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Trends in Cancer
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When breaks get hot: inflammatory signaling in BRCA1/2-mutant cancers

Open AccessPublished:January 06, 2022DOI:https://doi.org/10.1016/j.trecan.2021.12.003

      Highlights

      • Inflammatory signaling and genomic instability are hallmarks of cancer.
      • Cancer-associated DNA repair defects, including defects in homologous recombination repair, lead to cytoplasmic DNA.
      • Genomic instability fuels inflammatory signaling, triggering both tumor-suppressive as well as tumor-promoting traits.
      • Through largely unknown mechanisms, tumor cells rewire inflammatory signaling to prevent immune clearance.
      • Unique tumor microenvironment features of genomically unstable tumors may promote immunotherapy resistance
      • Therapeutic targeting of immune-suppressive mechanisms in genomically instable cancer may potentiate immune checkpoint inhibition.
      Genomic instability and inflammation are intricately connected hallmark features of cancer. DNA repair defects due to BRCA1/2 mutation instigate immune signaling through the cGAS/STING pathway. The subsequent inflammatory signaling provides both tumor-suppressive as well as tumor-promoting traits. To prevent clearance by the immune system, genomically instable cancer cells need to adapt to escape immune surveillance. Currently, it is unclear how genomically unstable cancers, including BRCA1/2-mutant tumors, are rewired to escape immune clearance. Here, we summarize the mechanisms by which genomic instability triggers inflammatory signaling and describe adaptive mechanisms by which cancer cells can ‘fly under the radar’ of the immune system. Additionally, we discuss how therapeutic activation of the immune system may improve treatment of genomically instable cancers.

      Keywords

      BRCA1/2 and genomic instability in cancer

      Genomic instability (see Glossary) is a trait of tumor cells that is observed in the majority of cancers. However, the level of genomic instability ranges significantly between tumors. High levels of genome instability are associated with hereditary or somatic mutations in DNA repair genes and oncogene-induced replication stress [
      • Negrini S.
      • et al.
      Genomic instability an evolving hallmark of cancer.
      ]. In particular, defective repair of double-stranded DNA breaks (DSBs) and DNA crosslinks due to mutations in homologous recombination (HR) genes [e.g., Breast cancer 1, early onset (BRCA1) and Breast cancer 2, early onset (BRCA2)] or Fanconi anemia (FA) genes yields tumors with extensive genomic instability (Figure 1A ). These tumors are characterized by focal genomic deletions and amplifications as well as complex genomic rearrangements [
      • Liu X.
      • et al.
      Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer.
      ,
      • Davies H.
      • et al.
      HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.
      ]. Notably, genomic instability drives intratumor heterogeneity and enables rapid acquisition of the genomic aberrations that drive therapy failure [
      • Gerlinger M.
      • Swanton C.
      How Darwinian models inform therapeutic failure initiated by clonal heterogeneity in cancer medicine.
      ].
      Figure 1
      Figure 1Cancer predisposition and cellular defects associated with BRCA1/2 mutation.
      (A) Inherited BRCA1/2 mutations confer predisposition to a range of cancers, predominantly breast and ovarian cancer. (B) Genome maintenance functions of BRCA1/2. Beyond their canonical role in homologous recombination repair, BRCA1 and BRCA2 are involved in the protection of stalled replication forks and repair by the Fanconi anemia pathway. (C) BRCA1/2 inactivation leads to defective DNA repair and consequent usage of error-prone DNA repair pathways, collapse of stalled forks, and defective completion of crosslink repair. Abbreviations: AltEJ, alternative end-joining; NHEJ, non-homologous end-joining; SSA, single-strand annealing.
      BRCA1 and BRCA2 are key regulators of DNA maintenance through HR [
      • Wyman C.
      • et al.
      Homologous recombination-mediated double-strand break repair.
      ]. BRCA1 functions in the initiation of HR by controlling DNA-end resection at DNA breaks, whereas BRCA2 functions downstream in HR, in loading the RAD51 recombinase to facilitate the actual recombination process (Figure 1B) [
      • Chen C.-C.
      • et al.
      Homology-directed repair and the role of BRCA1, BRCA2, and related proteins in genome integrity and cancer.
      ]. Besides these canonical roles, BRCA1 and BRCA2 function in DNA crosslink repair as part of the FA complex [
      • Bunting S.F.
      • et al.
      BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair.
      ] and were shown to protect nascent DNA at stalled replication forks from nucleolytic degradation (Figure 1B) [
      • Schlacher K.
      • et al.
      Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.
      ,
      • Schlacher K.
      • et al.
      A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.
      ].
      The relevance of BRCA1 and BRCA2 for genome maintenance and cellular viability became evident from genetic studies in mice. Specifically, loss of Brca1 or Brca2 leads to accumulation of DNA lesions, a consequent cell cycle arrest, and early embryonal death (Figure 1C) [
      • Sharan S.K.
      • et al.
      Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2.
      ,
      • Hakem R.
      • et al.
      The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse.
      ,
      • Tutt A.
      • et al.
      Absence of Brca2 causes genome instability by chromosome breakage and loss associated with centrosome amplification.
      ,
      • Hakem R.
      • et al.
      Developmental studies of Brca1 and Brca2 knock-out mice.
      ,
      • Liu C.Y.
      • et al.
      Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimplantation development.
      ,
      • Gowen L.C.
      • et al.
      Brca1 deficiency results in early embryonic lethality characterized by neuroepithelial abnormalities.
      ]. These findings were in apparent contrast with the observed tumor predisposition of BRCA mutation carriers and the full loss of BRCA1 or BRCA2 in the ensuing tumors (Figure 1A) [
      • Elledge S.J.
      • Amon A.
      The BRCA1 suppressor hypothesis: an explanation for the tissue-specific tumor development in BRCA1 patients.
      ]. This BRCA paradox was partly explained when Trp53 was conditionally inactivated along with Brca1/2, which allows cells to survive with damaged DNA and ultimately promotes tumor onset [
      • Liu X.
      • et al.
      Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer.
      ,
      • Jonkers J.
      • et al.
      Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.
      ,
      • Xu X.
      • et al.
      Conditional mutation of Brca 1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation.
      ]. The resulting tumors showed basal-like characteristics, including recurrent genomic features such as MYC amplification and RB1 loss [
      • Liu X.
      • et al.
      Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer.
      ]. Moreover, these tumors displayed the complex genomic rearrangements that resemble those of human BRCA1/2-mutant cancers [
      • Liu X.
      • et al.
      Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer.
      ,
      • Curtis C.
      • et al.
      The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.
      ,
      • Cancer Genome Atlas Network
      Comprehensive molecular portraits of human breast tumours.
      ,
      • Holstege H.
      • et al.
      Cross-species comparison of aCGH data from mouse and human BRCA1- and BRCA2-mutated breast cancers.
      ,
      • Hollern D.P.
      • et al.
      A mouse model featuring tissue-specific deletion of p53 and Brca1 gives rise to mammary tumors with genomic and transcriptomic similarities to human basal-like breast cancer.
      ].
      Loss-of-function mutations in BRCA1/2, as well as mutations in other HR genes, lead to a profound defect in genome maintenance [
      • Lord C.J.
      • Ashworth A.
      BRCAness revisited.
      ], generically termed BRCAness. Defective HR yields aberrant genomes that are enriched for several mutational signatures, including base substitution signatures (SBS3, SBS8), indel signatures (ID6, ID8), and rearrangement signatures (RS1, RS3, and RS5) [
      • Davies H.
      • et al.
      HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.
      ,
      • Alexandrov L.B.
      • et al.
      The repertoire of mutational signatures in human cancer.
      ,
      • Hwang T.
      • et al.
      Defining the mutation signatures of DNA polymerase θ in cancer genomes.
      ,
      • Nguyen L.
      • et al.
      Pan-cancer landscape of homologous recombination deficiency.
      ]. These genomic alterations can be explained by the usage of alternative, nonconservative DNA repair mechanisms to repair DNA DSBs (Figure 1C) [
      • Davies H.
      • et al.
      HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.
      ,
      • Stok C.
      • et al.
      Shaping the BRCAness mutational landscape by alternative double-strand break repair, replication stress and mitotic aberrancies.
      ]. In particular, the usage of non-homologous end-joining, polymerase Theta (POLQ)-mediated end-joining (also referred to as alternative end-joining), or single-strand annealing leads to deletions and translocations that are characteristic of HR-deficient cancers [
      • Davies H.
      • et al.
      HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.
      ,
      • Alexandrov L.B.
      • et al.
      The repertoire of mutational signatures in human cancer.
      ,
      • Hwang T.
      • et al.
      Defining the mutation signatures of DNA polymerase θ in cancer genomes.
      ,
      • Nik-Zainal S.
      • et al.
      Landscape of somatic mutations in 560 breast cancer whole-genome sequences.
      ,
      • Póti Á.
      • et al.
      Correlation of homologous recombination deficiency induced mutational signatures with sensitivity to PARP inhibitors and cytotoxic agents.
      ].
      In addition to the effects of defective DSB repair, genome integrity in BRCA1/2-mutant cancers is affected by defective protection of stalled replication forks [
      • Schlacher K.
      • et al.
      Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11.
      ]. This defect is independent of the HR defect [
      • Daza-Martin M.
      • et al.
      Isomerization of BRCA1–BARD1 promotes replication fork protection.
      ] and leads to fork collapse and persistent DNA breaks. Specific to BRCA1-mutant cells, re-replication of tracts of DNA adjacent to stalled forks leads to tandem duplications [
      • Willis N.A.
      • et al.
      Mechanism of tandem duplication formation in BRCA1-mutant cells.
      ], which are a recurrent feature of BRCA1-mutant cancers [
      • Nik-Zainal S.
      • et al.
      Landscape of somatic mutations in 560 breast cancer whole-genome sequences.
      ,
      • Menghi F.
      • et al.
      The tandem duplicator phenotype is a prevalent genome-wide cancer configuration driven by distinct gene mutations.
      ,
      • Menghi F.
      • Liu E.T.
      Reply to Watkins et al.: Whole-genome sequencing-based identification of diverse tandem duplicator phenotypes in human cancers.
      ]. The effects of BRCA1/2 mutation on genome integrity will likely extend beyond the effects of impaired HR and fork protection, as BRCA1 and BRCA2 (and multiple other HR genes) have been described to perform multiple other functions in tumor suppression, including roles for BRCA1 in regulating gene expression and chromatin remodeling [
      • Savage K.I.
      • et al.
      Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.
      ,
      • Filipponi D.
      • et al.
      Wip1 controls global heterochromatin silencing via ATM/BRCA1-dependent DNA methylation.
      ,
      • Zhu Q.
      • et al.
      BRCA1 tumour suppression occurs via heterochromatin-mediated silencing.
      ].
      Recently, inflammatory signaling was identified as a consequence of genomic instability, including in BRCA1/2-mutant cancers. Tumor-cell intrinsic inflammatory signaling can activate immune cells in the tumor microenvironment (TME) and has been linked to responses to immune checkpoint inhibitors. Although important features to respond to cancer immunotherapy are clearly present in genomically instable cancers, patients with such cancers paradoxically only minimally benefit from immune checkpoint inhibitors. Apparently, genomically instable cancers somehow evade immune eradication. To expand benefit of cancer immunotherapy to patients with genomically instable cancers, it is crucial to understand how these cancers have adapted to inflammatory signaling and to investigate if and how these adaptive mechanisms can be therapeutically targeted. We review the mechanisms that drive inflammatory signaling in response to genomic instability caused by BRCA1/2 inactivation. We also discuss alterations in these tumors to evade immune clearance and options to improve treatment of these cancers.

      BRCA1/2 mutation, inflammatory signaling, and immune activation

      In line with their DNA repair defects and consequent tumor mutational burden (TMB), BRCA1/2 mutation status in high-grade serous ovarian cancer associates with a predicted higher neoantigen load and improved overall survival [
      • Strickland K.C.
      • et al.
      Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer.
      ]. In this study, neoantigen load correlates with HR deficiency, even in non-BRCA mutations. Similarly, BRCA1/2 mutation in breast cancer associates with increased TMB and increased expression of the PD1/PDL-1 immune checkpoint components, albeit only in BRCA1-mutant cancers [
      • Wen W.X.
      • Leong C.-O.
      Association of BRCA1- and BRCA2-deficiency with mutation burden, expression of PD-L1/PD-1, immune infiltrates, and T cell-inflamed signature in breast cancer.
      ]. Even so, this increased TMB does not reach the scale of that observed in tumors classically responsive to immune checkpoint blockade: microsatellite unstable colorectal cancer, melanoma, and non-small cell lung cancer [
      • Lawrence M.S.
      • et al.
      Mutational heterogeneity in cancer and the search for new cancer-associated genes.
      ]. It is likely that the increased immunogenicity of BRCA1/2-mutant cancers cannot solely be attributed to a moderately elevated TMB, as HR mutations are predictors of response to immune checkpoint inhibitors, independent of TMB [
      • Hsiehchen D.
      • et al.
      DNA repair gene mutations as predictors of immune checkpoint inhibitor response beyond tumor mutation burden.
      ].
      Beyond mutational burden, inflammatory signaling appears to strongly associate with immunogenicity, likely in part because it is a direct read-out of immune cell activation. BRCA1/2 mutation in ovarian cancer associates with expression of immune-related genes, including type II interferon gamma (IFNγ) and TNFR [
      • Strickland K.C.
      • et al.
      Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer.
      ]. BRCA1 mutation is associated with the immunoreactive subtype of high-grade serous ovarian cancers, which are characterized by high levels of tumor-infiltrating immune cells [
      • George J.
      • et al.
      Nonequivalent gene expression and copy number alterations in high-grade serous ovarian cancers with BRCA1 and BRCA2 mutations.
      ]. Also, BRCA1/2 status is associated with increased abundance of CD3+ and CD4+ tumor-infiltrating lymphocytes [
      • McAlpine J.N.
      • et al.
      BRCA1 and BRCA2 mutations correlate with TP53 abnormalities and presence of immune cell infiltrates in ovarian high-grade serous carcinoma.
      ] and increased expression of the immune checkpoint molecules PD1/PDL1 [
      • Strickland K.C.
      • et al.
      Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer.
      ,
      • Wieser V.
      • et al.
      BRCA1/2 and TP53 mutation status associates with PD-1 and PD-L1 expression in ovarian cancer.
      ]. In a separate study only focusing on BRCA1 status, increased levels of DNA damage in BRCA1-mutant ovarian cancers were associated with elevated stimulator of interferon genes (STING) levels, increased STAT1 signaling, and increased T cell infiltrate [
      • Bruand M.
      • et al.
      Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.
      ]. Moreover, the tumor-associated inflammation in BRCA1-mutant tumors is a favorable prognostic feature [
      • van Verschuer V.M.T.
      • et al.
      Tumor-associated inflammation as a potential prognostic tool in BRCA1/2-associated breast cancer.
      ,
      • de Boo L.
      • et al.
      Tumour-infiltrating lymphocytes (TILs) and BRCA-like status in stage III breast cancer patients randomised to adjuvant intensified platinum-based chemotherapy versus conventional chemotherapy.
      ].
      Analysis of gene expression in patient samples with BRCA1/2 or FA mutations yielded a gene set signature, which was highly enriched for genes involved in immune signaling [
      • Mulligan J.M.
      • et al.
      Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer.
      ]. Lymphocyte infiltration only partially explains the expression of immune-related genes, pointing towards cell intrinsic inflammatory signaling [
      • Mulligan J.M.
      • et al.
      Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer.
      ]. Subsequent studies have revealed that inactivation of BRCA1, BRCA2, or FANCD2 results in tumor-cell intrinsic inflammatory signaling, involving secretion of proinflammatory cytokines CXCL10, CCL5, and TNF-α and attraction of immune cells [
      • Parkes E.E.
      • et al.
      Activation of STING-dependent innate immune signaling by S-phase-specific DNA damage in breast cancer.
      ,
      • Heijink A.M.
      • et al.
      BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity.
      ]. Inflammatory signaling in these cells likely stems from the DNA lesions that arise due to their defective DNA maintenance, as similar effects are observed in response to DNA damaging agents [
      • Parkes E.E.
      • et al.
      Activation of STING-dependent innate immune signaling by S-phase-specific DNA damage in breast cancer.
      ,
      • Sistigu A.
      • et al.
      Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy.
      ,
      • Legrier M.E.
      • et al.
      Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer.
      ]. These findings are in line with observations in genetically engineered mouse models. Combined tissue-specific loss of Trp53 and Brca1 leads to development of cancers with signatures associated with higher levels of Th2 cells, T-regulatory cells (Tregs), central memory cells, and exhausted T cells, as well as elevated expression of immune checkpoint genes, Pd1 and Ctla4 [
      • Hollern D.P.
      • et al.
      A mouse model featuring tissue-specific deletion of p53 and Brca1 gives rise to mammary tumors with genomic and transcriptomic similarities to human basal-like breast cancer.
      ].
      Whereas BRCA1/2 mutation and other HR deficiencies are all characterized by severe genomic instability, not all HR deficiencies are equal in terms of subsequent effects on the TME. For instance, gene expression analysis revealed that ovarian cancers with mutant or hypermethylated BRCA1 but not BRCA2-mutant cancers are associated with the immunoreactive subtype [
      • George J.
      • et al.
      Nonequivalent gene expression and copy number alterations in high-grade serous ovarian cancers with BRCA1 and BRCA2 mutations.
      ]. In a study of BRCA-mutant breast cancers, distinct features of the immune TME of BRCA1- versus BRCA2-mutant were identified, with increased immunosuppressive tumor-associated macrophages in Brca1-null mouse models and increased immune checkpoint expression in BRCA1-mutant breast cancer [
      • Samstein R.M.
      • et al.
      Mutations in BRCA1 and BRCA2 differentially affect the tumor microenvironment and response to checkpoint blockade immunotherapy.
      ]. A pan-cancer study of BRCA1- versus BRCA2-mutant cancers treated with immune checkpoint blockade suggests benefit in BRCA2-mutant disease only [
      • Zhou Z.
      • Li M.
      Evaluation of BRCA1 and BRCA2 as indicators of response to immune checkpoint inhibitors.
      ]. Whether similar patterns could be observed in response to other treatments such as chemotherapy is not known.
      Some studies have instead observed BRCA1/2-mutant cancers to be poorly immunogenic compared with BRCA1/2-wild type. Analysis of breast cancers in patients with a germline BRCA mutation or clonal loss of heterozygosity found these tumors to have a lower immune gene expression score compared with BRCA1/2-wild type or tumors with subclonal loss of heterozygosity, correlating with reduced immune infiltration on immunohistochemical analysis of germline BRCA1-mutant breast cancers [
      • Kraya A.A.
      • et al.
      Genomic signatures predict the immunogenicity of BRCA-deficient breast cancer.
      ]. These observations add granularity to the association of BRCA1/2 mutation and immune infiltration, indicating that detailed molecular analysis is needed to clearly understand this complex relationship. Moreover, intratumoral heterogeneity plays a role in enabling immune evasion in BRCA-mutant cancers. Using digital pathology to define morphological diversity in ovarian cancer, a significant association between low BRCA1 expression and increased diversification was noted [
      • Heindl A.
      • et al.
      Microenvironmental niche divergence shapes BRCA1-dysregulated ovarian cancer morphological plasticity.
      ]. Further morphological examination revealed a lack of tumor invasion by CD3+ T cells in morphologically diverse regions, with a suggestion of active exclusion of these cells from tumor nests by upregulation of the immune checkpoint galectin-3 [
      • Heindl A.
      • et al.
      Microenvironmental niche divergence shapes BRCA1-dysregulated ovarian cancer morphological plasticity.
      ]. The relationship between BRCA1/2 mutation and a favorable immune environment is far from straightforward, with genetic and spatial heterogeneity contributing to a spectrum of immunogenicity in genomically unstable cancers.

      Mechanisms of inflammatory signaling in response to genomic instability

      BRCA1/2-mutant cancers almost invariably harbor inactivating TP53 mutations [
      • Curtis C.
      • et al.
      The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups.
      ], therefore, these cells have incomplete cell cycle control and frequently transmit DNA damage into mitosis [
      • Schoonen P.M.
      • et al.
      Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells.
      ,
      • Feng W.
      • Jasin M.
      BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination.
      ]. These mitotic DNA lesions are positive for the replication stress marker FANCD2 [
      • Schoonen P.M.
      • et al.
      Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells.
      ] and likely represent ‘joint DNA molecules’, either due to incomplete DNA replication or unresolved intermediates of repair attempts. Consequently, BRCA1/2 inactivation leads to elevated numbers of mitotic chromatin bridges and ultrafine DNA bridges [
      • Schoonen P.M.
      • et al.
      Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells.
      ,
      • Laulier C.
      • et al.
      The relative efficiency of homology-directed repair has distinct effects on proper anaphase chromosome separation.
      ] and results in 53BP1 nuclear bodies in the subsequent G1 cells [
      • Feng W.
      • Jasin M.
      BRCA2 suppresses replication stress-induced mitotic and G1 abnormalities through homologous recombination.
      ].
      Unresolved mitotic DNA damage frequently results in micronuclei [
      • Löbrich M.
      • Jeggo P.A.
      The impact of a negligent G2/M checkpoint on genomic instability and cancer induction.
      ,
      • Lewis C.W.
      • Golsteyn R.M.
      Cancer cells that survive checkpoint adaptation contain micronuclei that harbor damaged DNA.
      ], which are small DNA-containing structures surrounded by a single lipid bilayer and are not part of the main nucleus (Figure 2). Micronuclei that originate from DNA lesions that are transmitted into mitosis typically contain acentric chromosome fragments. Chromatin in micronuclei is unable to support faithful DNA replication and DNA repair, leading to additional DNA damage, including local chromosome shattering (i.e., ‘chromotrypsis’) [
      • Umbreit N.T.
      • et al.
      Mechanisms generating cancer genome complexity from a single cell division error.
      ,
      • Crasta K.
      • et al.
      DNA breaks and chromosome pulverization from errors in mitosis.
      ,
      • Zhang C.-Z.
      • et al.
      Chromothripsis from DNA damage in micronuclei.
      ]. The nuclear lamina of micronuclei is not properly organized and frequently ruptures, leading to the release of micronucleus DNA into the cytoplasm (Figure 2) [
      • Hatch E.M.
      • et al.
      Catastrophic nuclear envelope collapse in cancer cell micronuclei.
      ].
      Figure 2
      Figure 2Mechanisms of inflammatory signaling in response to BRCA1/2 inactivation.
      A schematic overview is provided of the various potential routes by which defective homologous recombination can lead to cytoplasmic DNA and trigger downstream inflammatory signaling pathways and immune cell activation.
      To respond to microbial pathogens, cells have evolved an innate immune response, involving cytosolic DNA and RNA sensors, including the DNA sensor cyclic GMP-AMP synthase (cGAS), the RNA sensor retinoic acid-inducible gene-I (RIG-I) and the Toll-like DNA/RNA receptors [
      • Chen Q.
      • et al.
      Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing.
      ,
      • Chabanon R.M.
      • et al.
      PARP inhibition enhances tumor cell-intrinsic immunity in ERCC1-deficient non-small cell lung cancer.
      ]. Like microbial DNA, ‘self’ DNA that ends up in the cytosol following missegregation of chromosomes during mitosis is also recognized by cGAS [
      • Harding S.M.
      • et al.
      Mitotic progression following DNA damage enables pattern recognition within micronuclei.
      ,
      • Mackenzie K.J.
      • et al.
      cGAS surveillance of micronuclei links genome instability to innate immunity.
      ,
      • Bakhoum S.F.
      • et al.
      Chromosomal instability drives metastasis through a cytosolic DNA response.
      ]. Activated cGAS subsequently catalyzes the production of cyclic 2′3′ GAMP, which triggers STING-dependent inflammatory signaling, including the production of type-1 IFNs [
      • Chen Q.
      • et al.
      Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing.
      ]. Under physiological conditions, cytoplasmic DNA can be effectively degraded by the TREX1 nuclease [
      • Mohr L.
      • et al.
      ER-directed TREX1 limits cGAS activation at micronuclei.
      ]. Additionally, DNA:RNA hybrids can be processed by RNase H1/H2, whereas genome-embedded ribonucleotides can be hydrolyzed by RNAseH2, which prevents the accumulation of cytoplasmic nucleic acids and activation of the cGAS/STING pathway [
      • Mackenzie K.J.
      • et al.
      cGAS surveillance of micronuclei links genome instability to innate immunity.
      ,
      • Shen Y.J.
      • et al.
      Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells.
      ,
      • Mackenzie K.J.
      • et al.
      Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response.
      ,
      • Pokatayev V.
      • et al.
      RNase H2 catalytic core Aicardi-Goutières syndrome-related mutant invokes cGAS-STING innate immune-sensing pathway in mice.
      ]. When the amount of cytoplasmic DNA is overwhelming, for instance, when a micronucleus is induced upon BRCA1/2 inactivation, the cGAS/STING pathway is triggered (Figure 2) [
      • Parkes E.E.
      • et al.
      Activation of STING-dependent innate immune signaling by S-phase-specific DNA damage in breast cancer.
      ,
      • Heijink A.M.
      • et al.
      BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity.
      ,
      • Reisländer T.
      • et al.
      BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors.
      ]. Micronucleus formation upon missegregation of chromosome fragments leads to recruitment and activation of cGAS [
      • Harding S.M.
      • et al.
      Mitotic progression following DNA damage enables pattern recognition within micronuclei.
      ]. Micronucleus rupture strongly enhances this process, as was elegantly shown by cGAS recruitment along with leakage of cytoplasmic markers into micronuclei and the loss of nuclear markers out of micronuclei [
      • Mackenzie K.J.
      • et al.
      cGAS surveillance of micronuclei links genome instability to innate immunity.
      ]. In line with this observation, a split-GFP reporter for cGAS shows highest activity in micronuclei [
      • Li T.
      • et al.
      Phosphorylation and chromatin tethering prevent cGAS activation during mitosis.
      ], and cGAS can be activated directly by purified micronuclei [
      • Mohr L.
      • et al.
      ER-directed TREX1 limits cGAS activation at micronuclei.
      ]. However, additional sites of cGAS activation may exist (see Outstanding questions) and various layers of regulation are emerging that control cGAS activity.
      Chromatin [
      • Zierhut C.
      • et al.
      The cytoplasmic DNA sensor cGAS promotes mitotic cell death.
      ,
      • Kujirai T.
      • et al.
      Structural basis for the inhibition of cGAS by nucleosomes.
      ,
      • Pathare G.R.
      • et al.
      Structural mechanism of cGAS inhibition by the nucleosome.
      ,
      • Michalski S.
      • et al.
      Structural basis for sequestration and autoinhibition of cGAS by chromatin.
      ,
      • Boyer J.A.
      • et al.
      Structural basis of nucleosome-dependent cGAS inhibition.
      ] and the post-translational modification status of cGAS [
      • Li T.
      • et al.
      Phosphorylation and chromatin tethering prevent cGAS activation during mitosis.
      ] are increasingly recognized to determine the ability of cGAS to be activated by DNA. In addition, it was recently suggested that cGAS-dependent inflammatory signaling requires DNA stretching at mitotic chromatin bridges, rather than micronucleus formation per se [
      • Flynn P.J.
      • et al.
      Chromatin bridges, not micronuclei, activate cGAS after drug-induced mitotic errors in human cells.
      ]. However, these findings were done using antimitotic drugs or spindle-assembly checkpoint perturbation instead of genomic instability due to defective DNA repair. Yet, mitotic chromatin bridges are also frequently observed in HR-deficient cancer cells [
      • Schoonen P.M.
      • et al.
      Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells.
      ,
      • Laulier C.
      • et al.
      The relative efficiency of homology-directed repair has distinct effects on proper anaphase chromosome separation.
      ] and these structures may contribute to cGAS/STING activation.
      Cytoplasmic DNA can also arise from deprotected stalled replication forks, as observed in SAMHD1-deficient cells [
      • Coquel F.
      • et al.
      SAMHD1 acts at stalled replication forks to prevent interferon induction.
      ]. Whether DNA fragments from stalled replication forks in BRCA1/2-mutant cells also migrate into the cytoplasm is currently unclear. More recently, DNA fragments derived from R-loops were shown to end up in the cytoplasm of repair-deficient pancreatic cells [
      • Chatzidoukaki O.
      • et al.
      R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage.
      ]. The observation that DNA repair defects in differentiated tissues lead to cytoplasmic DNA fragments suggests that undergoing mitosis is not required to yield cytoplasmic DNA and trigger inflammatory responses [
      • Chatzidoukaki O.
      • et al.
      R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage.
      ].
      Once STING is activated, canonical NF-κB signaling is instigated through the IkK complex, leading to RelA/p50 translocation to the nucleus, where it transactivates canonical NF-κB targets, predominantly involved in immune responses [
      • Gilmore T.D.
      Introduction to NF-kappaB: players, pathways, perspectives.
      ] (Figure 2). Additionally, STING activates noncanonical NF-κB signaling, through the p100/RelB complex. Processing of p100 into p52, which associates with RelB, leads to a transcriptional response that is mostly linked to increased survival, through transactivation of antiapoptotic genes and epithelial-to-mesenchymal transition [
      • Gilmore T.D.
      Introduction to NF-kappaB: players, pathways, perspectives.
      ]. Finally, STING signaling leads to TANK-binding kinase 1 (TBK1)-dependent interferon regulatory factor-3 (IRF3) phosphorylation and subsequent transactivation of IRF3 target genes. Prominent among IRF3 target genes are type-1 IFNs, which induce autocrine and paracrine signaling. Type-1 (α and β) or type-2 (γ) IFNs induce pleiotropic effects on the physiology of cells, through the transactivation of a broad repertoire of target genes, collectively called ‘interferon-stimulated genes’ (ISGs). A major downstream consequence of IFN signaling is widespread immunomodulation. Binding of IFNs to the ubiquitously expressed IFNα/β receptor (IFNAR) ultimately results in phosphorylation and activation of the JAK/STAT pathway. Whereas JAK-mediated phosphorylation of the STAT1 transcription factor predominantly leads to growth suppressing and proapoptotic effects, STAT3 promotes proliferation, while it prevents apoptosis [
      • Stephanou A.
      • et al.
      Opposing actions of STAT-1 and STAT-3 on the Bcl-2 and Bcl-x promoters.
      ]. The repertoire of STAT expression thus determines the ultimate outcome of IFN signaling. Early on, IFNs were shown to stimulate multiple aspects of the innate immune system, including activation of macrophages and natural killer (NK) cells [
      • Denis M.
      Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates.
      ,
      • Gidlund M.
      • et al.
      Enhanced NK cell activity in mice injected with interferon and interferon inducers.
      ] and maturation of dendritic cells (DCs) [
      • Luft T.
      • et al.
      Type I IFNs enhance the terminal differentiation of dendritic cells.
      ]. Of note, through production of various soluble factors, NK cells interact with other immune cells, to promote efficient adaptive immune responses, beyond their role in innate immunity [
      • Vitale M.
      • et al.
      NK-dependent DC maturation is mediated by TNFalpha and IFNgamma released upon engagement of the NKp30 triggering receptor.
      ,
      • Morandi B.
      • et al.
      NK cells provide helper signal for CD8+ T cells by inducing the expression of membrane-bound IL-15 on DCs.
      ]. IFNs also directly support the proliferation and activity of certain T cell subsets, including the proliferation of CD8+ T cells [
      • Tough D.F.
      • et al.
      Induction of bystander T cell proliferation by viruses and type I interferon in vivo.
      ] and development of T-helper (TH)-1 cells, while restraining TH2 cell development [
      • Huber J.P.
      • Farrar J.D.
      Regulation of effector and memory T-cell functions by type I interferon.
      ]. As such, type-1 IFNs, among other innate cytokines, are considered important signals in shaping the effector and memory T cell pool.

      Tumor-intrinsic adaptation mechanisms to escape immune clearance

      Constitutive cGAS activation in BRCA1/2-mutant cancer presents a challenge to tumor development, as cGAS/STING pathway activation typically results in IFN signaling and, thus, immune-mediated tumor cell clearance. Therefore, BRCA1/2-mutant tumor cells need to dampen the IFN response that is triggered by genomic instability to sustain their growth and evade the immune system. Although our knowledge on how exactly cancer cells evade clearance by the immune system is incomplete, a number of mechanisms have been described (Figure 3, Figure 4). Understanding these mechanisms and the subsequent tumor-promoting effects may enable future stratification of cGAS/STING-targeting therapies in the clinical setting.
      Figure 3
      Figure 3Adaptation mechanisms of BRCA1/2-mutant cancer cells to survive inflammatory signaling.
      An overview is provided of tumor-cell intrinsic and extrinsic mechanisms by which BRCA1/2-mutant cells evade clearance by the immune system. Abbreviations: altEJ, alternative end-joining; EJ, end-joining; HR, homologous recombination; TME, tumor microenvironment.
      Figure 4
      Figure 4Therapeutic opportunities to enhance immune clearance of BRCA1/2-mutant cancers.
      Several therapeutic strategies are highlighted that could enhance the immune clearance of BRCA1/2-mutant cancer cells. Whereas PARP inhibition or cell cycle checkpoint perturbation can be used to enhance the load of cytoplasmic DNA, STING agonist treatment can be used to directly activate inflammatory signaling. Immune checkpoint inhibitors and ENPP1 inhibition can be used to activate immune cells in the tumor microenvironment.
      A key mechanism that suppresses cGAS/STING signaling is the same mechanism by which HR-defective cells limit their genome instability: restoration or rewiring of DNA repair will prevent the generation of cytoplasmic DNA and will decrease the cues that trigger inflammatory signaling. For instance, POLQ-mediated repair [
      • Ceccaldi R.
      • et al.
      Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair.
      ] is upregulated in HR-defective tumors and was recently established as a therapeutic vulnerability of BRCA1/2-mutant cancers [
      • Zatreanu D.
      • et al.
      Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.
      ]. POLQ inhibition yields micronuclei and IFN signaling [
      • Wang A.
      • et al.
      Abstract I14: Polymerase theta synthetic lethal interaction in homologous recombination-deficient pancreatic ductal adenocarcinoma.
      ], illustrating that utilization of alternative repair pathways in BRCA1/2 mutant cancers prevents excessive missegregation of chromosome fragments and the accumulation of cytoplasmic DNA. Likewise, BRCA1/2-mutant cells depend on Cip2A and TopBp1, which form a complex with Mdc1 to tether chromosome fragments during mitosis, preventing the generation of micronuclei [
      • Adam S.
      • et al.
      CIP2A is a prime synthetic-lethal target for BRCA-mutated cancers.
      ,
      • De Marco Zompit M.
      • et al.
      The CIP2A-TOPBP1 complex safeguards chromosomal stability during mitosis.
      ]. In addition, several mechanisms have been described by which HR-deficient cells can manage with defective replication fork protection, including the inactivation of PAXIP1 [
      • Ray Chaudhuri A.
      • et al.
      Replication fork stability confers chemoresistance in BRCA-deficient cells.
      ] and EZH2 [
      • Rondinelli B.
      • et al.
      EZH2 promotes degradation of stalled replication forks by recruiting MUS81 through histone H3 trimethylation.
      ]. Whether restored fork protection in BRCA1/2-mutant cancers affects cGAS/STING signaling is unknown.
      Besides preventing cytoplasmic DNA, several enzymes degrade cytoplasmic DNA and thereby suppress an IFN response, including TREX1 and RNAseH1. However, TREX1 and RNAseH1-mediated degradation of cytoplasmic DNA does not appear to be a significant compensatory mechanism in BRCA1/2-mutant cancers [
      • Mohr L.
      • et al.
      ER-directed TREX1 limits cGAS activation at micronuclei.
      ,
      • Yang Y.-G.
      • et al.
      Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease.
      ]. Another mechanism by which IFN signaling could be suppressed is the downregulation of DNA sensors or their effector proteins, such as cGAS and STING. While it is striking that cGAS and STING are rarely mutated across cancers [
      • Bakhoum S.F.
      • et al.
      Chromosomal instability drives metastasis through a cytosolic DNA response.
      ,
      • Konno H.
      • et al.
      Suppression of STING signaling through epigenetic silencing and missense mutation impedes DNA damage mediated cytokine production.
      ], promoter methylation of both cGAS and STING results in their downregulation in many solid tumors [
      • Konno H.
      • et al.
      Suppression of STING signaling through epigenetic silencing and missense mutation impedes DNA damage mediated cytokine production.
      ]. Treatment of melanoma and ovarian cell lines with demethylating agents results in rescue of cGAS and/or STING expression [
      • Falahat R.
      • et al.
      Epigenetic reprogramming of tumor cell-intrinsic STING function sculpts antigenicity and T cell recognition of melanoma.
      ,
      • De Queiroz N.M.G.P.
      • et al.
      Ovarian cancer cells commonly exhibit defective STING signaling which affects sensitivity to viral oncolysis.
      ]. Moreover, virus-driven cancers, such as human papilloma virus-driven cervical cancers, downregulate cGAS/STING signaling via direct binding of viral onco-proteins to STING [
      • Lau A.
      • et al.
      DNA tumor virus oncogenes antagonize the cGAS-STING DNA-sensing pathway.
      ]. However, it is not currently known how often these epigenetic or other silencing events occur in BRCA1/2-mutant or genomically unstable cancers; whether cGAS/STING signaling persists in BRCA1/2-mutant cancers by accident or through active mechanisms remains an important question.
      Important cell intrinsic mechanisms that modulate inflammatory signaling involve common tumor suppressor genes and oncogenes. TP53 mutations affecting the DNA binding or tetramerization domains result in binding of p53 to TBK1, preventing STING/TBK1/IRF3 activation and subsequent IFN signaling [
      • Ghosh M.
      • et al.
      Mutant p53 suppresses innate immune signaling to promote tumorigenesis.
      ], reflecting a potential mechanism of immunosuppression in TP53-mutant cancers. Additionally, common driver oncogenes may exert their tumorigenic effect through immune suppression. Early on, N-MYC expression was linked to downregulation of MHC-1, which possibly prevents neoantigen-mediated immune clearance [
      • Bernards R.
      • et al.
      N-myc amplification causes down-modulation of MHC class I antigen expression in neuroblastoma.
      ]. More recently, C-MYC, in conjunction with MIZ1, was shown to directly repress IFN gene expression in tumor cells, in a model of KRAS/CMYC-driven pancreatic cancer [
      • Muthalagu N.
      • et al.
      Repression of the type I interferon pathway underlies MYC- and KRAS-dependent evasion of NK and B cells in pancreatic ductal adenocarcinoma.
      ,
      • Sodir N.M.
      • et al.
      MYC instructs and maintains pancreatic adenocarcinoma phenotype.
      ]. C-MYC appears to act as a generic suppressor of inflammatory signaling, as it also suppresses IFN signaling in a model of BRCA1-mutant breast cancer [
      • Zimmerli D.
      • et al.
      MYC promotes immune-suppression in TNBC via inhibition of IFN signaling.
      ]. C-MYC-mediated suppression of IFN signaling likely reflects a clinically relevant mechanism, with C-MYC being frequently amplified in BRCA1/2-mutant cancers [
      • Annunziato S.
      • et al.
      Comparative oncogenomics identifies combinations of driver genes and drug targets in BRCA1-mutated breast cancer.
      ]. In addition to these cell intrinsic changes, oncogene expression has been linked to extensive modulation of the TME.

      Modulation of the microenvironment to escape immune clearance

      The TME of BRCA1/2-mutant tumors is closely linked to the native environment in which a tumor arises, illustrated by site-specific characteristics of tissue-resident macrophages [
      • Ham S.
      • et al.
      The impact of the cancer microenvironment on macrophage phenotypes.
      ]. However, a common characteristic across sites is the increased immune infiltration in breast, ovarian, and prostate BRCA1/2-mutant tumors [
      • Lord C.J.
      • Ashworth A.
      BRCAness revisited.
      ,
      • Soslow R.A.
      • et al.
      Morphologic patterns associated with BRCA1 and BRCA2 genotype in ovarian carcinoma.
      ,
      • Calagua C.
      • et al.
      A subset of localized prostate cancer displays an immunogenic phenotype associated with losses of key tumor suppressor genes.
      ]. The composition of this immune infiltrate plays a crucial role in tumor progression and therapeutic response. Like other cancers, BRCA1/2-mutant tumors can modulate their TME to avoid immune clearance and harness tumor-promoting inflammation.
      Tumor cells actively modify the TME to suppress immune responses, through secretion of immune-suppressive cytokines. For instance, production of immune-suppressive cytokines (e.g., IL-10) prevents maturation of DCs [
      • Matsuda M.
      • et al.
      Interleukin 10 pretreatment protects target cells from tumor- and allo-specific cytotoxic T cells and downregulates HLA class I expression.
      ]. Also, amplification of C-MYC promotes the secretion of CCL9 and IL-23, leading to suppression of immune responses in the local microenvironment [
      • Kortlever R.M.
      • et al.
      Myc cooperates with Ras by programming inflammation and immune suppression.
      ]. Conversely, C-MYC overexpression leads to suppression of IFN signaling, along with decreased secretion of proinflammatory cytokines [
      • Muthalagu N.
      • et al.
      Repression of the type I interferon pathway underlies MYC- and KRAS-dependent evasion of NK and B cells in pancreatic ductal adenocarcinoma.
      ,
      • Sodir N.M.
      • et al.
      MYC instructs and maintains pancreatic adenocarcinoma phenotype.
      ,
      • Zimmerli D.
      • et al.
      MYC promotes immune-suppression in TNBC via inhibition of IFN signaling.
      ]. Consequently, C-MYC overexpression depletes immune cells from the TME of BRCA1-mutant tumors and prevents killing of organoids derived from BRCA1-mutant cancers by T cells [
      • Zimmerli D.
      • et al.
      MYC promotes immune-suppression in TNBC via inhibition of IFN signaling.
      ].
      A role of constitutive cGAS/STING activation in recruiting immune-suppressive cells has been described in various cancers, although it is currently not characterized in BRCA-mutant cancers. However, it has been reported that radiation therapy results in STING-dependent recruitment of myeloid-derived suppressor cells (MDSCs) [
      • Liang H.
      • et al.
      Host STING-dependent MDSC mobilization drives extrinsic radiation resistance.
      ], suggesting a potential role in genomically unstable tumors. In addition, STING activation is required for PD-L1 upregulation in response to DNA damaging treatment [
      • Parkes E.E.
      • et al.
      Activation of STING-dependent innate immune signaling by S-phase-specific DNA damage in breast cancer.
      ,
      • Grabosch S.
      • et al.
      Cisplatin-induced immune modulation in ovarian cancer mouse models with distinct inflammation profiles.
      ,
      • Wang W.
      • et al.
      Upregulation of PD-L1 via HMGB1-activated IRF3 and NF-κB contributes to UV radiation-induced immune suppression.
      ] and STING agonists have been reported to upregulate PD-L1 [
      • Fu J.
      • et al.
      STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade.
      ,
      • Corrales L.
      • et al.
      The host STING pathway at the interface of cancer and immunity.
      ], suggesting that the increased PD-L1 expression observed in BRCA1/2-mutant cancers could be related to cGAS/STING pathway activation.
      Constitutive activation of the cGAS/STING pathway appears to result in rewiring of downstream signaling, with preferential activation of the noncanonical NF-κB/RelB pathway as opposed to TBK1/IRF3 signaling. Specifically, activation of RelB is associated with a paucity of IFN production in chromosomally unstable cells [
      • Bakhoum S.F.
      • et al.
      Chromosomal instability drives metastasis through a cytosolic DNA response.
      ]. Similarly, STING-dependent RelB signaling results in suppression of IFN production in DCs following radiation treatment and antagonizes IRF3/canonical NF-κB pathways [
      • Hou Y.
      • et al.
      Non-canonical NF-κB antagonizes STING sensor-mediated DNA sensing in radiotherapy.
      ]. Earlier reports suggested that the TNF receptor-associated factor TRAF3 could mediate noncanonical NF-κB activation downstream of STING in response to cytosolic DNA [
      • Abe T.
      • Barber G.N.
      Cytosolic-DNA-mediated, STING-dependent proinflammatory gene induction necessitates canonical NF-κB activation through TBK1.
      ]. However, the role of TRAF3 in downstream modulation of the STING pathway remains unclear. Noncanonical NF-κB activation itself has both pro- and antitumorigenic effects, leading to chronic tumor-promoting inflammation and mediating the development of tertiary lymphoid structure development, associated with an improved response to immune checkpoint blockade [
      • Yu H.
      • et al.
      Targeting NF-κB pathway for the therapy of diseases: mechanism and clinical study.
      ,
      • Cabrita R.
      • et al.
      Tertiary lymphoid structures improve immunotherapy and survival in melanoma.
      ,
      • Helmink B.A.
      • et al.
      B cells and tertiary lymphoid structures promote immunotherapy response.
      ]. Additionally, it remains unknown how chronic STING-mediated RelB activation shapes the TME and modifies host immune responses. Given the central role of cGAS/STING signaling in the tumor immune microenvironment and response to immune-targeting therapies, identifying the event(s) responsible for pathway choice downstream of STING in genomically unstable cancer remains a key question for the field.
      An intriguing mechanism of TME modulation downstream of STING involves the transmembrane pyrophosphatase ENPP1. ENPP1 is a negative regulator of cGAS signaling and was found to be upregulated in chromosomally unstable tumors [
      • Li J.
      • et al.
      Metastasis and immune evasion from extracellular cGAMP hydrolysis.
      ]. ENPP1 hydrolyzes cyclic dinucleotides, including 2′3′cGAMP, resulting in pA(3′5′)pG and subsequent AMP production [
      • Kato K.
      • et al.
      Structural insights into cGAMP degradation by ecto-nucleotide pyrophosphatase phosphodiesterase 1.
      ]. Further breakdown of AMP by NT5E (CD73) generates immunosuppressive adenosine within the TME [
      • Li J.
      • et al.
      Metastasis and immune evasion from extracellular cGAMP hydrolysis.
      ]. Therefore, ENPP1 upregulation results in restriction of 2′3′cGAMP transfer to host immune cells, preventing STING activation and IFN signaling. The increased adenosine levels amplify this immunosuppressive effect and promote tumor metastasis [
      • Vijayan D.
      • et al.
      Targeting immunosuppressive adenosine in cancer.
      ]. The expression of ENPP1 in cancers with genomic instability due to BRCA1/2 mutation requires further investigation and represents a potential therapeutic target [
      • Carozza J.A.
      • et al.
      Structure-aided development of small-molecule inhibitors of ENPP1, the extracellular phosphodiesterase of the immunotransmitter cGAMP.
      ,
      • Baird J.
      • et al.
      MV-626, a potent and selective inhibitor of ENPP1 enhances STING activation and augments T-cell mediated anti-tumor activity in vivo.
      ].
      Constitutive cGAS/STING activity and PD-L1 upregulation in BRCA1/2-mutant tumors could render them highly sensitive to immune checkpoint blockade. In addition, current evidence based on TMB [
      • Zhou Z.
      • Li M.
      Evaluation of BRCA1 and BRCA2 as indicators of response to immune checkpoint inhibitors.
      ] and DNA repair deficiency [
      • Hsiehchen D.
      • et al.
      DNA repair gene mutations as predictors of immune checkpoint inhibitor response beyond tumor mutation burden.
      ] suggests that increased response rates could be expected in BRCA1/2-mutant tumors. In contrast, the majority of patients with BRCA1/2-mutant tumors do not derive any clinical benefit from immune checkpoint blockade. Indeed, prospective trials of PARP inhibition and anti-PD-1 in platinum-resistant ovarian cancer [
      • Konstantinopoulos P.A.
      • et al.
      Single-arm phases 1 and 2 trial of niraparib in combination with pembrolizumab in patients with recurrent platinum-resistant ovarian carcinoma.
      ] and advanced triple-negative breast cancer (TNBC) [
      • Vinayak S.
      • et al.
      Abstract PD5-02: durability of clinical benefit with niraparib + pembrolizumab in patients with advanced triple-negative breast cancer beyond BRCA: (TOPACIO/Keynote-162).
      ] have not demonstrated improved responses in BRCA1/2-mutant tumors, although patient numbers were small. Potential reasons for this outcome may include treatment at an advanced stage, with weakened immune responses in pretreated disease [
      • Hiam-Galvez K.J.
      • et al.
      Systemic immunity in cancer.
      ,
      • Shaked Y.
      The pro-tumorigenic host response to cancer therapies.
      ]. Additionally, TME features play an important role in intrinsic immunotherapy resistance.

      Myeloid and lymphoid cells in the BRCA-mutant TME

      BRCA1-mutant breast cancers contain increased tumor-promoting immunosuppressive macrophages compared with BRCA-wild-type disease [
      • Mehta A.K.
      • et al.
      Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer.
      ]. Single-cell analysis of BRCA1-mutant TNBCs additionally demonstrated increased infiltration of CD4+ Tregs and exhausted (PD-1+) T cells, indicating an immunosuppressed TME [
      • Mehta A.K.
      • et al.
      Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer.
      ]. Other studies have similarly reported increased infiltration of Tregs in BRCA1/2-mutant breast cancers [
      • Li Y.
      • et al.
      Comprehensive analysis of regulatory factors and immune-associated patterns to decipher common and BRCA1/2 mutation-type-specific critical regulation in breast cancer.
      ]. Moreover, in a study of BRCA2-mutant prostate cancer, increased Tregs were identified in early-stage disease compared with BRCA-wild type [
      • Jenzer M.
      • et al.
      The BRCA2 mutation status shapes the immune phenotype of prostate cancer.
      ]. In general, genomic instability arising from HR deficiency promotes immune evasion via ‘M2’-like immunosuppressive polarization of macrophages and influx of Tregs, particularly as instability progresses towards aneuploidy with chromosomal arm or whole chromosomal copy number alterations [
      • Davoli T.
      • et al.
      Tumor aneuploidy correlates with markers of immune evasion and with reduced response to immunotherapy.
      ]. cGAS/STING pathway activation in genomically unstable cancers may play a role in this immunosuppressive skewing of the TME via upregulation of ENPP1 and subsequent increased adenosine levels [
      • Li J.
      • et al.
      Metastasis and immune evasion from extracellular cGAMP hydrolysis.
      ]. Additionally, downstream STING-mediated responses, such as upregulation of CCL2, CCL7, and CCL12, result in infiltration of MDSCs, which promote immunosuppression and therapeutic resistance [
      • Liang H.
      • et al.
      Host STING-dependent MDSC mobilization drives extrinsic radiation resistance.
      ]. Thus, TME features of BRCA1/2-mutant cancers tip the balance in favor of immune-resistance and may account for the poor response rates to immune checkpoint blockade observed in these tumors. As immune editing in genomically unstable cancers is an iterative process, it is not clear how the immune microenvironment of early-stage BRCA1/2-mutant cancers compares with metastatic disease. The studies cited earlier have primarily been conducted on early-stage disease (where resection specimens and genetic data are readily available). Further longitudinal analysis of BRCA1/2-mutant primary cancers and metastases with a focus on the immune TME would enable design of immune-targeting strategies in both early- and late-stage genomically unstable cancers.

      Metabolic and microenvironment immune evasion in BRCA-mutant cancers

      BRCA1/2-mutant tumors also alter immunometabolism to suppress immune responses. In DNA repair-deficient breast cancers with constitutive cGAS/STING pathway activation, indoleamine 2,3-dioxygenase 1 (IDO1) was found to be among the top upregulated genes [
      • Mulligan J.M.
      • et al.
      Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer.
      ,
      • Parkes E.E.
      • et al.
      Activation of STING-dependent innate immune signaling by S-phase-specific DNA damage in breast cancer.
      ]. Consistently, in high-grade TNBC, IDO1 is coexpressed in 70% of tumor cell PD-L1 positive cases, with a trend towards increased coexpression in BRCA1/2-mutant cancer [
      • Dill E.A.
      • et al.
      IDO expression in breast cancer: an assessment of 281 primary and metastatic cases with comparison to PD-L1.
      ]. IDO1 catabolizes tryptophan, resulting in anergy, cell cycle arrest, or apoptosis of T cells [
      • Munn D.H.
      • et al.
      GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2, 3-dioxygenase.
      ]. Additional immunosuppressive effects include IL6 production and expansion of protumorigenic MDSCs [
      • Smith C.
      • et al.
      IDO is a nodal pathogenic driver of lung cancer and metastasis development.
      ]. STING activity in the TME induces IDO1, suggesting a targetable mechanism of immune escape in BRCA1/2-mutant tumors. Enthusiasm for IDO1 inhibition was dampened in the clinical trial setting following a Phase III study that did not demonstrate improved responses using combination IDO1 inhibitor and anti-PD-1 treatment, compared with PD-1 targeting alone [
      • Long G.V.
      • et al.
      Epacadostat plus pembrolizumab versus placebo plus pembrolizumab in patients with unresectable or metastatic melanoma (ECHO-301/KEYNOTE-252): a phase 3, randomised, double-blind study.
      ]. However, stratification of patients, selecting those with STING-active, BRCA1/2-mutant tumors, may be fruitful for future studies using combination IDO1-targeting approaches.
      Tumors are complex collections of cells, comprising not just tumor and immune cells. Modifications within the tumor vasculature, such as vascular endothelial growth factor (VEGF)-driven angiogenesis, promote tumor growth and immunosuppression by promotion of Treg and MDSC activity [
      • Rahma O.E.
      • Hodi F.S.
      The intersection between tumor angiogenesis and immune suppression.
      ]. VEGF-A upregulation further correlates with the presence of BRCA1/2 mutations [
      • Ruscito I.
      • et al.
      Characterisation of tumour microvessel density during progression of high-grade serous ovarian cancer: clinico-pathological impact (an OCTIPS Consortium study).
      ]. A direct link between BRCA1 loss, STING activity, and VEGF-A upregulation was recently reported, whereby a Brca1-knockout model of ovarian cancer demonstrated increased sensitivity to combination immune checkpoint blockade in a Sting-null background, associated with reduced VEGF activity [
      • Bruand M.
      • et al.
      Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.
      ]. In contrast, acute stimulation of STING using exogeneous agonists resulted in reduced CD31+ vessel density, demonstrating the differing effects of constitutive versus acute STING pathway stimulation [
      • Yang H.
      • et al.
      STING activation reprograms tumor vasculatures and synergizes with VEGFR2 blockade.
      ]. Recently, the combination of PARP inhibition and the anti-VEGF agent bevacizumab was approved as maintenance treatment for BRCA-mutant or homologous recombination deficiency ovarian cancer [
      • Ray-Coquard I.
      • et al.
      Olaparib plus bevacizumab as first-line maintenance in ovarian cancer.
      ]. Taken together, treatment of inflamed BRCA1/2-mutant tumors with antiangiogenics may overcome intrinsic STING-mediated immunosuppression, permitting response to immune targeting agents.
      Mechanical properties of the TME can influence chromosomal instability. Cellular stresses caused by 2D cell culture induces increased chromosomal instability, which is rescued in 3D model systems and is dependent on integrins [
      • Knouse K.A.
      • et al.
      Chromosome segregation fidelity in epithelia requires tissue architecture.
      ]. Additionally, cancer cells cultured using stiff hydrogels have increased chromosomal instability [
      • López-Carrasco A.
      • et al.
      Impact of extracellular matrix stiffness on genomic heterogeneity in MYCN-amplified neuroblastoma cell line.
      ]. The composition of the extracellular matrix, which determines microenvironmental characteristics of the tumor, such as stiffness, is mediated by cancer-associated fibroblasts (CAFs) [
      • DeLeon-Pennell K.Y.
      • et al.
      Fibroblasts: the arbiters of extracellular matrix remodeling.
      ]. Potential variations in fibroblast phenotypes between BRCA-wild type and BRCA1/2-mutant cancers are not clearly defined. Recent data suggests there are key alterations between fibroblasts in BRCA-wild type and BRCA-mutant pancreatic cancers, with the latter containing increased clusterin-expressing CAFs associated with inflammatory gene expression [
      • Shaashua L.
      • et al.
      BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking CLU+ CAF expression with HSF1 signaling.
      ]. However, potential links between STING signaling and subsequent effects on the mechanical TME are unclear. Given the close relationship between immune cell exclusion and extracellular matrix composition [
      • Pickup M.W.
      • et al.
      The extracellular matrix modulates the hallmarks of cancer.
      ], the mechanical properties of the TME represent a further barrier to immunotherapy for exploration in genomically unstable cancers.
      These direct links between genomic characteristics of tumor cells and subsequent immune TME support a layered approach to patient stratification for combination immune targeting treatments and may be beneficial in the development of personalized immunotherapy. Understanding the immunosuppressive mechanisms adopted by cGAS/STING active cancers may enable the identification of novel therapeutic strategies for clinical exploration.

      Clinical implications

      In line with a profound role in modulating immune function, tumor-cell intrinsic IFN signaling is a key determinant in the response to immune checkpoint inhibitors. Functional genetic CRISPR/Cas9 screens in cocultures of tumor cells and T cells identified multiple IFN signaling components to be required for effective immune checkpoint inhibitor response [
      • Manguso R.T.
      • et al.
      In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.
      ,
      • Patel S.J.
      • et al.
      Identification of essential genes for cancer immunotherapy.
      ]. Conversely, a common mechanism to evade immune clearance, including for BRCA1/2-mutant tumors, involves suppression of IFN signaling [
      • Bakhoum S.F.
      • Cantley L.C.
      The multifaceted role of chromosomal instability in cancer and its microenvironment.
      ]. Consequently, approaches to therapeutically increase inflammatory signaling to increase benefit of immune checkpoint inhibitors have been proposed and preclinically tested.
      PARP inhibitors exacerbate the accumulation of cytoplasmic DNA, the subsequent activation of cGAS/STING, and immune reactivity in BRCA1-mutant or otherwise HR-deficient tumor models [
      • Chabanon R.M.
      • et al.
      PARP inhibition enhances tumor cell-intrinsic immunity in ERCC1-deficient non-small cell lung cancer.
      ,
      • Pantelidou C.
      • et al.
      PARP inhibitor efficacy depends on CD8+ T-cell recruitment via intratumoral STING pathway activation in BRCA-deficient models of triple-negative breast cancer.
      ]. Notably, the therapeutic effects of PARP inhibition were in large part dependent on T cells, stressing the involvement of immune cells in the in vivo effects of PARP inhibition BRCA-deficient tumors [
      • Pantelidou C.
      • et al.
      PARP inhibitor efficacy depends on CD8+ T-cell recruitment via intratumoral STING pathway activation in BRCA-deficient models of triple-negative breast cancer.
      ]. As a logical follow-up approach, the combination of PARP inhibitors with immune checkpoint inhibitors was tested. In agreement with earlier findings, PARP inhibition in a BRCA1-deficient humanized mouse TNBC xenograft model resulted in increased T cells in the TME and elevated IFN signaling [
      • Wang Z.
      • et al.
      Niraparib activates interferon signaling and potentiates anti-PD-1 antibody efficacy in tumor models.
      ]. Of note, synergistic effects were also observed in HR-proficient models in this study.
      Approaches combining PARP inhibition with immune checkpoint blockers have been evaluated in clinical trials involving patients with BRCA1/2-mutant breast cancer, with ongoing studies in BRCA2-mutant castration-resistant prostate cancer (NCT02484404)i, BRCA2-mutant bladder cancer (NCT02553642ii, NCT01928394iii, NCT02108652)iv, and BRCA1/2-mutant ovarian cancer (NCT02657889)v. Interestingly, the activity of PD1/CTLA4 inhibitors is also under investigation in the absence of PARP inhibitors in HR-deficient cancers (NCT02985957)vi. These approaches aim to prime local immune responses, employing the targeted effects of PARP inhibition, to enable subsequent improved responses to immunotherapy. However, in a similar manner to radiotherapy priming for immune responses, it is likely that optimization of scheduling and dose of PARP inhibition will be indicated. Of note, several approaches have been explored to increase the therapeutic effects of PARP inhibitors, including cell cycle checkpoint inhibitors. Targeting of the WEE1, ATR, and CHK1 kinases promotes entry into mitosis with unresolved DNA lesions and exacerbates the effects of PARP inhibition in BRCA1/2-mutant cancer cells [
      • Schoonen P.M.
      • et al.
      Progression through mitosis promotes PARP inhibitor-induced cytotoxicity in homologous recombination-deficient cancer cells.
      ,
      • Fang Y.
      • et al.
      Sequential therapy with PARP and WEE1 inhibitors minimizes toxicity while maintaining efficacy.
      ,
      • Kim H.
      • et al.
      Combining PARP with ATR inhibition overcomes PARP inhibitor and platinum resistance in ovarian cancer models.
      ,
      • Kim H.
      • et al.
      Targeting the ATR/CHK1 axis with PARP inhibition results in tumor regression in BRCA-mutant ovarian cancer models.
      ]. Combined ATR and PARP inhibition further promotes IFN signaling in BRCA1/2-mutant cancer cells [
      • Schoonen P.M.
      • et al.
      Premature mitotic entry induced by ATR inhibition potentiates olaparib inhibition-mediated genomic instability, inflammatory signaling, and cytotoxicity in BRCA2-deficient cancer cells.
      ], although the in vivo consequences of such combination approaches need to be explored.
      STING agonists have been proposed as effective treatments for BRCA1/2-mutant cancers, by restoring IFN signaling in the TME. In a preclinical model of Brca1-deficient breast cancer, STING agonist treatment led to increased IFN-dependent recruitment of cytotoxic T cells and subsequent tumor response [
      • Pantelidou C.
      • et al.
      STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer.
      ]. STING agonists are typically analogs of 2′3′cGAMP, delivered intratumorally to activate local immune responses. While STING agonism may be effective in BRCA1/2-mutant tumors due to intrinsic inflammation, chronic adaptation to inflammatory signaling may present additional barriers to therapeutic efficacy. Preventing generic activation of STING in the TME by using next-generation STING agonists targeted to antigen-presenting cells [
      • Jang S.C.
      • et al.
      ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance.
      ] may overcome these barriers. However, these approaches do not yet address factors preventing recruitment of immune cells to the TME for activation.

      Concluding remarks

      Similar to other defining characteristics of cancer, phenotypes related to BRCA1/2 mutation exist on a spectrum, with further complexity added by site-specific TME features. This complexity prevents an uncomplicated correlation between BRCA1/2 mutations, cGAS-STING activation, the immune TME, and treatment response. However, insight into the immune-evading mechanisms common to STING-active, BRCA1/2-mutant tumors is improving apace with evolving techniques of immune phenotyping of tumors. Combined, these new insights will enable personalized combination approaches of immune-targeting agents, alongside treatments targeting resistance mechanisms, to ultimately provide the greatest future improvements in patient outcomes (see Outstanding questions).
      Which aspects of defective DNA repair in BRCA1/2-mutant cancers trigger inflammatory signaling? Research involving separation-of-function mutants of BRCA1/2 and combinations of mutations that rescue specific repair defects is warranted to address this.
      Which DNA/chromatin features determine cGAS/STING activation and inflammatory signaling? It remains incompletely clear what the exact cue is for inflammatory signaling in HR-deficient cells and what the contribution is of micronuclei, micronucleus rupture, mitotic chromosome bridges, or mitosis-independent routes.
      To what extent do different sources of genome instability lead to specific or distinct inflammatory signaling profiles? A comprehensive analysis involving various genome maintenance defects, including DNA repair defects, CIN, and telomere erosion is required.
      What are the key differences between acute versus chronic inflammatory signaling? What are the downstream events that determine dominance of IRF3 or noncanonical NF-κB pathway signaling?
      What is the contribution of the various DNA sensing pathways in driving inflammatory signaling in cancers? It remains unclear how the different DNA sensors (cGAS, RIG-I, inflammasome, TLRs) collaborate or are active in different contexts.
      Can immune checkpoint therapy be used to clear early-stage cancer lesions with genome instability?
      Does BRCA1/2 haploinsufficiency trigger inflammatory signaling, and does anticancer immune clearance play a role in tumor development?
      To what extent are the mechanisms that BRCA1/2-mutant tumors employ to evade the immune system reversible?
      How can inflammatory signaling be reinstated therapeutically, in a way that promotes antitumor immunity?

      Acknowledgments

      This work was supported by an ERC Consolidator grant to M.A.T.M.v.V. ( ERC CoS 682421 ).

      Declaration of interests

      No interests are declared.

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      Glossary

      BRCAness
      defective homologous recombination repair, phenocopying BRCA1 or BRCA2 mutation.
      Breast cancer 1, early onset (BRCA1)
      DNA repair protein, involved in initial steps of HR repair. Germline mutations in the BRCA1 gene lead to predisposition to early onset breast and ovarian cancer.
      Breast cancer 2, early onset (BRCA2)
      DNA repair protein, involved in recruitment of Rad51 in HR repair. Germline mutations in the BRCA2 gene lead to predisposition to early onset breast and ovarian cancer.
      Cyclic GMP-AMP synthase (cGAS)
      sensor of cytoplasmic DNA and activator of STING.
      Fanconi anemia (FA)
      inherited disorder caused by mutation of one of the FA genes, leading to a defect in the repair of DNA crosslinks.
      Genomic instability
      defects in genome maintenance leading to progressive accumulation of structural and numerical changes to the genome.
      Homologous recombination (HR)
      pathway for the repair of DNA DSBs and stalled replication forks using a homologous template DNA. HRR acts predominantly in S/G2 phase of the cell cycle.
      IFNα
      type I interferon-α; a cytokine that activates transcription in response to triggers of the innate immune response.
      Interferon regulatory factor-3 (IRF3)
      transcription factor that, upon activation by TBK1, transactivates cytokines and type-1 interferon genes.
      JAK/STAT
      signal transduction pathway, involving Janus kinases (JAK) and STAT transcription factors, controlling immunity, proliferation, and cell survival.
      Micronucleus
      entire chromosome or fragment thereof that forms separate nuclear structure upon missegregation during mitosis.
      Retinoic acid-inducible gene-I (RIG-I)
      cytosolic pattern recognition receptor, responsible for the type-1 interferon response.
      Stimulator of interferon genes (STING)
      adaptor protein that is activated by cyclic GMP-AMP and enhances TBK1 activity.
      TANK-binding kinase 1 (TBK1)
      protein kinase downstream of STING to trigger innate immune responses.
      Tumor-infiltrating lymphocytes
      lymphocyte-derived immune cells that reside in the tumor microenvironment.
      Tumor mutational burden (TMB)
      total amount of DNA mutations present in the genome of a cancer.
      Type II interferon gamma (IFNγ)
      cytokine critical for immune activation and defense against viral infection.