DNA Repair Pathways Research Articles

The role of DNA topoisomerase 1α (AtTOP1α) in regulating arabidopsis meiotic recombination and chromosome segregation.

Meiosis is a critical process in sexual reproduction, and errors during this cell division can significantly impact fertility. Successful meiosis relies on the coordinated action of numerous genes involved in DNA replication, strand breaks, and subsequent rejoining. DNA topoisomerase enzymes play a vital role by regulating DNA topology, alleviating tension during replication and transcription. To elucidate the specific function of DNA topoisomerase 1α ( ) in male reproductive development of Arabidopsis thaliana, we investigated meiotic cell division in Arabidopsis flower buds. Combining cytological and biochemical techniques, we aimed to reveal the novel contribution of to meiosis. Our results demonstrate that the absence of leads to aberrant chromatin behavior during meiotic division. Specifically, the top1α1 mutant displayed altered heterochromatin distribution and clustered centromere signals at early meiotic stages. Additionally, this mutant exhibited disruptions in the distribution of 45s rDNA signals and a reduced frequency of chiasma formation during metaphase I, a crucial stage for genetic exchange. Furthermore, the atm-2×top1α1 double mutant displayed even more severe meiotic defects, including incomplete synapsis, DNA fragmentation, and the presence of polyads. These observations collectively suggest that plays a critical role in ensuring accurate meiotic progression, promoting homologous chromosome crossover formation, and potentially functioning in a shared DNA repair pathway with ATAXIA TELANGIECTASIA MUTATED (ATM) in Arabidopsis microspore mother cells.

Modulation of blood-tumor barrier transcriptional programs improves intra-tumoral drug delivery and potentiates chemotherapy in GBM.

Glioblastoma (GBM) is the most common malignant primary brain tumor. GBM has an extremely poor prognosis and new treatments are badly needed. Efficient drug delivery to GBM is a major obstacle as the blood-brain barrier (BBB) prevents passage of the majority of cancer drugs into the brain. It is also recognized that the blood-brain tumor barrier (BTB) in the growing tumor represents a challenge. The BTB is heterogeneous and poorly characterized, but similar to the BBB it can prevent therapeutics from reaching effective intra-tumoral doses, dramatically hindering their potential. Here, we identified a 12-gene signature associated with the BTB, with functions related to vasculature development, morphogenesis and cell migration. We identified CDH5 as a core molecule in this set and confirmed its over-expression in GBM vasculature using spatial transcriptomics of GBM patient specimens. We found that the indirubin-derivative, 6-bromoindirubin acetoxime (BIA), could downregulate CDH5 and other BTB signature genes, causing endothelial barrier disruption in endothelial monolayers and BBB 3D spheroids in vitro. Treatment of tumor-bearing mice with BIA enabled increased intra-tumoral accumulation of the BBB non-penetrant chemotherapeutic drug cisplatin and potentiated cisplatin-mediated DNA damage by targeting DNA repair pathways. Finally, using an injectable BIA nanoparticle formulation, PPRX-1701, we significantly improved the efficacy of cisplatin in patient-derived GBM xenograms and prolonged their survival. Overall, our work reveals potential targets at the BTB for improved chemotherapy delivery and the bifunctional properties of BIA as a BTB modulator and potentiator of chemotherapy, supporting its further development.

Loss of tet methyl cytosine dioxygenase 3 (TET3) enhances cardiac fibrosis via modulating the DNA damage repair response

BackgroundCardiac fibrosis is the hallmark of all forms of chronic heart disease. Activation and proliferation of cardiac fibroblasts are the prime mediators of cardiac fibrosis. Existing studies show that ROS and inflammatory cytokines produced during fibrosis not only signal proliferative stimuli but also contribute to DNA damage. Therefore, as a prerequisite to maintain sustained proliferation in fibroblasts, activation of distinct DNA repair mechanism is essential.ResultIn this study, we report that TET3, a DNA demethylating enzyme, which has been shown to be reduced in cardiac fibrosis and to exert antifibrotic effects does so not only through its demethylating activity but also through maintaining genomic integrity by facilitating error-free homologous recombination (HR) repair of DNA damage. Using both in vitro and in vivo models of cardiac fibrosis as well as data from human heart tissue, we demonstrate that the loss of TET3 in cardiac fibroblasts leads to spontaneous DNA damage and in the presence of TGF-β to a shift from HR to the fast but more error-prone non-homologous end joining repair pathway. This shift contributes to increased fibroblast proliferation in a fibrotic environment. In vitro experiments showed TET3’s recruitment to H2O2-induced DNA double-strand breaks (DSBs) in mouse cardiac fibroblasts, promoting HR repair. Overexpressing TET3 counteracted TGF-β-induced fibroblast proliferation and restored HR repair efficiency. Extending these findings to human cardiac fibrosis, we confirmed TET3 expression loss in fibrotic hearts and identified a negative correlation between TET3 levels, fibrosis markers, and DNA repair pathway alteration.ConclusionCollectively, our findings demonstrate TET3’s pivotal role in modulating DDR and fibroblast proliferation in cardiac fibrosis and further highlight TET3 as a potential therapeutic target.Graphical abstractSchematic representation illustrating the role of TET3 in modulating the DDR response in healthy and fibrotic fibroblasts.

Synergistic Cytotoxicity of Histone Deacetylase and Poly-ADP Ribose Polymerase Inhibitors and Decitabine in Breast and Ovarian Cancer Cells: Implications for Novel Therapeutic Combinations.

Breast and ovarian cancers pose significant therapeutic challenges. We explored the synergistic cytotoxicity of histone deacetylase inhibitors (HDACis), poly(ADP-ribose) polymerase inhibitors (PARPis), and decitabine in breast (MDA-MB-231 and MCF-7) and ovarian (HEY-T30 and SKOV-3) cancer cell lines that were exposed to HDACi (panobinostat or vorinostat), PARPi (talazoparib or olaparib), decitabine, or their combinations. HDACi, PARPi, and decitabine combinations had synergistic cytotoxicity (assessed by MTT and clonogenic assays) in all cell lines (combination index < 1). Clonogenic assays confirmed the sensitivity of breast and ovarian cancer cell lines to the three-drug combinations (panobinostat, talazoparib, and decitabine; panobinostat, olaparib, and decitabine; vorinostat, talazoparib, and decitabine; vorinostat, olaparib, and decitabine). Cell proliferation was inhibited by 48-70%, and Annexin V positivity was 42-59% in all cell lines exposed to the three-drug combinations. Western blot analysis showed protein PARylation inhibition, caspase 3 and PARP1 cleavage, and c-MYC down-regulation. The three-drug combinations induced more DNA damage (increased phosphorylation of histone 2AX) than the individual drugs, impaired the DNA repair pathways, and altered the epigenetic regulation of gene expression. These results indicate that HDACi, PARPi, and decitabine combinations should be further explored in these tumor types. Further clinical validation is warranted to assess their safety and efficacy.

The USP1 Inhibitor KSQ-4279 Overcomes PARP Inhibitor Resistance in Homologous Recombination-Deficient Tumors.

Defects in DNA repair pathways play a pivotal role in tumor evolution and resistance to therapy. At the same time, they create vulnerabilities that render tumors dependent on the remaining DNA repair processes. This phenomenon is exemplified by the clinical activity of PARP inhibitors in tumors with homologous recombination (HR) repair defects, such as tumors with inactivating mutations in BRCA1 or BRCA2. However, the development of resistance to PARP inhibitors in BRCA-mutant tumors represents a high unmet clinical need. In this study, we identified deubiquitinase ubiquitin-specific peptidase-1 (USP1) as a critical dependency in tumors with BRCA mutations or other forms of HR deficiency and developed KSQ-4279, the first potent and selective USP1 inhibitor to enter clinical testing. The combination of KSQ-4279 with a PARP inhibitor was well tolerated and induced durable tumor regression across several patient-derived PARP-resistant models. These findings indicate that USP1 inhibitors represent a promising therapeutic strategy for overcoming PARP inhibitor resistance in patients with BRCA-mutant/HR-deficient tumors and support continued testing in clinical trials. Significance: KSQ-4279 is a potent and selective inhibitor of USP1 that induces regression of PARP inhibitor-resistant tumors when dosed in combination with PARP inhibitors, addressing an unmet clinical need for BRCA-mutant tumors.

ERCC1 and ERCC2 Polymorphisms Predict the Efficacy and Toxicity of Platinum-Based Chemotherapy in Small Cell Lung Cancer.

Standard first-line chemotherapy in small cell lung cancer (SCLC) is based on the platinum plus etoposide combination. Despite a high objective response rate, responses are not durable and chemotherapy-induced toxicity may compromise treatment. Genetic variants in genes involved in the DNA-repair pathways and in etoposide metabolization could predict treatment efficacy and safety and help personalize platinum-based chemotherapy. Germline polymorphisms in XRCC1, ERCC1, ERCC2, ABCB1, ABCC3, UGT1A1 and GSTP1 genes were investigated in 145 patients with SCLC. The tumor expression of ERCC1 was determined using immunohistochemistry, and the tumor expression of ERCC1-XPF was determined via a proximity ligation assay. Survival analyses showed a statistically significant association between the ERCC1 rs11615 variant and median progression-free survival (PFS) in patients with limited-stage (LS) SCLC (multivariate: hazard ratio 3.25, [95% CI 1.38-7.70]; p = 0.007). Furthermore, we observed differences between the ERCC1-XPF complex and median PFS in LS-SCLC, although statistical significance was not reached (univariate: positive expression 10.8 [95% CI 4.09-17.55] months versus negative expression 13.3 [95% CI 7.32-19.31] months; p = 0.06). Safety analyses showed that the ERCC2 rs1799793 variant was significantly associated with the risk of grade ≥ 3 thrombocytopenia in the total cohort (multivariate: odds ratio 3.15, [95% CI 1.08-9.17]; p = 0.04). Our results provide evidence that ERCC1 and ERCC2 variants may predict the efficacy and safety of platinum-based chemotherapy in SCLC patients. LS-SCLC patients may benefit most from ERCC1 determination, but prospective studies are needed.

Germline variant profiling of CHEK2 sequencing variants in breast cancer patients

The cell cycle checkpoint kinase 2 (CHEK2) is a tumor suppressor gene coding for a protein kinase with a role in the cell cycle and DNA repair pathways. Variants within CHEK2 are associated with an increased risk of developing breast, colorectal, prostate and several other types of cancer. Comprehensive genetic risk assessment leads to early detection of hereditary cancer and provides an opportunity for better survival. Multigene panel screening can identify the presence of pathogenic variants in hereditary cancer predisposition genes (HCPG), including CHEK2. Multigene panels, however, also result in large quantities of genetic data some of which cannot be interpreted and are classified as variants of uncertain significance (VUS). A VUS provides no information for use in medical management and leads to ambiguity in genetic counseling. In the absence of variant segregation data, in vitro functional analyses can be used to clarify variant annotations, aiding in accurate clinical management of patient risk and treatment plans. In this study, we performed whole exome sequencing (WES) to investigate the prevalence of germline variants in 210 breast cancer (BC) patients and conspicuously among the many variants in HCPGs that we found, we identified 16 individuals with non-synonymous or frameshift CHEK2 variants, sometimes along with additional variants within other BC susceptibility genes. Using this data, we investigated the prevalence of these CHEK2 variants in African American (AA) and Caucasian (CA) populations identifying the presence of two novel frameshift variants, c.1350delA (p.Val451Serfs*18) and c.1528delC (p.Gln510Argfs*3) and a novel missense variant, c262C>T (p.Pro88Ser). Along with the current clinical classifications, we assembled available experimental data and computational predictions of function for these CHEK2 variants, as well as explored the role these variants may play in polygenic risk assessment.

The DNA repair pathway as a therapeutic target to synergize with trastuzumab deruxtecan in HER2-targeted antibody–drug conjugate–resistant HER2-overexpressing breast cancer

BackgroundAnti-HER2 therapies, including the HER2 antibody–drug conjugates (ADCs) trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), have led to improved survival outcomes in patients with HER2-overexpressing (HER2+) metastatic breast cancer. However, intrinsic or acquired resistance to anti-HER2–based therapies remains a clinical challenge in these patients, as there is no standard of care following disease progression. The purpose of this study was to elucidate the mechanisms of resistance to T-DM1 and T-DXd in HER2+ BC patients and preclinical models and identify targets whose inhibition enhances the antitumor activity of T-DXd in HER2-directed ADC-resistant HER2+ breast cancer in vitro and in vivo.MethodsTargeted DNA and whole transcriptome sequencing were performed in breast cancer patient tissue samples to investigate genetic aberrations that arose after anti-HER2 therapy. We generated T-DM1 and T-DXd–resistant HER2+ breast cancer cell lines. To elucidate their resistance mechanisms and to identify potential synergistic kinase targets for enhancing the efficacy of T-DXd, we used fluorescence in situ hybridization, droplet digital PCR, Western blotting, whole-genome sequencing, cDNA microarray, and synthetic lethal kinome RNA interference screening. In addition, cell viability, colony formation, and xenograft assays were used to determine the synergistic antitumor effect of T-DXd combinations.ResultsWe found reduced HER2 expression in patients and amplified DNA repair–related genes in patients after anti-HER2 therapy. Reduced ERBB2 gene amplification in HER2-directed ADC–resistant HER2+ breast cancer cell lines was through DNA damage and epigenetic mechanisms. In HER2-directed ADC–resistant HER2+ breast cancer cell lines, our non-biased RNA interference screening identified the DNA repair pathway as a potential target within the canonical pathways to enhance the efficacy of T-DXd. We validated that the combination of T-DXd with ataxia telangiectasia and Rad3-related inhibitor, elimusertib, led to significant breast cancer cell death in vitro (P < 0.01) and in vivo (P < 0.01) compared to single agents.ConclusionsThe DNA repair pathways contribute to HER2-directed ADC resistance. Our data justify exploring the combination treatment of T-DXd with DNA repair–targeting drugs to treat HER2-directed ADC–resistant HER2+ breast cancer in clinical trials.

From rest to repair: Safeguarding genomic integrity in quiescent cells

Quiescence is an important non-pathological state in which cells pause cell cycle progression temporarily, sometimes for decades, until they receive appropriate proliferative stimuli. Quiescent cells make up a significant proportion of the body, and maintaining genomic integrity during quiescence is crucial for tissue structure and function. While cells in quiescence are spared from DNA damage associated with DNA replication or mitosis, they are still exposed to various sources of endogenous DNA damage, including those induced by normal transcription and metabolism. As such, it is vital that cells retain their capacity to effectively repair lesions that may occur and return to the cell cycle without losing their cellular properties. Notably, while DNA repair pathways are often found to be downregulated in quiescent cells, emerging evidence suggests the presence of active or differentially regulated repair mechanisms. This review aims to provide a current understanding of DNA repair processes during quiescence in mammalian systems and sheds light on the potential pathological consequences of inefficient or inaccurate repair in quiescent cells.

Cell cycle arrest combined with CDK1 inhibition suppresses genome-wide mutations by activating alternative DNA repair genes during genome editing

Cells regularly repair numerous mutations. However, the effect of CRISPR/Cas9-induced dsDNA breaks on the repair processes of naturally occurring genome-wide mutations is unclear. In this study, we used TSCE5 cells with the heterozygous thymidine kinase genotype (TK+/-) to examine these effects. We strategically inserted the target sites for guide RNA (gRNA)/Cas9 and I-SceI into the functional allele and designed the experiment such that deletions of > 81bp or base substitutions within exon five disrupted the TK gene, resulting in a TK-/- genotype. TSCE5 cells in the resting state exhibited 16 genome-wide mutations that affected cellular functions. After gRNA/Cas9 editing, these cells produced 859 mutations, including 67 high-impact variants that severely affected cellular functions under standard culture conditions. Mutation profile analysis indicated a significant accumulation of C to A substitutions, underscoring the widespread induction of characteristic mutations by gRNA/Cas9. In contrast, gRNA/Cas9-edited cells under conditions of S∼G2/M arrest and cyclin-dependent kinase 1 inhibition showed only five mutations. Transcriptomic analysis revealed the downregulation of DNA replication genes and upregulation of alternative DNA repair genes, such as zinc finger protein 384 (ZNF384) and dual specificity phosphatase, under S∼G2/M conditions. Additionally, activation of nucleotide and base excision repair gene, including O-6-methylguanine-DNA methyltransferase and xeroderma pigmentosum complementation group C, was observed. This study highlights the profound impact of CRISPR/Cas9 editing on genome-wide mutation processes and underscores the emergence of novel DNA repair pathways. Finally, our findings provide significant insights into the maintenance of genome integrity during genome editing.

Targeting DNA damage repair mechanism by using RAD50-silencing siRNA nanoparticles to enhance radiotherapy in triple negative breast cancer

Radiotherapy (RT) is one of major therapeutic modalities in combating breast cancer. In RT, ionizing radiation is employed to induce DNA double-strand breaks (DSBs) as a primary mechanism that causes cancer cell death. However, the induced DNA damage can also trigger the activation of DNA repair mechanisms, reducing the efficacy of RT treatment. Given the pivotal role of RAD50 protein in the radiation-responsive DNA repair pathways involving DSBs, we developed a novel polymer-lipid based nanoparticle formulation containing RAD50-silencing RNA (RAD50-siRNA-NPs) and evaluated its effect on the RAD50 downregulation as well as cellular and tumoral responses to ionizing radiation using human triple-negative breast cancer as a model. The RAD50-siRNA-NPs successfully preserved the activity of the siRNA, facilitated its internalization by cancer cells via endocytosis, and enabled its lysosomal escape. The nanoparticles significantly reduced RAD50 expression, whereas RT alone strongly increased RAD50 levels at 24 h. Pretreatment with RAD50-siRNA-NPs sensitized the cancer cells to RT with ∼2-fold higher level of initial DNA DSBs as determined by a γH2AX biomarker and a 2.5-fold lower radiation dose to achieve 50 % colony reduction. Intratumoral administration of RAD50-siRNA-NPs led to a remarkable 53 % knockdown in RAD50. The pretreatment with RAD50-siRNA-NPs followed by RT resulted in approximately a 2-fold increase in DNA DSBs, a 4.5-fold increase in cancer cell apoptosis, and 2.5-fold increase in tumor growth inhibition compared to RT alone. The results of this work demonstrate that RAD50 silencing by RAD50-siRNA-NPs can disrupt RT-induced DNA damage repair mechanisms, thereby significantly enhancing the radiation sensitivity of TNBC MDA-MB-231 cells in vitro and in orthotopic tumors as measured by colony forming and tumor regrowth assays, respectively.

Celastrol induces DNA damage and cell death in BCR-ABL T315I-mutant CML by targeting YY1 and HMCES

BackgroundChronic myeloid leukemia (CML) is driven primarily by the constitutively active BCR-ABL fusion oncoprotein. Although the development of tyrosine kinase inhibitors has markedly improved the prognosis of CML patients, it remains a significant challenge to overcome drug-resistant mutations, such as the T315I mutation of BCR-ABL, and achieve treatment-free remission in the clinic. PurposeThe identification of new intervention targets beyond BCR-ABL could provide new perspectives for future research and therapeutic intervention. A network pharmacology analysis was conducted to identify the most promising natural product with anti-CML activity. Celastrol was selected for further analysis to gain insights into its mechanism of action (MoA), with the aim of identifying potential new intervention targets for BCR-ABL T315I-mutant CML. MethodsTranscriptomic and proteomic analyses were conducted to systematically investigate the molecular MoA of celastrol in K562T315I cells. To identify the target proteins of celastrol, mass spectrometry-coupled cellular thermal shift assay (MS-CETSA) was carried out, followed by validations with genetic knockdown and overexpression, cell proliferation assay, comet assay, Western blotting, celastrol probe-based in situ labeling and pull-down assay, molecular docking, and biolayer interferometry. ResultsOur multi-omics analyses revealed that celastrol primarily induces DNA damage accumulation and the unfolded protein response in K562T315I cells. Among the twelve most potential celastrol targets, experimental evidence demonstrated that the direct interaction of celastrol with YY1 and HMCES increases the levels of DNA damage, leading to cell death. ConclusionThis study represents the first investigation utilizing a proteome-wide label-free target deconvolution approach, MS-CETSA, to identify the protein targets of celastrol. This study also develops a new systems pharmacology strategy. The findings provide new insights into the multifaceted mechanisms of celastrol and, more importantly, highlight the potential of targeting proteins in DNA damage and repair pathways, particularly YY1 and HMCES, to combat drug-resistant CML.

BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during Caenorhabditis elegans meiosis.

The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinated interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.

Disparate pathways for extrachromosomal DNA biogenesis and genomic DNA repair.

Oncogene amplification on extrachromosomal DNA (ecDNA) is a pervasive driver event in cancer, yet our understanding of how ecDNA forms is limited. Here, we couple a CRISPR-based method for ecDNA induction with extensive characterization of newly formed ecDNA to examine their biogenesis. We find that DNA circularization is efficient, irrespective of 3D genome context, with formation of 800kb, 1 Mb, and 1.8 Mb ecDNAs reaching or exceeding 15%. We show non-homologous end joining and microhomology-mediated end joining both contribute to ecDNA formation, while inhibition of DNA-PKcs and ATM have opposing impacts on ecDNA formation. EcDNA and the corresponding chromosomal excision scar can form at significantly different rates and respond differently to DNA-PKcs and ATM inhibition. Taken together, our results support a model of ecDNA formation in which double strand break ends dissociate from their legitimate ligation partners prior to joining of illegitimate ends to form the ecDNA and excision scar.

Nanoplastics exposure simplifies the network structure of sea cucumber (Apostichopus japonicus) gut microbiota and improves cluster randomness

Nanoplastics (NPs) are abundant in ocean environments, leading to environmental pollution and notable disruptions to the physiological functions of marine animals. To investigate the toxic effects of NPs on echinoderms, specifically sea cucumbers (Apostichopus japonicus), they were exposed to varying concentrations of NPs (0, 102, 104 particles/L) for 14 d. Subsequently, the 102 particles/L exposure group was purified for 35 d to elucidate the impact of both NPs exposure and purification on the intestinal bacteria structure and function. The results showed that the richness and variety of intestinal bacteria in sea cucumbers significantly reduced under NPs exposure, and then they could be restored to the pre-exposure treatment state after 35 d of purification. With the increase of NPs exposure concentration in the environment, the intestinal core bacteria gradually changed from Firmicutes and Proteobacteria to Pseudoalteromonas and Vibrio. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database annotated that the gut microbiota of sea cucumbers was significantly downregulated in the glycosylation, carbohydratic and amino acid metabolic pathways (P < 0. 05), exogenous substance biodegradation and metabolism, DNA replication and repair pathways were significantly up-regulated (P < 0.05) under the exposure of NPs. In addition, nanoplastics exposure simplified the symbiotic network relationships of the gut bacteria, reduced the selective effect of host on the intestinal bacteria, and increased stochasticity. In conclusion, waterborne NPs can adversely affect the structure and function of sea cucumber intestinal bacteria, with these effects persisting for a duration. However, as the purification time lengthens, these adverse effects gradually diminish. This study aims to provide some theoretical basis for the biotoxic effects of NPs.

Low-dose ionizing radiation-induced RET/PTC1 rearrangement via the non-homologous end joining pathway to drive thyroid cancer.

Thyroid cancer incidence increases worldwide annually, primarily due to factors such as ionizing radiation (IR), iodine intake, and genetics. Papillary carcinoma of the thyroid (PTC) accounts for about 80% of thyroid cancer cases. RET/PTC1 (coiled-coil domain containing 6 [CCDC6]-rearranged during transfection) rearrangement is a distinctive feature in over 70% of thyroid cancers who exposed to low doses of IR in Chernobyl and Hiroshima‒Nagasaki atomic bombings. This study aims to elucidate mechanism between RET/PTC1 rearrangement and IR in PTC. N-thy-ori-3-1 cells were subjected to varying doses of IR (2/1/0.5/0.2/0.1/0.05Gy) of IR at different days, and result showed low-dose IR-induced RET/PTC1 rearrangement in a dose-dependent manner. RET/PTC1 has been observed to promote PTC both in vivo and in vitro. To delineate the role of different DNA repair pathways, SCR7, RI-1, and Olaparib were employed to inhibit non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ), respectively. Notably, inhibiting NHEJ enhanced HR repair efficiency and reduced IR-induced RET/PTC1 rearrangement. Conversely, inhibiting HR increased NHEJ repair efficiency and subsequent RET/PTC1 rearrangement. The MMEJ did not show a markable role in this progress. Additionally, inhibiting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) decreased the efficiency of NHEJ and thus reduced IR-induced RET/PTC1 rearrangement. To conclude, the data suggest that NHEJ, rather than HR or MMEJ, is the critical cause of IR-induced RET/PTC1 rearrangement. Targeting DNA-PKcs to inhibit the NHEJ has emerged as a promising therapeutic strategy for addressing IR-induced RET/PTC1 rearrangement in PTC.

Biologic and Clinical Analysis of Childhood Gamma Delta T-ALL Identifies LMO2/STAG2 Rearrangements as Extremely High Risk.

Acute lymphoblastic leukemia expressing the gamma delta T-cell receptor (γδ T-ALL) is a poorly understood disease. We studied 200 children with γδ T-ALL from 13 clinical study groups to understand the clinical and genetic features of this disease. We found age and genetic drivers were significantly associated with outcome. γδ T-ALL diagnosed in children under 3 years of age was extremely high-risk and enriched for genetic alterations that result in both LMO2 activation and STAG2 inactivation. Mechanistically, using patient samples and isogenic cell lines, we show that inactivation of STAG2 profoundly perturbs chromatin organization by altering enhancer-promoter looping, resulting in deregulation of gene expression associated with T-cell differentiation. High-throughput drug screening identified a vulnerability in DNA repair pathways arising from STAG2 inactivation, which can be targeted by poly(ADP-ribose) polymerase inhibition. These data provide a diagnostic framework for classification and risk stratification of pediatric γδ T-ALL. Significance: Patients with acute lymphoblastic leukemia expressing the gamma delta T-cell receptor under 3 years old or measurable residual disease ≥1% at end of induction showed dismal outcomes and should be classified as having high-risk disease. The STAG2/LMO2 subtype was enriched in this very young age group. STAG2 inactivation may perturb chromatin conformation and cell differentiation and confer vulnerability to poly(ADP-ribose) polymerase inhibition.

SPIN1 facilitates chemoresistance and HR repair by promoting Tip60 binding to H3K9me3

The tandem Tudor-like domain-containing protein Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. However, the involvement of SPIN1 in DNA damage repair has remained unclear. Our study shows that SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR), and facilitates homologous recombination (HR)-mediated DNA damage repair. SPIN1 promotes H3K9me3 accumulation at DNA damage sites and enhances the interaction between H3K9me3 and Tip60, thereby promoting the activation of ATM and HR repair. We also show that SPIN1 increases chemoresistance. These findings reveal a novel role for SPIN1 in the activation of H3K9me3-dependent DNA repair pathways, and suggest that SPIN1 may contribute to cancer chemoresistance by modulating the efficiency of double-strand break (DSB) repair.

Ruthenium drug BOLD-100 regulates BRAFMT colorectal cancer cell apoptosis through AhR/ROS/ATR signaling axis modulation.

Patients with class I V600EBRAF-mutant (MT) colorectal cancer (CRC) have a poor prognosis and their response to combined anti-BRAF/EGFR inhibition remains limited. There is clearly an unmet need in further understanding the biology of V600EBRAFMT CRC. We have used differential gene expression of BRAFWT and MT CRC cells to identify pathways underpinning BRAFMT CRC. We tested a panel of molecularly/genetically subtyped CRC cells for their sensitivity to the Unfolded Protein Response (UPR) activator BOLD-100. To identify novel combination strategies for BOLD-100, we performed RNA sequencing and high-throughput drug screening. Pathway enrichment analysis identified that the UPR and DNA repair pathways were significantly enriched in BRAFMT CRC. We found that oncogenic BRAF plays a crucial role in mediating response to BOLD-100. Using a systems biology approach, we identified V600EBRAFMT-dependent activation of the replication stress response kinase ATR as a key mediator of resistance to BOLD-100. Further analysis identified acute increases in BRAFMT-dependent-reactive oxygen species (ROS) levels following treatment with BOLD-100 that was demonstrated to promote ATR/CHK1 activation and apoptosis. Furthermore, activation of ROS/ATR/CHK1 following BOLD-100 was found to be mediated through the AHR transcription factor and CYP1A1. Importantly, pharmacological blockade of this resistance pathway with ATR inhibitors synergistically increased BOLD-100-induced apoptosis and growth inhibition in BRAFMT models. These results unveil possible novel therapeutic opportunity for BRAFMT CRC. Implications: BOLD-100 induces BRAFMT-dependent replication stress, and targeted strategies against replication stress (eg. by using ATR inhibitors) in combination with BOLD-100 may serve as a potential novel therapeutic strategy for clinically aggressive BRAFMT CRC.

FANCD2–FANCI surveys DNA and recognizes double- to single-stranded junctions

DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2–FANCI (D2–I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion1. However, D2–I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation2–4. At present, it is unclear how DNA crosslinks are recognized and how D2–I functions in replication fork protection. Here, using single-molecule imaging, we show that D2–I is a sliding clamp that binds to and diffuses on double-stranded DNA. Notably, sliding D2–I stalls on encountering single-stranded–double-stranded (ss–ds) DNA junctions, structures that are generated when replication forks stall at DNA lesions5. Using cryogenic electron microscopy, we determined structures of D2–I on DNA that show that stalled D2–I makes specific interactions with the ss–dsDNA junction that are distinct from those made by sliding D2–I. Thus, D2–I surveys dsDNA and, when it reaches an ssDNA gap, it specifically clamps onto ss–dsDNA junctions. Because ss–dsDNA junctions are found at stalled replication forks, D2–I can identify sites of DNA damage. Therefore, our data provide a unified molecular mechanism that reconciles the roles of D2–I in the recognition and protection of stalled replication forks in several DNA repair pathways.