RAD52-dependent mitotic DNA synthesis is required for genome stability in Cyclin E1-overexpressing cells.

Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.

FIRRM and FIGNL1: partners in the regulation of homologous recombination.

DNA repair through homologous recombination (HR) is of vital importance for maintaining genome stability and preventing tumorigenesis. RAD51 is the core component of HR, catalyzing the strand invasion and homology search. Here, we highlight recent findings on FIRRM and FIGNL1 as regulators of the dynamics of RAD51.

cGAS-STING pathway expression correlates with genomic instability and immune cell infiltration in breast cancer.

Genomic instability, as caused by oncogene-induced replication stress, can lead to the activation of inflammatory signaling, involving the cGAS-STING and JAK-STAT pathways. Inflammatory signaling has been associated with pro-tumorigenic features, but also with favorable response to treatment, including to immune checkpoint inhibition. In this study, we aim to explore relations between inflammatory signaling, markers of replication stress, and immune cell infiltration in breast cancer. Expression levels of cGAS-STING signaling components (STING, phospho-TBK1, and phospho-STAT1), replication stress markers (γH2AX and pRPA), replication stress-related proto-oncogenes (Cyclin E1 and c-Myc) and immune cell markers (CD20, CD4, and CD57) are determined immunohistochemically on primary breast cancer samples (n = 380). RNA-sequencing data from TCGA (n = 1082) and METABRIC (n = 1904) are used to calculate cGAS-STING scores. pTBK1, pSTAT1 expression and cGAS-STING pathway scores are all increased in triple-negative breast cancers compared to other subtypes. Expression of γH2AX, pRPA, Cyclin E1, c-Myc, and immune cell infiltration positively correlate with p-STAT1 expression (P < 0.001). Additionally, we observe significant positive associations between expression of pTBK1 and γH2AX, pRPA, c-Myc, and number of CD4+ cells and CD20+ cells. Also, cGAS-STING scores are correlated with genomic instability metrics, such as homologous recombination deficiency (P < 0.001) and tumor mutational burden (P < 0.01). Moreover, data from the I-SPY2 clinical trial (n = 71) confirms that higher cGAS-STING scores are observed in breast cancer patients who responded to immunotherapy combined with chemotherapy. In conclusion, the cGAS-STING pathway is highly expressed in TNBCs and is correlated with genomic instability and immune cell infiltration.

EXO1 protects BRCA1-deficient cells against toxic DNA lesions.

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.

The Fanconi anemia core complex promotes CtIP-dependent end-resection to drive homologous recombination at DNA double-strand breaks.

Homologous Recombination (HR) is a high-fidelity repair mechanism of DNA Double-Strand Breaks (DSBs), which are induced by irradiation, genotoxic chemicals or physiological DNA damaging processes. DSBs are also generated as intermediates during the repair of interstrand crosslinks (ICLs). In this context, the Fanconi anemia (FA) core complex, which is effectively recruited to ICLs, promotes HR-mediated DSB-repair. However, whether the FA core complex also promotes HR at ICL-independent DSBs remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen with cells carrying the DSB-repair reporter DSB-Spectrum. Using isogenic cell-line models, we validated the HR-function of FANCL and Ube2T, and demonstrated a similar function for their ubiquitination-substrate FANCD2. We further show that FANCL and Ube2T are directly recruited to DSBs and are required for the accumulation of FANCD2 at these break sites. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of the nuclease CtIP at DSBs, and consequently for optimal end-resection and Rad51 loading. CtIP overexpression rescues HR in FANCL-deficient cells, validating that FANCL primarily regulates HR by promoting CtIP recruitment. Together, these data demonstrate that the FA core complex and FANCD2 have a dual genome maintenance function by promoting repair of DSBs as well as the repair of ICLs.

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics.

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.

MND1 and PSMC3IP control PARP inhibitor sensitivity in mitotic cells.

The PSMC3IP-MND1 heterodimer promotes meiotic D loop formation before DNA strand exchange. In genome-scale CRISPR-Cas9 mutagenesis and interference screens in mitotic cells, depletion of PSMC3IP or MND1 causes sensitivity to poly (ADP-Ribose) polymerase inhibitors (PARPi) used in cancer treatment. PSMC3IP or MND1 depletion also causes ionizing radiation sensitivity. These effects are independent of PSMC3IP/MND1's role in mitotic alternative lengthening of telomeres. PSMC3IP- or MND1-depleted cells accumulate toxic RAD51 foci in response to DNA damage, show impaired homology-directed DNA repair, and become PARPi sensitive, even in cells lacking both BRCA1 and TP53BP1. Epistasis between PSMC3IP-MND1 and BRCA1/BRCA2 defects suggest that abrogated D loop formation is the cause of PARPi sensitivity. Wild-type PSMC3IP reverses PARPi sensitivity, whereas a PSMC3IP p.Glu201del mutant associated with D loop defects and ovarian dysgenesis does not. These observations suggest that meiotic proteins such as MND1 and PSMC3IP have a greater role in mitotic DNA repair.

WEE1 inhibitor adavosertib in combination with carboplatin in advanced TP53 mutated ovarian cancer: A biomarker-enriched phase II study.

OBJECTIVE: In the first part of this phase II study (NCT01164995), the combination of carboplatin and adavosertib (AZD1775) was shown to be safe and effective in patients with TP53 mutated platinum-resistant ovarian cancer (PROC). Here, we present the results of an additional safety and efficacy cohort and explore predictive biomarkers for resistance and response to this combination treatment. METHODS: This is a phase II, open-label, non-randomized study. Patients with TP53 mutated PROC received carboplatin AUC 5 mg/ ml·min intravenously and adavosertib 225 mg BID orally for 2.5 days in a 21-day cycle. The primary objective is to determine the efficacy and safety of carboplatin and adavosertib. Secondary objectives include progression-free survival (PFS), changes in circulating tumor cells (CTC) and exploration of genomic alterations. RESULTS: Thirty-two patients with a median age of 63 years (39-77 years) were enrolled and received treatment. Twenty-nine patients were evaluable for efficacy. Bone marrow toxicity, nausea and vomiting were the most common adverse events. Twelve patients showed partial response (PR) as best response, resulting in an objective ORR of 41% in the evaluable patients (95% CI: 23%-61%). The median PFS was 5.6 months (95% CI: 3.8-10.3). In patients with tumors harboring CCNE1 amplification, treatment efficacy was slightly but not significantly better. CONCLUSIONS: Adavosertib 225 mg BID for 2.5 days and carboplatin AUC 5 could be safely combined and showed anti-tumor efficacy in patients with PROC. However, bone marrow toxicity remains a point of concern, since this is the most common reason for dose reductions and dose delays.

Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51.

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.

MYC promotes immune-suppression in triple-negative breast cancer via inhibition of interferon signaling.

The limited efficacy of immune checkpoint inhibitor treatment in triple-negative breast cancer (TNBC) patients is attributed to sparse or unresponsive tumor-infiltrating lymphocytes, but the mechanisms that lead to a therapy resistant tumor immune microenvironment are incompletely known. Here we show a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of TNBC proficient or deficient of breast cancer type 1 susceptibility gene (BRCA1), MYC overexpression dramatically decreases lymphocyte infiltration in tumors, along with immune signature remodelling. MYC-mediated suppression of inflammatory signalling induced by BRCA1/2 inactivation is confirmed in human TNBC cell lines. Moreover, MYC overexpression prevents the recruitment and activation of lymphocytes in both human and mouse TNBC co-culture models. Chromatin-immunoprecipitation-sequencing reveals that MYC, together with its co-repressor MIZ1, directly binds promoters of multiple interferon-signalling genes, resulting in their downregulation. MYC overexpression thus counters tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist via suppressing induction of interferon signalling. Together, our data reveal that MYC suppresses innate immunity and facilitates tumor immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs.

Processing DNA lesions during mitosis to prevent genomic instability.

Failure of cells to process toxic double-strand breaks (DSBs) constitutes a major intrinsic source of genome instability, a hallmark of cancer. In contrast with interphase of the cell cycle, canonical repair pathways in response to DSBs are inactivated in mitosis. Although cell cycle checkpoints prevent transmission of DNA lesions into mitosis under physiological condition, cancer cells frequently display mitotic DNA lesions. In this review, we aim to provide an overview of how mitotic cells process lesions that escape checkpoint surveillance. We outline mechanisms that regulate the mitotic DNA damage response and the different types of lesions that are carried over to mitosis, with a focus on joint DNA molecules arising from under-replication and persistent recombination intermediates, as well as DNA catenanes. Additionally, we discuss the processing pathways that resolve each of these lesions in mitosis. Finally, we address the acute and long-term consequences of unresolved mitotic lesions on cellular fate and genome stability.

cGAS-STING drives the IL-6-dependent survival of chromosomally instable cancers.

Chromosomal instability (CIN) drives cancer cell evolution, metastasis and therapy resistance, and is associated with poor prognosis1. CIN leads to micronuclei that release DNA into the cytoplasm after rupture, which triggers activation of inflammatory signalling mediated by cGAS and STING2,3. These two proteins are considered to be tumour suppressors as they promote apoptosis and immunosurveillance. However, cGAS and STING are rarely inactivated in cancer4, and, although they have been implicated in metastasis5, it is not known why loss-of-function mutations do not arise in primary tumours4. Here we show that inactivation of cGAS-STING signalling selectively impairs the survival of triple-negative breast cancer cells that display CIN. CIN triggers IL-6-STAT3-mediated signalling, which depends on the cGAS-STING pathway and the non-canonical NF-κB pathway. Blockade of IL-6 signalling by tocilizumab, a clinically used drug that targets the IL-6 receptor (IL-6R), selectively impairs the growth of cultured triple-negative breast cancer cells that exhibit CIN. Moreover, outgrowth of chromosomally instable tumours is significantly delayed compared with tumours that do not display CIN. Notably, this targetable vulnerability is conserved across cancer types that express high levels of IL-6 and/or IL-6R in vitro and in vivo. Together, our work demonstrates pro-tumorigenic traits of cGAS-STING signalling and explains why the cGAS-STING pathway is rarely inactivated in primary tumours. Repurposing tocilizumab could be a strategy to treat cancers with CIN that overexpress IL-6R.

An mRNA expression-based signature for oncogene-induced replication-stress.

Oncogene-induced replication stress characterizes many aggressive cancers. Several treatments are being developed that target replication stress, however, identification of tumors with high levels of replication stress remains challenging. We describe a gene expression signature of oncogene-induced replication stress. A panel of triple-negative breast cancer (TNBC) and non-transformed cell lines were engineered to overexpress CDC25A, CCNE1 or MYC, which resulted in slower replication kinetics. RNA sequencing analysis revealed a set of 52 commonly upregulated genes. In parallel, mRNA expression analysis of patient-derived tumor samples (TCGA, n = 10,592) also revealed differential gene expression in tumors with amplification of oncogenes that trigger replication stress (CDC25A, CCNE1, MYC, CCND1, MYB, MOS, KRAS, ERBB2, and E2F1). Upon integration, we identified a six-gene signature of oncogene-induced replication stress (NAT10, DDX27, ZNF48, C8ORF33, MOCS3, and MPP6). Immunohistochemical analysis of NAT10 in breast cancer samples (n = 330) showed strong correlation with expression of phospho-RPA (R = 0.451, p = 1.82 × 10-20) and γH2AX (R = 0.304, p = 2.95 × 10-9). Finally, we applied our oncogene-induced replication stress signature to patient samples from TCGA (n = 8,862) and GEO (n = 13,912) to define the levels of replication stress across 27 tumor subtypes, identifying diffuse large B cell lymphoma, ovarian cancer, TNBC and colorectal carcinoma as cancer subtypes with high levels of oncogene-induced replication stress.

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

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.

Genomic instability, inflammatory signaling and response to cancer immunotherapy.

Genomic and chromosomal instability are hallmarks of cancer and shape the genomic composition of cancer cells, thereby determining their behavior and response to treatment. Various genetic and epigenetic alterations in cancer have been linked to genomic instability, including DNA repair defects, oncogene-induced replication stress, and spindle assembly checkpoint malfunction. A consequence of genomic and chromosomal instability is the leakage of DNA from the nucleus into the cytoplasm, either directly or through the formation and subsequent rupture of micronuclei. Cytoplasmic DNA subsequently activates cytoplasmic DNA sensors, triggering downstream pathways, including a type I interferon response. This inflammatory signaling has pleiotropic effects, including enhanced anti-tumor immunity and potentially results in sensitization of cancer cells to immune checkpoint inhibitors. However, cancers frequently evolve mechanisms to avoid immune clearance, including suppression of inflammatory signaling. In this review, we summarize inflammatory signaling pathways induced by various sources of genomic instability, adaptation mechanisms that suppress inflammatory signaling, and implications for cancer immunotherapy.

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma.

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.

Deposition Bias of Chromatin Proteins Inverts under DNA Replication Stress Conditions.

Following DNA replication, equal amounts of chromatin proteins are distributed over sister chromatids by re-deposition of parental chromatin proteins and deposition of newly synthesized chromatin proteins. Molecular mechanisms balancing the allocation of new and old chromatin proteins remain largely unknown. Here, we studied the genome-wide distribution of new chromatin proteins relative to parental DNA template strands and replication initiation zones using the double-click-seq. Under control conditions, new chromatin proteins were preferentially found on DNA replicated by the lagging strand machinery. Strikingly, replication stress induced by hydroxyurea or curaxin treatment and inhibition of ataxia telangiectasia and Rad3-related protein (ATR) or p53 inactivation inverted the observed chromatin protein deposition bias to the strand replicated by the leading strand polymerase in line with previously reported effects on replication protein A occupancy. We propose that asymmetric deposition of newly synthesized chromatin proteins onto sister chromatids reflects differences in the processivity of leading and lagging strand synthesis.

Identification and Validation of Esophageal Squamous Cell Carcinoma Targets for Fluorescence Molecular Endoscopy.

Dysplasia and intramucosal esophageal squamous cell carcinoma (ESCC) frequently go unnoticed with white-light endoscopy and, therefore, progress to invasive tumors. If suitable targets are available, fluorescence molecular endoscopy might be promising to improve early detection. Microarray expression data of patient-derived normal esophagus (n = 120) and ESCC samples (n = 118) were analyzed by functional genomic mRNA (FGmRNA) profiling to predict target upregulation on protein levels. The predicted top 60 upregulated genes were prioritized based on literature and immunohistochemistry (IHC) validation to select the most promising targets for fluorescent imaging. By IHC, GLUT1 showed significantly higher expression in ESCC tissue (30 patients) compared to the normal esophagus adjacent to the tumor (27 patients) (p < 0.001). Ex vivo imaging of GLUT1 with the 2-DG 800CW tracer showed that the mean fluorescence intensity in ESCC (n = 17) and high-grade dysplasia (HGD, n = 13) is higher (p < 0.05) compared to that in low-grade dysplasia (LGD) (n = 7) and to the normal esophagus adjacent to the tumor (n = 5). The sensitivity and specificity of 2-DG 800CW to detect HGD and ESCC is 80% and 83%, respectively (ROC = 0.85). We identified and validated GLUT1 as a promising molecular imaging target and demonstrated that fluorescent imaging after topical application of 2-DG 800CW can differentiate HGD and ESCC from LGD and normal esophagus.

A synthetic lethal screen identifies HDAC4 as a potential target in MELK overexpressing cancers.

Maternal embryonic leucine zipper kinase (MELK) is frequently overexpressed in cancer, but the role of MELK in cancer is still poorly understood. MELK was shown to have roles in many cancer-associated processes including tumor growth, chemotherapy resistance, and tumor recurrence. To determine whether the frequent overexpression of MELK can be exploited in therapy, we performed a high-throughput screen using a library of Saccharomyces cerevisiae mutants to identify genes whose functions become essential when MELK is overexpressed. We identified two such genes: LAG2 and HDA3. LAG2 encodes an inhibitor of the Skp, Cullin, F-box containing (SCF) ubiquitin-ligase complex, while HDA3 encodes a subunit of the HDA1 histone deacetylase complex. We find that one of these synthetic lethal interactions is conserved in mammalian cells, as inhibition of a human homolog of HDA3 (Histone Deacetylase 4, HDAC4) is synthetically toxic in MELK overexpression cells. Altogether, our work identified a novel potential drug target for tumors that overexpress MELK.