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Disruption of the CDKN2A (INK4A/ARF) and B (INK4B) genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFβ selectively induces Arf at mouse Cdkn2a in eye development and cultured fibroblasts. As TGFβ signaling is often derailed in cancer development or progression, we investigated its control of CDKN2A/B in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of ARF and INK4A in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFβ induced ARF mRNA and protein, and ARF knockdown limited TGFβ-mediated growth suppression. TGFβ-mediated ARF mRNA induction required SMAD2/3, p38MAPK, and SP1, and ARF mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFβ by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of ARF and INK4B by TGFβ, but not by oncogenic RAS. Implications: Our findings reveal new molecular mechanisms by which CDKN2A/B regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.

A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267–284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). GLIPR2 knockout resulted in less compact Golgi structures, which was also observed in autophagy-inducing conditions such as amino acid starvation or Tat-BECN1 peptide treatment. Importantly, the binding of GLIPR2 to purified PtdIns3K-C1 inhibited the in vitro lipid kinase activity of PtdIns3K-C1. Moreover, the tissues of glipr2 knockout mice had increased basal autophagic flux as well as increased recruitment of the PtdIns3P-binding protein, WIPI2. Taken together, our findings demonstrate that GLIPR2 is a negative regulator of PtdIns3K-C1 activity and basal autophagy. Abbreviations: ATG14: autophagy related 14; Baf A1: bafilomycin A1; BARA: β-α repeated, autophagy-specific; CQ: chloroquine; GFP: green fluorescent protein; GLIPR2: GLI pathogenesis related 2; HBSS: Hanks’ balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBS: phosphate-buffered saline; PtdIns3K-C1: phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; SEM: standard error of the mean; WIPI2: WD repeat domain, phosphoinositide interacting 2

Chromatin remodeling plays a critical role in tumor suppression as demonstrated by 20% of human cancers bearing inactivating mutations in SWI/SNF chromatin remodeling complex members. Mutations in different SWI/SNF subunits drive a variety of adult and pediatric tumor types, including non-small cell lung cancers, rhabdoid tumors, medulloblastomas, and ovarian cancers. Small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is an aggressive subtype of ovarian cancer occurring in young women. Nearly all (>98%) SCCOHTs have inactivating mutations inSMARCA4, which encodes 1 of 2 mutually exclusive catalytic subunits of the SWI/SNF complex. Less than half of SCCOHT patients survive 5 years despite aggressive surgery and multimodal chemotherapy. Empirical support for effective SCCOHT treatments is scarce, in part because of the poor understanding of SCCOHT tumorigenesis. To gain insight into the functional consequences of SWI/SNF subunit loss, we defined SWI/SNF composition and its protein-protein interactions (PPIs) by immunoprecipitation and mass spectrometry (IP-MS) of SWI/SNF subunits in 3 SCCOHT cell lines. Comparing these results to a cell line containing a wild-type SWI/SNF complex, the interaction of most canonical core SWI/SNF subunits was observed in all SCCOHT cell lines at a lower abundance. The SCCOHT SWI/SNF also lacked ATPase module subunits and showed a drastic reduction in PBAF-specific subunit interactions. The wild-type and SCCOHT SWI/SNF subunits immunoprecipitated a shared set of 26 proteins, including core SWI/SNF subunits and RNA processing proteins. We observed 131 proteins exclusively interacting with the wild-type SWI/SNF complex including isoform-specific SWI/SNF subunits, members of the NuRD complex, and members of the MLL3/4 complex. We observed 60 PPIs exclusive to the SCCOHT residual SWI/SNF shared in at least 2 of the 3 SCCOHT cell lines, including many proteins involved in RNA processing. Differential interactions with the residual SWI/SNF complex in SCCOHT may further elucidate altered functional consequences of SMARCA4 mutations in these tumors as well as identify synthetic lethal targets that translate to other SWI/SNF-deficient tumors.

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins – p16(lnk4a), which blocks cyclin-dependent kinase activity, and p19(Arf), which is best known as a positive regulator of the p53 tumor suppressor – and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts (MEFs) generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFβ-2-dependent induction of the Arf reporter in the eye - a process essential for normal eye development - does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.

Chromatin remodeling plays a critical role in tumor suppression as demonstrated by 20% of human cancers bearing inactivating mutations in SWI/SNF chromatin remodeling complex members. Mutations in different SWI/SNF subunits drive a variety of adult and pediatric tumor types, including non-small cell lung cancers, rhabdoid tumors, medulloblastomas, and ovarian cancers. Small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is an aggressive subtype of ovarian cancer occurring in young women. Nearly all (>98%) SCCOHTs have inactivating mutations in SMARCA4, which encodes 1 of 2 mutually exclusive catalytic subunits of the SWI/SNF complex. Less than half of SCCOHT patients survive 5 years despite aggressive surgery and multimodal chemotherapy. Empirical support for effective SCCOHT treatments is scarce, in part because of the poor understanding of SCCOHT tumorigenesis. To gain insight into the functional consequences of SWI/SNF subunit loss, we defined SWI/SNF composition and its protein-protein interactions (PPIs) by immunoprecipitation and mass spectrometry (IP-MS) of SWI/SNF subunits in 3 SCCOHT cell lines. Comparing these results to a cell line containing a wild-type SWI/SNF complex, the interaction of most canonical core SWI/SNF subunits was observed in all SCCOHT cell lines at a lower abundance. The SCCOHT SWI/SNF also lacked ATPase module subunits and showed a drastic reduction in PBAF-specific subunit interactions. The wild-type and SCCOHT SWI/SNF subunits immunoprecipitated a shared set of 26 proteins, including core SWI/SNF subunits and RNA processing proteins. We observed 131 proteins exclusively interacting with the wild-type SWI/SNF complex including isoform-specific SWI/SNF subunits, members of the NuRD complex, and members of the MLL3/4 complex. We observed 60 PPIs exclusive to the SCCOHT residual SWI/SNF shared in at least 2 of the 3 SCCOHT cell lines, including many proteins involved in RNA processing. Differential interactions with the residual SWI/SNF complex in SCCOHT may further elucidate altered functional consequences of SMARCA4 mutations in these tumors as well as identify synthetic lethal targets that translate to other SWI/SNF-deficient tumors. Citation Format: Elizabeth Raupach, Krystine Garcia-Mansfield, Ritin Sharma, Apurva Hegde, Victoria David-Dirgo, Yemin Wang, Chae Young Shin, Lan Tao, Salvatore Facista, Rayvon Moore, Jessica Lang, Victoria Zismann, Krystal Orlando, Monique Spillman, Anthony Karnezis, Lynda Bennett, David Huntsman, Jeffrey Trent, William Hendricks, Bernard Weissman, Patrick Pirrotte. Novel functional insights revealed by distinct protein-protein interactions of the residual SWI/SNF complex in SMARCA4-deficient small cell carcinoma of the ovary, hypercalcemic type [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-038.

SummaryThe analysis of patient blood transcriptional profiles offers a means to investigate the immunological mechanisms relevant to human diseases on a genome-wide scale. In addition, such studies provide a basis for the discovery of clinically relevant biomarker signatures. We designed a strategy for microarray analysis that is based on the identification of transcriptional modules formed by genes coordinately expressed in multiple disease data sets. Mapping changes in gene expression at the module level generated disease-specific transcriptional fingerprints that provide a stable framework for the visualization and functional interpretation of microarray data. These transcriptional modules were used as a basis for the selection of biomarkers and the development of a multivariate transcriptional indicator of disease progression in patients with systemic lupus erythematosus. Thus, this work describes the implementation and application of a methodology designed to support systems-scale analysis of the human immune system in translational research settings.

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder characterized by marked loss of subcutaneous adipose tissue from the extremities and trunk but by excess fat deposition in the head and neck. The disease is frequently associated with profound insulin resistance, dyslipidemia, and diabetes. We have localized a gene for FPLD to chromosome 1q21-q23, and it has recently been proposed that nuclear lamin A/C is altered in FPLD, on the basis of a novel missense mutation (R482Q) in five Canadian probands. This gene had previously been shown to be altered in autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD) and in dilated cardiomyopathy and conduction-system disease. We examined 15 families with FPLD for mutations in lamin A/C. Five families harbored the R482Q alteration that segregated with the disease phenotype. Seven families harbored an R482W alteration, and one family harbored a G465D alteration. All these mutations lie within exon 8 of the lamin A/C gene-an exon that has also been shown to harbor different missense mutations that are responsible for EDMD-AD. Mutations could not be detected in lamin A/C in one FPLD family in which there was linkage to chromosome 1q21-q23. One family with atypical FPLD harbored an R582H alteration in exon 11 of lamin A. This exon does not comprise part of the lamin C coding region. All mutations in FPLD affect the globular C-terminal domain of the lamin A/C protein. In contrast, mutations responsible for dilated cardiomyopathy and conduction-system disease are observed in the rod domain of the protein. The FPLD mutations R482Q and R482W occurred on different haplotypes, indicating that they are likely to have arisen more than once.

Background Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. Methodology/Principal Findings We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. Conclusions/Significance This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

Systemic onset juvenile idiopathic arthritis (SoJIA) represents up to 20% of juvenile idiopathic arthritis. We recently reported that interleukin (IL) 1 is an important mediator of this disease and that IL-1 blockade induces clinical remission. However, lack of specificity of the initial systemic manifestations leads to delays in diagnosis and initiation of therapy. To develop a specific diagnostic test, we analyzed leukocyte gene expression profiles of 44 pediatric SoJIA patients, 94 pediatric patients with acute viral and bacterial infections, 38 pediatric patients with systemic lupus erythematosus (SLE), 6 patients with PAPA syndrome, and 39 healthy children. Statistical group comparison and class prediction identified genes differentially expressed in SoJIA patients compared with healthy children. These genes, however, were also changed in patients with acute infections and SLE. An analysis of significance across all diagnostic groups identified 88 SoJIA-specific genes, 12 of which accurately classified an independent set of SoJIA patients with systemic disease. Transcripts that changed significantly in patients undergoing IL-1 blockade were also identified. Thus, leukocyte transcriptional signatures can be used to distinguish SoJIA from other febrile illnesses and to assess response to therapy. Availability of early diagnostic markers may allow prompt initiation of therapy and prevention of disabilities.