Homologous Recombination Research Articles

Abstract C046: PARP inhibitors as immune modulators in metastatic ovarian cancer treatment

Abstract Epithelial ovarian carcinoma (EOC) is one of the top five causes of cancer-related death in women. Most patients with EOC achieve initial remission after primary or interval cytoreductive surgery combined with platinum-taxane chemotherapy. However, mutations in BRCA1 or BRCA2 genes, which lead to homologous recombination (HR) defects, play a crucial role in platinum sensitivity and justify the use of poly (ADP-ribose) polymerase (PARP) inhibitors as maintenance therapy, commonly linked to extended progression-free survival (PFS). Besides their direct cytotoxic and cytostatic effects, PARP inhibitors (PARPi) have shown significant immunostimulatory properties by disrupting DNA repair in cancer cells, and opening possibilities for synergy with immune checkpoint inhibitors (ICIs). In this study, we investigate the immunomodulatory effects of PARPi using multiparametric flow cytometry, multiplexed immunolabeling, single-cell transcriptomics, and functional assays in an experimental BR5Brca1-/- syngeneic mouse model and human EOC tumor samples. We examine the molecular and cellular mechanisms that can be exploited to develop more effective combination therapies. PARPi may increase the mutational burden in EOCs due to unresolved DNA damage and the release of damage-associated molecular patterns (DAMPs), thereby increasing T-cell infiltration. In addition, PARPi appear to promote potent type I interferon (IFN) secretion through the activation of cGAMP synthase and the stimulator of the interferon genes (STING) pathway. In combination with ICIs, PARPi showed a beneficial effect on the balance between adaptive anti-tumor immunity and innate myeloid components, leading to an improved cytotoxic T-cell response, as observed in mouse models and HGSOC tumor samples. These observations emphasize the role of strategically designed combinations of PARPi and immunotherapeutic agents, which could be the key to overcoming immunosuppression in the EOC microenvironment, thereby improving clinical outcomes. Citation Format: Sarka Vosahlikova, Peter Holicek, Irena Moserova, Michal Hensler, Romana Mikyskova, Lenka Kasikova, Josef Pasuvka, Jana Drozenova, Katerina Mojzisova, Marek Kovar, Iain McNiesh, Michael Halaska, Lukas Rob, Sandra Orsulic, Milan Reinis, Lorenzo Galluzzi, Radek Spisek, Jitka Palich Fucikova. PARP inhibitors as immune modulators in metastatic ovarian cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C046.

The recombination landscape of the barn owl, from families to populations.

Homologous recombination is a meiotic process that generates diversity along the genome and interacts with all evolutionary forces. Despite its importance, studies of recombination landscapes are lacking due to methodological limitations and limited data. Frequently used approaches include linkage mapping based on familial data that provides sex-specific broad-scale estimates of realized recombination and inferences based on population LD that reveal a more fine scale resolution of the recombination landscape, albeit dependent on the effective population size and the selective forces acting on the population. In this study, we use a combination of these two methods to elucidate the recombination landscape for the Afro-European barn owl (Tyto alba). We find subtle differences in crossover placement between sexes that leads to differential effective shuffling of alleles. LD based estimates of recombination are concordant with family-based estimates and identify large variation in recombination rates within and among linkage groups. Larger chromosomes show variation in recombination rates, while smaller chromosomes have a universally high rate which shapes the diversity landscape. We find that recombination rates are correlated with gene content, genetic diversity and GC content. We find no conclusive differences in the recombination landscapes between populations. Overall, this comprehensive analysis enhances our understanding of recombination dynamics, genomic architecture, and sex-specific variation in the barn owl, contributing valuable insights to the broader field of avian genomics.

Microbial species and intraspecies units exist and are maintained by ecological cohesiveness coupled to high homologous recombination.

Recent genomic analyses have revealed that microbial communities are predominantly composed of persistent, sequence-discrete species and intraspecies units (genomovars), but the mechanisms that create and maintain these units remain unclear. By analyzing closely-related isolate genomes from the same or related samples and identifying recent recombination events using a novel bioinformatics methodology, we show that high ecological cohesiveness coupled to frequent-enough and unbiased (i.e., not selection-driven) horizontal gene flow, mediated by homologous recombination, often underlie these diversity patterns. Ecological cohesiveness was inferred based on greater similarity in temporal abundance patterns of genomes of the same vs. different units, and recombination was shown to affect all sizable segments of the genome (i.e., be genome-wide) and have two times or greater impact on sequence evolution than point mutations. These results were observed in both Salinibacter ruber, an environmental halophilic organism, and Escherichia coli, the model gut-associated organism and an opportunistic pathogen, indicating that they may be more broadly applicable to the microbial world. Therefore, our results represent a departure compared to previous models of microbial speciation that invoke either ecology or recombination, but not necessarily their synergistic effect, and answer an important question for microbiology: what a species and a subspecies are.

USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity.

PARP inhibitors (PARPi) represent a game-changing treatment for patients with ovarian cancer with tumors deficient for the homologous recombination (HR) pathway treated with platinum (Pt)-based therapy. PARPi exert their cytotoxic effect by both trapping PARP1 on the damaged DNA and by restraining its enzymatic activity (PARylation). How PARP1 is recruited and trapped at the DNA damage sites and how resistance to PARPi could be overcome are still matters of investigation. Here, we described PARP1 as a substrate of the deubiquitinase USP1. At molecular level, USP1 binds PARP1 to remove its K63-linked polyubiquitination and controls PARP1 chromatin trapping and PARylation activity, regulating sensitivity to PARPi. In both Pt/PARPi-sensitive and -resistant cells, USP1/PARP1 combined blockade enhances replicative stress, DNA damage, and cell death. Our work dissected the biological interaction between USP1 and PARP1 and recommended this axis as a promising and powerful therapeutic choice for not only sensitive but also chemoresistant patients with ovarian cancer irrespective of their HR status.

PARG inhibitor sensitivity correlates with accumulation of single-stranded DNA gaps in preclinical models of ovarian cancer

Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARylated proteins. Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA-damaging agents. PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as cancer patients with germline or somatic BRCA1/2-mutations. PARP inhibitors kill BRCA-deficient cancer cells by increasing single-stranded DNA gaps (ssGAPs) during replication. Here, we report that, like PARP inhibitor (PARPi), PARG inhibitor (PARGi) treatment also causes an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-phase-specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands and induced ssGAPs, in sensitive cells but not in resistant cells. PARGi also caused accumulation of PAR at the replication forks and at the ssDNA sites in sensitive cells. Additionally, PARGi exhibited monotherapy activity in specific HR-deficient, as well as HR-proficient, patient-derived, or patient-derived xenograft (PDX)-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssGAPs. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssGAPs due to OFP defects during replication. Regardless of the BRCA/HRD-status, the induction of ssGAPs in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids (PDOs) may be a useful model system for testing PARGi sensitivity and functional biomarkers.

Participation of retroelements in chromoanagenesis in cancer development

Purpose of the study: to determine the role of retroelements in chromoanagenesis mechanisms in cancer etiopathogenesis.Material and Methods. The search for relevant sources was carried out in the Scopus, Web of Science, PubMed, Elibrary systems, including publications from February 2002 to December 2023. Of the 864 scientifc articles found, 60 were used to write a systematic review.Results. According to original works and meta-analyses results, the cause of complex chromosomal rearrangements during cancer development may be retroelement pathological activation. Chromoanagenesis involves LINE1, SVA, Alu, HERV, which cause double-stranded DNA breaks, insertions in tumor suppressor genes region, the formation of chimeric oncogenes due to retroelement use as new promoters, and function as molecular “band-aids” in non-homologous end junctions and form bridges of distal DNA fragments. Global structural rearrangements of chromosomes observed during chromoanagenesis may be consequences of retroelements activation, which participate in non-allelic homologous recombination and in microhomology-mediated joining of ends characteristic. Certain types of neoplasms, such as colon cancer, are characterized by both high levels of chromothripsis and retroelement activity. In head and neck squamous cell carcinoma, chromoplexy is specifc, the sources of sequences at the breakpoints of which are retroelements. During chromoanagenesis, activation of proto-oncogenes and inactivation of tumor suppressor genes are observed, which is also a consequence of retroelement activation. This is due to the presence of retroelement sequences in proto-oncogenes promoter regions and introns (which become the basis for chimeric oncogene formation) and hot spots of insertional mutagenesis in tumor suppressor genes (transpositions into these regions inactivate these genes).Conclusion. The results obtained on the driver effect of retroelements in chromothripsis, chromoplexy and chromoanasynthesis mechanisms, which are the basis for the formation of clonal evolution of tumors, indicate promise of targeted therapy aimed at silencing the activity of retroelements in cancer patients treatment. For this purpose, it is possible to use microRNAs complementary to retroelements, which are also involved in tumor development, as tools.

Leveraging homologous recombination deficiency for sarcoma : Unravelling homologous recombination repair deficiency and therapeutic opportunities in soft tissue and bone sarcoma.

Homologous recombination deficiency (HRD) in tumors correlates with poor prognosis and metastases development. Determining HRD is of major clinical relevance as it can be treated with PARP inhibitors (PARPi). HRD remains poorly investigated in sarcoma, arare and heterogeneous cancer of mesenchymal origin. We aimed (i)to investigate predictive biomarkers of HRD in several independent sarcoma cohorts using across-functional strategy by combining genomic, transcriptomic and phenotypic approaches and (ii)to evaluate the therapeutic potential of PARPi and DNA damage response (DDR)-based therapies ex vivo. We performed acomprehensive genomic and transcriptomic characterization of sarcoma using datasets from The Cancer Genome Atlas (TCGA) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET), and our own bone and soft tissue sarcoma cohorts. We evaluated PARP1/2 and WEE1 inhibition ex vivo in patient-derived sarcoma cell models as monotherapy and in combination with chemotherapeutic agents to identify synergistic effects. Firstly, we identified genomic traits of HRD in asubset of sarcomas associated with molecular alterations in homologous recombination repair (HRR) pathway genes and high chromosomal instability. Secondly, we identified and validated distinct SARC-HRD transcriptional signatures that predicted sensitivity to PARPi. Finally, we showed functional defects in HRR in sarcoma cells that were associated with functional dependency towards PARPi and WEE1i and support the clinical use of RAD51 as apredictive biomarker for PARPi sensitivity. We provide apersonalized oncological approach to potentially improve the treatment of sarcoma patients. We encourage the evaluation of gene expression signatures to enhance the identification of patients who might benefit from DDR-based therapies.

Single-Nucleus RNA Sequencing Reveals the Transcriptome Profiling of Ovarian Cells in Adolescent Cyprinus carpio

The common carp (Cyprinus carpio) is a crucial freshwater species cultivated worldwide for food consumption. Female carp have better growth performance than males, which fascinates scholars to uncover the mechanism of gonadal differentiation and produce mono-sex populations. However, the mechanism of ovarian development at single-cell resolution is limited. Here, we conducted single-nucleus RNA sequencing in adolescent common carp ovaries. Our study obtained transcriptional profiles of 13,155 nuclei and revealed 13 distinct cell clusters in the ovaries, including three subtypes of germ cells and four subtypes of granulosa cells. We subsequently performed pseudotime trajectory analysis to delineate potential mechanisms underlying the development of germ cells and granulosa cells. We identified 1250 dynamic expression genes in germ cells and 1815 in granulosa cells (q-value < 0.01), including zp3, eif4a2 and aspm in germ cells and fshr and esr1 in granulosa cells. The functional annotation showed that dynamic expression genes in germ cells were involved in sperm–egg recognition and some terms related to meiosis, such as sister chromatid segregation and homologous recombination. Genes expressed dynamically in granulosa cells were related to the TGF-β signaling pathway, response to gonadotropin, and development of primary female sexual characteristics. In addition, the dynamic genes expressed in granulosa cells might relate to the complex communication between different cell types. In summary, our study provided a transcriptome profile of common carp ovaries at single-nucleus resolution, and we further revealed the potential cell type-specific mechanisms underlying oogenesis and the differentiation of granulosa cells, which will facilitate breeding all-female common carp populations.

Abstract B008: Evaluating ctDNA as a risk-stratifying biomarker in non-metastatic triple-negative breast cancer treated with neoadjuvant chemotherapy

Abstract Introduction: Patients with triple-negative breast cancer (TNBC) who respond well to neoadjuvant chemotherapy (NACT) generally experience favorable outcomes. However, those with residual disease after NACT face a higher risk of relapse and exhibit varying outcomes. Therefore, additional tools for assessing relapse risk are critical for guiding personalized treatment strategies. We aim to investigate the role of circulating tumor DNA (ctDNA) in conjunction with homologous recombination deficiency (HRD) as a risk-stratifying biomarker in TNBC patients with residual disease. Methods: TNBC were classified as HRD-Hereditary or Sporadic using multi-gene genetic testing. Comprehensive Genomic Profiling (CGP) was performed and tumor acquired small variants were tracked in plasma samples by a personalized-amplicontarget-sequencing (PATS) approach. Plasma samples were collected at six key time points: before treatment (BT), after the first block of NACT (ab1Neo), post-completion of NACT (aNeo), after tumor resection (aSur), and at two monitoring intervals every three months (M3m and M6m). Results: Of 105 TNBC patients tested for germline variants, 26% were classified as HRD-Hereditary, primarily due to BRCA1 mutations. CGP was performed on 73 tumors, with 12.5% displaying high tumor mutation burden (TMB), not associated with HRD- Hereditary status. Tumor-specific variants were identified in 95% (69/73) of cases, with TP53 variants being the most frequent (84%). Regarding response to NACT, 55% (58/105) achieved pathological complete response (RCB=0), while 11% (12/105) had RCB=I, 16% (17/105) RCB=II, 10% (11/105) RCB=III and 7% (7/105) unavailable RCB score. ctDNA analysis was feasible for 62% (65/105) of cases. Positive ctDNA (ctDNApos) was detected in 66.2% (43/65) at BT, 48.4% (30/62) at ab1Neo, 25.4% (15/59) at aNeo, 32.8% (20/61) at aSur, 18.6% (11/59) at M3m and 28.6% (14/49) at M6m. Interestingly, sporadic tumor patients exhibited significantly higher ctDNApos rates compared with HRD-Hereditary tumors at BT, aNeo, and aSur. No significant association was found between ctDNApos at any timepoint and RCB scores. Strong negative predictive value of RCB=0/1 and ctDNAneg aSur for progression was observed (94.2% - 65/69 and 85.4% - 35/41, respectively; mean follow-up 39.7 months, median 36.9 months). In contrast, the positive predictive value for both RCB=2/3 and ctDNApos aSur was relatively low (50% - 14/28; 50% - 10/20, respectively). However, when ctDNApos at aSur were combined with RCB=2/3, the PPV improved to 77.8%. Moreover, ctDNApos was significantly associated with clinical progression at BT (p=0.0359) and aSur (p=0.0053) and consistently anticipated progression detected by standard imaging, with a mean lead-time of 4.7 months. Patients with ctDNApos also demonstrated worse progression-free survival, strongly suggesting its potential as a prognostic marker. Conclusions: ctDNA appears to be a promising risk-stratifying biomarker for TNBC patients with residual cancer, offering valuable insights for tailoring patient management and improving outcomes. Citation Format: Rafael Canfield Brianese, Karina Miranda Santiago, Nathalia de Angelis de Carvalho, Marina de Brot Andrade, Giovana Tardin Torrezan, Vladmir Claudio Cordeiro de Lima, Solange Moraes Sanches, Maria Nirvana da Cruz Formiga, Fabiana Baroni Alves Makdissi, Dirce Maria Carraro. Evaluating ctDNA as a risk-stratifying biomarker in non-metastatic triple-negative breast cancer treated with neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B008.

Multiple paralogues and recombination mechanisms contribute to the high incidence of 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder. Why the incidence of 22q11.2DS is much greater than that of other genomic disorders remains unknown. Short read sequencing cannot resolve the complex segmental duplicon structure to provide direct confirmation of the hypothesis that the rearrangements are caused by nonallelic homologous recombination between the low copy repeats on Chromosome 22 (LCR22s). To enable haplotype-specific assembly and rearrangement mapping in LCR22 regions, we combined fiber-FISH optical mapping with whole genome (ultra-)long read sequencing or rearrangement-specific long-range PCR on 24 duos (22q11.2DS patient and parent-of-origin) comprising several different LCR22-mediated rearrangements. Unexpectedly, we demonstrate that not only different paralogous segmental duplicon but also palindromic AT-rich repeats (PATRR) are driving 22q11.2 rearrangements. In addition, we show the existence of two different inversion polymorphisms preceding rearrangement, and somatic mosaicism. The existence of different recombination sites and mechanisms in paralogues and PATRRs which are copy number expanding in the human population are a likely explanation for the high 22q11.2DS incidence.

Metabolically inducing defects in DNA repair sensitizes BRCA-wild-type cancer cells to replication stress.

Metabolic reprogramming from oxidative respiration to glycolysis is generally considered to be advantageous for tumor initiation and progression. However, we found that breast cancer cells forced to perform glycolysis acquired a vulnerability to PARP inhibitors. Small-molecule inhibition of mitochondrial respiration-using glyceollin I, metformin, or phenformin-induced overproduction of the oncometabolite lactate, which acidified the extracellular milieu and repressed the expression of homologous recombination (HR)-associated DNA repair genes. These serial events created so-called "BRCAness," in which cells exhibit an HR deficiency phenotype despite lacking germline mutations in HR genes such as BRCA1 and BRCA2, and, thus, sensitized the cancer cells to clinically available poly(ADP-ribose) polymerase inhibitors. The increase in lactate repressed HR-associated gene expression by decreasing histone acetylation. These effects were selective to breast cancer cells; normal epithelial cells retained HR proficiency and cell viability. These mechanistic insights into the BRCAness-prone properties of breast cancer cells support the therapeutic utility and cancer cell-specific potential of mitochondria-targeting drugs.

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

DNA damage response inhibitors in cancer therapy: lessons from the past, current status and future implications.

The DNA damage response (DDR) is a network of proteins that coordinate DNA repair and cell-cycle checkpoints to prevent damage being transmitted to daughter cells. DDR defects lead to genomic instability, which enables tumour development, but they also create vulnerabilities that can be used for cancer therapy. Historically, this vulnerability has been taken advantage of using DNA-damaging cytotoxic drugs and radiotherapy, which are more toxic to tumour cells than to normal tissues. However, the discovery of the unique sensitivity of tumours defective in the homologous recombination DNA repair pathway to PARP inhibition led to the approval of six PARP inhibitors worldwide and to a focus on making use of DDR defects through the development of other DDR-targeting drugs. Here, we analyse the lessons learnt from PARP inhibitor development and how these may be applied to new targets to maximize success. We explore why, despite so much research, no other DDR inhibitor class has been approved, and only a handful have advanced to later-stage clinical trials. We discuss why more reliable predictive biomarkers are needed, explore study design from past and current trials, and suggest alternative models for monotherapy and combination studies. Targeting multiple DDR pathways simultaneously and potential combinations with anti-angiogenic agents or immune checkpoint inhibitors are also discussed.

DDDR-30. ENHANCED CHEMO AND RADIO-SENSITIZATION OF GLIOMAS BY HSP90 INHIBITION WITH JIN-001, A NOVEL BLOOD-BRAIN BARRIER PENETRANT HSP90 INHIBITOR

Abstract BACKGROUND HSP90, an abundant molecular chaperone, plays a pivotal role as a key regulator in the growth and survival of malignant cells and has been a therapeutic target in various cancers. We have previously reported a strong role for HSP90 inhibitors to disable DNA damage repair mechanisms and induce cell death. Here, we determined the effects of JIN-001 mediated HSP90 inhibition on alkylator and radiation induced DNA damage. Method: Using a panel of patient-derived glioma stem cells (GSCs) we determined the synergy scores for cell viability of JIN-001 (0.1µM to 0.5µM) in combination with radiation (RT) and Temozolomide (TMZ). We also examined cell cycle dynamics and cell death of the combination using flow cytometry (PI and PI/Annexin method) and cell viability assays. Additionally, organotypic human glioma slice cultures treated with JIN-001 and chemoradiation were analyzed for changes in gene and protein expression as well as cell viability in situ. Ongoing in-vivo studies are being conducted to further elucidate drug combination efficacy in mouse models. RESULTS JIN-001 sensitized the cells to chemoradiation leading to cell cycle arrest and cell death which was higher in the combination compared to single agent. Significant changes in expression of downstream molecules of homologous recombination pathway was noted (ATM, pATM, CHK1, pCHK1, ATR, pATR) in the cell lines and confirmed in human glioma slice culture. Based on the synergy score from four cell lines, an effective dose for combination of JIN-001+RT+TMZ was identified; Results of ongoing ex-vivo human glioma gene expression changes and in-vivo mouse studies will be presented. CONCLUSION Through a comprehensive analysis, we demonstrated that HSP90 inhibitor JIN-001 sensitizes glioma cells to radio-chemotherapy including in human glioma tissue. Our study underscores the potential of combining JIN-001 with standard therapies in glioma treatment in future clinical trials for patients with glioblastoma.

CTIM-32. PHASE I/II STUDY OF STEREOTACTIC RADIOSURGERY WITH CONCURRENT OLAPARIB FOLLOWED BY ADJUVANT DURVALUMAB AND PHYSICIAN’S CHOICE SYSTEMIC THERAPY IN SUBJECTS WITH BREAST CANCER BRAIN METASTASES (SOLARA)

Abstract BACKGROUND Despite progress in the treatment of brain metastasis for HER2+ breast cancer, outcomes for patients with HER2-negative breast cancer brain metastases remain poor. Preclinical studies show inhibitors of poly(ADP-ribose) polymerase (PARP) are effective in combination with radiation therapy as a DNA damage response inhibitor. Triple-negative breast cancer (TNBC) has higher rates of homologous recombination deficiency compared to other subtypes, and together with HER2-negative, BRCA-mutated breast cancer would be particularly sensitive to PARP inhibition. PARP inhibition has also demonstrated promising efficacy combined with immunotherapy in patients with germline BRCA-mutant and metastatic TNBC in clinical trials. In addition, immunotherapy with stereotactic radiosurgery (SRS) is associated with favorable intracranial control and survival in patients with brain metastases. We hypothesize that this biologically-driven combination will enhance local control of SRS-treated brain metastases through synergy with PARP inhibition, while controlling micrometastatic disease via potentiation of the immune response. METHODS We are conducting a multi-institution, Phase I/II trial of SRS plus olaparib, followed by durvalumab (with physician’s choice systemic therapy), for patients with TNBC (any BRCA status) or HER2-negative with BRCA-mutated (germline or somatic) breast cancer brain metastases [NCT04711824]. A total of 41 patients are planned for enrollment at 8 sites. The primary objectives are to evaluate safety and tolerability (Phase I) and determine intracranial disease control at 6 months (Phase II). Secondary objectives include assessing intracranial and global progression-free survival, overall survival, and intracranial and extracranial response rate. Exploratory objectives will assess potential biomarkers of treatment response, including changes in circulating tumor cells and DNA in blood and cerebrospinal fluid, germline and tumor mutations in DNA repair pathway genes, and PD-L1 expression, as well as quality of life and patient-reported outcomes. As of May 2024, phase I has been completed without dose-limiting toxicity, and phase II is currently enrolling. Funding/drug provision from AstraZeneca.

EPCO-32. PROSPECTIVE GERMLINE SEQUENCING OF PATIENTS WITH GLIOMAS, GLIONEURONAL OR NEURONAL TUMORS

Abstract BACKGROUND Several genetic syndromes have been linked to the development of central nervous system (CNS) tumors; however, the prevalence and clinical significance of germline pathogenic variants in this population remain unclear. METHODS 2,367 patients with a glioma, glioneuronal, or neuronal tumor (WHO grade 1-4) ages 0.2-93.8 years underwent tumor and matched normal sequencing with MSK-IMPACT. Germline variants and copy-number variants affecting 90 well-established cancer predisposing genes were analyzed; the presence of biallelic inactivation was determined using FACETS. RESULTS Eleven percent harbored germline pathogenic mutations in high-, moderate-, and low-penetrance genes. High- and moderate-penetrance genes included CHEK2 (n=32, 1.3%), BRCA2 (n=10; 0.4%), TP53 (n=9; 0.4%), NF1 (n=6; 0.2%), and mismatch repair (MMR) genes (n=20, 0.9%). Low-penetrance genes included monoallelic MUTYH (n=42; 1.8%) and the APC I1307K variant (n=28, 1.2%). Biallelic inactivation was found in all tumors from patients with germline TP53 and NF1 mutations, with lower rates in tumors from patients with a germline MMR alteration (53%). Biallelic inactivation was rare among patients with a germline mutation in homologous recombination pathway (HRD) genes (27%), and in the aforementioned low-penetrance APC (15.3%) and MUTYH (10.2%) variants. All patients with biallelic inactivation of an MMR gene were hypermutated. Biallelic inactivation of MMR genes and TP53 were observed in IDH-mutant astrocytoma and IDH-WT gliomas, while biallelic inactivation of NF1 and somatic IDH1/2 mutation were mutually exclusive. When examining the age at diagnosis among patients with germline alterations, tumors with biallelic inactivation were diagnosed at a younger age compared to tumors with monoallelic alterations (37.7 vs 51.6 years, p=0.002). CONCLUSION Clinical germline sequencing identifies a germline mutation in a high proportion of patients with CNS tumors. Biallelic inactivation was most commonly identified in tumors from patients with germline TP53 or NF1 mutations and were less common in patients with a germline MMR alteration.

DNAR-11. INHIBITION OF RAD52 ACTIVITY INDUCES DNA DAMAGE BY ACCUMULATING R-LOOPS IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) is one of the most devastating childhood cancers with a median survival of < 1year from diagnosis. There is a critical need for new therapeutics for improving treatment outcomes for DMG patients. We performed genome-wide CRISPR/Cas9 screening and identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition, using shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03, suppressed DMG cell proliferation. Western blotting revealed that RAD52 inhibition increased DNA double-starand breaks (DSBs) marker γH2AX while it decreased repair markers BRCA1 and RAD51. Also, RAD52 inhibition causes a sustained level of phosphorylated RAD50 and γH2AX in irradiated DMG cells over 24 hours. Immunocytochemistry (ICC) of γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. DNA repair assay showed that RAD52 inhibition suppressed DNA DSB repair through a homologous recombination pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Importantly, RAD52 inhibition increased the radiosensitivity of DMG cells. To understand the mechanism of RAD52 inhibition on DNA damage, we performed dot plot assay and ICC of S9.6 which recognizes DNA-RNA hybrids (also known as R-loops) and found accumulation of R-loop formation in DMG cells treated with RAD52 inhibitor. In addition, RAD52 inhibition decreased the expression of RNA polymerase II. Finally, the combination therapy of RAD52 inhibitor and radiation inhibited tumor growth and increased survival of mice bearing DMG patient-derived xenografts, outperforming either monotherapy. Together, our results highlight that RAD52 inhibition induces DNA damage by accumulating R-loops, results in reducing DMG cell proliferation, while also increase DMG radiosensitivty, and providing a rationale for developing combination therapy with radiation for the treatment of DMG.

BCAS2 and hnRNPH1 orchestrate alternative splicing for DNA double-strand break repair and synapsis in meiotic prophase I

Understanding the intricacies of homologous recombination during meiosis is crucial for reproductive biology. However, the role of alternative splicing (AS) in DNA double-strand breaks (DSBs) repair and synapsis remains elusive. In this study, we investigated the impact of conditional knockout (cKO) of the splicing factor gene Bcas2 in mouse germ cells, revealing impaired DSBs repair and synapsis, resulting in non-obstructive azoospermia (NOA). Employing crosslinking immunoprecipitation and sequencing (CLIP-seq), we globally mapped BCAS2 binding sites in the testis, uncovering its predominant association with 5’ splice sites (5’SS) of introns and a preference for GA-rich regions. Notably, BCAS2 exhibited direct binding and regulatory influence on Trp53bp1 (codes for 53BP1) and Six6os1 through AS, unveiling novel insights into DSBs repair and synapsis during meiotic prophase I. Furthermore, the interaction between BCAS2, hnRNPH1, and SRSF3 was discovered to orchestrate Trp53bp1 expression via AS, underscoring its role in meiotic prophase I DSBs repair. In summary, our findings delineate the indispensable role of BCAS2-mediated post-transcriptional regulation in DSBs repair and synapsis during male meiosis. This study provides a comprehensive framework for unraveling the molecular mechanisms governing the post-transcriptional network in male meiosis, contributing to the broader understanding of reproductive biology.

Allelic Variation in gtfB–gtfC Region of Natural Variant of Streptococcus mutans Without Biofilm Formation

Streptococcus mutans is primarily found in biofilms on tooth surfaces and is associated with the development of dental caries. S. mutans synthesizes water-insoluble glucan (WIG) using sucrose as a substrate, inducing the formation of three-dimensional biofilms. WIG is produced by glucosyltransferases (GTFs) encoded by the tandem and highly homologous gtfB and gtfC genes. Conversely, the homologous recombination of gtfB and gtfC readily happens, producing natural variants without WIG. These WIG− variants are thought to have ecologically pleiotropic functions; however, the molecular basis for their appearance is unclear. This study aimed to determine the sequence of the gtfB–gtfC regions of WIG− variants. We sequenced the gtfB–gtfC regions of 23 WIG− variants derived from S. mutans UA159 and revealed the presence of five alleles and four types of single homologous recombination patterns. Regardless of the allele type, the WIG− variants showed low biofilm formation and GTF activity. To the best of our knowledge, this is the first study to report the presence of alleles in WIG− variants. These findings provide important information for explaining the appearance of mechanisms in WIG− variants.

Gene Targeting via CRISPR/Cas9-Mediated DNA Double-Strand Break Induction in Rice.

Gene targeting (GT) is a precise genome editing tool to achieve desired modification of a target gene, e.g., introduction of point mutations, knock-in of a reporter gene, or swapping of a functional domain, through homologous recombination. However, due to its low frequency, it has proved difficult to establish a universal GT system. The availability of the CRISPR/Cas9 system for genome editing in plants has opened up possibilities to apply GT successfully to some plant species. Here, we provide protocols for CRISPR/Cas9-mediated DNA double-strand break (DSB)-induced GT in rice.