Regulatory Compartment Research Articles

Abstract 2860: High-resolution mapping of oncogenic structural changes in osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify cancer-specific oncogenic alterations for treatment of osteosarcomas. Osteosarcoma is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. The functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments to affect gene expression. SVs can also lead to the formation of extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers yet they remain poorly understood. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), we studied SVs in osteosarcoma with the goal of elucidating their contribution to cancer development. We used a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies as well as genomically via whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in osteosarcoma, we integrated OGM and WGS data for all 11 cell lines to describe detailed genome-wide SVs including ecDNA formation. PDX lines assayed using OGM exhibited between 106 and 704 translocations each (versus zero detected in germline controls) and we identified dozens of potential ecDNA amplicons, including one amplicon which may explain exceptionally high expression of YAP1 and BIRC3 transcripts. Further, we used HiC to describe the topological environment resulting from these SVs and observed potential enhancer hijacking events affecting dozens of known oncogenes including MYC and CDK4. HiC interaction maps among six cell lines reveal genomic compartments and TAD boundaries in detail, with 4,167 unique TAD boundaries identified among six cell lines using 50kb genomic bins. Despite the observed complexity of these genomes, we note that 71% of TAD boundaries are consistent in at least four of the six HiC assayed cell lines, and 44% are present in all six lines. Changing TAD boundaries are associated with numerous SVs and/or copy number discrepancies, and are associated with genomic regions that contain genes with dysregulated RNA transcript levels. Overall, OGM and HiC modalities add considerably to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew Scott Clugston, Stan Leung, Eunice Fuentes, Leanne Sayles, Megan Ostrowski, Coco Wu, Christina Curtis, Zhicheng Ma, Yanding Zhao, Georgi K. Marinov, Vijay Ramani, Marcus Breese, Alejandro Sweet-Cordero. High-resolution mapping of oncogenic structural changes in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2860.

Helios as a Potential Biomarker in Systemic Lupus Erythematosus and New Therapies Based on Immunosuppressive Cells.

The Helios protein (encoded by the IKZF2 gene) is a member of the Ikaros transcription family and it has recently been proposed as a promising biomarker for systemic lupus erythematosus (SLE) disease progression in both mouse models and patients. Helios is beginning to be studied extensively for its influence on the T regulatory (Treg) compartment, both CD4+ Tregs and KIR+/Ly49+ CD8+ Tregs, with alterations to the number and function of these cells correlated to the autoimmune phenomenon. This review analyzes the most recent research on Helios expression in relation to the main immune cell populations and its role in SLE immune homeostasis, specifically focusing on the interaction between T cells and tolerogenic dendritic cells (tolDCs). This information could be potentially useful in the design of new therapies, with a particular focus on transfer therapies using immunosuppressive cells. Finally, we will discuss the possibility of using nanotechnology for magnetic targeting to overcome some of the obstacles related to these therapeutic approaches.

Current Concepts in the Diagnosis and Management of Adult Primary Immune Thrombocytopenia: Our Personal View.

Primary immune thrombocytopenia (ITP) is an acquired blood disorder that causes a reduction in circulating platelets with the potential for bleeding. The incidence of ITP is slightly higher in adults and affects more women than men until 60 years, when males are more affected. Despite advances in basic science, primary ITP remains a diagnosis of exclusion. The disease is heterogeneous in its clinical behavior and response to treatment. This reflects the complex underlying pathophysiology, which remains ill-understood. Platelet destruction plays a role in thrombocytopenia, but underproduction is also a major contributing factor. Active ITP is a proinflammatory autoimmune disease involving abnormalities within the T and B regulatory cell compartments, along with several other immunological abnormalities. Over the last several years, there has been a shift from using immunosuppressive therapies for ITP towards approved treatments, such as thrombopoietin receptor agonists. The recent COVID-19 pandemic has hastened this management shift, with thrombopoietin receptor agonists becoming the predominant second-line treatment. A greater understanding of the underlying mechanisms has led to the development of several targeted therapies, some of which have been approved, with others still undergoing clinical development. Here we outline our view of the disease, including our opinion about the major diagnostic and therapeutic challenges. We also discuss our management of adult ITP and our placement of the various available therapies.

Abstract 214: Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma

Abstract We have leveraged long-read and spatial sequencing modalities to identify patient-specific oncogenic alterations for treatment of pediatric osteosarcomas. Osteosarcoma (OS) is the most common primary bone malignancy in children and young adults, with over 500 cases identified each year in the United States. OS is archetypal of cancers driven by aneuploidy and structural rearrangement rather than point mutations. These structural variants (SVs) can alter gene expression stoichiometrically, but the functional consequences of many SVs stem from their effect on multidimensional genome organization. SVs can enable enhancer hijacking, alter boundaries of topologically associated domains (TADs), and move gene loci to different regulatory compartments affecting transcription. SVs can also form extrachromosomal DNA amplicons (ecDNA), which can be megabases in size and can incorporate both genes and regulatory elements to result in substantial increases in gene transcription and intratumor heterogeneity. Complex SVs may have profound implications for prognosis and treatment in OS and other cancers. By incorporating long-read optical genome mapping (OGM) and chromatin conformation capture sequencing (HiC), our goal is to detect complex tumor-specific SVs that will inform and refine pediatric OS subtypes, revealing new tumor-specific chemotherapy treatment options. We have generated a unique panel of 11 OS patient-derived xenograft (PDX) cell lines which have been previously characterized regarding response to targeted therapies using whole genome (WGS), RNA (RNA-seq), and chromatin accessibility (ATAC-seq) sequencing. To understand the complex genomic reorganization that occurs in OS, we have integrated OGM and WGS data for six cell lines, and OGM, WGS, and HiC data for two. SVs were detected using OGM, HiC, and WGS modalities, and we observed 59-61% concordance between SVs called using OGM and HiC, versus 22-45% when comparing with WGS calls. PDX lines assayed using OGM exhibit between 5,135 and 7,514 SVs each, and translocation numbers ranged from 212 to 1,422. These lines exhibit between dozens of potential ecDNA amplicons, including one amplicon which may explain that line’s specific and exceptionally high expression of YAP1 and BIRC2 transcripts. HiC interaction maps for two PDX lines reveal genomic compartments and TAD boundaries in detail, with 3,713 unique TAD boundaries identified between them using 50kb genomic bins. We observe that 60% of these boundaries are consistent between these lines, and 27% exhibit differences that suggest boundary loss or gain. These changing boundaries are associated with numerous SVs and/or copy number discrepancies, and with genomic regions containing genes with dysregulated RNA transcript levels. Incorporating OGM and HiC modalities adds substantially to our ability to detect oncogenic SVs and the genes they affect. Citation Format: Andrew S. Clugston, Megan Ostrowski, Coco Wu, Leanne Sayles, Stanley G. Leung, Vijay Ramani, E. Alejandro Sweet-Cordero, Marcus R. Breese. Patient-specific mapping of oncogenic structural changes in pediatric osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 214.

044 IL-2C treatment expands regulatory T cells in vivo and arrests development of murine alopecia areata

Alopecia Areata (AA) is a common autoimmune disease characterized by the loss of immune privilege of the hair follicle. CD8+ T cells have been identified as the main effectors in AA, however, little is known about the role of regulatory T cells (Tregs) and why they fail to control disease. The imbalance of Tregs: effectors is thought to influence the development of many autoimmune diseases. Enrichment of the regulatory compartment has been gaining momentum in the field and currently there are many ongoing clinical trials involving Treg therapy as a treatment for autoimmune diseases.

Recipient Tregs: Can They Be Exploited for Successful Hematopoietic Stem Cell Transplant Outcomes?

Human and mouse CD4+FoxP3+ T cells (Tregs) comprise non-redundant regulatory compartments which maintain self-tolerance and have been found to be of potential therapeutic usefulness in autoimmune disorders and transplants including allogeneic hematopoietic stem cell transplantation (allo-HSCT). There is substantial literature interrogating the application of donor derived Tregs for the prevention of graft versus host disease (GVHD). This Mini-Review will focus on the recipient’s Tregs which persist post-transplant. Although treatment in patients with low dose IL-2 months post-HSCT are encouraging, manipulating Tregs in recipients early post-transplant is challenging, in part likely an indirect consequence of damage to the microenvironment required to support Treg expansion of which little is understood. This review will discuss the potential for manipulating recipient Tregs in vivo prior to and after HSCT (fusion proteins, mAbs). Strategies that would circumvent donor/recipient peripheral blood harvest, cell culture and ex-vivo Treg expansion will be considered for the translational application of Tregs to improve HSCT outcomes.

Mediator complex maintains peripheral T cell tolerance through enforcement of the quiescence module

Abstract T cell tolerance has traditionally been thought to be maintained at stages after T cell receptor mediated activation either through anergy, exhaustion, contraction or T Regulatory cell mediated dominant suppression. Prior to these mechanisms, however, T cell quiescence is the main factor maintaining T cell tolerance. We have identified Mediator Complex Subunit 1 (Med1), as a novel factor responsible for enforcing T cell quiescence. Mice with T cell specific deletion of Med1 display increased levels of T cell activation despite having a normally functional T Regulatory cell compartment. Med1-deficient T cells were more responsive to T cell receptor mediated activation. Bone marrow chimeras revealed Med1 controls T cell quiescence in a cell intrinsic manner. Bulk and single cell transcriptome sequencing revealed Med1’s transcriptional control over key quiescence factors. By analyzing previously published chromatin immunoprecipitation sequencing we found Med1 controlled a transcription factor network rather than direct quiescence molecules. Med1 is a novel factor responsible for actively enforcing the transcriptional network maintaining T cell quiescence. Supported by T32DK007169 R01CA257520 R01CA232347

Evidence of immunometabolic dysregulation and airway dysbiosis in athletes susceptible to respiratory illness.

SummaryBackgroundRespiratory tract infection (RTI) is a leading cause of training and in-competition time-loss in athlete health. The immune factors associated with RTI susceptibility remain unclear. In this study, we prospectively characterise host immune factors in elite athletes exhibiting RTI susceptibility.MethodsPeripheral blood lymphocyte flow cytometry phenotyping and 16S rRNA microbial sequencing of oropharyngeal swabs was performed in a prospective elite athlete cohort study (n = 121). Mass cytometry, peripheral blood mononuclear cell (PBMC) stimulation and plasma metabolic profiling was performed in age-matched highly-susceptible (HS) athletes (≥4RTI in last 18 months) (n = 22) compared to non-susceptible (NS) (≤1RTI in last 18 months) (n = 23) athletes. Findings were compared to non-athletic healthy controls (HC) (n = 19).FindingsAthletes (n = 121) had a reduced peripheral blood memory T regulatory cell compartment compared to HC (p = 0.02 (95%CI:0.1,1.0)) and reduced upper airway bacterial biomass compared to HC (p = 0.032, effect size r = 0.19). HS athletes (n = 22) had lower circulating memory T regulatory cells compared to NS (n = 23) athletes (p = 0.005 (95%CI:-1.5,-0.15)) and HC (p = 0.002 (95%CI:-1.9,-0.3) with PBMC microbial stimulation assays revealing a T-helper 2 skewed immune response compared to HC. Plasma metabolomic profiling showed differences in sphingolipid pathway metabolites (a class of lipids important in infection and inflammation regulation) in HS compared to NS athletes and HC, with sphingomyelin predictive of RTI infection susceptibility (p = 0.005).InterpretationAthletes susceptible to RTI have reduced circulating memory T regulatory cells, metabolic dysregulation of the sphingolipid pathway and evidence of upper airway bacterial dysbiosis.FundingThis study was funded by the English Institute of Sport (UK).

Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells.

A subset of T regulatory cells (Tregs), identified by TIRC7 (T cell immune response cDNA 7) expression is designated as Immune Regulatory 1 Cells (IR1 cells). TIRC7 is an immune checkpoint inhibitor, co-localized with the T- cell receptor, HLA-DR and CTLA-4 during T-cell activation, which delivers regulatory signals via binding to its ligand, HLA-DR α2 domain. IR1 cells express FOXP3, and multiple other markers associated with immune suppression. They constitute as much as 10% of Tregs. IR1 cells strongly inhibit proliferation in mixed lymphocyte reactions, where they express high levels of IL-10. Ex vivo expansion of Tregs over 2 weeks in the presence of an agonist TIRC7 antibody disproportionately expands the IR1 Treg subset, while maintaining high expression of suppressive markers including CD39, IL-10, LAP and GARP. Ex vivo expanded IR1 cells are a potent, homogeneous, stable set of suppressor Tregs with the potential to modulate immune dysregulation. The characteristics of IR1 cells suggest a therapeutic advantage over polyclonal Tregs for therapeutic interventions. Early restoration of immune homeostasis using IR1 cells has the potential to fundamentally alter the natural history of conditions characterized by abnormalities in the T regulatory cell compartment.

Age-Associated Characteristics of CD4+ T-Cell Composition in Patients with Atherosclerosis

Background. We aimed to analyze the contents of the main CD4+ T-cell subsets in patients with atherosclerosis (AS) depending on age. Methods. Male patients with coronary and/or carotid AS, who are non-smokers, and who are receiving statins were divided into three age groups (I—<55 y.o. (n = 23), II—55–64 y.o. (n = 42), III—≥65 y.o. (n = 46)). Leukocyte phenotyping was performed by direct immunofluorescence and flow cytometry. For intracellular cytokine detection, blood mononuclear cells were pre-activated with phorbol 12-myristate 13-acetate and ionomycin in the presence of an intracellular vesicle transport blocker monensin. Results. The groups did not differ in traditional CVD risk factors and AS severity. The content of CD4+ T-cells was lower in group III and II than in group I. The content of CD4+CD25high Treg was lower in group III than in groups I and II. No differences in the quantities of the primed CD39+CD45RA− and CD278high Treg, CD4+INFγ+ Th1, CD4+IL17+ Th17, and CD4+IL17+INFγ+ Th1/17 were observed. There were negative correlations between the values of CD4+ T-cells, CD4+CD45RA+ T-cells, CD4+CD25high Treg, CD4+CD25highCD45RA+ Treg, and age. Conclusion. In patients with AS, the age-related depletion of naive CD4+ T-cells also extends to the regulatory compartment. This phenomenon should be considered when studying the impact of the immune cells on the progression of AS.

Regulatory SNPs: Altered Transcription Factor Binding Sites Implicated in Complex Traits and Diseases.

The vast majority of the genetic variants (mainly SNPs) associated with various human traits and diseases map to a noncoding part of the genome and are enriched in its regulatory compartment, suggesting that many causal variants may affect gene expression. The leading mechanism of action of these SNPs consists in the alterations in the transcription factor binding via creation or disruption of transcription factor binding sites (TFBSs) or some change in the affinity of these regulatory proteins to their cognate sites. In this review, we first focus on the history of the discovery of regulatory SNPs (rSNPs) and systematized description of the existing methodical approaches to their study. Then, we brief the recent comprehensive examples of rSNPs studied from the discovery of the changes in the TFBS sequence as a result of a nucleotide substitution to identification of its effect on the target gene expression and, eventually, to phenotype. We also describe state-of-the-art genome-wide approaches to identification of regulatory variants, including both making molecular sense of genome-wide association studies (GWAS) and the alternative approaches the primary goal of which is to determine the functionality of genetic variants. Among these approaches, special attention is paid to expression quantitative trait loci (eQTLs) analysis and the search for allele-specific events in RNA-seq (ASE events) as well as in ChIP-seq, DNase-seq, and ATAC-seq (ASB events) data.

Therapeutic induction of tolerogenic dendritic cells via aryl hydrocarbon receptor signaling.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs), which sample the exogenous and endogenous cues to control adaptive immunity, balancing effector and regulatory components of the immune response. Multiple subsets of DCs, such as plasmacytoid and conventional DCs, have been defined based on specific phenotypic markers, functions and regulatory transcriptional programs. Tolerogenic DCs (tolDCs) have been functionally defined based on their ability to expand the regulatory T-cell compartment and suppress immune responses. However, it is still unclear whether tolDCs represent a homogeneous population, a specific DC subset and/or a heterogeneous collection of DC activation/maturation states. The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) has been shown to control transcriptional programs associated to tolDCs. In this review, we discuss the role of AHR in the control of tolDCs, and also AHR-targeted approaches for the therapeutic induction of tolDCs in autoimmune diseases and allergy.

Regulation of membrane NMDA receptors by dynamics and protein interactions.

Understanding neurotransmitter system crosstalk in the brain is a major challenge in neurobiology. Several intracellular and genomic cascades have been identified in this crosstalk. However, the discovery that neurotransmitter receptors are highly diffusive in the plasma membrane of neurons, where they form heterocomplexes with other proteins, has profoundly changed our view of neurotransmitter signaling. Here, we review new insights into neurotransmitter crosstalk at the plasma membrane. We focus on the membrane organization and interactome of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) that plays a central role in excitatory synaptic and network physiology and is involved in the etiology of several major neuropsychiatric disorders. The nanoscale organization and dynamics of NMDAR is a key regulatory process for glutamate synapse transmission, plasticity, and crosstalk with other neurotransmitter systems, such as the monoaminergic ones. The plasma membrane appears to be a prime regulatory compartment for spatial and temporal crosstalk between neurotransmitter systems in the healthy and diseased brain. Understanding the molecular mechanisms regulating membrane neurotransmitter receptor crosstalk will likely open research avenues for innovative therapeutical strategies.

Abstract B61: The genotype of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immune checkpoint therapies

Abstract Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) DNA repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in homologous recombination-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune/stromal analysis using flow cytometry, single-cell RNASeq (scRNASeq) transcriptomic, and immunofluorescence imaging analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean Smith, David Pepin, Raghav Mohan, Tian Xia, Ferenc Reinhardt, Tony Chavarria, Esmee Hoefsmit, Vera Poort, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Paula T. Hammond, Benjamin G. Neel, Robert A. Weinberg. The genotype of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immune checkpoint therapies [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B61.

A Brazilian Zika virus isolate preferentially induces T follicular helper cell responses while suppressing Th1 immunity

Abstract Zika virus (ZIKV) is a mosquito-borne pathogen that caused a series of increasingly severe outbreaks in Micronesia, French Polynesia, and South and Central America. Recent work from our laboratory has shown that, compared to a pre-epidemic isolate (ZIKV-CDN), a Brazilian ZIKV isolate (ZIKV-BR) possesses a novel capacity to suppress antigen-specific CD8 T cell responses, resulting in sustained infection. However, it is unknown whether ZIKV-BR also modulates CD4 T cell immunity. Thus, we investigated the CD4 T cell response to infection with each ZIKV isolate. Our data demonstrate that the CD4 T cell response to ZIKV-BR is reduced in magnitude compared to the response induced by ZIKV-CDN. Further, CD4 T cells are less polarized to the Th1 subtype, express less T-Bet, and are functionally impaired, as they produce less IFN-γ following ex vivo restimulation. Although we observed no alterations in the Th2, Th17 or T regulatory cell compartments, we observed a striking accumulation of PD-1hiCXCR5+ T follicular helper (Tfh) cells 10 days post-infection with ZIKV-BR. This response correlated with an enhanced germinal center B cell response, and increased detection of germinal center formation by confocal microscopy. Future studies will aim to determine the mechanism through which ZIKV-BR infection enhances the Tfh response, and the implications of promoting Tfh responses while dampening Th1 responses for antiviral immunity. Together, our data suggest that contemporary ZIKV strains have evolved to modulate CD4 T cell responses and this could provide a model for interrogating the signals required for Tfh development.

Abstract A29: The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies

Abstract Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood, and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in homologous recombination-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, Myc, Cyclin E1 (CCNE1), Akt2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune/stromal analysis using flow cytometry, single-cell RNA sequencing, and transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and regulatory compartments between HR-proficient and -deficient tumors within the primary and metastatic tumors themselves and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean G. Smith, David Pepin, Raghav Mohan, Esmee Hoefsmit, Tian Xia, Ferenc Reinhardt, Tony E. Chavarria, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Benjamin G. Neel, Robert A. Weinberg. The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A29.

Abstract PR04: The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies

Abstract High-grade serous ovarian cancer (HGSOC) is the most frequent and most aggressive histologic subtype of ovarian cancer. The cornerstone of the existing treatment of HGSOC is DNA-damaging chemotherapy; however, practically all patients eventually develop a progressive disease and the 5-year survival is only 40%. Immunotherapy would seem to be an attractive alternative treatment to chemotherapy, yet existing immunotherapies perform poorly in ovarian cancer, with only ~10% of patients responding to checkpoint blockade. Why this is the case remains poorly understood and there is a pressing need to understand the underlying biology of immune evasion in ovarian cancer. One critical area of interest is the role of homology-dependent DNA repair (HR) in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. Hence, we have modeled the biology of ovarian cancer using patient-relevant mutational landscapes in an immune-proficient, syngeneic mouse model in order to help us identify the contribution of common driver mutations to the immune repertoire in the tumor microenvironment, and thus to responses of HGSOC tumors to immunotherapy. We hypothesize that the immune composition and gene expression signatures of the resulting tumors will vary based on the combination of genetic alterations and the DNA repair proficiency of the transformed cells. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of co-occurring mutations observed in HR-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations which are TP53, BRCA1, PTEN, MYC, Cyclin E1 (CCNE1), AKT2, and Kras to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor and DNA-damaging agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP inhibition in comparison to HR-proficient cells. Initial immune /stromal analysis using flow cytometry, scRNAseq transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and -deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease. Citation Format: Sonia Iyer, Shuang Zhang, Anniina Farkkila, Sean Smith, David Pepin, Raghav Mohan, Ferenc Reinhardt, Tian Xia, Tony E. Chavarria, Esmee Hoefsmit, Shailja Pathania, Yunlan Zhou, Kevin M. Elias, Benjamin G. Neel, Robert A. Weinberg. The genomic architecture of serous carcinomas shapes the tumor microenvironment and modulates responses to targeted and immunotherapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR04. doi:10.1158/1535-7163.TARG-19-PR04

Expansion of the CD4+ effector T-cell repertoire characterizes peanut-allergic patients with heightened clinical sensitivity

Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, whereas others suffer systemic reactions to 10 mg or less. Current diagnostic testing only partially predicts this clinical heterogeneity. We sought to identify characteristics of the peanut-specific CD4+ T-cell response in peanut-allergic patients that correlate with high clinical sensitivity. We studied the T-cell receptor β-chain (TCRβ) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRβ sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. TCRβ analysis of the CD154+ and CD154- fractions revealed more than 6000 complementarity determining region 3 sequences and motifs that were significantly enriched in the activated cells and 17% of the sequences were shared between peanut-allergic individuals, suggesting strong convergent selection of peanut-specific clones. These clones were more numerous among the reactive patients, and this expansion was identified within effector, but not regulatory T-cell populations. The transcriptional profile of CD154+ T cells in the reactive group skewed toward a polarized TH2 effector phenotype, and expression of TH2 cytokines strongly correlated with peanut-specific IgE levels. There were, however, also non-TH2-related differences in phenotype. Furthermore, the ratio of peanut-specific clones in the effector versus regulatory T-cell compartment, which distinguished the clinical groups, was independent of specific IgE concentration. Expansion of the peanut-specific effector T-cell repertoire is correlated with clinical sensitivity, and this observation may be useful to inform our assessment of disease phenotype and to monitor disease longitudinally.

Infection History Determines Susceptibility to Unrelated Diseases.

Epidemiological data suggest that previous infections can alter an individual's susceptibility to unrelated diseases. Nevertheless, the underlying mechanisms are not completely understood. Substantial research efforts have expanded the classical concept of immune memory to also include long-lasting changes in innate immunity and antigen-independent reactivation of adaptive immunity. Collectively, these processes provide possible explanations on how acute infections might induce long-term changes that also affect immunity to unrelated diseases. Here, we review lasting changes the immune compartment undergoes upon infection and how infection experience alters the responsiveness of immune cells towards universal signals. This heightened state of alert enhances the ability of the immune system to combat even unrelated infections but may also increase susceptibility to autoimmunity. At the same time, infection-induced changes in the regulatory compartment may dampen subsequent immune responses and promote pathogen persistence. The concepts presented here outline how infection-induced changes in the immune system may affect human health.

Effect of Disease-Associated Germline Mutations on Structure Function Relationship of DNA Methyltransferases.

Despite a large body of evidence supporting the role of aberrant DNA methylation in etiology of several human diseases, the fundamental mechanisms that regulate the activity of mammalian DNA methyltransferases (DNMTs) are not fully understood. Recent advances in whole genome association studies have helped identify mutations and genetic alterations of DNMTs in various diseases that have a potential to affect the biological function and activity of these enzymes. Several of these mutations are germline-transmitted and associated with a number of hereditary disorders, which are potentially caused by aberrant DNA methylation patterns in the regulatory compartments of the genome. These hereditary disorders usually cause neurological dysfunction, growth defects, and inherited cancers. Biochemical and biological characterization of DNMT variants can reveal the molecular mechanism of these enzymes and give insights on their specific functions. In this review, we introduce roles and regulation of DNA methylation and DNMTs. We discuss DNMT mutations that are associated with rare diseases, the characterized effects of these mutations on enzyme activity and provide insights on their potential effects based on the known crystal structure of these proteins.