Identification of pan-cancer/testis genes and validation of therapeutic targeting in triple-negative breast cancer: Lin28a-based and Siglece-based vaccination induces antitumor immunity and inhibits metastasis.

BACKGROUND: Cancer-testis (CT) genes are targets for tumor antigen-specific immunotherapy given that their expression is normally restricted to the immune-privileged testis in healthy individuals with aberrant expression in tumor tissues. While they represent targetable germ tissue antigens and play important functional roles in tumorigenesis, there is currently no standardized approach for identifying clinically relevant CT genes. Optimized algorithms and validated methods for accurate prediction of reliable CT antigens (CTAs) with high immunogenicity are also lacking. METHODS: Sequencing data from the Genotype-Tissue Expression (GTEx) and The Genomic Data Commons (GDC) databases was used for the development of a bioinformatic pipeline to identify CT exclusive genes. A CT germness score was calculated based on the number of CT genes expressed within a tumor type and their degree of expression. The impact of tumor germness on clinical outcome was evaluated using healthy GTEx and GDC tumor samples. We then used a triple-negative breast cancer mouse model to develop and test an algorithm that predicts epitope immunogenicity based on the identification of germline sequences with strong major histocompatibility complex class I (MHCI) and MHCII binding affinities. Germline sequences for CT genes were synthesized as long synthetic peptide vaccines and tested in the 4T1 triple-negative model of invasive breast cancer with Poly(I:C) adjuvant. Vaccine immunogenicity was determined by flow cytometric analysis of in vitro and in vivo T-cell responses. Primary tumor growth and lung metastasis was evaluated by histopathology, flow cytometry and colony formation assay. RESULTS: We developed a new bioinformatic pipeline to reliably identify CT exclusive genes as immunogenic targets for immunotherapy. We identified CT genes that are exclusively expressed within the testis, lack detectable thymic expression, and are significantly expressed in multiple tumor types. High tumor germness correlated with tumor progression but not with tumor mutation burden, supporting CTAs as appealing targets in low mutation burden tumors. Importantly, tumor germness also correlated with markers of antitumor immunity. Vaccination of 4T1 tumor-bearing mice with Siglece and Lin28a antigens resulted in increased T-cell antitumor immunity and reduced primary tumor growth and lung metastases. CONCLUSION: Our results present a novel strategy for the identification of highly immunogenic CTAs for the development of targeted vaccines that induce antitumor immunity and inhibit metastasis.

Microenvironment-dependent growth of Sezary cells in humanized IL-15 mice.

Sezary syndrome (SS) is a rare, aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) that lacks adequate therapeutic options and representative small-animal models. Here, we demonstrate that IL-15 is a critical CTCL growth factor. Importantly, an immunodeficient knock-in mouse model genetically engineered to express human IL-15 uniquely supported the growth of SS patient samples relative to conventional immunodeficient mouse strains. SS patient-derived xenograft (PDX) models recapacitated key pathological features of the human disease, including skin infiltration and spread of leukemic cells to the periphery, and maintained the dependence on human IL-15 upon serial in vivo passaging. Detailed molecular characterization of the engrafted cells by single-cell transcriptomic analysis revealed congruent neoplastic gene expression signatures but distinct clonal engraftment patterns. Overall, we document an important dependence of Sezary cell survival and proliferation on IL-15 signaling and the utility of immunodeficient humanized IL-15 mice as hosts for SS - and potentially other T and NK cell-derived hematologic malignancies - PDX model generation. Furthermore, these studies advocate the thorough molecular understanding of the resultant PDX models to maximize their translational impact.

Intratumoral dendritic cell-CD4+ T helper cell niches enable CD8+ T cell differentiation following PD-1 blockade in hepatocellular carcinoma.

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+CH25H+IL-21+PD-1+CD4+ T helper cells ("CXCL13+ TH") and Granzyme K+ PD-1+ effector-like CD8+ T cells, whereas terminally exhausted CD39hiTOXhiPD-1hiCD8+ T cells dominated in nonresponders. CD4+ and CD8+ T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1+TCF-1+ (Progenitor-exhausted) CD8+ T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8+ T cell differentiation occurs upon ICB. We found that these Progenitor CD8+ T cells interact with CXCL13+ TH within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ TH control the differentiation of tumor-specific Progenitor exhasuted CD8+ T cells following ICB.

Identification of pan-cancer/testis genes and validation of therapeutic targeting in triple-negative breast cancer: Lin28a- and Siglece-based vaccination induces anti-tumor immunity and inhibits metastasis.

Background: Cancer-testis (CT) genes are targets for tumor antigen-specific immunotherapy given that their expression is normally restricted to the immune-privileged testis in healthy individuals with aberrant expression in tumor tissues. While they represent targetable germ-tissue antigens and play important functional roles in tumorigenesis, there is currently no standardized approach for identifying clinically relevant CT genes. Optimized algorithms and validated methods for accurate prediction of reliable CT antigens with high immunogenicity are also lacking. Methods: Sequencing data from the Genotype-Tissue Expression (GTEx) and The Genomic Data Commons (GDC) databases was utilized for the development of a bioinformatic pipeline to identify CT exclusive genes. A CT germness score was calculated based on the number of CT genes expressed within a tumor type and their degree of expression. The impact of tumor germness with clinical outcome was evaluated using healthy GTEx and GDC tumor samples. We then used a triple-negative breast cancer mouse model to develop and test an algorithm that predicts epitope immunogenicity based on the identification of germline sequences with strong MHCI and MHCII binding affinities. Germline sequences for CT genes were synthesized as long synthetic peptide vaccines and tested in the 4T1 triple-negative model of invasive breast cancer with Poly(I:C) adjuvant. Vaccine immunogenicity was determined by flow cytometric analysis of in vitro and in vivo T cell responses. Primary tumor growth and lung metastasis was evaluated by histopathology, flow cytometry and colony formation assay. Results: We developed a new bioinformatic pipeline to reliably identify CT exclusive genes as immunogenic targets for immunotherapy. We identified CT genes that are exclusively expressed within the testis, lack detectable thymic expression, and are significantly expressed in multiple tumor types. High tumor germness correlated with tumor progression but not with tumor mutation burden, supporting CT antigens as appealing targets in low mutation burden tumors. Importantly, tumor germness also correlated with markers of anti-tumor immunity. Vaccination of 4T1 tumor bearing mice with Siglece and Lin28a antigens resulted in increased T cell anti-tumor immunity and reduced primary tumor growth and lung metastases. Conclusion: Our results present a novel strategy for the identification of highly immunogenic CT antigens for the development of targeted vaccines that induce anti-tumor immunity and inhibit metastasis.

Cancer cell-derived type I interferons instruct tumor monocyte polarization.

Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.

Proteogenomic identification of Hepatitis B virus (HBV) genotype-specific HLA-I restricted peptides from HBV-positive patient liver tissues.

The presentation of virus-derived peptides by HLA class I molecules on the surface of an infected cell and the recognition of these HLA-peptide complexes by, and subsequent activation of, CD8+ cytotoxic T cells provides an important mechanism for immune protection against viruses. Recent advances in proteogenomics have allowed researchers to discover a growing number of unique HLA-restricted viral peptides, resulting in a rapidly expanding repertoire of targets for immunotherapeutics (i.e. bispecific antibodies, engineered T-cell receptors (TCRs), chimeric antigen receptor T-cells (CAR-Ts)) to infected tissues. However, genomic variability between viral strains, such as Hepatitis-B virus (HBV), in combination with differences in patient HLA alleles, make it difficult to develop therapeutics against these targets. To address this challenge, we developed a novel proteogenomics approach for generating patient-specific databases that enable the identification of viral peptides based on the viral transcriptomes sequenced from individual patient liver samples. We also utilized DNA sequencing of patient samples to identify HLA genotypes and assist in target selection. Liver samples from 48 HBV infected patients, primarily from Asia, were examined to reconstruct patient-specific HBV genomes, identify regions within the human chromosomes targeted by HBV integrations and obtain a comprehensive view of HBV peptide epitopes using our HLA class-I (HLA-I) immunopeptidomics discovery platform. Two previously reported HLA associated HBV-derived peptides, HLA-A02 binder FLLTRILTI (S194-202) from the large surface antigen and HLA-A11 binder STLPETTVVRR (C141-151) from the capsid protein were validated by our discovery platform, but both were detected at very low frequencies. In addition, we identified and validated, using heavy peptide analogues, novel strain-specific HBV-HLA associated peptides, such as GSLPQEHIVQK (P606-616) and variants. Overall, our novel approach can guide the development of bispecific antibody, TCR-T, or CAR-T based therapeutics for the treatment of HBV-related HCC and inform vaccine development.

Common and rare variant associations with clonal haematopoiesis phenotypes.

Clonal haematopoiesis involves the expansion of certain blood cell lineages and has been associated with ageing and adverse health outcomes1-5. Here we use exome sequence data on 628,388 individuals to identify 40,208 carriers of clonal haematopoiesis of indeterminate potential (CHIP). Using genome-wide and exome-wide association analyses, we identify 24 loci (21 of which are novel) where germline genetic variation influences predisposition to CHIP, including missense variants in the lymphocytic antigen coding gene LY75, which are associated with reduced incidence of CHIP. We also identify novel rare variant associations with clonal haematopoiesis and telomere length. Analysis of 5,041 health traits from the UK Biobank (UKB) found relationships between CHIP and severe COVID-19 outcomes, cardiovascular disease, haematologic traits, malignancy, smoking, obesity, infection and all-cause mortality. Longitudinal and Mendelian randomization analyses revealed that CHIP is associated with solid cancers, including non-melanoma skin cancer and lung cancer, and that CHIP linked to DNMT3A is associated with the subsequent development of myeloid but not lymphoid leukaemias. Additionally, contrary to previous findings from the initial 50,000 UKB exomes6, our results in the full sample do not support a role for IL-6 inhibition in reducing the risk of cardiovascular disease among CHIP carriers. Our findings demonstrate that CHIP represents a complex set of heterogeneous phenotypes with shared and unique germline genetic causes and varied clinical implications.

Multiancestry exome sequencing reveals INHBE mutations associated with favorable fat distribution and protection from diabetes.

Body fat distribution is a major, heritable risk factor for cardiometabolic disease, independent of overall adiposity. Using exome-sequencing in 618,375 individuals (including 160,058 non-Europeans) from the UK, Sweden and Mexico, we identify 16 genes associated with fat distribution at exome-wide significance. We show 6-fold larger effect for fat-distribution associated rare coding variants compared with fine-mapped common alleles, enrichment for genes expressed in adipose tissue and causal genes for partial lipodystrophies, and evidence of sex-dimorphism. We describe an association with favorable fat distribution (p = 1.8 × 10-09), favorable metabolic profile and protection from type 2 diabetes (~28% lower odds; p = 0.004) for heterozygous protein-truncating mutations in INHBE, which encodes a circulating growth factor of the activin family, highly and specifically expressed in hepatocytes. Our results suggest that inhibin ßE is a liver-expressed negative regulator of adipose storage whose blockade may be beneficial in fat distribution-associated metabolic disease.

A single-cell map of dynamic chromatin landscapes of immune cells in renal cell carcinoma.

A complete chart of the chromatin regulatory elements of immune cells in patients with cancer and their dynamic behavior is necessary to understand the developmental fates and guide therapeutic strategies. Here, we map the single-cell chromatin landscape of immune cells from blood, normal tumor-adjacent kidney tissue and malignant tissue from patients with early-stage clear cell renal cell carcinoma (ccRCC). We catalog the T cell states dictated by tissue-specific and developmental-stage-specific chromatin accessibility patterns, infer key chromatin regulators and observe rewiring of regulatory networks in the progression to dysfunction in CD8+ T cells. Unexpectedly, among the transcription factors orchestrating the path to dysfunction, NF-κB is associated with a pro-apoptotic program in late stages of dysfunction in tumor-infiltrating CD8+ T cells. Importantly, this epigenomic profiling stratified ccRCC patients based on a NF-κB-driven pro-apoptotic signature. This study provides a rich resource for understanding the functional states and regulatory dynamics of immune cells in ccRCC.

Absence of central tolerance in Aire-deficient mice synergizes with immune-checkpoint inhibition to enhance antitumor responses.

The endogenous anti-tumor responses are limited in part by the absence of tumor-reactive T cells, an inevitable consequence of thymic central tolerance mechanisms ensuring prevention of autoimmunity. Here we show that tumor rejection induced by immune checkpoint blockade is significantly enhanced in Aire-deficient mice, the epitome of central tolerance breakdown. The observed synergy in tumor rejection extended to different tumor models, was accompanied by increased numbers of activated T cells expressing high levels of Gzma, Gzmb, Perforin, Cxcr3, and increased intratumoural levels of Cxcl9 and Cxcl10 compared to wild-type mice. Consistent with Aire's central role in T cell repertoire selection, single cell TCR sequencing unveiled expansion of several clones with high tumor reactivity. The data suggest that breakdown in central tolerance synergizes with immune checkpoint blockade in enhancing anti-tumor immunity and may serve as a model to unmask novel anti-tumor therapies including anti-tumor TCRs, normally purged during central tolerance.

Interactive analysis and assessment of single-cell copy-number variations.

We present Ginkgo (http://qb.cshl.edu/ginkgo), a user-friendly, open-source web platform for the analysis of single-cell copy-number variations (CNVs). Ginkgo automatically constructs copy-number profiles of cells from mapped reads and constructs phylogenetic trees of related cells. We validated Ginkgo by reproducing the results of five major studies. After comparing three commonly used single-cell amplification techniques, we concluded that degenerate oligonucleotide-primed PCR is the most consistent for CNV analysis.

Identification of PHLPP1 as a tumor suppressor reveals the role of feedback activation in PTEN-mutant prostate cancer progression.

Hyperactivation of the PI 3-kinase/AKT pathway is a driving force of many cancers. Here we identify the AKT-inactivating phosphatase PHLPP1 as a prostate tumor suppressor. We show that Phlpp1-loss causes neoplasia and, on partial Pten-loss, carcinoma in mouse prostate. This genetic setting initially triggers a growth suppressive response via p53 and the Phlpp2 ortholog, and reveals spontaneous Trp53 inactivation as a condition for full-blown disease. Surprisingly, the codeletion of PTEN and PHLPP1 in patient samples is highly restricted to metastatic disease and tightly correlated to deletion of TP53 and PHLPP2. These data establish a conceptual framework for progression of PTEN mutant prostate cancer to life-threatening disease.

p53: a new player in reproduction.

The roles of the p53 protein in tumor suppression have been firmly established. However, the functions of this protein under normal conditions or in the absence of stress, if any, have remained a mystery. In humans, some alleles containing a functional single nucleotide polymorphism in the p53 gene and its negative regulator, the Mdm2 gene, are under positive selection over evolutionary time frames, suggesting that the p53 pathway might have important functions that are optimized and selected for by evolutionary or reproductive pressures. Indeed, a recent study demonstrated a new function for the p53 protein in the regulation of maternal reproduction in mice, through transcriptional regulation of leukemia inhibitory factor (LIF), a novel p53 target gene. Sufficient uterine LIF levels are essential for the implantation of blastocysts or early embryos into the uterus. p53 deficient (p53(-/-)) female mice have a reduced pregnancy rate and litter size, due to impaired implantation resulting from decreased uterine LIF levels. Administration of LIF to pregnant p53(-/-) mice restored maternal reproduction by improving implantation. An association has been reported between women carrying the p53 codon 72 polymorphism (a proline to arginine change) with recurrent implantation failure, suggesting a similar function for p53 in humans. These findings of a new function for the p53 protein in reproduction may help to explain the observed evolutionary selection of some alleles of the p53 and Mdm2 genes. This may also be an excellent example of antagonistic pleiotrophy.