Repair DNA Double-strand Breaks Research Articles

PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ.

Human DNA-dependent protein kinase catalytic subunit deficiency: a comprehensive review and update

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has an essential role in the non-homologous end-joining pathway that repairs DNA double-strand breaks in V(D)J recombination involved in the expression of T- and B-cell receptors. Whereas homozygous mutations in PRKDC define the scid mouse, a model that has been widely used in biology, human mutations in PRKDC are extremely rare and the disease spectrum has not been described so far. To provide an update on the genetics, clinical spectrum, immunological profile, and therapy of DNA-PKcs deficiency in human. The clinical, biological, and treatment data from the 6 cases published to date and from 1 new patient were obtained and analyzed. Rubella PCR was performed on available granuloma material. We report on 7 patients; Six patients displayed the autosomal recessive p.L3062R mutation in PRKDC gene encoding DNA-PKcs. Atypical severe combined immunodeficiency with inflammatory lesions, granulomas, and autoimmunity was the predominant clinical manifestation (n=5/7). Rubella viral strain was detected in the granuloma of 1 patient over the 2 tested. T-cell counts, including naïve CD4+CD45RA+ T cells and T-cell function were low at diagnosis for 6 patients. For most patients with available values naïve CD4+CD45RA+ T cells decreased over time (n=5/6). Hematopoietic stem cell transplantation (HSCT) was performed in 5 patients, of whom 4 are still alive without transplant-related morbidity. Sustained T- and B-cell reconstitution was respectively observed for 4 and 3 patients, after a median follow-up of 8 years (range 3-16 y). DNA-PKcs deficiency mainly manifests as an inflammatory disease with granuloma and autoimmune features, along with severe infections.

ARID1A is involved in DNA double-strand break repair in gastric cancer.

Defects in DNA damage repair can cause genetic mutations, which in turn can cause different types of cancers. Chromatin remodeling complexes, which help repair damaged DNA, can cause the chromatin structure to change as a result of DNA damage. ARID1A may play a role in the process of DNA damage repair, and arid1a may be related to the occurrence and development of gastric cancer (GC). This study aimed to investigate the mechanism of ARID1A regulating the DNA damage repair of gastric adenocarcinoma cell lines AGS and SGC-7901 and its effect on migration, proliferation and apoptosis. The expression of ARID1A plasmid was detected by Western blot and real-time polymerase chain reaction (PCR). The effect of etoposide (ETO) on the survival rate of AGS and SGC-7901 gastric adenocarcinoma cell lines was detected by MTT assay. The DNA double-strand break model was established by ETO and then passed through the comet assay and immunofluorescence co-localization to observe DNA damage; western blot method was used to detect the effect of ARID1A on the expression of related proteins in DNA damage repair pathway in gastric adenocarcinoma cells; scratch test and colony formation experiments were used to observe ARID1A migration and proliferation of gastric adenocarcinoma cells. The flow cytometry was used to detect the effect of ARID1A on apoptosis of gastric adenocarcinoma cells. The expression of mRNA and protein was increased after transfection of ARID1A plasmid. ETO was confirmed by MTT assay to inhibit cell survival in a dose-dependent manner. After the DNA double-strand break model was established by ETO, the expression levels of phospho-ataxia telangiectasia mutated (p-ATM) protein increased in the overexpressed ARID1A group. Meanwhile, the overexpressed ARID1A group had a shortened tail moment, and γ-H2AX and ARID1A co-localized in the DNA damage site of the nucleus. The over-expressed ARID1A group had weaker wound healing ability, reduced number of clone formation, and increased apoptosis rate. ARID1A may repair DNA double-strand breaks caused by ETO by p-ATM pathway; ARID1A can inhibit the migration and proliferation of gastric adenocarcinoma cells and promote apoptosis. Our findings indicate that ARID1A could serve as a therapeutic target and biomarker for GC patients.

Inhibition of key DNA double strand break repair protein kinases enhances radiosensitivity of head and neck cancer cells to X-ray and proton irradiation

Ionising radiation (IR) is widely used in cancer treatment, including for head and neck squamous cell carcinoma (HNSCC), where it induces significant DNA damage leading ultimately to tumour cell death. Among these lesions, DNA double strand breaks (DSBs) are the most threatening lesion to cell survival. The two main repair mechanisms that detect and repair DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR). Among these pathways, the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA dependent protein kinase catalytic subunit (DNA-Pkcs) play key roles in the sensing of the DSB and subsequent coordination of the downstream repair events. Consequently, targeting these kinases with potent and specific inhibitors is considered an approach to enhance the radiosensitivity of tumour cells. Here, we have investigated the impact of inhibition of ATM, ATR and DNA-Pkcs on the survival and growth of six radioresistant HPV-negative HNSCC cell lines in combination with either X-ray irradiation or proton beam therapy, and confirmed the mechanistic pathway leading to cell radiosensitisation. Using inhibitors targeting ATM (AZD1390), ATR (AZD6738) and DNA-Pkcs (AZD7648), we observed that this led to significantly decreased clonogenic survival of HNSCC cell lines following both X-ray and proton irradiation. Radiosensitisation of HNSCC cells grown as 3D spheroids was also observed, particularly following ATM and DNA-Pkcs inhibition. We confirmed that the inhibitors in combination with X-rays and protons led to DSB persistence, and increased micronuclei formation. Cumulatively, our data suggest that targeting DSB repair, particularly via ATM and DNA-Pkcs inhibition, can exacerbate the impact of ionising radiation in sensitising HNSCC cell models.

Induced Pluripotent Stem Cell-Derived Fibroblasts Efficiently Engage Senescence Pathways but Show Increased Sensitivity to Stress Inducers.

The risk of aberrant growth of induced pluripotent stem cell (iPSC)-derived cells in response to DNA damage is a potential concern as the tumor suppressor genes TP53 and CDKN2A are transiently inactivated during reprogramming. Herein, we evaluate the integrity of cellular senescence pathways and DNA double-strand break (DSB) repair in Sendai virus reprogrammed iPSC-derived human fibroblasts (i-HF) compared to their parental skin fibroblasts (HF). Using transcriptomics analysis and a variety of functional assays, we show that the capacity of i-HF to enter senescence and repair DSB is not compromised after damage induced by ionizing radiation (IR) or the overexpression of H-RASV12. Still, i-HF lines are transcriptionally different from their parental lines, showing enhanced metabolic activity and higher expression of p53-related effector genes. As a result, i-HF lines generally exhibit increased sensitivity to various stresses, have an elevated senescence-associated secretory phenotype (SASP), and cannot be immortalized unless p53 expression is knocked down. In conclusion, while our results suggest that i-HF are not at a greater risk of transformation, their overall hyperactivation of senescence pathways may impede their function as a cell therapy product.

The impact of developmental stage, tissue type, and sex on DNA double-strand break repair in Drosophila melanogaster.

Accurate repair of DNA double-strand breaks (DSBs) is essential for the maintenance of genome integrity, as failure to repair DSBs can result in cell death. The cell has evolved two main mechanisms for DSB repair: non-homologous end-joining (NHEJ) and homology-directed repair (HDR), which includes single-strand annealing (SSA) and homologous recombination (HR). While certain factors like age and state of the chromatin are known to influence DSB repair pathway choice, the roles of developmental stage, tissue type, and sex have yet to be elucidated in multicellular organisms. To examine the influence of these factors, DSB repair in various embryonic developmental stages, larva, and adult tissues in Drosophila melanogaster was analyzed through molecular analysis of the DR-white assay using Tracking across Indels by DEcomposition (TIDE). The proportion of HR repair was highest in tissues that maintain the canonical (G1/S/G2/M) cell cycle and suppressed in both terminally differentiated and polyploid tissues. To determine the impact of sex on repair pathway choice, repair in different tissues in both males and females was analyzed. When molecularly examining tissues containing mostly somatic cells, males and females demonstrated similar proportions of HR and NHEJ. However, when DSB repair was analyzed in male and female premeiotic germline cells utilizing phenotypic analysis of the DR-white assay, there was a significant decrease in HR in females compared to males. This study describes the impact of development, tissue-specific cycling profile, and, in some cases, sex on DSB repair outcomes, underscoring the complexity of repair in multicellular organisms.

Abstract 1112: Modulated dNTP pools and their influence on DNA repair mechanisms and apoptosis in therapy resistant cancers

Abstract Glioblastoma (GBM) is a deadly primary tumor of the brain, accounting for almost half of all diagnosed malignant gliomas. Ionizing radiation (IR) and some chemotherapeutics strategically target cancer cells by causing DNA double-strand breaks (DSBs). However, cancers like GBM often develop resistance to DSB-inducing therapeutics due to dysregulated DNA repair. Homologous Recombination (HR) is an error-free DSB-repair process consisting of proteins that are often dysregulated in GBM, causing overactive DNA-repair, therapy resistance, and poor patient outcomes. Proteins involved in the HR-repair process have been extensively investigated; however, therapeutic resistance remains common. Here, we provide an alternative approach to target DSB-resistant GBM by modulating the dNTP pool instead of solely targeting DNA-repair proteins. Our goal is to understand the interplay between dNTP pools and DSB- repair. dNTPs are a necessary constituent to repair DSBs; however, the role of the dNTP pool in DNA damage repair is not fully understood. Previous research shows that the dNTP pool remains unchanged after inducing DSBs, highlighting the importance of a tightly regulated dNTP pool. Using a neutral comet assay, we quantified accumulated double-stranded breaks in different treatment groups. We then analyzed these groups using immunofluorescent microscopy to examine distinct proteins involved in HR-mediated repair. We carried out RNA sequencing analysis to determine differential gene expression. We quantified cell viability and apoptotic cell populations via flow cytometry and Alamar blue assays. We determined protein interactions using immunoblotting and immunoprecipitation assays. We found that increasing the dNTP pool prior to IR treatment led to impaired DNA DSB repair in GBM cells as determined by a neutral comet assay. When analyzing key proteins involved in HR via immunofluorescence, we observed a significant decrease in RPA70 localization to the DNA damage site, indicating a disruption in end resection, a necessary phase of HR. Our RNA sequencing results show that increasing the dNTP pool in LN229 GBM cells prior to IR treatment caused significant changes in gene expression when compared to IR treatment alone. The most impacted pathways most were apoptosis, p53 cell cycle arrest, and DNA repair. The pathway analysis has identified genes of interest in the synergistic dNTP + IR treatment model. Significant increases in apoptosis and decreases in viability were observed 48 hours after inducing DSBs in LN229 cells with elevated dNTP pools. Not only do these data suggest that HR efficiency is reduced due to end resection impairment, but they also show that as a result, cell viability is destabilized due to prolonged DNA damage, resulting in apoptosis. The identification of this impairment may be essential in finding novel therapeutic approaches to sensitize therapy resistant GBM. Citation Format: Dominique Monroe, Mercy Kehinde-Ige, Edidiong Usoro, Arilyn Williams, Emily Steinbeck, Shelby Aycox, Huidong Shi, Waaqo Daddacha. Modulated dNTP pools and their influence on DNA repair mechanisms and apoptosis in therapy resistant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1112.

RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double-strand breaks, making it challenging to generate knock-in events. In this study, we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrated stable and heritable knock-ins in wheat in 1% of the primary transformants. Taken together, our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.

Genetic And Environmental Associations Of Nonspecific Chromosomal Aberrations.

Nonspecific structural chromosomal aberrations (CAs) are found in around 1% of circulating lymphocytes from healthy individuals but the frequency may be higher after exposure to carcinogenic chemicals or radiation. CAs have been used in the monitoring of persons exposed to genotoxic agents and radiation. Previous studies on occupationally exposed individuals have shown associations between the frequency of CAs in peripheral blood lymphocytes and subsequent cancer risk. The cause for CA formation are believed to be unrepaired or insufficiently repaired DNA double-strand breaks or other DNA damage, and additionally telomere shortening. CAs include chromosome (CSAs) and chromatid type aberrations (CTAs). In the present review, we first describe the types of CAs, the conventional techniques used for their detection and some aspects of interpreting the results. We then focus on germline genetic variation in the frequency and type of CAs measured in a genome-wide association study (GWAS) in healthy individuals in relation to occupational and smoking-related exposure compared to non-exposed referents. The associations (at p<10-5) on 1473 healthy individuals were broadly classified in candidate genes from functional pathways related to DNA damage response/repair, including PSMA1, UBR5, RRM2B, PMS2P4, STAG3L4, BOD1, COPRS and FTO; another group included genes related to apoptosis, cell proliferation, angiogenesis and tumorigenesis, COPB1, NR2C1, COPRS, RHOT1, ITGB3, SYK, and SEMA6A; a third small group mapped to genes KLF7, SEMA5A and ITGB3 which were related to autistic traits, known to manifest frequent CAs. Dedicated studies on 153 DNA repair genes showed associations for some 30 genes, expression of which could be modified by the implicated variants. We finally point out that monitoring of CAs is so far the only method of assessing cancer risk in healthy human populations, and the use of the technology should be made more attractive by developing automated performance steps and incorporating artificial intelligence methods into the scoring.

Germline specific genes increase DNA double-strand break repair and radioresistance in lung adenocarcinoma cells

In principle, germline cells possess the capability to transmit a nearly unaltered set of genetic material to infinite future generations, whereas somatic cells are limited by strict growth constraints necessary to assure an organism’s physical structure and eventual mortality. As the potential to replicate indefinitely is a key feature of cancer, we hypothesized that the activation of a “germline program” in somatic cells can contribute to oncogenesis. Our group recently described over one thousand germline specific genes that can be ectopically expressed in cancer, yet how germline specific processes contribute to the malignant properties of cancer is poorly understood. We here show that the expression of germ cell/cancer (GC) genes correlates with malignancy in lung adenocarcinoma (LUAD). We found that LUAD cells expressing more GC genes can repair DNA double strand breaks more rapidly, show higher rates of proliferation and are more resistant to ionizing radiation, compared to LUAD cells that express fewer GC genes. In particular, we identified the HORMA domain protein regulator TRIP13 to be predominantly responsible for this malignant phenotype, and that TRIP13 inhibition or expression levels affect the response to ionizing radiation and subsequent DNA repair. Our results demonstrate that GC genes are viable targets in oncology, as they induce increased radiation resistance and increased propagation in cancer cells. Because their expression is normally restricted to germline cells, we anticipate that GC gene directed therapeutic options will effectively target cancer, with limited side effects besides (temporary) infertility.

Ku80 is indispensable for repairing DNA double-strand breaks at highly methylated sites in human HCT116 cells

DNA double-strand breaks (DSBs) are harmful to mammalian cells and a few of them can cause cell death. Accumulating DSBs in these cells to analyze their genomic distribution and their potential impact on chromatin structure is difficult. In this study, we used CRISPR to generate Ku80-/- human cells and arrested the cells in G1 phase to accumulate DSBs before conducting END-seq and Nanopore analysis. Our analysis revealed that DNA with high methylation level accumulates DSB hotspots in Ku80-/- human cells. Furthermore, we identified chromosome structural variants (SVs) using Nanopore sequencing and observed a higher number of SVs in Ku80-/- human cells. Based on our findings, we suggest that the high efficiency of Ku80 knockout in human HCT116 cells makes it a promising model for characterizing SVs in the context of 3D chromatin structure and studying the alternative-end joining (Alt-EJ) DSB repair pathway.

Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases.

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

An Efficient CRISPR/Cas9 Genome Editing System for a Ganoderma lucidum Cultivated Strain by Ribonucleoprotein Method.

The CRISPR/Cas9 system has become a popular approach to genome editing. Compared with the plasmid-dependent CRISPR system, the ribonucleoprotein (RNP) complex formed by the in vitro assembly of Cas9 and single-guide RNA (sgRNA) has many advantages. However, only a few examples have been reported and the editing efficiency has been relatively low. In this study, we developed and optimized an RNP-mediated CRISPR/Cas9 genome editing system for the monokaryotic strain L1 from the Ganoderma lucidum cultivar 'Hunong No. 1'. On selective media containing 5-fluoroorotic acid (5-FOA), the targeting efficiency of the genomic editing reached 100%. The editing efficiency of the orotidine 5'-monophosphate decarboxylase gene (ura3) was greater than 35 mutants/107 protoplasts, surpassing the previously reported G. lucidum CRISPR systems. Through insertion or substitution, 35 mutants introduced new sequences of 10-569 bp near the cleavage site of ura3 in the L1 genome, and the introduced sequences of 22 mutants (62.9%) were derived from the L1 genome itself. Among the 90 mutants, 85 mutants (94.4%) repaired DNA double-strand breaks (DSBs) through non-homologous end joining (NHEJ), and five mutants (5.6%) through microhomology-mediated end joining (MMEJ). This study revealed the repair characteristics of DSBs induced by RNA-programmed nuclease Cas9. Moreover, the G. lucidum genes cyp512a3 and cyp5359n1 have been edited using this system. This study is of significant importance for the targeted breeding and synthetic metabolic regulation of G. lucidum.

Clonal Medicine Targeting DNA Damage Response Eradicates AML

Clonal diversity of acute myeloid leukemia (AML) plays a key role in poor therapeutic outcomes. AML cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, AML cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous AML-driving mutations [ FLT3(ITD), TETmut, DNMT3Amut, AML1-ETO, PML-RARa , IDH1mut, cKITmut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual AML clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol AML cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of AML at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all AML clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused clonal attrition and eradicated the disease in vitro and in vivo. For example, in AML-MD2 sample 2 clones carrying EZH2(V679M) + TET2(L1721W) and EZH2(V679M) + TET2(L1721W) + FLT3(D835Y) were resistant/less responsive to ATRi but sensitive to PARPi, ATMi and RAD52i. Conversely, three other clones carrying EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y), EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y) + EZH2(E54*) and EZH2(V679M) + TET2(L1721W) + RUNX1(D160Y)EZH2(E54*) + BCOR1(R1334Tfs*32) + NRAS(G13R) were sensitive to ATRi while less responsive to PARPi, ATMi and RAD52i. Based on this observation, we hypothesized that simultaneous treatment with ATRi + PARPi or ATRi + ATMi should result in elimination of all AML-MD2 clones. In agreement with this hypothesis, in vitro treatment with these combinations was 20 - 30x more effective in eliminating clonogenic growth of Lin-CD34+ AML-MD2 cells when compared to individual inhibitors. This effect was associated with abundant accumulation of DSBs. Combinations of these inhibitors were only modestly toxic to normal hematopoietic cells. Moreover, combination of DDRi displaying similar clonal targeting specificity was only 2x better than individual inhibitors. Next, humanized NRGS immunodeficient mice bearing primary AML-MD2 xenograft were treated with vehicle, PARPi, ATRi and the combination of these drugs. We found that individual treatments reduced the number of hCD45+ AML-MD2 cells in bone marrow and spleen by approximately 2x and 3x, respectively. Remarkably, the combination of PARPi + ATRi eliminated more than 99% of leukemia cells in bone marrows of 7/10 mice and in spleens of 10/10 mice while no obvious toxicity was observed. Clonal targeting by DDR inhibitors, however, may not be applicable to all AML samples. For example, we identified AML patient samples, where clones displayed similar sensitivity pattern or were resistant to DDRi treatment. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of AML to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of AML in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in AML clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

Adaptive changes in the DNA damage response during skeletal muscle cell differentiation

DNA double-strand breaks (DSBs) trigger specialized cellular mechanisms that collectively form the DNA damage response (DDR). In proliferating cells, the DDR serves the function of mending DNA breaks and satisfying the cell-cycle checkpoints. Distinct goals exist in differentiated cells that are postmitotic and do not face cell-cycle checkpoints. Nonetheless, the distinctive requirements and mechanistic details of the DDR in differentiated cells are still poorly understood. In this study, we set an in vitro differentiation model of human skeletal muscle myoblasts into multinucleated myotubes that allowed monitoring DDR dynamics during cell differentiation. Our results demonstrate that myotubes have a prolonged DDR, which is nonetheless competent to repair DSBs and render them significantly more resistant to cell death than their progenitors. Using live-cell microscopy and single-molecule kinetic measurements of transcriptional activity, we observed that myotubes respond to DNA damage by rapidly and transiently suppressing global gene expression and rewiring the epigenetic landscape of the damaged nucleus. Our findings provide novel insights into the DDR dynamics during cellular differentiation and shed light on the strategy employed by human skeletal muscle to preserve the integrity of the genetic information and sustain long-term organ function after DNA damage.

Clinical response of pancreatic cancer bearing a germline BRCA2 p.I3169M fs*48 variant for platinum-based drug and PARP inhibitor.

Pancreatic cancer is a malignancy with a high mortality rate, accounting for 37 000 people annually in Japan. It is rarely diagnosed in a resectable state, and effective medicines for its advanced stage are scarce. Some pancreatic cancer is hereditary, and ~10% have germline mutations of Breast cancer 1/2 (BRCA1/2). BRCA1/2 are key molecules involved in homologous recombination to repair DNA double-strand break. Platinum-based drugs and poly Adenosine diphosphate ribose (ADP) ribose polymerase inhibitors that induce synthetic lethality would be theoretically effective in patients with loss-of-function mutations in BRCA1/2. Strictly speaking, some discrepancy between the pathogenicity of BRCA1/2 and their drug sensitivity might be expected. Hence, we report that platinum-based anticancer agents and poly ADP ribose polymerase inhibitors were effective against pancreatic cancer bearing BRCA2 p.I3169M fs*48.

Clonal Targeting of DNA Damage Response Pathways Eradicates Myeloproliferative Neoplasms

Clonal diversity of myeloproliferative neoplasms (MPN) plays a key role in poor therapeutic outcomes. MPN cells usually accumulate spontaneous DNA damage including highly toxic DNA double-strand breaks (DSBs) induced by metabolic products and replication stress. To repair numerous DSBs and survive, MPN cells activate the DNA damage response (DDR) involving the pathways that sense (ATM and ATR kinases) and repair (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, PARP1/Polq-dependent microhomology-mediated end-joining = MMEJ) DSBs. Thus, DDR is a legitimate therapeutic target. Numerous MPN-driving mutations [ JAK2(V617F), TETmut, DNMT3Amut, IDH1mut] regulate DDR and affect the sensitivity of leukemia cells to DDR inhibitors (DDRi). The genetic landscape of malignant clones in a patient may be complicated since individual clones can carry multiple mutations. Thus, individual MPN clones may respond differently to DDRi depending on their mutational profile. In our experimental protocol MPN cells from individual patients were treated with various DDRi followed by single-cell targeted DNA sequencing (sctDNA-seq) to integrate patient's leukemia clonal composition with response to the inhibitors. We developed a sctDNA-seq myeloid platform interrogating up to 1394 genetic variants of 54 known leukemia driver genes, which unraveled the clonal landscape of MPN at a single-cell resolution before and after the treatment. Based on these results we designed a “clonal attack”, a patient-tailored combination of DDRi targeting all MPN clones in a patient sample. Here we show that DDRi simultaneously attacking different clones caused MPN clonal attrition in vitro. For example, phylogenetic tree analysis of MPN patient P349 sample detected linear multi-clonal architecture with 3 Lin-CD34+ clones carrying specific sets of mutations. sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. The clone carrying KMT2A(L2373H) + SETBP1(H1100R) was more sensitive to ATRi than RAD52i, conversely clones with KMT2A(L2373H) alone and KMT2A(L2373H) + SETBP1(H1100R) + FLT3(R834L) were more sensitive to RAD52i than ATRi. Based on this observation, we hypothesized that simultaneous treatment with ATRi+RAD52i should result in elimination of all 3 MPN P349 clones. Remarkably, the combination of RAD52i + ATRi was &amp;gt;100x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells (all clonogenic cells were eradicated). On the other hand, combination of RAD52i + ATMi, the two DDR inhibitors displaying similar pattern of clonal targeting was only 2x better than individual inhibitors. Phylogenetic tree analysis of Lin-CD34+ cells from another MPN patient P350 sample showed linear multi-clonal architecture with 4 clones. Again, sctDNA-seq followed by fish plot analysis revealed clonal similarities and differences in response to DDRi. All 4 clones displayed similar sensitivity to RAD52i and ATMi, but they responded differently to PARPi and ATRi. Clones carrying TET2(P363L) + NRAS(G12D) were more sensitive to ATRi than PARPi, whereas clones with TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) and TET2(P363L) + NRAS(G12D) + DNMT3A(W330C) + IDH2(R132C) responded better to PARPi than ATRi. Importantly, the combination of PARPi + ATRi was &amp;gt;9x more effective in inhibiting clonogenic growth of Lin-CD34+ P349 cells, whereas RAD52i + ATMi was only 2x better than individual inhibitors. In conclusion, we postulate that sctDNA-seq combined with in vitro DDRi sensitivity testing (sctDNA-seq/DDRi) is a powerful tool to interrogate clonal sensitivity of MPN to these agents. This clonal medicine approach may become a novel therapeutic regimen to overcome clonal complexity of MPN in a cohort of patients. The “clonal attack” by DDR inhibitors shifts the paradigm of genotoxic therapies from those using non-discriminative cytotoxic drugs to those selectively attacking DDR vulnerabilities in MPN clones with minimal harm to normal cells. Since clonal heterogeneity and DNA damage are hallmarks of cancer, the “clonal attack” may be broadly applicable to the quest for cancer cure.

A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae.

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions(SEIs) and double-Holliday junctions(dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.

AZD-7648, a DNA-PK Inhibitor, Induces DNA Damage, Apoptosis, and Cell Cycle Arrest in Chronic and Acute Myeloid Leukemia Cells.

The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML (HEL, HL-60, KG-1, NB-4, and THP-1) cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor.