Predictive DNA damage signaling for low­dose ionizing radiation.

Numerous studies have attempted to develop biological markers for the response to radiation for broad and straightforward application in the field of radiation. Based on a public database, the present study selected several molecules involved in the DNA damage repair response, cell cycle regulation and cytokine signaling as promising candidates for low­dose radiation­sensitive markers. The HuT 78 and IM­9 cell lines were irradiated in a concentration­dependent manner, and the expression of these molecules was analyzed using western blot analysis. Notably, the activation of ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), p53 and H2A histone family member X (H2AX) significantly increased in a concentration­dependent manner, which was also observed in human peripheral blood mononuclear cells. To determine the radioprotective effects of cinobufagin, as an ATM and CHK2 activator, an in vivo model was employed using sub­lethal and lethal doses in irradiated mice. Treatment with cinobufagin increased the number of bone marrow cells in sub­lethal irradiated mice, and slightly elongated the survival of lethally irradiated mice, although the difference was not statistically significant. Therefore, KU60019, BML­277, pifithrin­α, and nutlin­3a were evaluated for their ability to modulate radiation­induced cell death. The use of BML­277 led to a decrease in radiation­induced p­CHK2 and γH2AX levels and mitigated radiation­induced apoptosis. On the whole, the present study provides a novel approach for developing drug candidates based on the profiling of biological radiation­sensitive markers. These markers hold promise for predicting radiation exposure and assessing the associated human risk.

BRCA1 safeguards genome integrity by activating chromosome asynapsis checkpoint to eliminate recombination-defective oocytes.

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.

Immunohistochemical findings and clinicopathological features of breast cancers with pathogenic germline mutations in Non-BRCA genes.

Deleterious germline mutations in multiple genes confer an increased breast cancer (BC) risk. Immunohistochemical (IHC) expression of protein products of mutated high-risk genes has not been investigated in BC. We hypothesized that pathogenic mutations may lead to an abnormal IHC expression pattern in the tumor cells. BCs with deleterious germline mutations in CHEK2, ATM, PALB2 & PTEN were identified. Immunohistochemistry was performed using Dako staining platform on formalin fixed paraffin embedded tumor tissue. Primary antibodies for PALB2 (ab202970), ATM [2C1(1A10)}, CHK2 (EPR4325), and PTEN (138G6) proteins were used for BCs with respective deleterious mutations. IHC expression was assessed in tumor and adjacent benign breast tissue. Total 27 BCs with 10 CHEK2, 9 ATM, 6 PALB2 & 2 PTEN deleterious germline mutations were identified. IHC staining was performed on 8 CHEK2, 7 ATM, 6 PALB2 & 2 PTEN cases. Abnormal CHEK2 IHC staining was identified in 7/8(88%) BCs. Three distinct CHK2 IHC patterns were noted: 1) Strong diffuse nuclear positivity (5 BC), 2) Null-pattern (2 BC), & 3) Normal breast-like staining in 1 BC Four of 5 (80%) strong CHK2 staining BC had missense CHEK2 mutations. Null-pattern was present with a missense & a frameshift mutation. Normal breast-like CHEK2 IHC staining pattern was present in 1 BC with CHEK2 frameshift mutation. Loss of nuclear/cytoplasmic PTEN IHC expression was noted in 2 in-situ carcinomas. Abnormal PTEN and CHK2 IHC were present in atypical ductal hyperplasia and flat epithelial atypia. ATM and PALB2 IHC expression patterns were similar in tumor cells and benign breast epithelium: mild to moderate intensity nuclear and cytoplasmic staining. We report abnormal CHEK2 IHC expression in 88% of BCs with pathogenic CHEK2 mutations. With PTEN and CHEK2 pathogenic mutations, abnormal IHC patterns are seen in early atypical proliferative lesions. IHC may be applied to identify CHEK2 & PTEN mutated BCs and precursor lesions.

Efficacy of Poly(ADP-ribose) Polymerase Inhibitors by Individual Genes in Homologous Recombination Repair Gene-Mutated Metastatic Castration-Resistant Prostate Cancer: A US Food and Drug Administration Pooled Analysis.

PURPOSE: We performed a pooled analysis of multiple trials of poly(ADP-ribose) polymerase inhibitors (PARPi) in metastatic castration-resistant prostate cancer (mCRPC) to investigate the efficacy of PARPi in each individual homologous recombination repair (HRR) mutated (m) gene. PATIENTS AND METHODS: We pooled patient-level data from trials of PARPi in mCRPC that reported mutation status in individual HRR genes. Any HRR gene with available data across all the randomized trials of PARPi in first-line mCRPC was selected. The hazard ratios (HRs; 95% CI) for radiographic progression-free survival (rPFS; by blinded independent review) and overall survival (OS) of a PARPi plus an androgen receptor pathway inhibitor (ARPI) relative to placebo plus an ARPI in the pool of three randomized trials in first-line mCRPC were calculated using Kaplan-Meier estimates and a Cox proportional hazards model. RESULTS: In ATMm (N = 268), rPFS HR was 1.05 (0.74 to 1.49) and OS HR was 1.18 (0.82 to 1.71). In BRCA1m (N = 64), rPFS HR was 0.51 (0.23 to 1.1) and OS HR was 0.74 (0.34 to 1.61). In BRCA2m (N = 422), rPFS HR was 0.31 (0.23 to 0.42) and OS HR was 0.66 (0.49 to 0.89). In CDK12m (N = 164), rPFS HR was 0.50 (0.32 to 0.80) and OS HR was 0.63 (0.39 to 0.99). In CHEK2m (N = 172), rPFS HR was 1.06 (0.67 to 1.66) and OS HR was 1.53 (0.95 to 2.46). In PALB2m (N = 41) rPFS HR was 0.52 (0.23 to 1.17) and OS HR was 0.78 (0.34 to 1.8). CONCLUSION: In this pooled analysis, benefit from PARPi appeared greatest for patients with BRCA1m, BRCA2m, CDK12m, and PALB2m. Given limitations of this exploratory analysis, the apparent lack of benefit from PARPi in patients with CHEK2m or ATMm should be further explored in future clinical trials.

CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.

Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer-rich regions and 38 spliceogenic variants.

Splicing is controlled by a large set of regulatory elements (SREs) including splicing enhancers and silencers, which are involved in exon recognition. Variants at these motifs may dysregulate splicing and trigger loss-of-function transcripts associated with disease. Our goal here was to study the alternatively spliced exons 8 and 10 of the breast cancer susceptibility gene CHEK2. For this purpose, we used a previously published minigene with exons 6-10 that produced the expected minigene full-length transcript and replicated the naturally occurring events of exon 8 [Δ(E8)] and exon 10 [Δ(E10)] skipping. We then introduced 12 internal microdeletions of exons 8 and 10 by mutagenesis in order to map SRE-rich intervals by splicing assays in MCF-7 cells. We identified three minimal (10-, 11-, 15-nt) regions essential for exon recognition: c.863_877del [ex8, Δ(E8): 75%] and c.1073_1083del and c.1083_1092del [ex10, Δ(E10): 97% and 62%, respectively]. Then 87 variants found within these intervals were introduced into the wild-type minigene and tested functionally. Thirty-eight of them (44%) impaired splicing, four of which (c.883G>A, c.883G>T, c.884A>T, and c.1080G>T) induced negligible amounts (<5%) of the minigene full-length transcript. Another six variants (c.886G>A, c.886G>T, c.1075G>A, c.1075G>T, c.1076A>T, and c.1078G>T) showed significantly strong impacts (20-50% of the minigene full-length transcript). Thirty-three of the 38 spliceogenic variants were annotated as missense, three as nonsense, and two as synonymous, underlying the fact that any exonic change is capable of disrupting splicing. Moreover, c.883G>A, c.883G>T, and c.884A>T were classified as pathogenic/likely pathogenic variants according to ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based criteria. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The heterogeneous cancer phenotype of individuals with biallelic germline pathogenic variants in CHEK2.

PURPOSE: Females with biallelic CHEK2 germline pathogenic variants (gPVs) more often develop multiple breast cancers than individuals with monoallelic CHEK2 gPVs. This study is aimed at expanding the knowledge on the occurrence of other malignancies. METHODS: Exome sequencing of individuals who developed multiple primary malignancies identified 3 individuals with the CHEK2 (NM_007194.4) c.1100del p.(Thr367MetfsTer15) loss-of-function gPV in a biallelic state. We collected the phenotypes of an additional cohort of individuals with CHEK2 biallelic gPVs (n = 291). RESULTS: In total, 157 individuals (53.4%; 157/294 individuals) developed ≥1 (pre)malignancy. The most common (pre)malignancies next to breast cancer were colorectal- (n = 19), thyroid- (n = 19), and prostate (pre)malignancies (n = 12). Females with biallelic CHEK2 loss-of-function gPVs more frequently developed ≥2 (pre)malignancies and at an earlier age compared with females biallelic for the CHEK2 c.470T>C p.(Ile157Thr) missense variant. Furthermore, 26 males (31%; 26/84 males) with CHEK2 biallelic gPVs developed ≥1 (pre)malignancies of 15 origins. CONCLUSION: Our study suggests that CHEK2 biallelic gPVs likely increase the susceptibility to develop multiple malignancies in various tissues, both in females and males. However, it is possible that a substantial proportion of individuals with CHEK2 biallelic gPVs is missed as diagnostic testing for CHEK2 often is limited to individuals who developed breast cancer.

CHEK2 Founder Variants and Thyroid Cancer Risk.

Background: Germline pathogenic variants in CHEK2 are associated with a moderate increase in the lifetime risk for breast cancer. Increased risk for other cancers, including non-medullary thyroid cancer (NMTC), has also been suggested. To date, data implicating CHEK2 variants in NMTC predisposition primarily derive from studies within Poland, driven by a splice site variant (c.444 + 1G>A) that is uncommon in other populations. In contrast, the predominant CHEK2 variants in non-Polish populations are c.1100del and c.470T>C/p.I157T, representing 61.1% and 63.8%, respectively, of all CHEK2 pathogenic variants in two large U.S.-based commercial laboratory datasets. To further delineate the impact of common CHEK2 variants on thyroid cancer, we aimed to investigate the association of three CHEK2 founder variants (c.444 + 1G>A, c.1100del, and c.470T>C/p.Ile157Thr) on NMTC susceptibility in three groups of unselected NMTC patients. Methods: The presence of three CHEK2 founder variants was assessed within three groups: (1) 1544 NMTC patients (and 1593 controls) from previously published genome-wide association study (GWAS) analyses, (2) 789 NMTC patients with germline exome sequencing (Oncology Research Information Exchange Network [ORIEN] Avatar), and (3) 499 NMTC patients with germline sequence data available in The Cancer Genome Atlas (TCGA). A case-control study design was utilized with odds ratios (ORs) calculated by comparison of all three groups with the Ohio State University GWAS control group. Results: The predominant Polish variant (c.444 + 1G>A) was present in only one case. The proportion of patients with c.1100del was 0.92% in the GWAS group, 1.65% in the ORIEN Avatar group, and 0.80% in the TCGA group. The ORs (with 95% confidence intervals [CIs]) for NMTC associated with c.1100del were 1.71 (0.73-4.29), 2.64 (0.95-7.63), and 2.5 (0.63-8.46), respectively. The proportion of patients with c.470T>C/p.I157T was 0.91% in the GWAS group, 0.76% in the ORIEN Avatar group, and 0.80% in the TCGA group, respectively. The ORs (with CIs) for NMTC associated with c.470T>C/p.I157T were 1.75 (0.74-4.39), 1.52 (0.42-4.96), and 2.31 (0.58-7.90), respectively. Conclusions: Our analyses of unselected patients with NMTC suggest that CHEK2 variants c.1100del and c.470T>C/p.I157T have only a modest impact on thyroid cancer risk. These results provide important information for providers regarding the relatively low magnitude of thyroid cancer risk associated with these CHEK2 variants.

Clinicopathologic and genetic analysis of invasive breast carcinomas in women with germline CHEK2 variants.

PURPOSE: Germline pathogenic variants in checkpoint kinase 2 (CHEK2) are associated with a moderately increased risk of breast cancer (BC). The spectrum of clinicopathologic features and genetics of these tumors has not been fully established. METHODS: We characterized the histopathologic and clinicopathologic features of 44 CHEK2-associated BCs from 35 women, and assessed responses to neoadjuvant chemotherapy. A subset of cases (n = 23) was additionally analyzed using targeted next-generation DNA sequencing (NGS). RESULTS: Most (94%, 33/35) patients were heterozygous carriers for germline CHEK2 variants, and 40% had the c.1100delC allele. Two patients were homozygous, and five had additional germline pathogenic variants in ATM (2), PALB2 (1), RAD50 (1), or MUTYH (1). CHEK2-associated BCs occurred in younger women (median age 45 years, range 25-75) and were often multifocal (20%) or bilateral (11%). Most (86%, 38/44) were invasive ductal carcinomas of no special type (IDC-NST). Almost all (95%, 41/43) BCs were ER + (79% ER + HER2-, 16% ER + HER2 + , 5% ER-HER2 +), and most (69%) were luminal B. Nottingham grade, proliferation index, and results of multiparametric molecular testing were heterogeneous. Biallelic CHEK2 alteration with loss of heterozygosity was identified in most BCs (57%, 13/23) by NGS. Additional recurrent alterations included GATA3 (26%), PIK3CA (226%), CCND1 (22%), FGFR1 (22%), ERBB2 (17%), ZNF703 (17%), TP53 (9%), and PPM1D (9%), among others. Responses to neoadjuvant chemotherapy were variable, but few patients (21%, 3/14) achieved pathologic complete response. Most patients (85%) were without evidence of disease at time of study (n = 34). Five patients (15%) developed distant metastasis, and one (3%) died (mean follow-up 50 months). CONCLUSION: Almost all CHEK2-associated BCs were ER + IDC-NST, with most classified as luminal B with or without HER2 overexpression. NGS supported the luminal-like phenotype and confirmed CHEK2 as an oncogenic driver in the majority of cases. Responses to neoadjuvant chemotherapy were variable but mostly incomplete.

Profiling of the genetic features of patients with breast, ovarian, colorectal and extracolonic cancers: Association to CHEK2 and PALB2 germline mutations.

BACKGROUND AND AIMS: Cancer predisposition goes beyond BRCA and DNA Mismatch Repair (MMR) genes since multi-gene panel testing has become the routine diagnostic tool for hereditary cancer suspicion (HCS) cases. CHEK2 and PALB2 are some of the foremost-mutated non-BRCA/MMR actionable genes in families with a significant familial aggregation. Therefore, the purpose of this work is to unravel which tumours other than breast, ovary or colorectal display the patients. MATERIALS AND METHODS: We have analysed 528 probands that meet the inclusion criteria for Hereditary Breast and Ovarian Cancer and Lynch Syndrome established by our Hereditary Cancer Regional Program with a customized 35 genes-panel by using Ion Torrent™ Technology. RESULTS: We have identified pathogenic variants (PVs) in 61 families (1.55%), of which more than half (31 probands) harboured PVs in CHEK2 and PALB2 genes. Ours results reveal that not only were PVs CHEK2 and PALB2 carriers more likely to have family history of cancer not limited to breast, ovarian or colorectal cancers, but also they are prone to other extracolonic cancers, noteworthy endometrial and gastric cancers. CONCLUSIONS: Multigene panel testing improves the chance of finding PVs in actionable genes in families with HCS. In addition, the coexistence of variants should be recorded to implement a polygenic risk algorithm that might explain the missing heritability in the aforementioned families.

Systematic Minigene-Based Splicing Analysis and Tentative Clinical Classification of 52 CHEK2 Splice-Site Variants.

BACKGROUND: Disrupted pre-mRNA splicing is a frequent deleterious mechanism in hereditary cancer. We aimed to functionally analyze candidate spliceogenic variants of the breast cancer susceptibility gene CHEK2 by splicing reporter minigenes. METHODS: A total of 128 CHEK2 splice-site variants identified in the Breast Cancer After Diagnostic Gene Sequencing (BRIDGES) project (https://cordis.europa.eu/project/id/634935) were analyzed with MaxEntScan and subsetted to 52 variants predicted to impact splicing. Three CHEK2 minigenes, which span all 15 exons, were constructed and validated. The 52 selected variants were then genetically engineered into the minigenes and assayed in MCF-7 (human breast adenocarcinoma) cells. RESULTS: Of 52 variants, 46 (88.5%) impaired splicing. Some of them led to complex splicing patterns with up to 11 different transcripts. Thirty-four variants induced splicing anomalies without any trace or negligible amounts of the full-length transcript. A total of 89 different transcripts were annotated, which derived from different events: single- or multi-exon skipping, alternative site-usage, mutually exclusive exon inclusion, intron retention or combinations of the abovementioned events. Fifty-nine transcripts were predicted to introduce premature termination codons, 7 kept the original open-reading frame, 5 removed the translation start codon, 6 affected the 5'UTR (Untranslated Region), and 2 included missense variations. Analysis of variant c.684-2A > G revealed the activation of a non-canonical TG-acceptor site and exon 6 sequences critical for its recognition. CONCLUSIONS: Incorporation of minigene read-outs into an ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based classification scheme allowed us to classify 32 CHEK2 variants (27 pathogenic/likely pathogenic and 5 likely benign). However, 20 variants (38%) remained of uncertain significance, reflecting in part the complex splicing patterns of this gene.

In silico and structure-based evaluation of deleterious mutations identified in human Chk1, Chk2, and Wee1 protein kinase.

Checkpoint kinases Chk1, Chk2, Wee1 are playing a key role in DNA damage response and genomic integrity. Cancer-associated mutations identified in human Chk1, Chk2, and Wee1 were retrieved to understand the function associated with the mutation and also alterations in the folding pattern. Therefore, an attempt has been made to identify deleterious effect of variants using in silico and structure-based approach. Variants of uncertain significance for Chk1, Chk2, and Wee1 were retrieved from different databases and four prediction servers were employed to predict pathogenicity of mutations. Further, Interpro, I-Mutant 3.0, Consurf, TM-align, and have (y)our protein explained were used for comprehensive study of the deleterious effects of variants. The sequences of Chk1, Chk2, and Wee1 were analyzed using Clustal Omega, and the three-dimensional structures of the proteins were aligned using TM-align. The molecular dynamics simulations were performed to explore the differences in folding pattern between Chk1, Chk2, Wee1 wild-type, and mutant protein and also to evaluate the structural integrity. Thirty-six variants in Chk1, 250 Variants in Chk2, and 29 in Wee1 were categorized as pathogenic using in silico prediction tools. Furthermore, 25 mutations in Chk1, 189 in Chk2, and 14 in Wee1 were highly conserved, possessing deleterious effect and also influencing the protein structure and function. These identified mutations may provide underlying genetic intricacies to serve as potential targets for therapeutic inventions and clinical management.

The benefit of adding polygenic risk scores, lifestyle factors, and breast density to family history and genetic status for breast cancer risk and surveillance classification of unaffected women from germline CHEK2 c.1100delC families.

To determine the changes in surveillance category by adding a polygenic risk score based on 311 breast cancer (BC)-associated variants (PRS311), questionnaire-based risk factors and breast density on personalized BC risk in unaffected women from Dutch CHEK2 c.1100delC families. In total, 117 unaffected women (58 heterozygotes and 59 non-carriers) from CHEK2 families were included. Blood-derived DNA samples were genotyped with the GSAMDv3-array to determine PRS311. Lifetime BC risk was calculated in CanRisk, which uses data from the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA). Women, were categorized into three surveillance groups. The surveillance advice was reclassified in 37.9 % of heterozygotes and 32.2 % of non-carriers after adding PRS311. Including questionnaire-based risk factors resulted in an additional change in 20.0 % of heterozygotes and 13.2 % of non-carriers; and a subanalysis showed that adding breast density on top shifted another 17.9 % of heterozygotes and 33.3 % of non-carriers. Overall, the majority of heterozygotes were reclassified to a less intensive surveillance, while non-carriers would require intensified surveillance. The addition of PRS311, questionnaire-based risk factors and breast density to family history resulted in a more personalized BC surveillance advice in CHEK2-families, which may lead to more efficient use of surveillance.

Germline testing of BRCA1, BRCA2, PALB2 and CHEK2 c.1100delC in 1514 triple negative familial and isolated breast cancers from a single centre, with extended testing of ATM, RAD51C and RAD51D in over 400.

BACKGROUND: The identification of germline pathogenic gene variants (PGVs) in triple negative breast cancer (TNBC) is important to inform further primary cancer risk reduction and TNBC treatment strategies. We therefore investigated the contribution of breast cancer associated PGVs to familial and isolated invasive TNBC. METHODS: Outcomes of germline BRCA1, BRCA2 and CHEK2_c.1100delC testing were recorded in 1514 women (743-isolated, 771-familial), and for PALB2 in 846 women (541-isolated, 305-familial), with TNBC and smaller numbers for additional genes. Breast cancer free controls were identified from Predicting Risk Of Cancer At Screening and BRIDGES (Breast cancer RIsk after Diagnostic GEne Sequencing) studies. RESULTS: BRCA1_PGVs were detected in 52 isolated (7.0%) and 195 (25.3%) familial cases (isolated-OR=58.9, 95% CI: 16.6 to 247.0), BRCA2_PGVs in 21 (2.8%) isolated and 67 (8.7%) familial cases (isolated-OR=5.0, 95% CI: 2.3 to 11.2), PALB2_PGVs in 9 (1.7%) isolated and 12 (3.9%) familial cases (isolated-OR=8.8, 95% CI: 2.5 to 30.4) and CHEK2_c.1100delC in 0 isolated and 3 (0.45%) familial cases (isolated-OR=0.0, 95% CI: 0.00 to 2.11). BRCA1_PGV detection rate was >10% for all familial TNBC age groups and significantly higher for younger diagnoses (familial: <50 years, n=165/538 (30.7%); ≥50 years, n=30/233 (12.9%); p<0.0001). Women with a G3_TNBC were more likely to have a BRCA1_PGV as compared with a BRCA2 or PALB2_PGV (p<0.0001). 0/743 isolated TNBC had the CHEK2_c.1100delC PGV and 0/305 any ATM_PGV, but 2/240 (0.83%) had a RAD51D_PGV. CONCLUSION: PGVs in BRCA1 are associated with G3_TNBCs. Familial TNBCs and isolated TNBCs <30 years have a >10% likelihood of a PGV in BRCA1. BRCA1_PGVs are associated with younger age of familial TNBC. There was no evidence for any increased risk of TNBC with CHEK2 or ATM PGVs.

CHEK2 deficiency increase the response to PD-1 inhibitors by affecting the tumor immune microenvironment.

Immune checkpoint blockade (ICB) therapy has improved treatment effects in multiple cancers. Gene mutations in the DNA damage repair pathway (DDR) may cause genomic instability and may relate to the efficacy of ICB. Checkpoint kinase 2 (CHEK2) and polymerase epsilon (POLE) are important genes in the DDR. In this study, we aimed to study the impact of CHEK2 deficiency mutations on the response to ICB. We found that tumors with CHEK2 mutations had a significantly higher tumor mutational burden (TMB) compared to those with CHEK2-WT in a pancancer database. We noted that CHEK2 deficiency mutations potentiated the anti-tumor effect of anti-PD-1 therapy in MC38 and B16 tumor-bearing mice with the decrease of tumor volume and tumor weight after anti-PD-1 treatment. Mechanistically, CHEK2 deficiency tumors were with the increased cytotoxic CD8+ T-cell infiltration, especially cytotoxic CD8+ T cells, and modulated the tumor-immune microenvironment with an upregulated immune inflammatory pathway and antigen presentation pathway after anti-PD-1 treatment. Furthermore, murine models with POLE mutations confirmed that CHEK2 deficiency shaped similar mutational and immune landscapes as POLE mutations after anti-PD-1 treatment. Taken together, our results demonstrated that CHEK2 deficiency mutations may increase the response to ICB (eg. anti-PD-1) by influencing the tumor immune microenvironment. This indicated that CHEK2 deficiency mutations were a potentially predictive biomarker and CHEK2 deficiency may potentiate response to immunotherapy.

CHEK2 is a potential prognostic biomarker associated with immune infiltration in clear cell renal cell carcinoma.

Checkpoint kinase 2 (CHEK2) plays a crucial role in responding to DNA damage and is linked to diverse cancer types. However, its significance in the prediction of prognosis and impacts on the immune status of clear cell renal cell carcinoma (ccRCC) remains unclear. This study aimed to identify the role of CHEK2 in prognosis and immune microenvironment of ccRCC. We analyzed transcriptome and clinicopathological data from the cancer genome atlas (TCGA) database and conducted functional enrichment analysis to explore molecular mechanisms. The relationship between CHEK2 and immune infiltration was evaluated, and drug sensitivity analysis was performed using the CellMiner database. The results showed that CHEK2 was an independent predictor of ccRCC prognosis and was closely associated with immune-related processes. Additionally, high expression of CHEK2 was linked to resistance to certain targeted drugs. These findings suggest that CHEK2 could serve as a biomarker for ccRCC, providing insights into tumor immune microenvironment alterations and immunotherapeutic response. Further investigation is needed to fully understand the potential of CHEK2 as a prognostic predictor and therapeutic target for ccRCC.

PTPN13 rs989902 and CHEK2 rs738722 are associated with esophageal cancer.

PURPOSE: Individual genetic background can play an essential role in determining the development of esophageal squamous cell carcinoma (ESCC). PTPN13 and CHEK2 play important roles in the pathogenesis of ESCC. This case-control study aimed to analyze the association between gene polymorphisms and ESCC susceptibility. METHODS: DNA was extracted from the peripheral blood of patients. The Agena MassARRAY platform was used for the genotyping. Statistical analysis was conducted using the chi-squared test or Fisher's exact test, logistic regression analysis, and stratification analysis. RESULTS: The 'G' allele of rs989902 (PTPN13) and the 'T' allele of rs738722 (CHEK2) were both associated with an increased risk of ESCC (rs989902: OR = 1.23, 95% CI = 1.02-1.47, p = 0.028; rs738722: OR = 1.28, 95% CI = 1.06-1.55, p = 0.011). Stratification analysis showed that SNPs (rs989902 and rs738722) were notably correlated with an increased risk of ESCC after stratification for age, sex, smoking, and drinking status. In addition, rs738722 might be associated with lower stage, while rs989902 had a lower risk of metastasis. CONCLUSION: Our findings display that PTPN13 rs989902 and CHEK2 rs738722 are associated with an increased risk of ESCC in the Chinese Han population.

The PP2A phosphatase counteracts the function of the 9-1-1 axis in checkpoint activation.

DNA damage elicits a checkpoint response depending on the Mec1/ATR kinase, which detects the presence of single-stranded DNA and activates the effector kinase Rad53/CHK2. In Saccharomyces cerevisiae, one of the signaling circuits leading to Rad53 activation involves the evolutionarily conserved 9-1-1 complex, which acts as a platform for the binding of Dpb11 and Rad9 (referred to as the 9-1-1 axis) to generate a protein complex that allows Mec1 activation. By examining the effects of both loss-of-function and hypermorphic mutations, here, we show that the Cdc55 and Tpd3 subunits of the PP2A phosphatase counteract activation of the 9-1-1 axis. The lack of this inhibitory function results in DNA-damage sensitivity, sustained checkpoint-mediated cell-cycle arrest, and impaired resection of DNA double-strand breaks. This PP2A anti-checkpoint role depends on the capacity of Cdc55 to interact with Ddc1 and to counteract Ddc1-Dpb11 complex formation by preventing Dpb11 recognition of Ddc1 phosphorylated on Thr602.

Rad53 regulates the lifetime of Rdh54 at homologous recombination intermediates.

Rdh54 is a conserved DNA translocase that participates in homologous recombination (HR), DNA checkpoint adaptation, and chromosome segregation. Saccharomyces cerevisiae Rdh54 is a known target of the Mec1/Rad53 signaling axis, which globally protects genome integrity during DNA metabolism. While phosphorylation of DNA repair proteins by Mec1/Rad53 is critical for HR progression little is known about how specific post translational modifications alter HR reactions. Phosphorylation of Rdh54 is linked to protection of genomic integrity but the consequences of modification remain poorly understood. Here, we demonstrate that phosphorylation of the Rdh54 C-terminus by the effector kinase Rad53 regulates Rdh54 clustering activity as revealed by single molecule imaging. This stems from phosphorylation dependent and independent interactions between Rdh54 and Rad53. Genetic assays reveal that loss of phosphorylation leads to phenotypic changes resulting in loss-of-heterozygosity (LOH) outcomes. Our data highlight Rad53 as a key regulator of HR intermediates through activation and attenuation of Rdh54 motor function.