Homologous Recombination Genes Research Articles

MEK inhibition enhances the antitumor effect of radiation therapy in NF1-deficient glioblastoma.

Individuals with neurofibromatosis type 1 (NF-1), an autosomal dominant neurogenetic and tumor predisposition syndrome, are susceptible to developing low-grade glioma (LGG) and, less commonly, high-grade glioma (HGG). These gliomas exhibit loss of the neurofibromin gene (NF1), and 10-15% of sporadic HGG have somatic NF1 alterations. Loss of NF1 leads to hyperactive RAS signaling, creating opportunity given the established efficacy of MEK inhibitors (MEKi) in plexiform neurofibromas and some individuals with LGG. We observed that NF1-deficient glioblastoma neurospheres were sensitive to the combination of a MEKi (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 those with intact NF1. In sensitive models, benefits were observed at least three 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.

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, 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 brain relative to body mass. Using existing transcriptome data from 34 mammalian species, we investigated the possible correlations between the expression of genes involved in NHEJ and encephalization, body mass 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 post-mitotic cells, double-strand breaks 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.

Intracellular delivery of oncolytic viruses with engineered Salmonella causes viral replication and cell death

Intracellular delivery of oncolytic viruses with engineered Salmonella causes viral replication and cell death

Abstract 1254: Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma

Abstract Refractory high-grade serous carcinoma (HGSC) is one of the most severe clinical problems in gynecology, but we have barely grasped the underlying mechanism of aggressiveness and chemo-resistance. Previous study has shown that homologous recombination genes, BRCA1/2-unmutated HGSC patients possess the potential to be in refractory condition, and their primary response to chemotherapy was heterogenous; thus, there can be subtype-specific determinant factors hatching refractoriness in BRCA1/2-unmutated HGSC patients. To explore the determinant factors, we performed gene set variation analysis and comprehensive log-rank tests on TCGA advanced serous ovarian cancer datasets. As a result, regulation of small GTPase signaling prominently correlated with poor prognosis uniquely in BRCA1/2-unmutated HGSC patients (p-value &amp;lt; 0.001), and eventually, pathway clustering analysis by variational Bayesian Gaussian mixture model discovered aberrant RAS/PI3K crosstalk as peculiar poor prognostic signature of BRCA1/2-unmutated HGSC patients. To elucidate the mechanism of poor progression triggered by RAS/PI3K, we utilized our syngeneic HGSC organoid models newly established from murine fallopian tube epithelium by making Myc overexpressing, and knocking out Trp53 and Rb1; as a supplement, the propriety of these genetic alterations to develop HGSC models was backed up by genetic profiles of high-grade serous ovarian cancer patients in AACR Project GENIE. After that, we knocked out Nf1 and Pten whose deletions were detected relatively with high frequency in HGSC to induce aberrant RAS/PI3K crosstalk in the primitive HGSC model. In-vivo evaluation and growth comparison indicated that aberrant RAS/PI3K crosstalk transformed Brca1/2-unmutated HGSC modeling cells into aggressive phenotypes such as poor survival (p-value &amp;lt; 0.01) and high growth rate (p-value &amp;lt; 0.001). Chemo-sensitivity assay also showed that the genetic manipulation made Brca1/2-unmutated HGSC modeling cells more tolerant to anti-tumor agents like carboplatin (p-value &amp;lt; 0.001), paclitaxel (p-value &amp;lt; 0.01), and olaparib (p-value &amp;lt; 0.01). To investigate biological processes that induce the aggressive and chemo-resistant phenotype, we performed gene set enrichment analysis among our HGSC models. The result showed that aberrant RAS/PI3K crosstalk induces cell cycle acceleration and downregulation of apoptosis in the HGSC cells, which backed up aggressive phenotype and chemo-resistance in our HGSC models. In conclusion, aberrant RAS/PI3K crosstalk transforms Brca1/2-unmutated HGSC modeling cells more aggressive and chemo-resistant, which was well recapitulated by our HGSC modeling organoids. The integration of our computational approach and experimental models will lead us to detect peculiar therapeutic targets against refractory HGSC. Citation Format: Tomohiro Tamura, Kenta Masuda, Shimpei Nagai, Keiyo Imaeda, Juntaro Yamasaki, Eiji Sugihara, Hiroyuki Nobusue, Yuji Otsuki, Rui Yamaguchi, Kazuhiro Sakurada, Wataru Yamagami, Hideyuki Saya, Osamu Nagano. Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma [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 1254.

Abstract 6551: Comprehensive genomic and transcriptomic analyses capture the effects of epigenetic therapy priming on immune checkpoint blockade response in non-small cell lung cancer

Abstract Introduction: Emergence of resistance to immune checkpoint blockade (ICB) mandates the development of strategies for ICB sensitization. Here we employed a multi-omic approach to understand the effects of epigenetic priming in re-shaping the tumor microenvironment, together with genomic drivers of therapeutic response of epigenetic therapy followed by ICB in non-small cell lung cancer (NSCLC). Methods: We performed whole exome sequencing (WES) on 39 baseline tumors and bulk RNA sequencing (RNAseq) on 18 serial tumors across 42 patients with metastatic immunotherapy-naïve NSCLC, who received azacitidine and entinostat followed by nivolumab (NCT01928576). WES was utilized to assess co-mutations, mutation signatures and genome-wide structural changes. RNAseq was utilized for gene and gene set enrichment analysis (GSEA) and repeat element analysis. Response after nivolumab initiation was assessed using RECIST 1.1 criteria. Patients who were progression-free at 6 months or alive at 2 years after initial treatment were defined as exceptional responders. Results: We found an enrichment in inactivating mutations in homologous recombination genes in radiographic responders (Fisher’s exact, p=0.0007). Higher germline and somatic HLA class II diversity (Mann-Whitney U-test, p=0.05 and p=0.03 respectively), and an enrichment of smoking and DNA damage mutational signatures were noted in radiographic responders (Mann-Whitney U-test, p=0.03 and p=0.15 respectively). Radiographically responding tumors harbored a higher number of mutations in genomic regions that were haploid or present in multiple copies (persistent tumor mutation burden; Mann-Whitney U-test, p=0.018). GSEA revealed an upregulation of inflammatory response gene sets post-epigenetic therapy in patients attaining an exceptional response. Antigen presentation, interferon-alpha, and interferon-gamma related gene sets were upregulated in exceptional responders post epigenetic priming (FDR-adjusted p=0.005, p=0.009, and p=0.011 respectively). Additionally, we observed an enrichment in interferon-alpha, cancer testis antigens, and interferon-gamma related gene sets in patients with a longer progression-free survival (FDR-adjusted p=0.007, p=0.011, and p=0.013 respectively). Repeat element analysis showed an increase in long terminal repeats on-therapy in exceptional responders, suggesting induction of repeat element expression with epigenetic therapy. A higher expression of MAGE family tumor associated antigens (MAGEA10, MAGEB1, MAGEB2) was noted post-epigenetic therapy in exceptional responders. Conclusions: Genomic and transcriptomic analyses reveal the potential of epigenetic-priming to re-shape the tumor microenvironment and highlight patients that could benefit from combination epigenetic therapy with immunotherapy. Citation Format: Blair V. Landon, Kristen A. Marrone, Michael J. Topper, Akshaya Annapragada, Hua-Ling Tsai, Archana Balan, Noushin Niknafs, Christopher Cherry, James R. White, Gavin Pereira, Vilmos Adleff, Chen Hu, Joanne Riemer, Margaret Fitzpatrick, Patrick Forde, Christine L. Hann, Ronan J. Kelly, David S. Ettinger, Benjamin Levy, Jorge Nieva, James Herman, Victor E. Velculescu, Stephen Baylin, Julie Brahmer, Valsamo Anagnostou. Comprehensive genomic and transcriptomic analyses capture the effects of epigenetic therapy priming on immune checkpoint blockade response in non-small cell lung cancer [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 6551.

Abstract 5606: The role of KMT2C in the homologous recombination repair in epithelial ovarian cancer

Abstract Background: Epithelial ovarian cancer (EOC) with homologous recombination (HR) deficiency is susceptible to PARP inhibitor therapy due to synthetic lethality. Identification of the patient population for PARP inhibitor therapy is imperative to reduce recurrence of EOC and improve the survival outcomes of patients. Current biomarkers for PARP inhibitor therapy consist of an array of genes involved in the HR repair pathway in EOC, including BRCA1, BRCA2 and PALB2. Through TCGA analysis, we found that the lysine (K)-specific methyltransferase 2C (KMT2C) gene is mutated in 10.3% of all EOC patients and considerably enriched in BRCA1 mutated EOC patients. However, the role of KMT2C in HR repair and PARP inhibitor sensitivity of EOC remains largely undefined. Methods: BRCA2-mutated PEO1 and BRCA2-wild type PEO1-NR cells were transfected with KMT2C siRNA and assayed for KMT2C protein expression by western blotting. DR-GFP (HR) and EJ5-GFP (NHEJ)-SKOV3 cell lines were co-transfected with the ISceI-expressing plasmid pCBA-ISceI and KMT2C siRNA. GFP-positive cells were analyzed by flow cytometry. SKOV3 cells were transfected with KMT2C siRNA and the expression of BRCA2 and Rad51 mRNA was determined using quantitative RT-PCR analysis. Results: siRNA-mediated knockdown of KMT2C resulted in marked down-regulation of KMT2C protein in PEO1 and PEO1-NR cells. KMT2C knockdown caused a significant decrease in ISceI-induced HR activity in DR-GFP-SKOV3 cells (p &amp;lt; 0.05). In contrast, KMT2C knockdown had no effects on ISceI-induced NHEJ activity in EJ5-GFP-SKOV3 cells. As a control, Rad51 knockdown caused complete suppression of HR activity and had no effects on NHEJ activity. Furthermore, quantitative RT-PCR analysis of key HR genes showed that KMT2C knockdown resulted in a moderate but significant down-regulation of BRCA2 (p &amp;lt; 0.05) and Rad51 (p &amp;lt; 0.01) expression in SKOV3 cells. Conclusion: Our results suggest that KMT2C partakes in the HR repair pathway in EOC. However, the contribution of KMT2C to HR activity is not rate-limiting. Based on the known function of KMT2C in histone modification, we speculate that KMT2C serves to positively regulate the transcription of HR genes and therefore promote HR activity. Future investigation into the exact roles of KMT2C in HR repair, etiology of EOC, and diagnostic biomarkers for the PARP inhibitor sensitivity of EOC is warranted. Citation Format: Mirielle C. Ma, Z. Ping Lin, Elena S. Ratner. The role of KMT2C in the homologous recombination repair in epithelial ovarian cancer [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 5606.

Abstract 7116: Targeting the CBP/p300 axis in lethal prostate cancer impacts DNA repair

Abstract Prostate cancer (PCa) is the 2nd leading cause of cancer related deaths in US men. PCa is an androgen dependent disease driven by the androgen receptor (AR). Currently, androgen-deprivation therapy (ADT) is the standard of care first-line therapy for metastatic PCa. Resistance to ADT uniformly leads to lethal disease, termed castration-resistant prostate cancer (CRPC). Thus, there is an unmet clinical need to identify and develop novel strategies to treat CRPC. CBP/p300 are potent co-activators for AR. High expression of CBP/p300 is associated with locally advanced disease and castration resistant function of AR, resulting in poor patient outcomes and further highlighting the need to discern the role of CBP/p300 to potentially develop therapeutic targets for precision medicine. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300. In this study, CBP/p300 mediated bromodomain activity is targeted by CCS1477 (inobrodib), a first-in-class bromodomain inhibitor developed by Cell Centric. CCS1477 treatment demonstrated effective inhibition in growth and clonogenicity assays. Inhibition of the CBP/p300 bromodomain with CCS1477 resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression in addition to inhibition of associated factors such as c-MYC and its downstream targets in PCa cell lines as well as patient derived xenograft (PDX) models. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC patient samples. The clinical significance of CBP/p300 expression in PCa has been investigated via elucidation of CBP/p300 transcriptional reprogramming and its role in DNA damage response (DDR) pathways. Specifically, findings revealed that CBP/p300 bromodomain suppression sensitizes to AR-dependent DNA-repair. Transcriptional mapping identified CBP/p300 as regulators of cell proliferation and DNA repair processes, which were functionally confirmed across several PCa model systems. To assess relevance, exogenous challenge with radiation revealed that CBP/p300 are required for AR-mediated DNA repair, and CBP/p300 expression is linked to DNA repair capacity in the clinical setting. Molecular analyses revealed that CBP/p300 facilitate double-strand break (DSB) repair efficiency via homologous recombination (HR) mediated DDR. Congruently, CBP/p300 strongly correlated with HR gene expression in PCa patient tissue. These collective findings reveal that CBP/p300 govern repair of DNA DSBs by regulating HR, thus modulating genome integrity and promoting CRPC growth. These studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Citation Format: Sumaira Sardar, Lakshmi Ravindranath, Christopher McNair, Saswati N. Chand, Wei Yuan, Denisa Bogdan, Jonathan Welti, Adam Sharp, Matthew Schiewer, Lisa Butler, Johann de Bono, Kris Frese, Nigel Brooks, Neil Pegg, Karen Knudsen, Ayesha A. Shafi. Targeting the CBP/p300 axis in lethal prostate cancer impacts DNA repair [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 7116.

Somatic mutations in four novel genes contribute to homologous recombination deficiency in breast cancer: a real-world clinical tumor sequencing study.

Breast cancers involving mutations in homologous recombination (HR) genes, most commonly BRCA1 and BRCA2 (BRCA1/2), respond well to PARP inhibitors and platinum-based chemotherapy. However, except for these specific HR genes, it is not clear which other mutations contribute to homologous recombination defects (HRD). Here, we performed next-generation sequencing of tumor tissues and matched blood samples from 119 breast cancer patients using the OncoScreen Plus panel. Genomic mutation characteristics and HRD scores were analyzed. In the HR genes, we found that BRCA1/2 and PLAB2 mutations were related to HRD. HRD was also detected in a subset of patients without germline or somatic mutations in BRCA1/2, PLAB2, or other HR-related genes. Notably, LRP1B, NOTCH3, GATA2, and CARD11 (abbreviated as LNGC) mutations were associated with high HRD scores in breast cancer patients. Furthermore, functional experiments demonstrated that silencing CARD11 and GATA2 impairs HR repair efficiency and enhances the sensitivity of tumor cells to olaparib treatment. In summary, in the absence of mutations in the HR genes, the sensitivity of tumor cells to PARP inhibitors and platinum-based chemotherapy may be enhanced in a subset of breast cancer patients with LNGC somatic mutations.

Abstract IA016: Molecular diversity of uterine serous carcinoma

Abstract Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer with a high recurrence rate and poor prognosis disproportionately affecting black women. Whole-exome-sequencing analysis revealed that up to 10% of USC harbor features of ultra/hypermutation due to a deficiency of polymerase e (POLE) and/or mismatched-repair genes (MMRd). These tumors are notable for having a high number of single nucleotide variants (SNV), no LOH segments or copy-number variants (CNV) and a peculiar genetic signature characterized by an increased rate of C &amp;gt; A transversions. In contrast, the overwhelming majority of USC (~ 90%) are POLE/MMR proficient, have a low number of SNV and demonstrate alteration in genes including TP53, PIK3CA mutation/amplification, ERBB2 amplification, CCNE1 amplification, FBXW7 mutation/deletion, PPP2R1A mutation, and somatic mutations involving homologous recombination genes. Frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling-complex, and TAF1, an element of the core TFIID transcriptional machinery are also detected in a significant number of USC. Somatic CNVs, a sign of genomic instability, are identified in the majority of USC with multiple copy number gains and copy number losses, with the most frequent events taking place in pathways similar to those found from analysis of SNVs. In addition to loss of TP53, frequent deletion of a small segment of chromosome 19 containing MBD3, a member of the NuRD-chromatin-modification-complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (encoding for the HER2/neu receptor) and CCNE1 (a target of FBXW7-mediated ubiquitination) are commonly detected. Overall, these findings suggest that both somatic point mutations (SNV) and somatic CNV play a major role in the pathogenesis of USC. Importantly, the frequent mutation of DNA damage, chromatin remodeling, cell cycle and cell proliferation pathways in USC including the overexpression/amplification of HER2/neu, CCNE1 and the tumor-associated calcium signal transducer 2 (TACSTD2/TROP2) genes, suggest novel targets for therapy against this lethal variant of endometrial cancer. Citation Format: Alessandro D. Santin. Molecular diversity of uterine serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA016.

Germline mutations of homologous recombination genes and clinical outcomes in pancreatic cancer: a multicenter study in Taiwan

BackgroundCancer susceptibility germline mutations are associated with pancreatic ductal adenocarcinoma (PDAC). However, the hereditary status of PDAC and its impact on survival is largely unknown in the Asian population.MethodsExome sequencing was performed on 527 blood samples from PDAC individuals and analyzed for mutations in 80 oncogenic genes. Pathogenic and likely pathogenic (P/LP) germline variants were diagnosed according to the ACMG variant classification categories. The association between germline homologous recombination gene mutations (gHRmut, including BAP1, BRCA1, BRCA2, PALB2, ATM, BLM, BRIP1, CHEK2, NBN, MUTYH, FANCA and FANCC) and the treatment outcomes was explored in patients with stage III/IV diseases treated with first-line (1L) platinum-based versus platinum-free chemotherapy.ResultsOverall, 104 of 527 (19.7%) patients carried germline P/LP variants. The most common mutated genes were BRCA2 (3.60%), followed by ATR (2.66%) and ATM (1.9%). After a median follow-up duration of 38.3-months (95% confidence interval, 95% CI 35.0–43.7), the median overall survival (OS) was not significantly different among patients with gHRmut, non-HR germline mutations, or no mutation (P = 0.43). Among the 320 patients with stage III/IV disease who received 1L combination chemotherapy, 32 (10%) had gHRmut. Of them, patients receiving 1L platinum-based chemotherapy exhibited a significantly longer median OS compared to those with platinum-free chemotherapy, 26.1 months (95% CI 12.7–33.7) versus 9.6 months (95% CI 5.9–17.6), P = 0.001. However, the median OS of patients without gHRmut was 14.5 months (95% CI 13.2–16.9) and 12.6 months (95% CI 10.8–14.7) for patients receiving 1L platinum-based and platinum-free chemotherapy, respectively (P = 0.22). These results were consistent after adjusting for potential confounding factors including age, tumor stage, performance status, and baseline CA 19.9 in the multivariate Cox regression analysis.ConclusionsOur study showed that nearly 20% of Taiwanese PDAC patients carried germline P/LP variants. The longer survival observed in gHRmut patients treated with 1L platinum-based chemotherapy highlights the importance of germline testing for all patients with advanced PDAC at diagnosis.

Clinical outcomes and molecular characteristics of patients with pancreatic acinar cell carcinoma.

688 Background: PACC is a rare malignancy originating from the exocrine pancreas, accounting for less than 1% of all pancreatic cancers. Due to the rarity of the disease entity, there is a paucity of data to guide treatment decisions. This retrospective study aims to provide additional clinical characteristics and outcomes to aid in future treatment decisions. Methods: Pathology records from Mayo Clinic (AZ, FL, MN) patients (pts) denoting acinar or mixed-acinar cell carcinoma were searched between 2002 to 2023 and selected for retrospective review. Pt demographics, treatment courses, and next generation sequencing (NGS) were collected. Results: A total of 62 pts (74% male, n = 46) were identified with median age 64 years old. Histologic subtypes were 67% pure acinar cell carcinoma (42/62), 24% mixed acinar-neuroendocrine (15/62), and 8% mixed acinar-adenocarcinoma (5/62). AJCC stages at diagnosis were 34% stage I/II (21/62), 16% stage III (10/62), and 50% stage IV (31/62). Early-stage disease was treated with upfront surgical resection (81%, 17/21). Locally advanced disease received neoadjuvant therapy (NAT) followed by surgical resection (80%, 8/10). FOLFIRINOX was most often chosen as NAT (65%, 11/17), while others received gemcitabine-based NAT. The R0 resection rate was 86%. The 3 and 5-year survival estimates for stage III were 90.9% (95% CI: 75.4 – 100%) and 67.3% (95% CI: 42.3 – 100%), respectively. The 3 and 5-year survival estimates for stage IV were 36.6% (95% CI: 18.6 – 71.9%) and 29.3% (95% CI: 13.1 – 65.5%), respectively. Median survival time for stage IV was 3 years. Somatic NGS testing was done in 24 pts with alterations found in: homologous recombination (HR) in 42% (10/24); RAF alterations in 25% (6/24); mismatch repair (MMR) in 13% (3/24; none of them with MMR germline mutations), 1 ETV6-NTRK3 fusion, and 1 FGFR mutation. Germline NGS testing was done in 21 pts and alterations in HR were seen in 33% (7/21) of pts. This led to additional therapeutic options in 46% (11/24) of pts. Conclusions: Unlike PDAC, PACC is a genomically diverse disease, with potentially targetable alterations in RAF, HRD, and MMR genes, offering additional therapeutic options for significant number of pts. In addition, more than one-third of the pts harbored germline mutations in HR genes. Therefore, somatic and germline testing should be considered for any pt diagnosed with PACC.

Abstract A010: Insights into the Impact of Fanconi Anemia Repair Genes on Immunotherapy Response in Solid Tumors

Abstract Title: Insights into the Impact of Fanconi Anemia Repair Genes on Immunotherapy Response in Solid Tumors Background: Fanconi anemia repair (FAR) genes have crucial involvement in determining immunogenicity of solid tumors due to their substantial involvement in DNA damage repair (DDR). Previous work has alluded to the significance of DDR pathways such as mismatch repair and homologous recombination genes in treatment guidance. Herein, we study the potential impact of FAR genes mutations on immunotherapy parameters in solid tumors. Methods: We used the MSK immunotherapy and Tumor Mutational Burden (TMB) dataset was used in our analysis, which includes mutational and clinical data for 1661 patients on immune checkpoint inhibitors (anti-PD-1/L1, anti-CTLA-4, or combinational therapy) from ten cancer types (bladder, breast, colorectal, esophagogastric, glioma, head and neck, melanoma, non- small cell lung, renal cell, and unknown primary cancer). All clinical ang genomic data was retrieved through the cBioPortal database. Patients were categorized into the FAR mutated group if they harbored a mutation in at least one of the FAR genes (FANCA, FANCC, BRCA2, BRIP1, PALB2, RAD51C, SLX4) included in the MSK-IMPACT targeted next-generation sequencing panel. The log rank test and the Wilcoxon test, Chi-squared test were used to compare overall survival (OS), continuous and categorical data between FAR mutated and wildtype groups. Results: The MSK TMB cohort included 220 (13%) patients with FAR mutations, of which melanoma comprised the highest percentage (26.4%) followed by non-small cell lung cancer (NSCLC; 19.6%). The most common altered FAR gene was BRCA2 (6%), followed by SLX4 (5%), while the least altered gene was RAD51C (0.7%). FAR mutated patients had significantly higher OS compared to the wildtype group (median OS (mOS): 29 vs 17 months, log rank p &amp;lt; 0.001). Among melanoma patients (n = 320), there was no significant difference in OS between the mutated and FAR groups, whereas the NSCLC (n = 350) and colorectal cancer (CRC, n = 110) subgroups had significantly higher OS in the FAR mutated groups (NSCLC - mOS: 19 vs 10 months, p = 0.0256, CRC – mOS: not reached vs 13 months, p &amp;l; 0.001). TMB was significantly higher in the FAR mutated group (median TMB: 22.3 vs 5.2, p &amp;lt; 0.001). Notably, the most frequent co-mutations in the FAR mutated group included CTLA4, STK19, E2F3, CD276, MYCL. Conclusion: Our analysis highlights the potential role of FAR genes mutations in predisposing solid tumor patients to ICI benefit, specifically in NSCLC and CRC patients. Insights into the potential interactions between FAR genes and other DDR pathways as well as their impact on tumor immune microenvironment parameters are needed in future research to understand the drivers of ICI benefit in this subset of patients. Citation Format: Ahmed M. Ashour, Amro M. Al-Omari, Mohammad S. Bani Amer, Zaid R. Kamal. Insights into the Impact of Fanconi Anemia Repair Genes on Immunotherapy Response in Solid Tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A010.

Abstract B009: Targeting the AR co-activator CBP/p300 axis in castration resistant prostate cancer Impacts DNA damage repair function

Abstract Prostate cancer (PCa) is the second leading cause of cancer related deaths in US men. PCa is an androgen dependent disease and the transcription factor, androgen receptor (AR), is crucial for the overall outcome. Currently, androgen-deprivation therapy (ADT) is the standard of care first-line therapy for metastatic PCa. Resistance to ADT almost uniformly leads to lethal disease, termed castration-resistant prostate cancer (CRPC). Thus, there is a significant unmet clinical need to identify and develop novel strategies to treat CRPC. CBP/p300 are potent co-activators for AR. High expression of CBP/p300 are associated with locally advanced disease and castration resistant function of AR; thus, resulting in poor patient outcome and further highlighting the need to discern the role of CBP/p300 to potentially develop novel therapeutic targets for precision medicine. Previous studies have relied on non-specific compounds and genetic silencing to target CBP/p300 and its associated transcriptional machinery. In this study, CBP/p300 mediated bromodomain activity is targeted by CCS1477 (Inobrodib), a first-in-class bromodomain inhibitor developed by Cell Centric. CCS1477 was shown to demonstrate effective inhibition in growth and clonogenicity assays. Inhibition of the CBP/p300 bromodomain with CCS1477 resulted in significant downregulation of AR-FL, AR-V7, and its targets’ mRNA expression in addition to inhibition of associated factors such as c-MYC and its downstream targets in PCa cell lines as well as patient derived xenograft (PDX) models. This study shows that CBP and p300 are highly expressed and correlate closely with AR gene expression and AR activity score in primary PCa and CRPC patient samples. The clinical significance of CBP/p300 expression in PCa has been investigated via elucidation of CBP/p300 transcriptional reprogramming and role in DNA damage response (DDR) pathways. Specifically, findings revealed that CBP/p300 bromodomain suppression sensitizes to AR-dependent DNA-repair. Transcriptional mapping identified CBP/p300 as regulators of cell proliferation and DNA repair processes, which were functionally confirmed across several PCa model systems. To assess relevance, exogenous challenge with radiation revealed that CBP/p300 bromodomain is required for AR-mediated DNA repair, and CBP/p300 expression is linked to DNA repair capacity in the clinical setting. Molecular analyses revealed that CBP/p300 facilitate double-strand break (DSB) repair efficiency via homologous recombination (HR) mediated DDR. Congruently, CBP/p300 strongly correlated with HR gene expression in PCa patient tissue. These collective findings reveal that CBP/p300 govern repair of DNA DSBs by regulating HR, thus modulating genome integrity, and promoting CRPC growth. In conclusion, these studies identify CBP/p300 as a driver of PCa tumorigenesis through coordinated control of critical transcriptional events and lay the groundwork to optimize therapeutic strategies for advanced PCa via CBP/p300 inhibition, potentially in combination with AR-directed therapies. Citation Format: Sumaira Sardar. Targeting the AR co-activator CBP/p300 axis in castration resistant prostate cancer Impacts DNA damage repair function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr B009.

Helicobacter pylori Infection and Pathogenic Variants in Homologous Recombination Genes in Gastric Cancer.

Helicobacter pylori Infection and Pathogenic Variants in Homologous Recombination Genes in Gastric Cancer.