RANK ligand converts the NCoR/HDAC3 co-repressor to a PGC1ß- and RNA-dependent co-activator of osteoclast gene expression.

The nuclear receptor co-repressor (NCoR) complex mediates transcriptional repression dependent on histone deacetylation by histone deacetylase 3 (HDAC3) as a component of the complex. Unexpectedly, we found that signaling by the receptor activator of nuclear factor κB (RANK) converts the NCoR/HDAC3 co-repressor complex to a co-activator of AP-1 and NF-κB target genes that are required for mouse osteoclast differentiation. Accordingly, the dominant function of NCoR/HDAC3 complexes in response to RANK signaling is to activate, rather than repress, gene expression. Mechanistically, RANK signaling promotes RNA-dependent interaction of the transcriptional co-activator PGC1ß with the NCoR/HDAC3 complex, resulting in the activation of PGC1ß and inhibition of HDAC3 activity for acetylated histone H3. Non-coding RNAs Dancr and Rnu12, which are associated with altered human bone homeostasis, promote NCoR/HDAC3 complex assembly and are necessary for RANKL-induced osteoclast differentiation in vitro. These findings may be prototypic for signal-dependent functions of NCoR in other biological contexts.

Predicting chronic postsurgical pain: current evidence and a novel program to develop predictive biomarker signatures.

ABSTRACT: Chronic pain affects more than 50 million Americans. Treatments remain inadequate, in large part, because the pathophysiological mechanisms underlying the development of chronic pain remain poorly understood. Pain biomarkers could potentially identify and measure biological pathways and phenotypical expressions that are altered by pain, provide insight into biological treatment targets, and help identify at-risk patients who might benefit from early intervention. Biomarkers are used to diagnose, track, and treat other diseases, but no validated clinical biomarkers exist yet for chronic pain. To address this problem, the National Institutes of Health Common Fund launched the Acute to Chronic Pain Signatures (A2CPS) program to evaluate candidate biomarkers, develop them into biosignatures, and discover novel biomarkers for chronification of pain after surgery. This article discusses candidate biomarkers identified by A2CPS for evaluation, including genomic, proteomic, metabolomic, lipidomic, neuroimaging, psychophysical, psychological, and behavioral measures. Acute to Chronic Pain Signatures will provide the most comprehensive investigation of biomarkers for the transition to chronic postsurgical pain undertaken to date. Data and analytic resources generatedby A2CPS will be shared with the scientific community in hopes that other investigators will extract valuable insights beyond A2CPS's initial findings. This article will review the identified biomarkers and rationale for including them, the current state of the science on biomarkers of the transition from acute to chronic pain, gaps in the literature, and how A2CPS will address these gaps.

Variant STAT4 and Response to Ruxolitinib in an Autoinflammatory Syndrome.

BACKGROUND: Disabling pansclerotic morphea (DPM) is a rare systemic inflammatory disorder, characterized by poor wound healing, fibrosis, cytopenias, hypogammaglobulinemia, and squamous-cell carcinoma. The cause is unknown, and mortality is high. METHODS: We evaluated four patients from three unrelated families with an autosomal dominant pattern of inheritance of DPM. Genomic sequencing independently identified three heterozygous variants in a specific region of the gene that encodes signal transducer and activator of transcription 4 (STAT4). Primary skin fibroblast and cell-line assays were used to define the functional nature of the genetic defect. We also assayed gene expression using single-cell RNA sequencing of peripheral-blood mononuclear cells to identify inflammatory pathways that may be affected in DPM and that may respond to therapy. RESULTS: Genome sequencing revealed three novel heterozygous missense gain-of-function variants in STAT4. In vitro, primary skin fibroblasts showed enhanced interleukin-6 secretion, with impaired wound healing, contraction of the collagen matrix, and matrix secretion. Inhibition of Janus kinase (JAK)-STAT signaling with ruxolitinib led to improvement in the hyperinflammatory fibroblast phenotype in vitro and resolution of inflammatory markers and clinical symptoms in treated patients, without adverse effects. Single-cell RNA sequencing revealed expression patterns consistent with an immunodysregulatory phenotype that were appropriately modified through JAK inhibition. CONCLUSIONS: Gain-of-function variants in STAT4 caused DPM in the families that we studied. The JAK inhibitor ruxolitinib attenuated the dermatologic and inflammatory phenotype in vitro and in the affected family members. (Funded by the American Academy of Allergy, Asthma, and Immunology Foundation and others.).

Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation.

The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.

Multi-Site Observational Study to Assess Biomarkers for Susceptibility or Resilience to Chronic Pain: The Acute to Chronic Pain Signatures (A2CPS) Study Protocol.

Chronic pain has become a global health problem contributing to years lived with disability and reduced quality of life. Advances in the clinical management of chronic pain have been limited due to incomplete understanding of the multiple risk factors and molecular mechanisms that contribute to the development of chronic pain. The Acute to Chronic Pain Signatures (A2CPS) Program aims to characterize the predictive nature of biomarkers (brain imaging, high-throughput molecular screening techniques, or "omics," quantitative sensory testing, patient-reported outcome assessments and functional assessments) to identify individuals who will develop chronic pain following surgical intervention. The A2CPS is a multisite observational study investigating biomarkers and collective biosignatures (a combination of several individual biomarkers) that predict susceptibility or resilience to the development of chronic pain following knee arthroplasty and thoracic surgery. This manuscript provides an overview of data collection methods and procedures designed to standardize data collection across multiple clinical sites and institutions. Pain-related biomarkers are evaluated before surgery and up to 3 months after surgery for use as predictors of patient reported outcomes 6 months after surgery. The dataset from this prospective observational study will be available for researchers internal and external to the A2CPS Consortium to advance understanding of the transition from acute to chronic postsurgical pain.

The breast pre-cancer atlas illustrates the molecular and micro-environmental diversity of ductal carcinoma in situ.

Microenvironmental and molecular factors mediating the progression of Breast Ductal Carcinoma In Situ (DCIS) are not well understood, impeding the development of prevention strategies and the safe testing of treatment de-escalation. We addressed methodological barriers and characterized the mutational, transcriptional, histological, and microenvironmental landscape across 85 multiple microdissected regions from 39 cases. Most somatic alterations, including whole-genome duplications, were clonal, but genetic divergence increased with physical distance. Phenotypic and subtype heterogeneity was frequently associated with underlying genetic heterogeneity and regions with low-risk features preceded those with high-risk features according to the inferred phylogeny. B- and T-lymphocytes spatial analysis identified three immune states, including an epithelial excluded state located preferentially at DCIS regions, and characterized by histological and molecular features of immune escape, independently from molecular subtypes. Such breast pre-cancer atlas with uniquely integrated observations will help scope future expansion studies and build finer models of outcomes and progression risk.

The unfolded protein response links tumor aneuploidy to local immune dysregulation.

Aneuploidy is a chromosomal abnormality associated with poor prognosis in many cancer types. Here, we tested the hypothesis that the unfolded protein response (UPR) mechanistically links aneuploidy and local immune dysregulation. Using a single somatic copy number alteration (SCNA) score inclusive of whole-chromosome, chromosome arm, and focal alterations in a pan-cancer analysis of 9,375 samples in The Cancer Genome Atlas (TCGA) database, we found an inverse correlation with a cytotoxicity (CYT) score across disease stages. Co-expression patterns of UPR genes changed substantially between SCNAlow and SCNAhigh groups. Pathway activity scores showed increased activity of multiple branches of the UPR in response to aneuploidy. The PERK branch showed the strongest association with a reduced CYT score. The conditioned medium of aneuploid cells transmitted XBP1 splicing and caused IL-6 and arginase 1 transcription in receiver bone marrow-derived macrophages and markedly diminished the production of IFN-γ and granzyme B in activated human T cells. We propose the UPR as a mechanistic link between aneuploidy and immune dysregulation in the tumor microenvironment.

An iPSC line derived from a human acute myeloid leukemia cell line (HL-60-iPSC) retains leukemic abnormalities and displays myeloid differentiation defects.

Cancer-derived iPSCs have provided valuable insight into oncogenesis, but human cancer cells can often be difficult to reprogram, especially in cases of complex genetic abnormalities. Here we report, to our knowledge, the first successful generation of an iPSC line from a human immortalized acute myeloid leukemia (AML) cell line, the cell line HL-60. This iPSC line retains a majority of the leukemic genotype and displays defects in myeloid differentiation, thus providing a tool for modeling and studying AML.

Mutational profiling of micro-dissected pre-malignant lesions from archived specimens.

BACKGROUND: Systematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA. METHODS: Using test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3-200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs. cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS). RESULTS: Using low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity. CONCLUSION: Alternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.

IRE1α regulates macrophage polarization, PD-L1 expression, and tumor survival.

In the tumor microenvironment, local immune dysregulation is driven in part by macrophages and dendritic cells that are polarized to a mixed proinflammatory/immune-suppressive phenotype. The unfolded protein response (UPR) is emerging as the possible origin of these events. Here we report that the inositol-requiring enzyme 1 (IRE1α) branch of the UPR is directly involved in the polarization of macrophages in vitro and in vivo, including the up-regulation of interleukin 6 (IL-6), IL-23, Arginase1, as well as surface expression of CD86 and programmed death ligand 1 (PD-L1). Macrophages in which the IRE1α/X-box binding protein 1 (Xbp1) axis is blocked pharmacologically or deleted genetically have significantly reduced polarization and CD86 and PD-L1 expression, which was induced independent of IFNγ signaling, suggesting a novel mechanism in PD-L1 regulation in macrophages. Mice with IRE1α- but not Xbp1-deficient macrophages showed greater survival than controls when implanted with B16.F10 melanoma cells. Remarkably, we found a significant association between the IRE1α gene signature and CD274 gene expression in tumor-infiltrating macrophages in humans. RNA sequencing (RNASeq) analysis showed that bone marrow-derived macrophages with IRE1α deletion lose the integrity of the gene connectivity characteristic of regulated IRE1α-dependent decay (RIDD) and the ability to activate CD274 gene expression. Thus, the IRE1α/Xbp1 axis drives the polarization of macrophages in the tumor microenvironment initiating a complex immune dysregulation leading to failure of local immune surveillance.

Two iPSC lines generated from the bone marrow of a relapsed/refractory AML patient display normal karyotypes and myeloid differentiation potential.

Using iPSCs to study cancer has been complicated by the fact that many cancer cells are difficult to reprogram, which has been attributed to the genomic abnormalities present. Acute Myeloid Leukemia (AML) is a complex disease that presents with various types of genomic aberrations that affect prognosis. Here we reprogrammed CD34+ cells from an AML patient containing a rare der(7)t(7;13) translocation associated with poor prognosis, who had relapsed and was refractory to current treatments. The generated AML-iPSCs displayed normal karyotypes and myeloid differentiation potential. These findings have implications for modeling and treating AML disease.

Extracellular vesicles produced in B cells deliver tumor suppressor miR-335 to breast cancer cells disrupting oncogenic programming in vitro and in vivo.

The successful implementation of miRNA (miR) therapies in humans will ultimately rely on the use of vehicles with improved cellular delivery capability. Here we tested a new system that leverages extracellular vesicles (EVs) laden with a tumor suppressor miRNA (miR-335) produced in B cells by plasmid DNA induction (iEVs). We demonstrate that iEVs-335 efficiently and durably restored the endogenous miR-335 pool in human triple negative breast cancer cells, downregulated the expression of the miR-335 target gene SOX4 transcription factor, and markedly inhibited tumor growth in vivo. Remarkably, iEVs-335 mediated transcriptional effects that persisted in tumors after 60 days post orthotopic implantation. Genome-wide RNASeq analysis of cancer cells treated in vitro with iEVs-335 showed the regulation of a discrete number of genes only, without broad transcriptome perturbations. This new technology may be ideally suited for therapies aimed to restore tumor suppressor miRNAs in cancer cells, disrupting the oncogenic program established after escape from miRNA control.

Massively Parallel Sequencing of Peritoneal and Splenic B Cell Repertoires Highlights Unique Properties of B-1 Cell Antibodies.

B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained VH11 and VH12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.

Systematic transcriptome analysis reveals tumor-specific isoforms for ovarian cancer diagnosis and therapy.

Tumor-specific molecules are needed across diverse areas of oncology for use in early detection, diagnosis, prognosis and therapy. Large and growing public databases of transcriptome sequencing data (RNA-seq) derived from tumors and normal tissues hold the potential of yielding tumor-specific molecules, but because the data are new they have not been fully explored for this purpose. We have developed custom bioinformatic algorithms and used them with 296 high-grade serous ovarian (HGS-OvCa) tumor and 1,839 normal RNA-seq datasets to identify mRNA isoforms with tumor-specific expression. We rank prioritized isoforms by likelihood of being expressed in HGS-OvCa tumors and not in normal tissues and analyzed 671 top-ranked isoforms by high-throughput RT-qPCR. Six of these isoforms were expressed in a majority of the 12 tumors examined but not in 18 normal tissues. An additional 11 were expressed in most tumors and only one normal tissue, which in most cases was fallopian or colon. Of the 671 isoforms, the topmost 5% (n = 33) ranked based on having tumor-specific or highly restricted normal tissue expression by RT-qPCR analysis are enriched for oncogenic, stem cell/cancer stem cell, and early development loci--including ETV4, FOXM1, LSR, CD9, RAB11FIP4, and FGFRL1. Many of the 33 isoforms are predicted to encode proteins with unique amino acid sequences, which would allow them to be specifically targeted for one or more therapeutic strategies--including monoclonal antibodies and T-cell-based vaccines. The systematic process described herein is readily and rapidly applicable to the more than 30 additional tumor types for which sufficient amounts of RNA-seq already exist.

BAP1 mutation is a frequent somatic event in peritoneal malignant mesothelioma.

BACKGROUND: Malignant mesothelioma (MM) arises from mesothelial cells that line the pleural, peritoneal and pericardial surfaces. The majority of MMs are pleural and have been associated with asbestos exposure. Previously, pleural MMs have been genetically characterized by the loss of BAP1 (40-60%) as well as loss of NF2 (75%) and CDKN2A (60%). The rare peritoneal form of MM occurs in ~10% cases. With only ~300 cases diagnosed in the US per year, its link to asbestos exposure is not clear and its mutational landscape unknown. METHODS: We analyzed the somatic mutational landscape of 12 peritoneal MM of epitheloid subtype using copy number analysis (N = 9), whole exome sequencing (N = 7) and targeted sequencing (N = 12). RESULTS: Peritoneal MM display few copy number alterations, with most samples having less than 10 Mbp total changes, mostly through deletions and no high copy number amplification. Chromosome band 3p21 encoding BAP1 is the most recurrently deleted region (5/9), while, in contrast to pleural MM, NF2 and CDKN2A are not affected. We further identified 87 non-silent mutations across 7 sequenced tumors, with a median of 8 mutated genes per tumor, resulting in a very low mutation rate (median 1.3 10(-6)). BAP1 was the only recurrently mutated gene (N = 3/7). In one additional case, loss of the entire chromosome 3 leaves a non-functional copy of BAP1 carrying a rare nonsense germline variant, thus suggesting a potential genetic predisposition in this patient. Finally, with targeted sequencing of BAP1 in 3 additional cases, we conclude that BAP1 is frequently altered through copy number losses (N = 3/12), mutations (N = 3/12) or both (N = 2/12) sometimes at a sub-clonal level. CONCLUSION: Our findings suggest a major role for BAP1 in peritoneal MM susceptibility and oncogenesis and indicate important molecular differences to pleural MM as well as potential strategies for therapy and prevention.

Transcriptome sequencing reveals potential mechanism of cryptic 3' splice site selection in SF3B1-mutated cancers.

Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (<10%) relative to nearby canonical 3'SSs, we identified ten genes that preferred out-of-frame cryptic 3'SSs. We show that cancers with mutations in the SF3B1 HEAT 5-9 repeats use cryptic 3'SSs downstream of the branch point and provide both a mechanistic model consistent with published experimental data and affected targets that will guide further research into the oncogenic effects of SF3B1 mutation.

Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments.

Accurate allele frequencies are important for measuring subclonal heterogeneity and clonal evolution. Deep-targeted sequencing data can contain PCR duplicates, inflating perceived read depth. Here we adapted the Illumina TruSeq Custom Amplicon kit to include single molecule tagging (SMT) and show that SMT-identified duplicates arise from PCR. We demonstrate that retention of PCR duplicate reads can imply clonal evolution when none exists, while their removal effectively controls the false positive rate. Additionally, PCR duplicates alter estimates of subclonal heterogeneity in tumor samples. Our method simplifies PCR duplicate identification and emphasizes their removal in studies of tumor heterogeneity and clonal evolution.

Genome-wide mutational landscape of mucinous carcinomatosis peritonei of appendiceal origin.

BACKGROUND: Mucinous neoplasms of the appendix (MNA) are rare tumors which may progress from benign to malignant disease with an aggressive biological behavior. MNA is often diagnosed after metastasis to the peritoneal surfaces resulting in mucinous carcinomatosis peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs, and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer and not based on molecular rationale. METHODS: We sequenced the whole exomes of 10 MCPs of appendiceal origin to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases. RESULTS: Our study demonstrates that MNA has a different molecular makeup than colorectal cancer. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (20/29) and are characterized by downstream PKA activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with colorectal cancer including gain of 1q (5/10), Wnt, and TGFß pathway alterations. In contrast, mutations in TP53 (1/10) and APC (0/10), common in colorectal cancer, are rare in MNA. Concurrent activation of the KRAS and GNAS mediated signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm. CONCLUSIONS: MNA genome-wide mutational analysis reveals genetic alterations distinct from colorectal cancer, in support of its unique pathophysiology and suggests new targeted therapeutic opportunities.

miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1.

We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome.