) ...hypermethylation PF group A (PFA), implicating epigenetic alterations in their pathogenesis. Further, histological grade does not reliably predict prognosis, highlighting the importance of developing more robust prognostic markers. We discovered global H3K27me3 reduction in a subset of these tumors (PF-ve ependymomas) analogous to H3K27M mutant gliomas. PF-ve tumors exhibited many clinical and biological similarities with PFA ependymomas. Genomic H3K27me3 distribution showed an inverse relationship with CpGi methylation, suggesting that CpGi hypermethylation drives low H3K27me3 in PF-ve ependymomas. Despite CpGi hypermethylation and global H3K27me3 reduction, these tumors showed DNA hypomethylation in the rest of the genome and exhibited increased H3K27me3 genomic enrichment at limited genomic loci similar to H3K27M mutant gliomas. Combined integrative analysis of PF-ve ependymomas with H3K27M gliomas uncovered common epigenetic deregulation of select factors that control radial glial biology, and PF radial glia in early human development exhibited reduced H3K27me3. Finally, H3K27me3 immunostaining served as a biomarker of poor prognosis and delineated radiologically invasive tumors, suggesting that reduced H3K27me3 may be a prognostic indicator in PF ependymomas.
Background
Immune cell expression profiling from patient samples is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, to identify ...exploratory biomarkers predictive of response, and to guide treatment selection and combination therapy strategies. LAG-3 is an inhibitory immune checkpoint that can suppress antitumor T-cell responses and targeting LAG-3, in combination with PD-1, is a rational approach to enhance antitumor immunity that has recently demonstrated clinical success. Here, we sought to identify human immune cell subsets that express LAG-3 and its ligands, to characterize the marker expression profile of these subsets, and to investigate the potential relationship between LAG-3 expressing subsets and clinical outcomes to immuno-oncology therapies.
Methods
Comprehensive high-parameter immunophenotyping was performed using mass and flow cytometry of tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) from two independent cohorts of samples from patients with various solid tumor types. Profiling of circulating immune cells by single cell RNA-seq was conducted on samples from a clinical trial cohort of melanoma patients treated with immunotherapy.
Results
LAG-3 was most highly expressed by subsets of tumor-infiltrating CD8 T central memory (TCM) and effector memory (TEM) cells and was frequently co-expressed with PD-1. We determined that these PD-1
+
LAG-3
+
CD8 memory T cells exhibited a unique marker profile, with greater expression of activation (CD69, HLA-DR), inhibitory (TIM-3, TIGIT, CTLA-4) and stimulatory (4-1BB, ICOS) markers compared to cells that expressed only PD-1 or LAG-3, or that were negative for both checkpoints. In contrast to tumors, LAG-3 expression was more limited in circulating immune cells from healthy donors and solid tumor patients. Additionally, we found abundant expression of the LAG-3 ligands MHC-II and galectin-3 in diverse immune cell types, whereas FGL1 and LSECtin were minimally expressed by immune cells in the tumor microenvironment (TME). Lastly, we found an inverse relationship between baseline and on-treatment levels of circulating LAG3 transcript-expressing CD8 memory T cells and response to combination PD-1 and CTLA-4 blockade in a clinical trial cohort of melanoma patients profiled by scRNAseq.
Conclusions
These results provide insights into the nature of LAG-3- and ligand-expressing immune cells within the TME, and suggest a biological basis for informing mechanistic hypotheses, treatment selection strategies, and combination immunotherapy approaches to support continued development of dual PD-1 and LAG-3 blockade.
PDZBase is a database that aims to contain all known PDZ-domain-mediated protein–protein interactions. Currently, PDZBase contains approximately 300 such interactions, which have been manually ...extracted from >200 articles. The database can be queried through both sequence motif and keyword-based searches, and the sequences of interacting proteins can be visually inspected through alignments (for the comparison of several interactions), or as residue-based diagrams including schematic secondary structure information (for individual complexes). Availability: http://icb.med.cornell.edu/services/pdz/start. Contact: pdzbase@med.cornell.edu.
BackgroundImmunotherapy (IO) has changed the treatment paradigm for patients with metastatic melanoma. IO combination therapy such as anti-PD1 nivolumab (NIVO), anti-CTLA4 ipilimumab (IPI) and ...anti-LAG3 relatlimab (RELA) have replaced chemotherapy as first line treatment. Although candidate biomarkers such as interferon gamma gene expression, BRAF mutation, PD-L1 tumor expression, high tumor mutational burden and CD8 T-cell infiltration have been explored for their predictive value, no definitive biomarkers for IO response have been identified. Our study explores novel exploratory biomarkers that can help us understand which metastatic melanoma patients will have an increased benefit from either anti-CTLA4 or anti-CTLA4/PD1 treatment.MethodsScRNAseq profiling of peripheral blood mononuclear cells (PBMCs) was performed on a subset of CheckMate-069 clinical trial (NCT01927419) samples, which included 52 patients with metastatic melanoma treated with either IPI (n=26) or NIVO + IPI (n=26), using the 5 prime 10x Genomics platform (total n=52, samples=104). PBMCs from patients were profiled at Baseline (C1D1) and after 2 cycles of therapy (C3D1) and separated as responders (R, n=20) or non-responders (NR, n=32). We deployed Seurat R package for cell phenotype determination including Tregs and interrogated drug response in R vs. NR, mechanisms of drug resistance/action, pharmacodynamic (PD) markers, and biomarker of response.ResultsScRNAseq analysis revealed that peripheral regulatory T-cells (Tregs) did not decrease post IO regardless of treatment type (IPI or NIVO+IPI) or response. In fact, the proportion of Treg cells increased post IO regardless of treatment or response, despite an observed decrease in their cell of origin proportion (CD4+ T-cells) (figure 1A). Although Tregs do not decrease, their ‘suppressive’ phenotype was down-modulated preferentially in responders as indicated by the selective downregulation of EZH2 and FOXP3 expression in responders (figure 1B). A lower Treg cell proportion at Baseline and higher CD8-central-memory cell proportion at C3D1 served as a predictive response biomarker of IPI and NIVO+IPI respectively. The ratio of CD8+Teffector memory to Treg also predicts outcome benefit to anti-CTLA4 and may serve as a potential predictive biomarker (figure 2).ConclusionsOur analysis serves to elucidate a missing piece of anti-CTLA4 therapy mechanism of action and identifies potential new biomarkers that can be applied to inform stratification for combination therapy trials. Although Tregs were not depleted, EZH2 was selectively upregulated in T-regs of non-responders upon IPI treatment indicating that a combination therapy of EZH2i+anti-CLTA4 may increase response rates. Additionally, CD8+effector memory/Treg cells ratio may serve as a new biomarker predictive of anti-CTLA4 benefit.AcknowledgementsWe acknowledge the contribution of Megan Wind-Rotolo and Evisa Gjini to the design of this work prior to their departure from the companyTrial RegistrationNCT01927419Study of Nivolumab (BMS-936558) Plus Ipilimumab Compared With Ipilimumab Alone in the Treatment of Previously Untreated, Unresectable, or Metastatic Melanoma (CheckMate 069)Ethics ApprovalAll subjects consented to this research.ConsentWritten informed consent was obtained by BMS from the patient for this exploratory research and publication of the resultsAbstract 624 Figure 1(A) Tregs cell proportion are not depleted upon either IPI or Nivo+IPI treatment regardless of response (B) Treg suppressive features are downregulated in responders and upregulated in non-responders. Colors denote different response groupsAbstract 624 Figure 2A higher CD8 effector memory cell to T-reg ratio at baseline predicts better outcome to Ipi
The complement system is thought to be involved in the pathogenesis of numerous neurological diseases. We previously reported that pre-treatment of murine cortico-hippocampal neuronal cultures with ...the complement derived anaphylatoxin C5a, protects against glutamate mediated apoptosis. Our present study with C5a receptor knock out (C5aRKO) mice corroborates that the deficiency of C5a renders C5aRKO mouse more susceptible to apoptotic injury in vivo. In this study we explored potential upstream mechanisms involved in C5a mediated neuroprotection in vivo and in vitro.
Based on evidence suggesting that reduced expression of glutamate receptor subunit 2 (GluR2) may influence apoptosis in neurons, we studied the effect of human recombinant C5a on GluR2 expression in response to glutamate neurotoxicity. Glutamate analogs were injected into C5aRKO mice or used to treat in vitro neuronal culture and GluR2 expression were assessed in respect with cell death.
In C5aRKO mice we found that the neurons are more susceptible to excitotoxicity resulting in apoptotic injury in the absence of the C5a receptor compared to WT control mice. Our results suggest that C5a protects against apoptotic pathways in neurons in vitro and in vivo through regulation of GluR2 receptor expression.
Complement C5a neuroprotects through regulation of GluR2 receptor subunit.
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Purpose: To compare gene expression profiles of chromophobe renal cell carcinoma (RCC) and benign oncocytoma, aiming at identifying
differentially expressed genes.
Experimental Design: Nine cases ...each of chromophobe RCC and oncocytoma were analyzed by oligonucleotide microarray. Candidate genes that showed
consistent differential expression were validated by reverse transcription-PCR using 25 fresh-frozen and 15 formalin-fixed,
paraffin-embedded tumor samples. Immunohistochemical analysis was also done for two selected gene products, claudin 8 and
MAL2.
Results: Unsupervised hierarchical clustering separated the chromophobe RCC and oncocytoma into two distinct groups. By a combination
of data analysis approaches, we identified 11 candidate genes showing consistent differential expression between chromophobe
RCC and oncocytoma. Five of these genes, AP1M2, MAL2, PROM2, PRSS8 , and FLJ20171 , were shown to effectively separate these two tumor groups by quantitative reverse transcription-PCR using fresh tissue samples,
with similar trends seen on formalin-fixed tissues. Immunohistochemical analysis revealed selective expression of MAL2 and
claudin 8 in distal renal tubules, with MAL2 antibody showing differential expression between chromophobe RCC and oncocytoma.
Functional analyses suggest that genes encoding tight junction proteins and vesicular membrane trafficking proteins, normally
expressed in distal nephrons, are retained in chromophobe RCC and lost or consistently down-regulated in oncocytoma, indicating
that these two tumor types, believed to be both derived from distal tubules, are likely distinctive in their histogenesis.
Conclusions: We showed that chromophobe RCC and oncocytoma are distinguishable by mRNA expression profiles and a panel of gene products
potentially useful as diagnostic markers were identified.
Vocal learning and neuronal replacement have been studied extensively in songbirds, but until recently, few molecular and genomic tools for songbird research existed. Here we describe new ...molecular/genomic resources developed in our laboratory. We made cDNA libraries from zebra finch (Taeniopygia guttata) brains at different developmental stages. A total of 11,000 cDNA clones from these libraries, representing 5,866 unique gene transcripts, were randomly picked and sequenced from the 3' ends. A web-based database was established for clone tracking, sequence analysis, and functional annotations. Our cDNA libraries were not normalized. Sequencing ESTs without normalization produced many developmental stage-specific sequences, yielding insights into patterns of gene expression at different stages of brain development. In particular, the cDNA library made from brains at posthatching day 30-50, corresponding to the period of rapid song system development and song learning, has the most diverse and richest set of genes expressed. We also identified five microRNAs whose sequences are highly conserved between zebra finch and other species. We printed cDNA microarrays and profiled gene expression in the high vocal center of both adult male zebra finches and canaries (Serinus canaria). Genes differentially expressed in the high vocal center were identified from the microarray hybridization results. Selected genes were validated by in situ hybridization. Networks among the regulated genes were also identified. These resources provide songbird biologists with tools for genome annotation, comparative genomics, and microarray gene expression analysis.
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