High-resolution functional mapping of RAD51C by saturation genome editing.

Pathogenic variants in RAD51C confer an elevated risk of breast and ovarian cancer, while individuals homozygous for specific RAD51C alleles may develop Fanconi anemia. Using saturation genome editing (SGE), we functionally assess 9,188 unique variants, including >99.5% of all possible coding sequence single-nucleotide alterations. By computing changes in variant abundance and Gaussian mixture modeling (GMM), we functionally classify 3,094 variants to be disruptive and use clinical truth sets to reveal an accuracy/concordance of variant classification >99.9%. Cell fitness was the primary assay readout allowing us to observe a phenomenon where specific missense variants exhibit distinct depletion kinetics potentially suggesting that they represent hypomorphic alleles. We further explored our exhaustive functional map, revealing critical residues on the RAD51C structure and resolving variants found in cancer-segregating kindred. Furthermore, through interrogation of UK Biobank and a large multi-center ovarian cancer cohort, we find significant associations between SGE-depleted variants and cancer diagnoses.

Neoadjuvant PARPi or chemotherapy in ovarian cancer informs targeting effector Treg cells for homologous-recombination-deficient tumors.

Homologous recombination deficiency (HRD) is prevalent in cancer, sensitizing tumor cells to poly (ADP-ribose) polymerase (PARP) inhibition. However, the impact of HRD and related therapies on the tumor microenvironment (TME) remains elusive. Our study generates single-cell gene expression and T cell receptor profiles, along with validatory multimodal datasets from >100 high-grade serous ovarian cancer (HGSOC) samples, primarily from a phase II clinical trial (NCT04507841). Neoadjuvant monotherapy with the PARP inhibitor (PARPi) niraparib achieves impressive 62.5% and 73.6% response rates per RECIST v.1.1 and GCIG CA125, respectively. We identify effector regulatory T cells (eTregs) as key responders to HRD and neoadjuvant therapies, co-occurring with other tumor-reactive T cells, particularly terminally exhausted CD8+ T cells (Tex). TME-wide interferon signaling correlates with cancer cells upregulating MHC class II and co-inhibitory ligands, potentially driving Treg and Tex fates. Depleting eTregs in HRD mouse models, with or without PARP inhibition, significantly suppresses tumor growth without observable toxicities, underscoring the potential of eTreg-focused therapeutics for HGSOC and other HRD-related tumors.

Proteogenomic analysis of chemo-refractory high-grade serous ovarian cancer.

To improve the understanding of chemo-refractory high-grade serous ovarian cancers (HGSOCs), we characterized the proteogenomic landscape of 242 (refractory and sensitive) HGSOCs, representing one discovery and two validation cohorts across two biospecimen types (formalin-fixed paraffin-embedded and frozen). We identified a 64-protein signature that predicts with high specificity a subset of HGSOCs refractory to initial platinum-based therapy and is validated in two independent patient cohorts. We detected significant association between lack of Ch17 loss of heterozygosity (LOH) and chemo-refractoriness. Based on pathway protein expression, we identified 5 clusters of HGSOC, which validated across two independent patient cohorts and patient-derived xenograft (PDX) models. These clusters may represent different mechanisms of refractoriness and implicate putative therapeutic vulnerabilities.

Tumor-reactive antibodies evolve from non-binding and autoreactive precursors.

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.

Locally produced autoantibodies in cancer.

The function and antigen-specificities of tumor-infiltrating B cells are mostly unknown. In a new study by Mazor et al., matrix metalloproteinase 14 (MMP14), a self-antigen that is overexpressed by ovarian cancers, is shown to drive B cell activation and autoantibody production in tertiary lymphoid structures (Mazor et al., 2022).

Mapping spatial organization and genetic cell-state regulators to target immune evasion in ovarian cancer.

The drivers of immune evasion are not entirely clear, limiting the success of cancer immunotherapies. Here we applied single-cell spatial and perturbational transcriptomics to delineate immune evasion in high-grade serous tubo-ovarian cancer. To this end, we first mapped the spatial organization of high-grade serous tubo-ovarian cancer by profiling more than 2.5 million cells in situ in 130 tumors from 94 patients. This revealed a malignant cell state that reflects tumor genetics and is predictive of T cell and natural killer cell infiltration levels and response to immune checkpoint blockade. We then performed Perturb-seq screens and identified genetic perturbations-including knockout of PTPN1 and ACTR8-that trigger this malignant cell state. Finally, we show that these perturbations, as well as a PTPN1/PTPN2 inhibitor, sensitize ovarian cancer cells to T cell and natural killer cell cytotoxicity, as predicted. This study thus identifies ways to study and target immune evasion by linking genetic variation, cell-state regulators and spatial biology.

Transforming lives through genetics.

Mary-Claire King's approach to genetics has had a major impact on breast and ovarian cancer and, more recently, mental illnesses including schizophrenia. Science writer Kendall Morgan talked with Mary-Claire, recipient of a 2021 Canada Gairdner International Award, about her life, her lengthy quest to discover the genetic basis of susceptibility to breast cancer, the struggles for women in science, and much more. An edited version of this conversation is presented below.

Ribosome ADP-ribosylation inhibits translation and maintains proteostasis in cancers.

Defects in translation lead to changes in the expression of proteins that can serve as drivers of cancer formation. Here, we show that cytosolic NAD+ synthesis plays an essential role in ovarian cancer by regulating translation and maintaining protein homeostasis. Expression of NMNAT-2, a cytosolic NAD+ synthase, is highly upregulated in ovarian cancers. NMNAT-2 supports the catalytic activity of the mono(ADP-ribosyl) transferase (MART) PARP-16, which mono(ADP-ribosyl)ates (MARylates) ribosomal proteins. Depletion of NMNAT-2 or PARP-16 leads to inhibition of MARylation, increased polysome association and enhanced translation of specific mRNAs, aggregation of their translated protein products, and reduced growth of ovarian cancer cells. Furthermore, MARylation of the ribosomal proteins, such as RPL24 and RPS6, inhibits polysome assembly by stabilizing eIF6 binding to ribosomes. Collectively, our results demonstrate that ribosome MARylation promotes protein homeostasis in cancers by fine-tuning the levels of protein synthesis and preventing toxic protein aggregation.

Interfaces of Malignant and Immunologic Clonal Dynamics in Ovarian Cancer.

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer.

Most high-grade serous ovarian cancer (HGSOC) patients develop resistance to platinum-based chemotherapy and recur, but 15% remain disease free over a decade. To discover drivers of long-term survival, we quantitatively analyzed the proteomes of platinum-resistant and -sensitive HGSOC patients from minute amounts of formalin-fixed, paraffin-embedded tumors. This revealed cancer/testis antigen 45 (CT45) as an independent prognostic factor associated with a doubling of disease-free survival in advanced-stage HGSOC. Phospho- and interaction proteomics tied CT45 to DNA damage pathways through direct interaction with the PP4 phosphatase complex. In vitro, CT45 regulated PP4 activity, and its high expression led to increased DNA damage and platinum sensitivity. CT45-derived HLA class I peptides, identified by immunopeptidomics, activate patient-derived cytotoxic T cells and promote tumor cell killing. This study highlights the power of clinical cancer proteomics to identify targets for chemo- and immunotherapy and illuminate their biological roles.

Heterogeneous Tumor-Immune Microenvironments among Differentially Growing Metastases in an Ovarian Cancer Patient.

We present an exceptional case of a patient with high-grade serous ovarian cancer, treated with multiple chemotherapy regimens, who exhibited regression of some metastatic lesions with concomitant progression of other lesions during a treatment-free period. Using immunogenomic approaches, we found that progressing metastases were characterized by immune cell exclusion, whereas regressing and stable metastases were infiltrated by CD8+ and CD4+ T cells and exhibited oligoclonal expansion of specific T cell subsets. We also detected CD8+ T cell reactivity against predicted neoepitopes after isolation of cells from a blood sample taken almost 3 years after the tumors were resected. These findings suggest that multiple distinct tumor immune microenvironments co-exist within a single individual and may explain in part the heterogeneous fates of metastatic lesions often observed in the clinic post-therapy. VIDEO ABSTRACT.

PARP Inhibitors for Cancer Therapy.

Rucaparib is an inhibitor of nuclear poly (ADP-ribose) polymerases (inhibition of PARP-1 > PARP-2 > PARP-3), following a similar drug, Olaparib. It disrupts DNA repair and replication pathways (and possibly transcription), leading to selective killing of cancer cells with BRCA1/2 mutations. Rucaparib is approved for recurrent ovarian cancers with germline or somatic mutations in BRCA1/2.

Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy.

Despite its success in several clinical trials, cancer immunotherapy remains limited by the rarity of targetable tumor-specific antigens, tumor-mediated immune suppression, and toxicity triggered by systemic delivery of potent immunomodulators. Here, we present a proof-of-concept immunomodulatory gene circuit platform that enables tumor-specific expression of immunostimulators, which could potentially overcome these limitations. Our design comprised de novo synthetic cancer-specific promoters and, to enhance specificity, an RNA-based AND gate that generates combinatorial immunomodulatory outputs only when both promoters are mutually active. These outputs included an immunogenic cell-surface protein, a cytokine, a chemokine, and a checkpoint inhibitor antibody. The circuits triggered selective T cell-mediated killing of cancer cells, but not of normal cells, in vitro. In in vivo efficacy assays, lentiviral circuit delivery mediated significant tumor reduction and prolonged mouse survival. Our design could be adapted to drive additional immunomodulators, sense other cancers, and potentially treat other diseases that require precise immunological programming.

Integrated Proteogenomic Characterization of Human High-Grade Serous Ovarian Cancer.

To provide a detailed analysis of the molecular components and underlying mechanisms associated with ovarian cancer, we performed a comprehensive mass-spectrometry-based proteomic characterization of 174 ovarian tumors previously analyzed by The Cancer Genome Atlas (TCGA), of which 169 were high-grade serous carcinomas (HGSCs). Integrating our proteomic measurements with the genomic data yielded a number of insights into disease, such as how different copy-number alternations influence the proteome, the proteins associated with chromosomal instability, the sets of signaling pathways that diverse genome rearrangements converge on, and the ones most associated with short overall survival. Specific protein acetylations associated with homologous recombination deficiency suggest a potential means for stratifying patients for therapy. In addition to providing a valuable resource, these findings provide a view of how the somatic genome drives the cancer proteome and associations between protein and post-translational modification levels and clinical outcomes in HGSC. VIDEO ABSTRACT.

Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.

Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

Ovarian tumors orchestrate distinct cellular compositions.

The determinants of T cell infiltration in tumors remain largely unknown. In a recent issue of Cancer Cell, Hornburg et al. use single-cell RNA sequencing to characterize the cellular compartments of the ovarian cancer microenvironment and shed light on how tumor, immune, and stromal cells interact and shape T cell infiltration.

ER Stress in Dendritic Cells Promotes Cancer.

XBP1 is part of the ER stress response, and when activated in cancer cells, it fosters tumor growth. In this issue of Cell, Cubillos-Ruiz et al. demonstrate that XBP1 in tumor-infiltrating dendritic cells blunts anti-tumor immunity. These findings further imply XBP1 as a relevant target for cancer therapy.

ER Stress Sensor XBP1 Controls Anti-tumor Immunity by Disrupting Dendritic Cell Homeostasis.

Dendritic cells (DCs) are required to initiate and sustain T cell-dependent anti-cancer immunity. However, tumors often evade immune control by crippling normal DC function. The endoplasmic reticulum (ER) stress response factor XBP1 promotes intrinsic tumor growth directly, but whether it also regulates the host anti-tumor immune response is not known. Here we show that constitutive activation of XBP1 in tumor-associated DCs (tDCs) drives ovarian cancer (OvCa) progression by blunting anti-tumor immunity. XBP1 activation, fueled by lipid peroxidation byproducts, induced a triglyceride biosynthetic program in tDCs leading to abnormal lipid accumulation and subsequent inhibition of tDC capacity to support anti-tumor T cells. Accordingly, DC-specific XBP1 deletion or selective nanoparticle-mediated XBP1 silencing in tDCs restored their immunostimulatory activity in situ and extended survival by evoking protective type 1 anti-tumor responses. Targeting the ER stress response should concomitantly inhibit tumor growth and enhance anti-cancer immunity, thus offering a unique approach to cancer immunotherapy.

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy.

There is a lack of effective predictive biomarkers to precisely assign optimal therapy to cancer patients. While most efforts are directed at inferring drug response phenotype based on genotype, there is very focused and useful phenotypic information to be gained from directly perturbing the patient's living cancer cell with the drug(s) in question. To satisfy this unmet need, we developed the Dynamic BH3 Profiling technique to measure early changes in net pro-apoptotic signaling at the mitochondrion ("priming") induced by chemotherapeutic agents in cancer cells, not requiring prolonged ex vivo culture. We find in cell line and clinical experiments that early drug-induced death signaling measured by Dynamic BH3 Profiling predicts chemotherapy response across many cancer types and many agents, including combinations of chemotherapies. We propose that Dynamic BH3 Profiling can be used as a broadly applicable predictive biomarker to predict cytotoxic response of cancers to chemotherapeutics in vivo.