Homologous Recombination Genes Research Articles

Gene editing by SSB/CRISPR-Cas9 ribonucleoprotein in bacteria

The application of CRISPR-Cas9 ribonucleoprotein (RNP) for gene editing is commonly used in plants and animals, but its application in bacteria has not been reported. In this study, we employed DNA single-strand binding protein (SSB) to construct an SSB/CRISPR-Cas9 RNP-editing system for non-homologous recombination and homologous recombination gene editing of the upp gene in bacteria. The RNP targeting the upp gene, along with SSB, was introduced into the protoplasts of Escherichia coli, Pseudomonas, and Bacillus subtilis. Transformants were obtained on plates containing 5-fluorouracil (5-FU) with gene editing efficiencies (percentage of transformants relative to the number of protoplasts) of 9.75 %, 5.02 %, and 8.37 %, respectively, and sequencing analysis confirmed 100 % non-homologous recombination. When RNP, SSB, and a 100-nucleotide single-stranded oligodeoxynucleotide (ssODN) donor were introduced into the protoplasts of these bacteria, transformants were obtained with editing efficiencies of 45.11 %, 30.13 %, and 27.18 %, respectively, and sequencing confirmed 100 % homologous recombination knockout of the upp gene. Additionally, introducing RNP, SSB, and a 100 base-pair double-stranded oligodeoxynucleotide (dsODN) donor containing a tetracycline resistance gene (tetR-dsODN) resulted in transformants on 5-FU plates with editing efficiencies of 35.94 %, 22.46 %, and 19.08 %, respectively, with sequencing confirming 100 % homologous recombination replacement of the upp gene with tetR. These results demonstrate that the SSB/CRISPR-Cas9 RNP system can efficiently, simply, and rapidly edit bacterial genomes without the need for plasmids. This study is the first to report the use of RNP-based gene editing in bacteria.

Multicenter non-interventional study of prevalence of homologous recombination gene mutations and approaches to treatment of metastatic castration-resistant prostate cancer in Russia (ADAM)

Multicenter non-interventional study of prevalence of homologous recombination gene mutations and approaches to treatment of metastatic castration-resistant prostate cancer in Russia (ADAM)

XRCC2 driven homologous recombination subtypes and therapeutic targeting in lung adenocarcinoma metastasis

Lung adenocarcinoma (LUAD) is a leading cause of cancer mortality, with many patients facing poor prognosis, particularly those with metastatic or drug-resistant tumors. Homologous recombination genes (HRGs) are crucial in tumor progression and therapy resistance, but their clinical significance in LUAD is not well understood. In this study, we systematically characterize key HRGs in LUAD patients, identifying two distinct HR subtypes associated with different outcomes and biological functions. We establish a 5-gene scoring system (XRCC2, RAD51, BRCA1, FANCA, and CHEK1) that reliably predicts patient outcomes and immunotherapy responses in LUAD. Bioinformatics analysis and clinical validation highlight XRCC2 as a crucial biomarker in LUAD. Functional investigations through in vivo and in vitro experiments reveal the role of XRCC2 in promoting lung cancer migration and invasion. Mechanistically, XRCC2 stabilizes vimentin (VIM) protein expression through deubiquitylation. We predict c-MYC as a potential regulator of XRCC2 and demonstrate that inhibiting c-MYC with compound 10058-F4 reduces XRCC2 and VIM expression. Preclinical studies show the synergistic inhibition of metastasis in vivo when combining 10058-F4 with doxorubicin (Dox). Our findings present a potential personalized predictive tool for LUAD prognosis, identifying XRCC2 as a critical biomarker. The c-Myc-XRCC2-VIM axis emerges as a promising therapeutic target for overcoming lung metastasis. This study provides valuable insights into LUAD, proposing a prognostic tool for further clinical validation and unveiling a potential therapeutic strategy for combating lung metastasis by targeting c-Myc-XRCC2-VIM.

CDC20 Holds Novel Regulation Mechanism in RPA1 during Different Stages of DNA Damage to Induce Radio-Chemoresistance.

Targeting CDC20 can enhance the radiosensitivity of tumor cells, but the function and mechanism of CDC20 on DNA damage repair response remains vague. To examine that issue, tumor cell lines, including KYSE200, KYSE450, and HCT116, were utilized to detect the expression, function, and underlying mechanism of CDC20 in radio-chemoresistance. Western blot and immunofluorescence staining were employed to confirm CDC20 expression and location, and radiation could upregulate the expression of CDC20 in the cell nucleus. The homologous recombination (HR) and non-homologous end joining (NHEJ) reporter gene systems were utilized to explore the impact of CDC20 on DNA damage repair, indicating that CDC20 could promote HR repair and radio/chemo-resistance. In the early stages of DNA damage, CDC20 stabilizes the RPA1 protein through protein-protein interactions, activating the ATR-mediated signaling cascade, thereby aiding in genomic repair. In the later stages, CDC20 assists in the subsequent steps of damage repair by the ubiquitin-mediated degradation of RPA1. CCK-8 and colony formation assay were used to detect the function of CDC20 in cell vitality and proliferation, and targeting CDC20 can exacerbate the increase in DNA damage levels caused by cisplatin or etoposide. A tumor xenograft model was conducted in BALB/c-nu/nu mice to confirm the function of CDC20 in vivo, confirming the in vitro results. In conclusion, this study provides further validation of the potential clinical significance of CDC20 as a strategy to overcome radio-chemoresistance via uncovering a novel role of CDC20 in regulating RPA1 during DNA damage repair.

Endogenous base damage as a driver of genomic instability in homologous recombination-deficient cancers

Homologous recombination (HR) is a high-fidelity DNA double-strand break (DSB) repair pathway. Both familial and somatic loss of function mutation(s) in various HR genes predispose to a variety of cancer types, underscoring the importance of error-free repair of DSBs in human physiology. While environmental sources of DSBs have been known, more recent studies have begun to uncover the role of endogenous base damage in leading to these breaks. Base damage repair intermediates often consist of single-strand breaks, which if left unrepaired, can lead to DSBs as the replication fork encounters these lesions. This review summarizes various sources of endogenous base damage and how these lesions are repaired. We highlight how conversion of base repair intermediates, particularly those with 5′or 3′ blocked ends, to DSBs can be a predominant source of genomic instability in HR-deficient cancers. We also discuss how endogenous base damage and ensuing DSBs can be exploited to enhance the efficacy of Poly (ADP-ribose) polymerase inhibitors (PARPi), that are widely used in the clinics for the regimen of HR-deficient cancers.

Alterations in the expression of homologous recombination repair (HRR) genes in breast cancer tissues considering germline BRCA1/2 mutation status.

Homologous recombination (HR) is a crucial DNA-repair mechanism, and its disruption can lead to the accumulation of mutations that initiate and promote cancer formation. The key HR genes, BRCA1 and BRCA2, are particularly significant as their germline pathogenic variants are associated with a hereditary predisposition to breast and/or ovarian cancer. The study was performed on 45 FFPE breast cancer tissues obtained from 24 and21 patients, with and without the germline BRCA1/2 mutation, respectively. The expression of 11 genes: BRCA1, BRCA2, ATM, BARD1, FANCA, FANCB, FANCI, RAD50, RAD51D, BRIP1, and CHEK2 was assessed using Custom RT2 PCR Array (Qiagen), and results were analysed using R. Cancer tissues from patients with BRCA1 or BRCA2 germline mutations displayed no significant differences in the expression of the selected HR genes compared to BRCA1 or BRCA2 wild-type cancer tissues. In BRCA1mut cancer tissues, BRCA1 expression was significantly higher than in BRCA2mut and BRCA wild-type cancer tissues. In cancer tissues harbouring either BRCA1 or BRCA2 germline mutations, no significant differences in expression were observed at the mRNA level of any tested HR genes, except BRCA1. However, the significant differences observed in BRCA1 expression between germline BRCA1mut,germline BRCA2mut and BRCA1/2wttissues may indicate a compensatory mechanism at the mRNA level to mitigate the loss of BRCA1 function in the cells.

Limitations of homologous recombination status testing and poly (ADP-ribose) polymerase inhibitor treatment in the current management of ovarian cancer.

Homologous recombination (HR) is a highly conserved DNA repair system, in which aberrations can lead to the accumulation of DNA damage and genomic scars known as homologous recombination deficiency (HRD). The identification of mutations in key genes (i.e., BRCA1, and BRCA2 (BRCA)) and the quantification of large-scale structural variants (e.g., loss of heterozygosity) are indicators of the HRD phenotype. HRD is a stable biomarker and remains unchanged during recurrence, but fails to reveal the molecular profile of tumor progression. Moreover, interpretation of the current HRD score lacks comprehensiveness, especially for the HR-proficient group. Poly (ADP-ribose) polymerase (PARP) enzymes play an important role in the repair of DNA single-strand breaks, the blockage of which using PARP inhibitors (PARPi) can generate synthetic lethality in cancer cells with HRD. Although numerous studies have demonstrated that the benefit of PARPi is substantial in ovarian cancer (OC) patients, the efficacy is limited by the development of resistance, and seems to be irrespective of HR and/or BRCA mutation status. Moreover, in addition to improving progression-free survival, long-term benefit as overall survival brought by PARPi for advanced, recurrent and refractory OC patients remains unclear. Therefore, further investigations are needed to uncover the role of HR genes beyond BRCA and their interactions with other oncogenic pathways, to determine the value of HRD in the recurrent setting, and to identify alternative strategies for the precise management of advanced, refractory OC patients.

Molecular consequences of acute versus chronic CDK12 loss in prostate carcinoma nominates distinct therapeutic strategies.

Genomic loss of the transcriptional kinase CDK12 occurs in ~6% of metastatic castration-resistant prostate cancers (mCRPC) and correlates with poor patient outcomes. Prior studies demonstrate that acute CDK12 loss confers a homologous recombination (HR) deficiency (HRd) phenotype via premature intronic polyadenylation (IPA) of key HR pathway genes, including ATM. However, mCRPC patients have not demonstrated benefit from therapies that exploit HRd such as inhibitors of polyADP ribose polymerase (PARP). Based on this discordance, we sought to test the hypothesis that an HRd phenotype is primarily a consequence of acute CDK12 loss and the effect is greatly diminished in prostate cancers adapted to CDK12 loss. Analyses of whole genome sequences (WGS) and RNA sequences (RNAseq) of human mCRPCs determined that tumors with biallelic CDK12 alterations (CDK12 BAL ) lack genomic scar signatures indicative of HRd, despite carrying bi-allelic loss and the appearance of the hallmark tandem-duplicator phenotype (TDP). Experiments confirmed that acute CDK12 inhibition resulted in aberrant polyadenylation and downregulation of long genes (including BRCA1 and BRCA2) but such effects were modest or absent in tumors adapted to chronic CDK12 BAL . One key exception was ATM, which did retain transcript shortening and reduced protein expression in the adapted CDK12 BAL models. However, CDK12 BAL cells demonstrated intact HR as measured by RAD51 foci formation following irradiation. CDK12 BAL cells showed a vulnerability to targeting of CDK13 by sgRNA or CDK12/13 inhibitors and in vivo treatment of prostate cancer xenograft lines showed that tumors with CDK12 BAL responded to the CDK12/13 inhibitor SR4835, while CDK12-intact lines did not. Collectively, these studies show that aberrant polyadenylation and long HR gene downregulation is primarily a consequence of acute CDK12 deficiency, which is largely compensated for in cells that have adapted to CDK12 loss. These results provide an explanation for why PARPi monotherapy has thus far failed to consistently benefit patients with CDK12 alterations, though alternate therapies that target CDK13 or transcription are candidates for future research and testing.

Predictive modeling of gene mutations for the survival outcomes of epithelial ovarian cancer patients.

Epithelial ovarian cancer (EOC) has a low overall survival rate, largely due to frequent recurrence and acquiring resistance to platinum-based chemotherapy. EOC with homologous recombination (HR) deficiency has increased sensitivity to platinum-based chemotherapy because platinum-induced DNA damage cannot be repaired. Mutations in genes involved in the HR pathway are thought to be strongly correlated with favorable response to treatment. Patients with these mutations have better prognosis and an improved survival rate. On the other hand, mutations in non-HR genes in EOC are associated with increased chemoresistance and poorer prognosis. For this reason, accurate predictions in response to treatment and overall survival remain challenging. Thus, analyses of 360 EOC cases on NCI's The Cancer Genome Atlas (TCGA) program were conducted to identify novel gene mutation signatures that were strongly correlated with overall survival. We found that a considerable portion of EOC cases exhibited multiple and overlapping mutations in a panel of 31 genes. Using logistical regression modeling on mutational profiles and patient survival data from TCGA, we determined whether specific sets of deleterious gene mutations in EOC patients had impacts on patient survival. Our results showed that six genes that were strongly correlated with an increased survival time are BRCA1, NBN, BRIP1, RAD50, PTEN, and PMS2. In addition, our analysis shows that six genes that were strongly correlated with a decreased survival time are FANCE, FOXM1, KRAS, FANCD2, TTN, and CSMD3. Furthermore, Kaplan-Meier survival analysis of 360 patients stratified by these positive and negative gene mutation signatures corroborated that our regression model outperformed the conventional HR genes-based classification and prediction of survival outcomes. Collectively, our findings suggest that EOC exhibits unique mutation signatures beyond HR gene mutations. Our approach can identify a novel panel of gene mutations that helps improve the prediction of treatment outcomes and overall survival for EOC patients.

Modulation of homologous recombination gene activity in breast tumor cells in an <i>in vitro</i> model

Introduction. It has been established that the presence of homologous recombination deficiency in a breast tumor is associated with the effectiveness of treatment. But despite the high chemosensitivity of the tumor to DNA-damaging agents, complete pathological responses to treatment are very rare. And this process may be based on a change in the somatic status of BRCA1, that is, a reversion and return of the wild-type allele occurs and the DNA repair function is restored.Aim. To evaluate changes in the presence of chromosomal aberrations and the expression profile of the main genes of homologous recombination in cell models of breast cancer under the influence of cisplatin and docetaxel.Materials and methods. The study was conducted on breast cancer tumor cell cultures: MCF-7, MDA-MB-231 and MDA-MB-468. A cell model of drug resistance was obtained for two drugs: cisplatin and docetaxel. RNA and DNA were isolated from cell suspension using the RNeasy Plus Mini Kit and QIAamp DNA Mini Kit (Qiagen, Germany), respectively. The expression level of homologous recombination genes was assessed using reverse transcription polymerase chain reaction. To assess the presence of chromosomal aberrations, microarray analysis was performed on DNA chips.Results. Restoration of normal copy number for the BRCA1, CDK12, CHEK1 and RAD51D genes in MCF-7 under the influence of cisplatin was shown. For BRCA2 and PALB2, amplifications were detected. A statistically significant increase in the expression of the BRCA1 (p = 0.04), BRCA2 (p = 0.02), PALB2 (p = 0.01) and RAD51D (p = 0.05) genes was also shown. MDAMB-231 shows that all identified loci with deletions, where the BRCA2, BARD1, CHEK2, PALB2 and RAD54L genes are localized, are restored to normal copy number by cisplatin. The appearance of amplifications was registered for BRCA1, BRIP1, FANCL, RAD51B, PARP1. A similar result was shown for docetaxel. An increase in the expression level is typical for the genes BRCA1 (p = 0.02), BRCA2 (p = 0.02), CHEK2 (p = 0.05), FANCL (p = 0.04), PALB2 (p = 0.05), RAD51C (p = 0.02), PARP1 (p = 0.02), which corresponds to the appearance of amplifications. In the MDA-MB-468 cell culture, an increase in the copy number of only the BRCA1 gene is observed. The effect of docetaxel has no effect on this cell culture. The level of BRCA1 expression increases in direct proportion to the duration of drug action.Conclusion. Thus, the study showed that under the influence of cisplatin, reversion of not only homologous recombination gene mutations, but also other disorders can occur.

DIPG-76. PEDIATRIC PATIENTS WITH DIFFUSE MIDLINE GLIOMA DEMONSTRATE AN UNEXPECTED PREVALENCE OF GERMLINE VARIANTS IN HOMOLOGOUS RECOMBINATION GENES

Abstract BACKGROUND Pediatric patients with Diffuse Midline Gliomas (DMG), H3K27M altered have a dismal prognosis and novel therapeutic approaches are urgently needed. Factors that drive development of pediatric DMG are unknown. METHODS To determine the prevalence of germline pathogenic/likely pathogenic variants (P/LPV) in DMG, we assembled an international cohort of 252 patients with germline whole genome or whole exome sequencing data, including diffuse intrinsic pontine glioma (DIPG; n=153), from Australian, European and North American centres. RESULTS We identified germline P/LPV in cancer predisposition genes in 7.5% (19/252) of patients, mainly homologous recombination (n=9; BRCA1, BRCA2, PALB2) and Fanconi anemia genes (n=4). Germline P/LPV in mismatch repair genes (MSH2, PMS2) were found in two patients. Two patients each had two separate germline P/LPV. The prevalence of germline P/LPV was not significantly different according to age, location of DMG nor H3K27M mutational status. Furthermore, tumor profiles differed, with absence of somatic drivers in the PI3K/mTOR pathway in patients with germline P/LPV compared to those without (P = 0.023). Knockdown of BRCA1 in DMG cell cultures sensitized tumor cells to PARP inhibition. Reflecting the potential therapeutic relevance of these findings, we describe one H3.3 K27M-mutant DMG patient with a pathogenic germline BRCA2 and FANCE variant and multiple recurrences, who was treated with a PARP inhibitor (olaparib) and immune checkpoint inhibitor, leading to a near complete radiological response after 4 months. CONCLUSION Our study is the largest series to date investigating germline P/LPV in cancer predisposition genes in DMG and provides new therapeutic insights. It is expected that these germline findings will also guide cascade testing for a proband’s relatives. Our data highlight the importance of germline testing in H3K27-altered DMG patients at diagnosis.

MEK Inhibition Enhances the Antitumor Effect of Radiotherapy in NF1-Deficient Glioblastoma.

Individuals with neurofibromatosis type 1, an autosomal dominant neurogenetic and tumor predisposition syndrome, are susceptible to developing low-grade glioma and less commonly high-grade glioma. These gliomas exhibit loss of the neurofibromin gene [neurofibromin type 1 (NF1)], and 10% to 15% of sporadic high-grade gliomas have somatic NF1 alterations. Loss of NF1 leads to hyperactive RAS signaling, creating opportunity given the established efficacy of MEK inhibitors in plexiform neurofibromas and some individuals with low-grade glioma. We observed that NF1-deficient glioblastoma neurospheres were sensitive to the combination of an MEK inhibitor (mirdametinib) with irradiation, as evidenced by synergistic inhibition of cell growth, colony formation, and increased cell death. In contrast, NF1-intact neurospheres were not sensitive to the combination, despite complete ERK pathway inhibition. No neurosphere lines exhibited enhanced sensitivity to temozolomide combined with mirdametinib. Mirdametinib decreased transcription of homologous recombination genes and RAD51 foci, associated with DNA damage repair, in sensitive models. Heterotopic xenograft models displayed synergistic growth inhibition to mirdametinib combined with irradiation in NF1-deficient glioma xenografts but not in those with intact NF1. In sensitive models, benefits were observed at least 3 weeks beyond the completion of treatment, including sustained phosphor-ERK inhibition on immunoblot and decreased Ki-67 expression. These observations demonstrate synergistic activity between mirdametinib and irradiation in NF1-deficient glioma models and may have clinical implications for patients with gliomas that harbor germline or somatic NF1 alterations.

Differences in RAD51 transcriptional response and cell cycle dynamics reveal varying sensitivity to DNA damage among Arabidopsis thaliana root cell types.

Throughout their lifecycle, plants are subjected to DNA damage from various sources, both environmental and endogenous. Investigating the mechanisms of the DNA damage response (DDR) is essential to unravel how plants adapt to the changing environment, which can induce varying amounts of DNA damage. Using a combination of whole-mount single-molecule RNA fluorescence in situ hybridization (WM-smFISH) and plant cell cycle reporter lines, we investigated the transcriptional activation of a key homologous recombination (HR) gene, RAD51, in response to increasing amounts of DNA damage in Arabidopsis thaliana roots. The results uncover consistent variations in RAD51 transcriptional response and cell cycle arrest among distinct cell types and developmental zones. Furthermore, we demonstrate that DNA damage induced by genotoxic stress results in RAD51 transcription throughout the whole cell cycle, dissociating its traditional link with S/G2 phases. This work advances the current comprehension of DNA damage response in plants by demonstrating quantitative differences in DDR activation. In addition, it reveals new associations with the cell cycle and cell types, providing crucial insights for further studies of the broader response mechanisms in plants.

Clinical utility of AlphaMissense in predicting pathogenicity of DNA damage repair genes in cancer.

10581 Background: Deficiencies in DNA damage repair (DDR) impact cancer predisposition and various treatment responses.Functional implications of missense variants in DDR genes remain elusive.AlphaMissense, a new AI-based method, accurately predicts the pathogenicity of missense variants, but its clinical utility requires validation. We evaluated the accuracy of AlphaMissense predictions by analyzing known mutational signatures, genomic characteristics, and therapeutic vulnerabilities of DDR-deficient tumors. Methods: We analyzed data from TCGA (n=8,178) and MSK-IMPACT targeted panel sequencing (n=56,695) to evaluate the performance of AlphaMissense in predicting clinically actionable DDR genes, including core homologous recombination (HR) genes ( BRCA1/2, PALB2, RAD51C), POLE, and ATM. Missense mutations were classified as known pathogenic by OncoKB or previous literature, newly pathogenic identified with AlphaMisense, or benign. Mutational signatures were computed using SigMA algorithm, classifying each signature as positive at >20%. The irradiated tumor control was evaluated in patients with ATM missense mutations who received radiotherapy. Results: Among 6,743 unique DDR missense mutations from MSK-IMPACT, 372 (3.5%) were known pathogenic, and 1,182 (17.5%) were new pathogenic mutations. In 205 tumors with bi-allelic core-HR missense mutations in breast, ovarian, pancreatic, and prostate cancers, 20 (9.8%) had new pathogenic mutations. Tumors with new pathogenic mutations more frequently exhibited HR-deficient signatures (65%) than wild-type (31.8%, p < .001), similar to tumors with known pathogenic mutations (62.8%, p = 1.0). In TCGA, HR-deficient signatures tended to be more frequent in tumors with new pathogenic missense mutations than in wild-type tumors (41.7% vs. 21.0%, p = .16). Of 1,010 tumors with POLE missense mutations from MSK-IMPACT, 300 (30%) had new pathogenic mutations. The presence of POLE signatures in tumors with new pathogenic mutations (11.9%) was not significantly different from those with benign mutations (4.3%, p = .19) and less frequent than in tumors with known pathogenic mutations (91.0%, p < .001). In MSK-IMPACT, tumors with new pathogenic ATM missense mutations were less likely to have TP53 mutations than benign missense mutations (32.7% vs. 56.6%, p < .001), consistent with bona-fide pathogenicity. Further, patients with new pathogenic ATM missense mutations showed increased radiosensitivity, characteristic of ATM deficiency, and improved irradiated tumor control compared to those with benign missense mutations (Hazard ratio 0.58, 95% Cl 0.35-0.95, p = .03). Conclusions: AlphaMissense can identify previously unknown pathogenic DDR missense mutations, but its accuracy is gene-dependent. AlphaMissense pathogenicity predictions for DDR genes can utilized to help classify mutations, but may not be sufficient on their own.

The Influence of Homologous Arm Length on Homologous Recombination Gene Editing Efficiency Mediated by SSB/CRISPR-Cas9 in Escherichia coli.

The precise editing of genes mediated by CRISPR-Cas9 necessitates the application of donor DNA with appropriate lengths of homologous arms and fragment sizes. Our previous development, SSB/CRISPR-Cas9, has demonstrated high efficiency in homologous recombination and non-homologous end joining gene editing within bacteria. In this study, we optimized the lengths and sizes of homologous arms of the donor DNA within this system. Two sets of donor DNA constructs were generated: one set comprised donors with only 10-100 bp homologous arms, while the other set included donors with homologous arms ranging from 10-100 bp, between which was a tetracycline resistance expression cassette (1439 bp). These donor constructs were transformed into Escherichia coli MG1655 cells alongside pCas-SSB/pTargetF-lacZ. Notably, when the homologous arms ranged from 10 to 70 bp, the transformation efficiency of non-selectable donors was significantly higher than that of selectable donors. However, within the range of 10-100 bp homologous arm lengths, the homologous recombination rate of selectable donors was significantly higher than that of non-selectable donors, with the gap narrowing as the homologous arm length increased. For selectable donor DNA with homologous arm lengths of 10-60 bp, the homologous recombination rate increased linearly, reaching a plateau when the homologous arm length was between 60-100 bp. Conversely, for non-selectable donor DNA, the homologous recombination rate increased linearly with homologous arm lengths of 10-90 bp, plateauing at 90-100 bp. Editing two loci simultaneously with 100 bp homologous arms, whether selectable or non-selectable, showed no difference in transformation or homologous recombination rates. Editing three loci simultaneously with 100 bp non-selectable homologous arms resulted in a 45% homologous recombination rate. These results suggest that efficient homologous recombination gene editing mediated by SSB/CRISPR-Cas9 can be achieved using donor DNA with 90-100 bp non-selectable homologous arms or 60-100 bp selectable homologous arms.

Digenic Inheritance of Mutations in Homologous Recombination Genes in Cancer Patients.

BRCA1, BRCA2, ATM, and CHEK2 are known cancer predisposition genes (CPGs), but tumor risk in patients with simultaneous pathogenic variants (PVs) in CPGs remains largely unknown. In this study, we describe six patients from five families with multiple cancers who coinherited a combination of PVs in these genes. PVs were identified using NGS DNA sequencing and were confirmed by Sanger. Families 1, 2, and 3 presented PVs in BRCA2 and ATM, family 4 in BRCA2 and BRCA1, and family 5 in BRCA2 and CHEK2. PVs were identified using NGS DNA sequencing and were confirmed by Sanger. The first family included patients with kidney, prostate, and breast cancer, in addition to pancreatic adenocarcinomas. In the second family, a female had breast cancer, while a male from the third family had prostate, gastric, and pancreatic cancer. The fourth family included a male with pancreatic cancer, and the fifth family a female with breast cancer. The early age of diagnosis and the development of multiple cancers in the reported patients indicate a very high risk of cancer in double-heterozygous patients associated with PVs in HR-related CPGs. Therefore, in families with patients who differ from other family members in terms of phenotype, age of diagnosis, or type of cancer, the cascade testing needs to include the study of other CPGs.

Abstract B012: BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors

Abstract Background: Bladder cancer (BC) remains a common and deadly disease, with 83,190 cases and 32,350 deaths projected in the U.S. in 2024. We previously showed bromodomain and extra-terminal protein inhibitors (BETi) are potent in multiple preclinical models of BC. Our bulk RNA-seq and RT-qPCR data showed BETi significantly reduces expression of homologous recombination (HR) genes, such as RAD51 and RBBP8, which could activate alternative DNA repair pathways including non-homologous end joining (NHEJ) and theta-mediated end joining (TMEJ). Specifically, DNA breaks are repaired through TMEJ by polymerase theta (Polθ) utilizing microhomology templates. Recently published data revealed Polθ inhibition is synthetically lethal when other DDR genes are concomitantly lost or inhibited. Thus, in this study, we sought to elucidate whether BETi-induced HR gene repression impacts TMEJ and POLQ expression, and to identify combinations with BETi that best leverage Polθ-mediated synthetic lethality. Methods: 5637 and J82 BC cells were pretreated with birabresib for 24 h and then transfected via electroporation with a Cas9 ribonucleoprotein, a gRNA targeting LBR2 and a 996 bp HR donor. Transfected cells were treated with birabresib for 24H, and DDR pathway function was assessed by digital droplet PCR. BC cells were synchronized at the G1/S transition with 2 mM thymidine incubated with birabresib alone. After 48 h, cells were harvested, and changes to target gene expression (i.e., POLQ) was assessed by RT-qPCR. Next, the same cells were treated with 8 ascending doses of the BETi birabresib (0.1–100 μM) alone or in combination with either the PARP inhibitor olaparib, CtIP inhibitor triapene, or RAD51 inhibitor RI-1 (10 nM–200 μM). After 72-96 h, CellTiter-Glo™ measured cell viability, and Compusyn v1 calculated combination index (CI) scores where <1.0 indicated synergism and >1.0 indicated antagonism. RT-qPCR was conducted to evaluate expression changes to POLQ after combination treatment. Results: After 48 h, birabresib significantly reduced TMEJ in both 5637 and J82 BC cells (49% reduction, P=0.008 and 41% reduction, P=0.004 both n=3), but did not significantly impact HR or NHEJ. POLQ mRNA expression was most reduced in synchronized 5637 and J82 BC cells after treatment with birabresib alone (46% reduction, P=0.006, n=3, and 38% reduction, P=0.001, n=3). Birabresib combined with olaparib was the most synergistic combination in both 5637 and J82 BC cells (CI = 0.83 and 0.29), but birabresib combined with triapene was also synergistic (CI = 0.91 and 0.90). In J82 cells, birabresib significantly reduced POLQ expression (27% reduction, P=0.008, n=3), but olaparib did not. In both 5637 and J82 cells combined birabresib and olaparib increased POLQ expression (41% increase and 5% increase, ns, n=3). Combined birabresib and triapene displayed a similar trend (46% increase and 17% increase, ns, n=3). Conclusions: These preliminary data support further exploration of POLQ expression increases caused by combined BETi+DDRi treatment in preclinical models of BC. Citation Format: Ryan M. Kemper, Bhavika C. Chirumamilla, Dennis A. Simpson, Manfred Meng, Gaorav P. Gupta, Daniel J. Crona. BET inhibition sensitizes preclinical models of bladder cancer to DDR inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B012.

Coevolution of non-homologous end joining efficiency and encephalization.

Double-strand breaks (DSB), the most difficult to repair DNA damage, are mainly repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). Previous studies seem to indicate that primates, and particularly humans, have a better NHEJ system. A distinctive feature of the primate lineage (beside longevity) is encephalization, i.e., the expansion of the brain relative to body mass (BM). Using existing transcriptome data from 34 mammalian species, we investigated the possible correlations between the expression of genes involved in NHEJ and encephalization, BM, and longevity. The same was done also for genes involved in the HR pathway. We found that, while HR gene expression is better correlated with longevity, NHEJ gene expression is strongly (and better) correlated with encephalization. Since the brain is composed of postmitotic cells, DSB repair should be mainly performed by NHEJ in this organ. Therefore, we interpret the correlation we found as an indication that NHEJ efficiency coevolved with encephalization.

The Macrophage migration inhibitory factor is a vital player in Pan-Cancer by functioning as a M0 Macrophage biomarker

BackgroundThe role of the macrophage migration inhibitory factor (MIF) has recently attracted considerable attention in cancer research; nonetheless, the insights provided by current investigations remain constrained. Our main objective was to investigate its role and the latent mechanisms within the pan-cancer realm. MethodsWe used comprehensive pan-cancer bulk sequencing data and online network tools to investigate the association between MIF expression and patient prognosis, genomic instability, cancer cell stemness, DNA damage repair, and immune infiltration. Furthermore, we validated the relationship between MIF expression and M0 macrophages using single-cell datasets, the SpatialDB database, and fluorescence staining. Additionally, we assessed the therapeutic response using the ROC plotter tool. ResultsWe observed the upregulation of MIF expression across numerous cancer types. Notably, elevated MIF levels were associated with a decline in genomic stability. We found a significant correlation between increased MIF expression and increased expression of mismatch repair genes, stemness features, and homologous recombination genes across diverse malignancies. Subsequently, through an analysis using ESTIMATE and cytokine results, we revealed the involvement of MIF in immune suppression. Then, we validated MIF as a hallmark of the M0 macrophages involved in tumor immunity. Our study suggests an association with other immune-inhibitory cellular populations and restraint of CD8 + T cells. In addition, we conducted a comparative analysis of MIF expression before and after treatment in three distinct sets of therapy responders and non-responders. Intriguingly, we identified notable disparities in MIF expression patterns in bladder urothelial carcinoma and ovarian cancer following particular therapeutic interventions. ConclusionComprehensive pan-cancer analysis revealed notable enrichment of MIF within M0 macrophages, exerting a profound influence on tumor-associated immunosuppression and the intricate machinery of DNA repair.

Clinical actionability of BRCA2 alterations in uterine leiomyosarcoma: a molecular tumor board case report and a cBioPortal comprehensive analysis.

Uterine leiomyosarcoma (uLMS) represents one of the most common sarcoma histotypes, demonstrating an overall dismal prognosis. Previous studies reported uLMS to carry recurrent somatic BRCA2 homozygous deletions, related to significant clinical benefits from the use of PARP inhibitors. To investigate the prevalence in uLMS of genomic alterations (alt) in BRCA2 and other homologous recombination (HR) and DNA damage response (DDR) genes, cBioPortal was accessed and data were retrieved from studies including pan-sarcoma histologies. HR-/DDR-genes included BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FANCA, FANCB, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCL, FANCM, NBN, PALB2, RAD51C, RAD51D, RAD50, and ATR. Only oncogenic/likely oncogenic alterations were included according to OncoKB. We reported a clinical case of a patient affected by a highly pretreated uLMS discussed at the European Institute of Oncology Molecular Tumor Board. A targeted next-generation sequencing panel demonstrated a somatic BRCA2 homozygous deletion (homDel). Upon access to Niraparib, a remarkable response of 15 months was observed before experiencing disease progression. In the genomic query, among 2393 cases, uLMS (n = 193) displayed 9 of all 31 BRCA2alt observed, representing the only sarcoma histotype showing an enrichment in BRCA2alt (4.66%; q < 0.001). All of 9 BRCA2alt were represented by homDel, which related to a high fraction of genome altered. uLMS displays a significant frequency of somatic BRCA2alt homDel. Considering their dismal prognosis, further investigation is warranted to test the use of PARPi in uLMS, and particularly in the setting of BRCA1/2 alterations.