DNA Repair Deficiency Research Articles

RepairSig: Deconvolution of DNA damage and repair contributions to the mutational landscape of cancer.

Cancer genomes accumulate a large number of somatic mutations resulting from a combination of stochastic errors in DNA processing, cancer-related aberrations of the DNA repair machinery, or carcinogenic exposures; each mutagenic process leaves a characteristic mutational signature. A key challenge is understanding the interactions between signatures, particularly as DNA repair deficiencies often modify the effects of other mutagens. Here, we introduce RepairSig, a computational method that explicitly models additive primary mutagenic processes; non-additive secondary processes, which interact with the primary processes; and a mutation opportunity, that is, the distribution of sites across the genome that are vulnerable to damage or preferentially repaired. We demonstrate that RepairSig accurately recapitulates experimentally identified signatures, identifies autonomous signatures of deficient DNA repair processes, and explains mismatch repair deficiency in breast cancer by de novo inference of both primary and secondary signatures from patient data. RepairSig is freely available for download at https://github.com/ncbi/RepairSig.

Immune checkpoint inhibitor sensitivity of DNA repair deficient tumors.

Faithful DNA replication is necessary to maintain genome stability and implicates a complex network with several pathways depending on DNA damage type: homologous repair, nonhomologous end joining, base excision repair, nucleotide excision repair and mismatch repair. Alteration in components of DNA repair machinery led to DNA damage accumulation and potentially carcinogenesis. Preclinical data suggest sensitivity to immune checkpoint inhibitors in tumors with DNA repair deficiency. Here, we review clinical studies that explored the use of immune checkpoint inhibitor in patient harboring tumor with DNA repair deficiency.

Nanoparticle-Encapsulated Camptothecin: Epigenetic Modulation in DNA Repair Mechanisms in Colon Cancer Cells.

Molecular crosstalk between the cellular epigenome and genome converge as a synergistic driver of oncogenic transformations. Besides other pathways, epigenetic regulatory circuits exert their effect towards cancer progression through the induction of DNA repair deficiencies. We explored this mechanism using a camptothecin encapsulated in β-cyclodextrin–EDTA–Fe3O4 nanoparticles (CPT-CEF)-treated HT29 cells model. We previously demonstrated that CPT-CEF treatment of HT29 cells effectively induces apoptosis and cell cycle arrest, stalling cancer progression. A comparative transcriptome analysis of CPT-CEF-treated versus untreated HT29 cells indicated that genes controlling mismatch repair, base excision repair, and homologues recombination were downregulated in these cancer cells. Our study demonstrated that treatment with CPT-CEF alleviated this repression. We observed that CPT-CEF exerts its effect by possibly affecting the DNA repair mechanism through epigenetic modulation involving genes of HMGB1, APEX1, and POLE3. Hence, we propose that CPT-CEF could be a DNA repair modulator that harnesses the cell’s epigenomic plasticity to amend DNA repair deficiencies in cancer cells.

Genomic instability in the naturally and prematurely aged myocardium

Genomic instability, the unresolved accumulation of DNA variants, is hypothesized as one of the contributors to the natural aging process. We assessed the frequency of unresolved DNA damage reaching the transcriptome of the murine myocardium during the course of natural aging and in hearts from four distinct mouse models of premature aging with established aging-related cardiac dysfunctions. RNA sequencing and variant calling based on total RNA sequencing was compared between hearts from naturally aging mice, mice with cardiomyocyte-specific deficiency of Ercc1, a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity, Tert-deficient mice with reduced telomere length, and a mouse model of human Hutchinson-Gilford progeria syndrome (HGPS). Our results demonstrate that no enrichment in variants is evident in the naturally aging murine hearts until 2 y of age from the HGPS mouse model or mice with reduced telomere lengths. In contrast, a dramatic accumulation of variants was evident in Ercc1 cardiomyocyte-specific knockout mice with deficient DNA repair machinery, in mice with reduced mitochondrial antioxidant capacity, and in the intestine, liver, and lung of naturally aging mice. Our data demonstrate that genomic instability does not evidently contribute to naturally aging of the mouse heart in contrast to other organs and support the contention that the endogenous DNA repair machinery is remarkably active to maintain genomic integrity in cardiac cells throughout life.

Utility of Homologous Recombination Deficiency Biomarkers Across Cancer Types.

PURPOSEHomologous recombination DNA repair deficiency (HRD) is associated with sensitivity to platinum and poly (ADP-ribose) polymerase inhibitors in certain cancer types, including breast, ovarian, pancreatic, and prostate. In these cancers, BRCA1/2 alterations and genomic scar signatures are useful indicators for assessing HRD. However, alterations in other homologous recombination repair (HRR)-related genes and their clinical significance in other cancer types have not been adequately and systematically investigated.METHODSWe obtained data sets of all solid tumors in The Cancer Genome Atlas and Cancer Cell Line Encyclopedia, and comprehensively analyzed HRR pathway gene alterations, their loss-of-heterozygosity status, and per-sample genomic scar scores, that is, the HRD score and mutational signature 3 ratio, DNA methylation profiles, gene expression profiles, somatic TP53 mutations, sex, and clinical or in vitro response to chemical exposure.RESULTSBiallelic alterations in HRR genes other than BRCA1/2 were also associated with elevated genomic scar scores. The association between HRR-related gene alterations and genomic scar scores differed significantly by sex and the presence of somatic TP53 mutations. HRD tumors determined by a combination of indices also showed HRD features in gene expression analysis and exhibited significantly higher sensitivity to DNA-damaging agents than non-HRD cases in both clinical samples and cell lines.CONCLUSIONThis study provides evidence for the usefulness of HRD analysis in all cancer types, improves chemotherapy decision making and its efficacy in clinical settings, and represents a substantial advancement in precision oncology.A comprehensive pan-cancer analysis on the clinical significance of homologous recombination deficiency

Comparable radiation sensitivity in p53 wild-type and p53 deficient tumor cells associated with different cell death modalities

Studies of radiation interaction with tumor cells often take apoptosis as the desired results. However, mitotic catastrophe and senescence are also promoted by clinically relevant doses of radiation. Furthermore, p53 is a well-known transcription factor that is closely associated with radiosensitivity and radiation-induced cell death. Therefore, we aimed to investigate the involvement of radiosensitivity, cell death modalities and p53 status in response to carbon-ion radiation (CIR) here. Isogenic human colorectal cancer cell lines HCT116 (p53+/+ and p53−/−) were irradiated with high-LET carbon ions. Cell survival was determined by the standard colony-forming assay. 53BP1 foci were visualized to identify the repair kinetics of DNA double-strand breaks (DSBs). Cellular senescence was measured by SA-β-Gal and Ki67 staining. Mitotic catastrophe was determined with DAPI staining. Comparable radiosensitivities of p53+/+ and p53−/− HCT116 colorectal cells induced by CIR were demonstrated, as well as persistent 53BP1 foci indicated DNA repair deficiency in both cell lines. Different degree of premature senescence in isogenic HCT116 colorectal cancer cells suggested that CIR-induced premature senescence was more dependent on p21 but not p53. Sustained upregulation of p21 played multifunctional roles in senescence enhancement and apoptosis inhibition in p53+/+ cells. p21 inhibition further increased radiosensitivity of p53+/+ cells. Complex cell death modalities rather than single cell death were induced in both p53+/+ and p53−/− cells after 5 Gy CIR. Mitotic catastrophe was predominant in p53−/− cells due to inefficient activation of Chk1 and Chk2 phosphorylation in combination with p53 null. Senescence was the major cell death mechanism in p53+/+ cells via p21-dependent pathway. Taken together, p21-mediated premature senescence might be used by tumor cells to escape from CIR-induced cytotoxicity, at least for a time.

RNF8-CDH1 Co-Expression Predicts Clinical Benefit of Chemoradiotherapy in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and exhibits an overall poor outcome. Due to the lack of targeted therapy, conventional systemic chemotherapy has been the main strategy for the treatment of TNBC. Further evidence has shown that combining radiation with chemotherapy is also a suitable treatment based on DNA repair deficiencies in patients with TNBC. However, the preferred treatment for metastatic TNBC remains unclear. Therefore, identification of biomarkers is an unmet need in personalized therapy for TNBC. RNF8 (ring finger protein 8) is a ubiquitin ligase implicated in TNBC metastasis; however, its role in TNBC pathogenesis is unclear. The purpose of the present study was to investigate the roles of the RNF8–CDH1(Cadherin 1) axis in node-positive TNBC patients. We found that the RNF8high/CDH1low index was significantly higher in patients with TNBC than in patients without TNBC. Furthermore, patients with an RNF8high/CDH1low index displayed poorer outcomes than those with an RNF8low-medium/CDH1medium-high index. Notably, as compared to patients with an RNF8low-medium/CDH1medium-high index, those with an RNF8high/CDH1low index had a poorer survival rate with chemotherapy treatment alone. The combination of radiation and chemotherapy resulted in a better survival rate than chemotherapy alone in patients with an RNF8high/CDH1low index. Taken together, the RNF8high/CDH1low index not only functions as a prognostic and therapeutic marker but may also act as a target in the development of anti-cancer agents for patients with TNBC.

Abstract 366: Identification of a clinically significant pan-cancer biomarker for homologous recombination deficiency

Abstract [Background] Genomic alterations in BRCA1/2 and the ‘genomic scar' signatures are associated with homologous recombination DNA repair deficiency (HRD) and serve as therapeutic biomarkers for sensitivity to platinum and PARP inhibitors in breast and ovarian cancers. However, the clinical significance of these biomarkers in other homologous recombination repair (HRR) pathway genes or in other cancer types is not fully understood. [Method] From the TCGA and the AACR GENIE project databases, we obtained clinical and genomic data of patients with multiple solid cancers, evaluated both locus-specific LOH in HRR pathway gene mutations and two genomic scar signature scores (HRD score and mutational signature 3), identified potential HRD cases, and examined the efficacy of DNA-damaging drugs in these HRD cases. [Results] Germline and somatic gene mutations in the HRR pathway genes with locus-specific LOH were significantly enriched in cases having high genomic scar scores, whereas those without the LOH were not enriched. Somatic mutations without the LOH were remarkably enriched in hypermutated cases and considered to be passenger mutations. The mutation rate and frequency with LOH in the HRR pathway genes varied widely by cancer type. When stratifying patients by gender and presence of TP53 mutation, the correlation between the two genomic scar scores and the resulting optimal cutoff values for biallelic HRR pathway gene alterations differed greatly. The potential HRD cases that were selected by the two genomic scar cutoffs and biallelic HRR pathway gene alterations showed significantly high gene expression-based HRD scores. In Cox proportional hazard multivariate analyses with covariates of age, stage, gender, HRD, and TP53 mutation, HRD was a good prognostic factor in the group with DNA-damaging agents, but a poor one in the group without the agents. [Conclusion] Increased attention on locus-specific LOH in HRR pathway gene alterations and the differences in genomic scar signatures stratified by gender and TP53 mutation is newly warranted in order to properly assess HRD in a pan-cancer manner. The results in the present study proved the clinical significance of HRD across cancer types and may contribute to the development of personalized medicine based on HRD. Citation Format: Shiro Takamatsu, JB Brown, Koji Yamanoi, Ken Yamaguchi, Junzo Hamanishi, Takashi Kobayashi, Masaki Mandai, Noriomi Matsumura. Identification of a clinically significant pan-cancer biomarker for homologous recombination deficiency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 366.

Abstract 2723: How BRCA1/2 mutations in TNBC affect TME and subsequently immune cell functions

Abstract Breast cancer type 1 and 2 susceptibility proteins (BRCA1/BRCA2) are well known breast cancer genes, mutations in which lead to defective homologous recombination repair (HRR). HR-based DNA repair deficiency (HRD) scores can be used to indicate DNA damage, genomic instability and may predict response to DNA damaging agents in BRCA1/2 mutated tumors. Tumors with a high HRD score caused by complete loss of BRCA1 or BRCA2 function locus-specific LOH are sensitive to DNA damage agents including platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). To understand the relationship between BRCA1/2 mutations and DNA damage in the tumor microenvironment (TME), we have characterized 107 BRCA1/2 tumors to determine HRD score using whole exome sequencing (WES) and simultaneously measured markers of DNA damage, PARP expression, tumor-infiltrating lymphocytes (TILs) and immune checkpoints to identify potential treatment targets on matching samples (n=47) using highly multiplexed fluorescence microscopy CO-Detection by indEXing (CODEX). We have established and validated a 40-plex breast cancer specific antibody panel consisting of markers to detect DNA damage, TILs and immune checkpoints to deeply profile how the TME is affected by BRCA1/2 mutations using CODEX. Computational image processing of CODEX data was performed to interrogate changes in number, size, morphology, and marker expression in tumor and immune cells. We have characterized BRCA1/2 tumors (n=47) on Tissue Microarrays (TMAs), and we have detected cytotoxic CD8+T and CD107a+NK cells in HRD low (<42) and HRD high groups. Interestingly, their frequency and cytolytic function (measured by Granzyme A and perforin transcript level) appear to be associated with HRD scores. For instance, HRD low groups showed increased cytotoxic CD8+T and CD107a+NK cells whereas HRD high groups revealed decreased cell numbers as well as cytolytic function. We observed that HRD high, LOH positive was associated with increased DNA damage marker expression in tumor cells (H3pSer28, pATM, yH2AX) as well as NK cells (pATM) whereas immune checkpoint protein levels were decreased. We have planned quantitative analysis which allow us to determine the percentage of cell subtypes as well as the spatial compartmentalization of cells to interrogate the tumor microenvironment associated with BRCA1/2 mutations. In conclusion, BRCA1/2 mutated tumors with high HRD score revealed upregulated DNA damage expression in immune cells suggesting that BRCA1/2 mutations can impact HRR in CD8+T cells and CD107a+ NK cells and subsequently affect their ability to produce Granzyme A and Perforin. Our findings will decipher the role of DNA damage in BRCA1/2 mutated tumor cells and immune cell types. Outcomes can potentially predict treatment responses such as DNA damage, PARPi and checkpoint inhibitor therapies in TNBC BRCA1/2 breast cancer. Citation Format: Dana Pueschl, Derek A. Oldrige, Jonathan Belman, Jake S. Shilan, Anupma Nayak, Bradley Wubbenhorst, John Pluta, Robert H. Vonderheide, Michael Feldman, Kara N. Maxwell, E. John Wherry, Susan M. Domchek, Katherine L. Nathanson. How BRCA1/2 mutations in TNBC affect TME and subsequently immune cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2723.

Association of Copy Number Variation Signature and Survival in Patients With Serous Ovarian Cancer

Tailoring therapeutic regimens to individual patients with ovarian cancer is informed by severity of disease using a variety of clinicopathologic indicators. Although DNA repair variations are increasingly used for therapy selection in ovarian cancer, molecular features are not widely used for general assessment of patient prognosis and disease severity. To distill a highly dynamic characteristic, signature of copy number variations (CNV), into a risk score that could be easily validated analytically or repurposed for use given existing US Food and Drug Administration (FDA)-approved multigene assays. This genetic association study used the Cancer Genome Atlas Ovarian Cancer database to assess for genome-wide survival associations agnostic to gene function. Regions enriched for significant associations were compared to associations from scrambled data. CNV associations were condensed into a risk score, which was internally validated using bootstrapping. The participants were patients with serous ovarian cancer (stages I-IV) diagnosed from 1992 to 2013. Statistical analysis was performed from April to July 2020. Overall survival (OS). Among 564 patients with serous ovarian cancer, the mean (SD) age was 59.7 (11.5) years; 34 (6%) identified as Black or African American. A total of 13 genome regions, comprising 14 alterations, were identified as significantly risk associated. Composite risk score was independent of total CNV burden, total mutational burden, BRCA status, and open-source genome-wide DNA repair deficiency signatures. Binned terciles yielded high-, standard-, and low-risk groups with respective median OS estimates of 2.9 (95% CI, 2.3-3.2) years, 4.1 (95% CI, 3.7-4.8) years, and 5.7 (95% CI, 4.7-7.4) years, respectively (P < .001). Associated 5-year survival estimates in each tercile were 15% (95% CI, 10%-22%), 36% (95% CI, 29%-46%), and 53% (95% CI, 45%-62%). The risk score had more discriminatory ability to prognosticate OS than age, clinical stage, grade, and race combined, and was strongly additive to significant clinical features (P < .001). Simulated adaptation of FDA-approved assays showed similar performance. Gene ontology analyses of identified regions showed an enrichment for regulatory miRNAs and protein kinase regulators. This study found that a CNV-based risk score is independent to and stronger than current or near-future ovarian cancer genomic biomarkers to prognosticate OS. CNV regions identified were not strongly associated with canonical ovarian cancer biological pathways, identifying candidates for future mechanistic investigations. External validation of the CNV risk score, especially in concert with more extensive clinical features, could be pursued via existing FDA-approved assays.

DNA Damage-Induced Neurodegeneration in Accelerated Ageing and Alzheimer's Disease.

DNA repair ensures genomic stability to achieve healthy ageing, including cognitive maintenance. Mutations on genes encoding key DNA repair proteins can lead to diseases with accelerated ageing phenotypes. Some of these diseases are xeroderma pigmentosum group A (XPA, caused by mutation of XPA), Cockayne syndrome group A and group B (CSA, CSB, and are caused by mutations of CSA and CSB, respectively), ataxia-telangiectasia (A-T, caused by mutation of ATM), and Werner syndrome (WS, with most cases caused by mutations in WRN). Except for WS, a common trait of the aforementioned progerias is neurodegeneration. Evidence from studies using animal models and patient tissues suggests that the associated DNA repair deficiencies lead to depletion of cellular nicotinamide adenine dinucleotide (NAD+), resulting in impaired mitophagy, accumulation of damaged mitochondria, metabolic derailment, energy deprivation, and finally leading to neuronal dysfunction and loss. Intriguingly, these features are also observed in Alzheimer’s disease (AD), the most common type of dementia affecting more than 50 million individuals worldwide. Further studies on the mechanisms of the DNA repair deficient premature ageing diseases will help to unveil the mystery of ageing and may provide novel therapeutic strategies for AD.

Synthetic lethality of LP-184, a next generation acylfulvene, in ex vivo PDX models with homologous recombination defects.

e15064 Background: The clinical success of PARP inhibitors (PARPi) in homologous recombination (HR) deficient (HRD+) solid tumors has broadened the scope of identifying additional agents and vulnerabilities in cancers with DNA repair deficiencies. However, with more than 40% of BRCA1/2-deficient patients failing to respond to PARPi or acquiring resistance with prolonged PARPi administration, newer agents are also needed. LP-184, an acylfulvene, is a prodrug activated by PTGR1. Threshold expression levels of PTGR1 are higher in several tumors, providing a window of specificity for its cytotoxic action. DNA damage inflicted by acylfulvene (AF) agents is reliant upon HR pathway genes including BRCA1 for correction and removal. We hypothesized that tumors with high PTGR1 expression and HR deficiency will therefore be uniquely targeted and demonstrate synthetic lethality when exposed to LP-184. Methods: We evaluated ex vivo antitumor activity of LP-184 in selected PDX models representing lung, pancreatic and prostate cancers with high PTGR1 and known HR defects. Dissociated tumor fragments were treated with LP-184 across a concentration range of 5 nM to 36 uM for 5 days. Cell viability was quantified by CellTiter Glo. LP-184 IC50s were compared with PARPi efficacy. We further confirmed the dependency of PTGR1 in HR deficient tumor cells by comparing LP-184 sensitivity in the BRCA2 deficient cell line CAPAN-1 and the ATM mutant cell line PANC03.27, with and without PTGR1 suppression following an engineered CRISPR knockout of PTGR1. We also analyzed TCGA data to estimate the percentage of tumors with elevated PTGR1 and co-occurring damaging mutations in a panel of 60 HR genes. Results: The mean LP-184 IC50 across 15 HRD+ PDX models tested was 288 nM (range 31 - 2900 nM). LP-184 turned out to be 6 - 340X more potent ex vivo than the PARPi Olaparib in these models. 9 of 15 models were associated with no clinical response to or initial response followed by progression on approved standard of care (SOC) agents. 6 of 15 models showed &lt; 10% tumor growth inhibition in vivo with SOC treatment. Regardless of cancer type, models with high-impact, loss-of-function mutations in ATM, ATR and BRCA1 showed exquisite sensitivity to LP-184 (mean IC50 ̃ 60 nM). CRISPRi-mediated stable suppression of PTGR1 in the pancreatic cancer cell lines CAPAN-1 and PANC03.27 entirely abrogated LP-184 sensitivity relative to isogenic parental cell lines. 17.6% of lung adenocarcinomas (n = 517), 4.5% of pancreatic adenocarcinomas (n = 179) and 9.6% of prostate adenocarcinomas (n = 498) displayed elevated PTGR1 along with damaging HR related mutations, and are likely to be responsive to LP-184 based on analysis of TCGA data. Conclusions: LP-184 is broadly effective in HRD+ tumors that may be less responsive to SOC including PARPi and could be useful clinically in a subset of tumors with high PTGR1 and HR defects.

Trial in progress: A phase 1b/2 study of the PARP inhibitor niraparib in combination with trastuzumab in patients with metastatic HER2+ breast cancer (TBCRC 050).

TPS1098 Background: Approximately 20% of breast cancers (BC) express the human epidermal growth factor receptor 2 (HER2). Although HER2-directed therapies result in improved patient outcome, resistance ultimately occurs. Poly (ADP-Ribose) polymerase (PARP) inhibitors are currently indicated in cancers that express germline mutations in the DNA repair proteins BRCA1/2 due to their synthetic lethality against the homologous recombination repair (HR) pathway. In addition to its role in DNA damage repair, PARP1 has also been implicated in other cellular functions, including co-activation of genes such as NF-κB, which regulate tumor proliferation and HER2 drug resistance. Our group identified that HER2+ BC overexpress the PARP1 and phospho-p65 protein. In HER2+ BC cells and animal models, PARP inhibitors initiated apoptosis independent of a DNA repair deficiency, via inhibition of NF-kB signaling. Key proteins (p65, IKK-α) of the NF-κB-mediated growth pathways were reduced and IκBα was increased in the presence of PARPi, implicating another oncologic pathway in which HER2+ BC cells may be dependent. Methods: The study is a phase 1b/2, multicenter, single arm clinical trial evaluating the safety and efficacy of niraparib 200 mg orally days 1-21 with trastuzumab 6 mg/kg (cycle 1 loading dose of 8 mg/kg) intravenously on day 1 of a 21-day cycle for patients with unresectable or metastatic HER2+ BC. Eligible patients include metastatic HER2+ BC, progression on at least 1 prior HER2-targeted therapy, measurable disease, ECOG PS 0-1, and LVEF ≥ 50%. Stable/treated CNS disease allowed. Prior PARPi and known germline BRCA 1/2 excluded. Forty patients will be enrolled at 7 US sites within the Translational Breast Cancer Research Consortium. The primary objectives are determining the dose-limiting toxicity (DLT) of the combination and assessing the objective response rate. The phase 1b cohort has been completed (N=6). Enrollment in phase 2 began February 2021 with a total accrual goal of 40. Gehan’s two-stage design will be used assuming the response rate is at least 24% and the response rate will be estimated with Clopper-Pearson exact method. Correlative aims include assessing blood and tissue biomarkers (e.g. PARP1, p65, phosphor-p65, let-7a miRNA, NF-kB, ctDNA, etc.) for association with clinical benefit and to predict response to therapy. Clinical trial information: NCT03368729 .

Decreased ATM Function Causes Delayed DNA Repair and Apoptosis in Common Variable Immunodeficiency Disorders

PurposeCommon variable immunodeficiency disorders (CVID) is characterized by low/absent serum immunoglobulins and susceptibility to bacterial infection. Patients can develop an infections-only phenotype or a complex disease course with inflammatory, autoimmune, and/or malignant complications. We hypothesized that deficient DNA repair mechanisms may be responsible for the antibody deficiency and susceptibility to inflammation and cancer in some patients.MethodsGermline variants were identified following targeted sequencing of n = 252 genes related to DNA repair in n = 38 patients. NanoString nCounter PlexSet assay measured gene expression in n = 20 CVID patients and n = 7 controls. DNA damage and apoptosis were assessed by flow cytometry in n = 34 CVID patients and n = 11 controls.ResultsTargeted sequencing supported enrichment of rare genetic variants in genes related to DNA repair pathways with novel and rare likely pathogenic variants identified and an altered gene expression signature that distinguished patients from controls and complex patients from those with an infections-only phenotype. Consistent with this, flow cytometric analyses of lymphocytes following DNA damage revealed a subset of CVID patients whose immune cells have downregulated ATM, impairing the recruitment of other repair factors, delaying repair and promoting apoptosis.ConclusionThese data suggest that germline genetics and altered gene expression predispose a subset of CVID patients to increased sensitivity to DNA damage and reduced DNA repair capacity.

Clinical Activity and Safety of Cediranib and Olaparib Combination in Patients with Metastatic Pancreatic Ductal Adenocarcinoma without BRCA Mutation.

Lessons Learned Cediranib and olaparib combination did not result in clinically meaningful activity in patients with metastatic pancreatic ductal adenocarcinoma without known BRCA mutation. BackgroundCediranib, a vascular endothelial growth factor receptor inhibitor, suppresses expression of BRCA1/2 and RAD51 inducing homologous recombination DNA repair deficiency (HRD) in several cancer cell lines and xenograft models [1]. Olaparib provides a clinical benefit in patients with metastatic pancreatic adenocarcinoma (mPDAC) with germline BRCA mutation (gBRCAmt) [2]. We hypothesized that cediranib induces HRD in the absence of gBRCAmt and synergizes with olaparib, resulting in an objective response in patients with mPDAC.MethodsPatients with mPDAC with at least one prior systemic chemotherapy were enrolled. Patients with known gBRCAmt were excluded. Patients took cediranib 30 mg daily and olaparib 200 mg twice daily, orally. The primary endpoint was objective response (OR) rate.ResultsNineteen patients received the study drugs. Seven patients came off treatment before the first restaging scan: six because of clinical progression and one because of an adverse event. No OR was observed. Six patients had stable disease (SD) as a best overall response. The median duration of SD was 3.1 months. The median overall survival was 3.4 months. Common treatment‐related adverse events were fatigue, hypertension, and diarrhea.ConclusionCediranib and olaparib combination did not result in clinically meaningful activity in patients with mPDAC without gBRCAmt.

Abstract PO-029: USP14 is a potential target to improve the efficacy of radiotherapy in combination with immune-checkpoint inhibitors in non-small cell lung cancer

Abstract Non-small cell lung cancer (NSCLC) represents ~85% of lung cancer cases and has a 5-year survival rate of ~23%. Thus, research aimed at determining more effective treatment strategies is critical. Immune checkpoint inhibitors (ICis), e.g. anti-PD-L1 antibodies, have transformed cancer treatment in recent years. Clinical trial studies have indicated that radiotherapy enhances the curative effect of ICis. However, most patients do not respond to immune-radiotherapy combinations. While best known to induce DNA double strand break (DSB)-mediated cytotoxicity, ionizing radiation (IR) induces an increase of cytosolic DNA and activates the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) signaling pathway that leads to the type 1 interferon (IFN) response, essential to achieve anti-tumor immunity in combination with ICis. Homologous recombination (HR) DNA repair deficiency is an important determinant of radiation response that can also increase the immune recognition of cancers by increasing the number of non-synonymous mutations, as well as by increasing the cGAS/STING-mediated type1 IFN response. We have shown an important role for ubiquitin specific protease (USP14) in regulating DNA repair (1, 2,3). Here, we identify a novel role of USP14 in regulating HR and the cGAS/STING pathway in response to IR in NSCLC. USP14 is a proteasomal deubiquitinase that we show to have a prominent role in NSCLC. Knockdown of USP14 (shUSP14) or its pharmacological inhibition (IU1 or VLX1570) led to: (i) radio-sensitization, as shown by reduced clonogenic survival in NSCLC cell lines, and delayed growth of NSCLC tumor xenografts in nude mice; increased (ii) HR-deficiency; (iii) chromosomal aberrations and micronuclei formation; as well as (iv) cGAS puncta in response to IR. Further, USP14 interacts with and deubiquitinates cGAS in IR-treated NSCLC cells. USP14-mediated deubiquitination of cGAS has been shown to inhibit cGAS protein turn-over and increase the levels of cGAS in response to virus infection. Unexpectedly, however, in response to IR shUSP14 NSCLC cells have significantly higher cytoplasmic cGAS puncta and type 1 IFN response. Overall, these findings identify USP14 as a potential target to enhance sensitization to immune-radiotherapy by both regulation of HR and direct regulation of cGAS ubiquitination, leading to type 1 IFN response. Understanding the mechanisms that regulate DSB repair and cGAS/STING pathways holds potential for developing strategies to enhance the effectiveness of immune-radiotherapy combinations in NSCLC.

Breast cancer type 1 and neurodegeneration: consequences of deficient DNA repair.

Numerous cellular processes, including toxic protein aggregation and oxidative stress, have been studied extensively as potential mechanisms underlying neurodegeneration. However, limited therapeutic efficacy targeting these processes has prompted other mechanisms to be explored. Previous research has emphasized a link between cellular senescence and neurodegeneration, where senescence induced by excess DNA damage and deficient DNA repair results in structural and functional changes that ultimately contribute to brain dysfunction and increased vulnerability for neurodegeneration. Specific DNA repair proteins, such as breast cancer type 1, have been associated with both stress-induced senescence and neurodegenerative diseases, however, specific mechanisms remain unclear. Therefore, this review explores DNA damage-induced senescence in the brain as a driver of neurodegeneration, with particular focus on breast cancer type 1, and its potential contribution to sex-specific differences associated with neurodegenerative disease.

Integrated mutational landscape analysis of uterine leiomyosarcomas

Uterine leiomyosarcomas (uLMS) are aggressive tumors arising from the smooth muscle layer of the uterus. We analyzed 83 uLMS sample genetics, including 56 from Yale and 27 from The Cancer Genome Atlas (TCGA). Among them, a total of 55 Yale samples including two patient-derived xenografts (PDXs) and 27 TCGA samples have whole-exome sequencing (WES) data; 10 Yale and 27 TCGA samples have RNA-sequencing (RNA-Seq) data; and 11 Yale and 10 TCGA samples have whole-genome sequencing (WGS) data. We found recurrent somatic mutations in TP53, MED12, and PTEN genes. Top somatic mutated genes included TP53, ATRX, PTEN, and MEN1 genes. Somatic copy number variation (CNV) analysis identified 8 copy-number gains, including 5p15.33 (TERT), 8q24.21 (C-MYC), and 17p11.2 (MYOCD, MAP2K4) amplifications and 29 copy-number losses. Fusions involving tumor suppressors or oncogenes were deetected, with most fusions disrupting RB1, TP53, and ATRX/DAXX, and one fusion (ACTG2-ALK) being potentially targetable. WGS results demonstrated that 76% (16 of 21) of the samples harbored chromoplexy and/or chromothripsis. Clinically actionable mutational signatures of homologous-recombination DNA-repair deficiency (HRD) and microsatellite instability (MSI) were identified in 25% (12 of 48) and 2% (1 of 48) of fresh frozen uLMS, respectively. Finally, we found olaparib (PARPi; P = 0.002), GS-626510 (C-MYC/BETi; P < 0.000001 and P = 0.0005), and copanlisib (PIK3CAi; P = 0.0001) monotherapy to significantly inhibit uLMS-PDXs harboring derangements in C-MYC and PTEN/PIK3CA/AKT genes (LEY11) and/or HRD signatures (LEY16) compared to vehicle-treated mice. These findings define the genetic landscape of uLMS and suggest that a subset of uLMS may benefit from existing PARP-, PIK3CA-, and C-MYC/BET-targeted drugs.

Endocrine-Disrupting Chemicals and Vitamin D Deficiency in the Pathogenesis of Uterine Fibroids.

Uterine fibroids (UFs) are the most prevalent gynecologic neoplasm, affecting 70-80% of women over their lifespan. Although UFs are benign they can become life-threatening and require invasive surgeries such as myomectomy and hysterectomy. Notwithstanding the significant negative influence UFs have on female reproductive health, very little is known about early events that initiate tumor development. Several risk factors for UFs have been identified including vitamin D deficiency, inflammation, DNA repair deficiency, and environmental exposures to endocrine-disrupting chemicals (EDCs). EDCs have come under scrutiny recently due to their role in UF development. Epidemiologic studies have found an association between increased risk for early UF diagnosis and in utero EDC exposure. Environmental exposure to EDCs during uterine development increases UF incidence in a UF animal model. Notably, several studies demonstrated that abnormal myometrial stem cells (MMSCs) are the cell origin for UFs development. Our recent studies demonstrated that early-life EDC exposure reprogrammed the MMSCs toward a pro-fibroid landscape and altered the DNA repair and inflammation pathways. Notably, Vitamin D3 (VITD3) as a natural compound shrank the UF growth concomitantly with the reversion of several abnormal biological pathways and ameliorated the developmental exposure-induced DNA damage and pro-inflammation pathway in primed MMSCs. This review highlights and emphasizes the importance of multiple pathway interactions in the context of hypovitaminosis D at the MMSCs level and provides proof-of-concept information that can help develop a safe, long-term, durable, and non-surgical therapeutic option for UFs.

MYC/NBS1-Mediated DNA Damage Response is Involved in the Inhibitory Effect of Hydroxysafflor Yellow A on Glioma Cells

BackgroundThe role of Hydroxysafflor Yellow A (HSYA) in glioma is less studied, this research determined the effect of HSYA on glioma cells.MethodsThe expressions of MYC and NBS1 in glioma tissues were detected by bioinformatics analysis and verified by RT-qPCR. The target relationship between MYC and NBS1 was predicted by bioinformatics. After treating the cells with HSYA, silenced MYC, or overexpressed NBS1, the viability, apoptosis, proliferation, invasion, migration, and DNA damage of the glioma cells were detected by MTT, flow cytometry, colony formation, transwell, wound healing, and γH2AX immunofluorescence assays, respectively. IC50 of HSYA in glioma cells was analyzed by Probit regression analysis. The expressions of MYC, NBS1, factors related to migration, invasion, apoptosis, and DNA damage of the glioma cells were determined by Western blot or RT-qPCR.ResultsMYC and NBS1 were high-expressed in glioma, and NBS1 was targeted by MYC. HSYA and siRNA targeting MYC inhibited the cell viability, proliferation, invasion, migration, and induced the cell apoptosis of glioma cells. HSYA upregulated the expressions of MYC, γH2AX, E-Cadherin, Bax, and Cleaved-PARP1, stimulated the activation of NBS1, MRE11, RAD50, and ATM, and downregulated the expressions of N-Cadherin and Bcl2 in glioma cells. SiMYC decreased the IC50 of HSYA in the glioma cells, enhanced the sensitivity of glioma cells to HSYA, and inhibited the activation of NBS1 and ATM. NBS1 overexpression reversed the effect of siRNA targeting MYC on glioma cells.ConclusionMYC silencing inhibited the DNA damage response via regulation of NBS1, leading to DNA repair deficiency, and subsequently enhanced the sensitivity of glioma cells to HSYA.