Systematic genetic analysis of the MHC region reveals mechanistic underpinnings of HLA type associations with disease.

The MHC region is highly associated with autoimmune and infectious diseases. Here we conduct an in-depth interrogation of associations between genetic variation, gene expression and disease. We create a comprehensive map of regulatory variation in the MHC region using WGS from 419 individuals to call eight-digit HLA types and RNA-seq data from matched iPSCs. Building on this regulatory map, we explored GWAS signals for 4083 traits, detecting colocalization for 180 disease loci with eQTLs. We show that eQTL analyses taking HLA type haplotypes into account have substantially greater power compared with only using single variants. We examined the association between the 8.1 ancestral haplotype and delayed colonization in Cystic Fibrosis, postulating that downregulation of RNF5 expression is the likely causal mechanism. Our study provides insights into the genetic architecture of the MHC region and pinpoints disease associations that are due to differential expression of HLA genes and non-HLA genes

Long read sequencing identifies complex structural variant landscape and recurrent TERT rearrangements in mucoepidermoid carcinoma.

Mucoepidermoid Carcinoma (MEC) is a common salivary malignant neoplasm. Approximately 60 % of MECs harbor translocations between CRTC1 or CRTC3 and MAML2, which are thought to drive disease pathogenesis. However, the precise structural mechanism driving this rearrangement remains uncharacterized. Here, we performed multi-omic and long read genomic sequencing, discovering a chain of alterations that created the CRTC1::MAML2 fusion, but also an unexpected MAML2 to MYBL1 rearrangement, suggesting that MYBL1 may play a larger role in salivary gland cancers than previously recognized. Furthermore, we discovered and validated recurrent TERT rearrangements and amplifications in MEC models. 5/5 MEC cell lines and 36/39 (92 %) primary MEC tumors harbored a TERT rearrangement or copy number amplification. Custom sequencing of the TERT locus confirmed translocation breakpoints in 13/33 (39 %) MECs, while exome sequencing confirmed frequent TERT amplifications. Critically, TERT knockdown in NCI-H292, a cell line with TERT promoter rearrangement, reduced clonogenic cell survival, supporting a critical role of this gene in MEC tumorigenesis. Overall, our data suggest that complex chromothripsis rearrangement mechanisms drive the formation of structural variation in CRTC1::MAML2 fusion positive and negative tumors and reveal highly recurrent structural variation driving TERT rearrangement in MEC

Molecular Analysis of Recurrent Translocations in Mucoepidermoid Carcinoma

Head and neck cancers include a diverse group of malignancies, and pathogenesis is driven by different recurring somatic mutations. In head and neck squamous cell carcinoma (HNSCC), these mutations include single nucleotide variants of several different genes, as well as HPV viral integration. By contrast, many salivary gland tumors are characterized by genomic translocations, resulting in frequent gene fusions. For example, mucoepidermoid carcinomas (MEC) have prevalent CRTC1-MAML2 fusions, while hyalinizing clear cell carcinomas (HCCC) have prevalent EWSR1-ATF1 fusions. Exploring the molecular phenotypes caused by these driver mutations and others will better explain the mechanisms of tumorigenesis and growth in salivary gland tumors, and is therefore necessary to identify potential targets for future patient treatments. In my thesis, I investigate the hypothesis that driver mutations, such as CRTC1-MAML2 and EWSR1-ATF1, alter transcription regulation in salivary gland tumors, varying based on tumor type, fusion status, and grade. In this thesis, I begin by using molecular techniques to differentiate two salivary gland tumors, MEC and HCCC, which are difficult to differentiate by standard histopathology approaches. Using RNA sequencing (RNAseq), I identify a 354 gene signature that differentiates both malignancies. These genes are significantly enriched for an ATF1 binding motif, consistent with the EWSR1-ATF1 fusion found in HCCC. These differentially expressed genes include IGF1R, SGK1, and SGK3, which are elevated in HCCC tumors. This, and other differentially expressed genes in this signature, describe examples of differing molecular pathology between MEC and HCCC. I then seek to further understand the genetic underpinning of MEC. Within MEC tumors, the most common somatic translocation forms the CRTC1-MAML2 fusion. I map the CRTC1-MAML2 breakpoint in four MEC-derived cell lines, via long-read sequencing. I also identify a series of genomic translocations leading to this fusion and uncover a TERT promoter rearrangement in NCI-H292. Subsequent TERT break apart FISH reveals TERT copy number increase and translocation events in all four cell lines tested. These experiments reveal complex genomic rearrangement leading to CRTC1-MAML2 formation and a novel TERT driver mutation. Thus, I discover and validate TERT as a novel MEC driver. While the CRTC1-MAML2 fusion is the most common MEC driver mutation, patients with CRTC1-MAML2, or less commonly CRTC3-MAML2, positive tumors have a better prognosis. Therefore, using RNAseq on 48 MEC tumors, I identify gene expression patterns associated with tumor CRTC1/3-MAML2 fusion status and grade. Gene expression signatures associated with fusion status are enriched for gene sets involving cellular respiration, including oxidative phosphorylation and the electron transport chain. Moreover, changes to T and B cell infiltration are associated with MAML2 fusion status and grade, respectively. Therefore, I perform spatial RNA sequencing to measure the effect of CRTC1-MAML2 activity throughout the MEC tumor microenvironment. I identify spatial overlap between CRTC1-MAML2 associated gene expression and many other transcripts, including VEGFA and CTNNB1. These data suggest that CRTC1-MAML2-associated gene expression affect a variety of biological processes throughout the tumor microenvironment. Overall, these data describe a pattern of gene regulation dependent on tumor type, fusion status, and grade. These gene expression changes, coupled with novel driver mutations, such as TERT translocation, affect multiple cancer phenotypes throughout the tumor microenvironment. These biological processes play a role in the molecular etiology of HCCC and MEC tumors, uncovering several pathways which are opportunities to advance targeted therapies, which may improve the survival of MEC and HCCC patients.PHDCellular & Molecular BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/176529/1/gensterb_1.pd

Protecting Tumors by Preventing Human Papilloma Virus Antigen Presentation: Insights from Emerging Bioinformatics Algorithms

Recent developments in bioinformatics technologies have led to advances in our understanding of how oncogenic viruses such as the human papilloma virus drive cancer progression and evade the host immune system. Here, we focus our review on understanding how these emerging bioinformatics technologies influence our understanding of how human papilloma virus (HPV) drives immune escape in cancers of the head and neck, and how these new informatics approaches may be generally applicable to other virally driven cancers. Indeed, these tools enable researchers to put existing data from genome wide association studies, in which high risk alleles have been identified, in the context of our current understanding of cellular processes regulating neoantigen presentation. In the future, these new bioinformatics approaches are highly likely to influence precision medicine-based decision making for the use of immunotherapies in virally driven cancers

Novel Transcriptional Activity and Extensive Allelic Imbalance in the Human MHC Region

Abstract The MHC region encodes HLA genes and is the most complex region in the human genome. The extensively polymorphic nature of the HLA hinders accurate localization and functional assessment of disease risk loci within this region. Using targeted capture sequencing and constructing individualized genomes for transcriptome alignment, we identified 908 novel transcripts within the human MHC region. These include 593 novel isoforms of known genes, 137 antisense strand RNAs, 119 novel long intergenic noncoding RNAs, and 5 transcripts of 3 novel putative protein-coding human endogenous retrovirus genes. We revealed allele-dependent expression imbalance involving 88% of all heterozygous transcribed single nucleotide polymorphisms throughout the MHC transcriptome. Among these variants, the genetic variant associated with Behçet’s disease in the HLA-B/MICA region, which tags HLA-B*51, is within novel long intergenic noncoding RNA transcripts that are exclusively expressed from the haplotype with the protective but not the disease risk allele. Further, the transcriptome within the MHC region can be defined by 14 distinct coexpression clusters, with evidence of coregulation by unique transcription factors in at least 9 of these clusters. Our data suggest a very complex regulatory map of the human MHC, and can help uncover functional consequences of disease risk loci in this region.</jats:p

Hypomethylation of <i>STAT1</i> and <i>HLA-DRB1</i> in CD8+ T cells is associated with type-I interferon dependent activation of CD4+ T cells in systemic lupus erythematosus

Abstract Background Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by autoantibody and type I interferon (IFN) production. The goal of this study was to explore possible pathogenic roles of CD8+ T cells in lupus through characterizing DNA methylation changes. Methods Genome-wide DNA methylation of SLE and matched healthy control CD8+ T cells was measured using Infinium Human Methylation 450k arrays. Cell surface expression of HLA-DRB1 on CD8+ T cells with and without IFNα was measured by flow cytometry. Co-incubation of IFNα-treated CD8+ T cells with autologous naïve CD4+ T cells was performed. Results SLE CD8+ T cells had 188 hypomethylated CpG sites compared to healthy controls. Among the most demethylated were sites associated with HLA-DRB1 (Δβ = −0.33) and STAT1 (Δβ = −0.15). The proportion of CD8+ T cells expressing HLA-DRB1 was significantly higher in SLE compared to controls. Treatment with IFNα upregulated cell surface expression of HLA-DRB1 on CD8+ T cells of SLE patients but not healthy controls, as measured by median fluorescence intensity. Co-incubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to increased expression of the stimulation marker CD69 on CD4+ T cells in SLE patients, but not in healthy controls. There was a significant increase in STAT1 mRNA levels in both SLE patients and controls with IFNα treatment. Conclusion HLA-DRB1 and STAT1 loci are hypomethylated and epigenetically poised for overexpression in SLE CD8+ T cells in the presence of type-I interferon. IFNα-treated lupus CD8+ T cells stimulate autologous CD4+ T cells in vitro. These data suggest a possible pathogenic role for CD8+ T cells that is dependent upon a high type-I interferon environment in SLE patients. </jats:sec

Protecting Tumors by Preventing Human Papilloma Virus Antigen Presentation: Insights from Emerging Bioinformatics Algorithms

Recent developments in bioinformatics technologies have led to advances in our understanding of how oncogenic viruses such as the human papilloma virus drive cancer progression and evade the host immune system. Here, we focus our review on understanding how these emerging bioinformatics technologies influence our understanding of how human papilloma virus (HPV) drives immune escape in cancers of the head and neck, and how these new informatics approaches may be generally applicable to other virally driven cancers. Indeed, these tools enable researchers to put existing data from genome wide association studies, in which high risk alleles have been identified, in the context of our current understanding of cellular processes regulating neoantigen presentation. In the future, these new bioinformatics approaches are highly likely to influence precision medicine-based decision making for the use of immunotherapies in virally driven cancers

Hypomethylation of <i>STAT1</i> and <i>HLA-DRB1</i> is associated with type-I interferon-dependent <i>HLA-DRB1</i> expression in lupus CD8+ T cells

ObjectiveWe examined genome-wide DNA methylation changes in CD8+ T cells from patients with lupus and controls and investigated the functional relevance of some of these changes in lupus.MethodsGenome-wide DNA methylation of lupus and age, sex and ethnicity-matched control CD8+ T cells was measured using the Infinium MethylationEPIC arrays. Measurement of relevant cell subsets was performed via flow cytometry. Gene expression was quantified by qPCR. Inhibiting STAT1 and CIITA was performed using fludarabine and CIITA siRNA, respectively.ResultsLupus CD8+ T cells had 188 hypomethylated CpG sites compared with healthy matched controls. Among the most hypomethylated were sites associated with HLA-DRB1. Genes involved in the type-I interferon response, including STAT1, were also found to be hypomethylated. IFNα upregulated HLA-DRB1 expression on lupus but not control CD8+ T cells. Lupus and control CD8+ T cells significantly increased STAT1 mRNA levels after treatment with IFNα. The expression of CIITA, a key interferon/STAT1 dependent MHC-class II regulator, is induced by IFNα in lupus CD8+ T cells, but not healthy controls. CIITA knockdown and STAT1 inhibition experiments revealed that HLA-DRB1 expression in lupus CD8+ T cells is dependent on CIITA and STAT1 signalling. Coincubation of naïve CD4+ T cells with IFNα-treated CD8+ T cells led to CD4+ T cell activation, determined by increased expression of CD69 and cytokine production, in patients with lupus but not in healthy controls. This can be blocked by neutralising antibodies targeting HLA-DR.ConclusionsLupus CD8+ T cells are epigenetically primed to respond to type-I interferon. We describe an HLA-DRB1+ CD8+ T cell subset that can be induced by IFNα in patients with lupus. A possible pathogenic role for CD8+ T cells in lupus that is dependent on a high type-I interferon environment and epigenetic priming warrants further characterisation.</jats:sec

Th1/Th17-mediated Immunity and Protection from Peripheral Neuropathy in Wildtype and IL10^–/– BALB/c Mice Infected with a Guillain–Barré Syndrome-associated <i> Campylobacter jejuni</i> Strain

Campylobacter jejuni is an important cause of bacterial gastroenteritis worldwide and is linked to Guillain–Barré syndrome (GBS), a debilitating postinfectious polyneuropathy. The immunopathogenesis of GBS involves the generation of antibodies that are cross reactive to C. jejuni lipooligosaccharide and structurally similar peripheral nerve gangliosides. Both the C. jejuni infecting strain and host factors contribute to GBS development. GBS pathogenesis is associated with Th2-mediated responses in patients. Moreover, induction of IgG1 antiganglioside antibodies in association with colonic Th2-mediated immune responses has been reported in C. jejuni-infected C57BL/6 IL10–/– mice at 4 to 6 wk after infection. We hypothesized that, due to their Th2 immunologic bias, BALB/c mice would develop autoantibodies and signs of peripheral neuropathy after infection with a GBS patient–derived strain of C. jejuni (strain 260.94). WT and IL10–/– BALB/c mice were orally inoculated with C. jejuni 260.94, phenotyped weekly for neurologic deficits, and euthanized after 5 wk. Immune responses were assessed as C. jejuni-specific and antiganglioside antibodies in plasma and cytokine production and histologic lesions in the proximal colon. Peripheral nerve lesions were assessed in dorsal root ganglia and their afferent nerve fibers by scoring immunohistochemically labeled macrophages through morphometry. C. jejuni 260.94 stably colonized both WT and IL10–/– mice and induced systemic Th1/Th17-mediated immune responses with significant increases in C. jejuni-specific IgG2a, IgG2b, and IgG3 plasma antibodies. However, C. jejuni 260.94 did not induce IgG1 antiganglioside antibodies, colitis, or neurologic deficits or peripheral nerve lesions in WT or IL10–/– mice. Both WT and IL10–/– BALB/c mice showed relative protection from development of Th2-mediated immunity and antiganglioside antibodies as compared with C57BL/6 IL10–/– mice. Therefore, BALB/c mice infected with C. jejuni 260.94 are not an effective disease model but provide the opportunity to study the role of immune mechanisms and host genetic background in the susceptibility to post infectious GBS.</jats:p

Microbe-Mediated Activation of Toll-like Receptor 2 Drives PDL1 Expression in HNSCC

As immunotherapies targeting the PDL1 checkpoint have become a mainstay of treatment for a subset of head and neck squamous cell carcinoma (HNSCC) patients, a detailed understanding of the mechanisms underlying PDL1-mediated immune evasion is needed. To elucidate factors regulating expression of PDL1 in HNSCC cells, a genome-wide CRISPR profiling approach was implemented to identify genes and pathways conferring altered PDL1 expression in an HNSCC cell line model. Our screen nominated several candidate PDL1 drivers, including Toll-like Receptor 2 (TLR2). Depletion of TLR2 blocks interferon-γ-induced PDL1 expression, and stimulation of TLR2 with either Staphylococcus aureus or a bacterial lipopeptide mimetic, Pam3CSK4, enhanced PDL1 expression in multiple models. The data herein demonstrate a role for TLR2 in modulating the expression of PDL1 in HNSCC models and suggest that microbiota may directly modulate immunosuppression in cancer cells. Our study represents a step toward disentangling the diverse pathways and stimuli regulating PDL1 expression in HNSCC and underscores a need for future work to characterize the complex microbiome in HNSCC patients treated with immunotherapy