Homologous Recombination Pathway Research Articles

In-silico Screening of Novel Drug Against Ovarian Cancer by Phytochemicals from Carica Papaya

Most of the women population suffer from many kinds of reproductive health issues. The cause of the diseases may be genetic or acquired causes. Ovarian cancer is a genetic aberration caused in females generation after generation. Various genes cause the genetic disorder, mainly BRCA 1 and BRCA 2. These major genes are present in chromosome 17q. 77% of the epithelial ovarian cancer tumor genes are in chromosome 17q. The disorder can be a germline mutation in females who express this disease before age 30. By DNA sequencing technology, the functioning of BRCA1 and BRCA2 can be discovered along with the involvement of the additional genes that compromise the homologous recombination (HR) pathway. Epithelial ovarian cancer (EOC) is the deadliest gynecological cancer and presents a major clinical challenge due to limited treatment options. Folate receptor alpha (FRα), encoded by the FOLR1 gene in chromosome 11q13.4, is an attractive therapeutic target due to its prevalent and high expression in EOC cells. The folate receptors FOLR1 and FOLR2 are overexpressed in multiple cancers. The overexpression of FOLR1 is often associated with increased cancer progression and poor patient prognosis. There is emerging evidence that FOLR1 is involved in signaling pathways that are independent of one-carbon metabolism.

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.

Abstract PO4-15-05: Genomic characterization and molecular predictive biomarkers for chemotherapy in a real-world population with metastatic triple-negative breast cancer

Abstract Background: The genomic landscape of metastatic triple-negative breast cancer (mTNBC) in a real-world population is poorly defined and despite recent advances in precision oncology, molecular biomarkers for chemotherapy are largely lacking. The presence of specific genomic alterations and alterations in oncogenic pathways have the potential to provide prognostic as well as predictive information for chemotherapy, which remains the backbone treatment strategy for mTNBC. Methods: We conducted next-generation sequencing (NGS) with the FoundationOne CDx panel on tissue from the primary tumor and/or metastasis of 112 consecutive patients with mTNBC that were treated between 1998 and 2019 at 4 different Swedish hospitals. Every genomic alteration was, if applicable, subdivided into 1 of 10 canonical oncogenic pathways and noted for its involvement in the homologous recombination pathway (HRP). Frequently altered genes and pathways were then correlated with overall survival (OS) and evaluated regarding their association with progression-free survival (PFS) and response rate (RR) in patients treated with different chemotherapy agents. Tumor samples of patients with rapid progression or exceptional response to chemotherapy underwent exploratory comparison with regards to frequently altered genes and pathways and known clinical prognostic factors were compared between the two patient groups. Results: After excluding 15 patients due to insufficient quantity/quality of tumor tissue or absence of clinical data, 97 patients were analyzed. The median age at diagnosis of metastatic disease was 61 (range: 28-90), 80 % had visceral disease and 26 % received platinum-based chemotherapy in the first line setting. The most frequently altered genes were: TP53 (82 %), RAD21 (25 %), PIK3CA (23 %), MYC (22 %) and BRCA 1 or 2 (16 %). The most frequently altered pathways were TP53 (86 %), PI3K (60 %), RTK/RAS (47 %) and cell cycle (36 %). In total, 26 % of patients had an alteration in the HRP. None of the most frequently occurring genomic alterations were associated with OS. Variants of clinical significance in the HRP and BRCA1/BRCA2 genes were associated with a longer PFS in patients treated with platinum-based chemotherapy in the first line setting (HR 0.12-0.84 and 0.12-0.92). Exceptional responders to chemotherapy exhibited a more favorable clinical profile than rapid progressors (median age 51.5 vs 66.5, median Charlson comorbidity index 0.5 vs 3) whereas some numerical difference in the genomic profiling in terms of genomic alterations in MYC and RAS/RTK pathways (more often in exceptional responders) could be observed. Conclusions: We found no genomic alteration with prognostic significance in a cohort of mTNBC patients treated with chemotherapy in a real-world setting. Somatic variants of clinical significance in BRCA 1 and 2 and other HRP-related genes seem to define subgroups of patients with mTNBC that respond favorably to platinum-based chemotherapy. Further research into the genomic landscape of tumors from patients with rapid progression or exceptional response to specific treatment strategies can provide insights into mechanisms of resistance and identify new predictive biomarkers. Citation Format: Erik Olsson, Henrik Lindman, Evangelos Digkas, Viktoria Thurfjell, Haidar Mir Ali, Ute Krüger, Anna-Karin Wennstig, Marie Sundqvist, Antonis Valachis. Genomic characterization and molecular predictive biomarkers for chemotherapy in a real-world population with metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-15-05.

Abstract PO2-08-11: The MAGIC study: Universal whole genome tumour and germline sequencing of newly diagnosed breast cancer identifies a high proportion of ER+ BRCA-like tumours

Abstract Background: Identification of germline mutations in hereditary breast cancer (HBC) genes can have profound benefits in the treatment of breast cancer (BC) and manging risk for patient and the family. In addition, sequencing of the tumours can reveal clinically relevant somatic features, such as homologous recombination deficiency (HRD) and mutational signatures, that can guide treatment which are not discernible by germline sequencing alone. We hypothesised that current standard of care where women diagnosed with BC are not routinely offered tumour sequencing is suboptimal as fails to exploit all therapeutic vulnerabilities which can be exploited in the treatment of BC. The MAGIC study is the first prospective trial in Australia of unselected invasive BCs in general oncology practice combining both germline and tumour sequencing. Methods: The MAGIC study included a total of 651 consecutive consented patients presenting with non-metastatic BC between June 2020 to March 2023. It included invasive BC, high-grade DCIS and pleomorphic LCIS. The sequencing was conducted in two phases; for phase one, 157 cases underwent whole genome sequencing (WGS) on both germline and matched BC DNA while for phase two, 494 cases underwent only germline whole exome sequencing. Germline variants were interrogated for pathogenic variants in BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51B, RAD51C, RAD51D, MLH1, MSH2, MSH6, PMS2, CDH1, PTEN, STK11, TP53 and NTHL1. Tumour homologous recombination (HR) repair deficiency was calculated using HRDetect (Nat Med 2017;23:517). For BCs showing a high HRDetect score ( >0.75) all HBC gene promoter CpG islands were assessed for hypermethylation using the Twist NGS Methylation system. Mutational signatures were calculated from somatic mutations identified from whole genome sequenced BCs using the DeconstructSig package in R (Genome Biol. 2016;17:31) and referenced against the COSMIC v2 signature catalogue. Results: Actionable germline variants in any HBC gene were identified in 7.7% of cases (7.8% of invasive and 6.1% of in situ BC) with 5.2% having an actionable mutation in an HR pathway gene (BRCA1, BRCA2, PALB2 and RAD51C). Most cases with pathogenic variants showed bi-allelic inactivation in the tumour. All BRCA1, BRCA2 and PALB2 tumours showed high HRDetect and/or HRD scores consistent with loss of HR repair function, indicating them to be the underlying hereditary driver of the BC. One BC with a pathogenic PMS2 variant showed retention of the wild-type allele with no evidence of a mismatch repair mutational signature suggesting PMS2 was not the driver of this BC. Of the 157 BC in phase one that underwent WGS and did not have a germline pathogenic HBC gene variant, 16% (18/117 invasive and 3/13 DCIS) had an HRDetect score of >0.7 indicative of a homologous recombination repair defect and therefore biologically a “BRCA-like” tumour. Of these 21 BRCA-like cases, only 3 invasive BCs had clear somatic bi-allelic inactivation of an HBC gene that could explain the high HRDetect score (2 BRCA1 and 1 BRCA2). One of the 21 BRCA-like cases had a germline variant of unknown significance (VUS) in an HBC gene (a BRCA2 missense variant in an ER+ tumour. The tumour showed loss of the wild-type allele). Of the BRCA-like cases, 71% were ER positive which included 3 invasive lobular cancers. Conclusion: Tumour sequencing identified three times as many cases that might be eligible for HR repair defect targeted therapy than germline testing alone (16% versus 5.2%). Unlike a previous study (Ann Oncol., 2019 30:1071-1079), the majority of the BRCA-like BCs were ER+ with no evidence of a VUS in a known HBC gene suggesting these are driven by novel germline or somatic mutations in genes involved in DNA HR repair. Citation Format: Ian Campbell, Lisa Devereux, Kirsten Hogg, Luxi Lal, Michelle Sinclair, Lesley Stafford, Magnus Zethoven, Anita Skandarajah, Paul James, Geoffrey Lindeman, Bruce Mann, Dilanka De Silva. The MAGIC study: Universal whole genome tumour and germline sequencing of newly diagnosed breast cancer identifies a high proportion of ER+ BRCA-like tumours [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-11.

Advancements in the Understanding of Small-Cell Neuroendocrine Cervical Cancer: Where We Stand and What Lies Ahead.

Small-cell neuroendocrine cervical carcinoma (SCNCC) is a rare yet aggressive gynecological malignancy associated with dismal clinical outcomes. Its rarity has led to a limited number of retrospective studies and an absence of prospective research, posing significant challenges for evidence-based treatment approaches. As a result, most gynecologic oncology centers have limited experience with this tumor, emphasizing the urgent need for a comprehensive review and summary. This article systematically reviews the pathogenesis, immunohistochemical and molecular characteristics, prognostic factors, and clinical management of gynecologic SCNCC. We specifically focused on reviewing the distinct genomic characteristics of SCNCC identified via next-generation sequencing technologies, including loss of heterozygosity (LOH), somatic mutations, structural variations (SVs), and microRNA alterations. The identification of these actionable genomic events offers promise for discovering new molecular targets for drug development and enhancing therapeutic outcomes. Additionally, we delve deeper into key clinical challenges, such as determining the optimal treatment modality between chemoradiation and surgery for International Federation of Gynecology and Obstetrics (FIGO) stage I phase patients within a precision stratification framework, as well as the role of targeted therapy within the homologous recombination (HR) pathway, immune checkpoint inhibitors (ICIs), and prophylactic cranial irradiation (PCI) in the management of SCNCC. Finally, we anticipate the utilization of multiple SCNCC models, including cancer tissue-originated spheroid (CTOS) lines and patient-derived xenografts (PDXs), to decipher driver events and develop individualized therapeutic strategies for clinical application.

Prominent transcriptomic changes in Mycobacterium intracellulare under acidic and oxidative stress.

Mycobacterium avium complex (MAC), including Mycobacterium intracellulare is a member of slow-growing mycobacteria and contributes to a substantial proportion of nontuberculous mycobacterial lung disease in humans affecting immunocompromised and elderly populations. Adaptation of pathogens in hostile environments is crucial in establishing infection and persistence within the host. However, the sophisticated cellular and molecular mechanisms of stress response in M. intracellulare still need to be fully explored. We aimed to elucidate the transcriptional response of M. intracellulare under acidic and oxidative stress conditions. At the transcriptome level, 80 genes were shown [FC] ≥ 2.0 and p < 0.05 under oxidative stress with 10mM hydrogen peroxide. Specifically, 77 genes were upregulated, while 3 genes were downregulated. In functional analysis, oxidative stress conditions activate DNA replication, nucleotide excision repair, mismatch repair, homologous recombination, and tuberculosis pathways. Additionally, our results demonstrate that DNA replication and repair system genes, such as dnaB, dinG, urvB, uvrD2, and recA, are indispensable for resistance to oxidative stress. On the contrary, 878 genes were shown [FC] ≥ 2.0 and p < 0.05 under acidic stress with pH 4.5. Among these genes, 339 were upregulated, while 539 were downregulated. Functional analysis highlighted nitrogen and sulfur metabolism pathways as the primary responses to acidic stress. Our findings provide evidence of the critical role played by nitrogen and sulfur metabolism genes in the response to acidic stress, including narGHIJ, nirBD, narU, narK3, cysND, cysC, cysH, ferredoxin 1 and 2, and formate dehydrogenase. Our results suggest the activation of several pathways potentially critical for the survival of M. intracellulare under a hostile microenvironment within the host. This study indicates the importance of stress responses in M. intracellulare infection and identifies promising therapeutic targets.

RecA-dependent or independent recombination of plasmid DNA generates a conflict with the host EcoKI immunity by launching restriction alleviation.

Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis.

Differential expression of DMD transcripts as a novel prognostic biomarker in histologically diverse mesotheliomas.

The identification of prognostic biomarkers is crucial for guiding treatment strategies in mesothelioma patients. The Duchenne muscular dystrophy (DMD) gene and its specific transcripts have been associated with patient survival in various tumours. In this study, we aimed to investigate the prognostic potential of DMD gene expression and its transcripts in mesothelioma patients. We analysed The Cancer Genome Atlas (TCGA) mesothelioma RNAseq, mutation, and clinical data to assess the association between DMD gene expression and its transcripts (Dp427, Dp71 splice variants) and mesothelioma survival. We also evaluated the specific Dp71 transcript as a unique prognostic biomarker across mesothelioma subtypes. Additionally, we performed differential gene expression analysis between high and low DMD gene/transcript expression groups. The analysis included 57 epithelioid, 23 biphasic, two sarcomatoid, and five not otherwise specified (NOS) histological subtypes of mesothelioma samples. Univariate analysis revealed that high expression of the DMD gene and its Dp71 transcript was significantly associated with shorter survival in mesothelioma patients (P=0.003 and P<0.001, respectively). In a multivariate analysis, the association between Dp71 expression and survival remained significant [hazard ratio (HR) 2.29, 95% confidence interval (CI): 1.24-4.23, P=0.008] across all mesothelioma patients, and also among patients with mesotheliomas without deep CDKN2A deletions (HR 3.58, 95% CI: 1.31-9.80, P=0.01). Pathway analysis revealed enrichment of cell cycle (P=3.01×10-4) and homologous recombination (P=0.01) pathways in differentially expressed genes (DEGs) between high and low Dp71 groups. Furthermore, there were correlations between Dp71 transcript expression and tumour microenvironment (TME) cells, including a weak positive correlation with macrophages (R=0.32, P=0.002) specifically M2 macrophages (R=0.34, P=0.001). Our findings indicate that the differential expression of specific DMD transcripts is associated with poor survival in mesothelioma patients. The specific Dp71 transcript can serve as a potential biomarker for predicting patient survival in diverse histological subtypes of mesothelioma. Further studies are needed to understand the role of specific dystrophin transcripts in cancer and TME cells, and their implications in the pathogenesis and progression of mesothelioma. Identifying patients at risk of poor survival based on DMD transcript expression can guide treatment strategies in mesothelioma, informing decisions regarding treatment intensity, follow-up schedules, eligibility for clinical trials, and ultimately, end-of-life care planning.

Abstract 7126: Synergistic combinations and tissue type-specificity of compounds targeting components of the DNA damage response pathway in 2D culture and 3D spheroid cell viability assays

Abstract Poly (ADP-ribose) polymerase (PARP) inhibitors have changed the landscape of treatment for homologous recombination (HR)-deficient cancers. PARP inhibitors are synthetically lethal with deficiencies in genes involved in the HR pathway, such as BRCA2, but resistance against PARP inhibitors is a major problem in the clinic. The use of new synergistic drug combinations targeting different components of the DNA damage response (DDR) pathway may overcome PARP inhibitor resistance. Sixteen small molecule inhibitors acting on different targets in the DDR pathway (including PARP, Polθ and ATR) were profiled in 2D culture and 3D spheroid cell viability assays. Intracellular ATP content was used as an indirect readout of cell number. Compound synergies were determined by profiling combinations of compounds in a 6-by-6 matrix, and quantifying synergistic effects using the Excess over Bliss score [1]. A pair of isogenic BRCA2-deficient and -proficient DLD-1 colon adenocarcinoma cell lines was used as a model of BRCA2-deficient tumors. In 2D cell viability assays, PARP inhibitors and the Polθ inhibitor ART558 were more active in BRCA2-deficient compared to BRCA2-proficient DLD-1 cells, whereas an ATR inhibitor (ceralasertib) was equally potent in both cell lines. Combining olaparib with ART558 in 2D culture and 3D spheroid studies showed synergistic effects in BRCA2-deficient DLD-1 cells. Moreover, olaparib synergized with ceralasertib in both BRCA2-deficient and -proficient DLD-1 cells. Different DDR-targeting inhibitors were profiled in over a hundred cancer cell lines from diverse tumor tissue origin in 2D assays. This showed a relatively high potency for PARP inhibitors in ovarian cancer cell lines compared to cell lines derived from other tissue types, which is consistent with the clinical application of PARP inhibitors for treatment of ovarian cancers. For ATR inhibitors and ART558, kidney and myeloid cancer cell lines were found to be most sensitive, respectively, suggesting potential therapeutic utility of ATR and Polθ inhibitors for treatment of these cancer types. The genomic determinants of the different sensitivities were investigated by comparing compound potencies in the cell viability assays with the mutation status of more than 500 genes involved in cancer or the DDR pathway in the cell lines. Additionally, cancer cell line sensitivity was correlated with the expression levels of more than 19,000 genes in these cell lines to identify novel RNA signatures capable of predicting response to DDR-targeting inhibitors. This study uncovers new synergistic drug combinations that increase the efficiency of PARP inhibitors, and provides new insights into the determinants of cancer cell line sensitivity to compounds targeting different components of the DDR pathway. [1] Liu et al. Stat. Biopharm. Res. 10, 112-22; 2018 Citation Format: Janneke J. Melis, Daphne J. Kluitmans, Jeffrey J. Kooijman, Jelle Dylus, Sabine Wetemans, Suzan Geerdink-Datema, Jeroen A. de Roos, Yvonne Grobben, Guido J. Zaman. Synergistic combinations and tissue type-specificity of compounds targeting components of the DNA damage response pathway in 2D culture and 3D spheroid cell viability assays [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 7126.

Abstract 7148: Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR

Abstract Flap endonuclease 1 (FEN1) is a structure-specific metallonuclease that cleaves 5’ DNA flaps during replication and repair. FEN1 is an attractive target for development of anticancer therapeutics because it is overexpressed in many tumor types and has a large number of synthetic lethality partners including genes in Homologous Recombination (HR) pathway (Mengwasser et al., 2019; Guo et al., 2020). Utilizing fragment-based drug discovery approach we identified a novel metal-binding pharmacophore that binds to the two magnesium ions in the FEN1 active site. Further elaboration using fragment growth strategies resulted in highly potent and selective inhibitors. The current lead (BSM-1516) is ~65-fold more potent against FEN1 than its related enzyme Exonuclease 1 (Exo1) in biochemical assays (IC50 of 7 nM and 460 nM, respectively), an improvement of more than an order of magnitude in selectivity compared to earlier efforts. FEN1 target engagement in live cells was validated by cellular thermal shift assay (CETSA EC50 of 24 nM). Inhibition of FEN1 led to its increased association with chromatin in S-phase cells and recruitment of PARP1 enzyme. In clonogenic assay, BRCA2-deficient DLD1 cells were ~15-fold more sensitive to FEN1 inhibition than their isogenic BRCA2-wild-type counterparts (EC50 of 350 nM and 5 µM, respectively), confirming the increased susceptibility of HR deficient cancer cells to FEN1 inhibition. Treatment of BRCA2-deficient but not wild-type DLD1 cells with BSM-1516 resulted in cell cycle arrest and dose-dependent decrease of S-phase BrdU incorporation into DNA. Cell cycle arrest of BRCA2-deficient DLD1 cells was accompanied by DNA damage signaling and by accumulation of chromatin-bound RPA32, a marker of ssDNA. To explore synthetic lethal interactions of FEN1 with other DNA repair genes we performed FEN1-inhibitor-anchored CRISPR screen. This analysis revealed that in addition to HR pathway inactivation, genomic perturbations in EXO1, USP1 and PARP1 genes sensitized cells to FEN1 inhibition. Synergistic relationships of BSM-1516 and its combination potential were further explored in viability studies with a panel of DDR inhibitors (n=25) in BRCA2-proficient and deficient cell lines. Strong synergy was identified with multiple drug classes that included inhibitors of USP1 (KSQ-4279), PARP (Olaparib, Niraparib, Talazoparib, AZD5305), PARG (PDD 00017273) and ATR (AZD6738, VE-822, Elimusertib). In vitro ADME assays and in vivo PK studies showed that BSM-1516 has properties suitable for in vivo testing, either as a single agent or in combination with synergistic DDR inhibitors, an investigation that is currently underway. Citation Format: Jason Munguia, Sanjay Agarwalla, Elaine Guo, Dave Martin, Junhua Fan, Dave Lonergan, David Puerta, Zachary Zimmerman, Konstantin Taganov. Small molecule inhibitor of FEN1 nuclease utilizing a novel metal-binding pharmacophore synergizes with inhibitors of USP1, PARP, PARG and ATR [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 7148.

Abstract 7595: PRMT5 inhibitor synergizes with PARP inhibitors in triple-negative breast cancer cells

Abstract Background: Treating triple-negative breast cancer (TNBC) patients, an aggressive subtype of breast cancer, is an ongoing clinical challenge. Small-molecule inhibitors targeting the poly ADP-ribose polymerase (PARP) DNA repair enzymes, have been approved for treating only a subset of TNBC patients with a specific genetic landscape (BRCA1 mutation). However, TNBC patients with wildtype BRCA1 (BRCA1WT) are still lacking targeted therapy and chemotherapy remains their main treatment strategy. High expression of protein arginine methyltransferase 5 (PRMT5) is linked to poor prognosis in breast cancer and PRMT5 alone or via its interacting proteins plays an oncogenic role in breast cancer. Recent evidence suggests that PRMT5 is a key player in regulating the repair of DNA double strand breaks (DSBs) either through homologous recombination (HR) or non-homologous end joining (NHEJ) depending on the PRMT5 expression level within the cell. This dependency of cancer cells on PRMT5 expression levels in deciding the repair pathway of choice can be exploited for developing therapies. We hypothesize that in BRCA1 wildtype TNBC cells, inhibiting PRMT5 activity will force the cells to rely on NHEJ rather than HR to repair DNA DSBs, and introducing PARP inhibitors in this setting will inactivate NHEJ as well, causing synthetic lethality and cell death. In this study, we evaluated the pre-clinical benefit of combining PARP inhibitors with a PRMT5 inhibitor in TNBC cells. Methods: Human TNBC cell lines with wildtype BRCA1 (MDA-MB-468, HCC1806) and mutant BRCA1 (MDA-MB-436, SUM149-PT) were treated with varying doses of two PARP inhibitors (Olaparib, Talazoparib) and a PRMT5 inhibitor (GSK3326595) as single agents or in combination. MTT-based cell viability assay was used to determine cytotoxicity of the treatments. mRNA expression was determined by RT-qPCR after drug treatment. Results and Conclusion: We report that PRMT5 inhibition synergizes with both Olaparib and Talazoparib, to potentiate cell death in two BRCA1WT but not in BRCA1mut TNBC cell lines. Furthermore, we show that inhibiting PRMT5 in BRCA1WT cells upregulates the mRNA expression of RAD50, a DNA damage sensing gene in the HR pathway. Our ongoing study is to delineate the molecular mechanism of action for the observed synergy and whether PRMT5 inhibition can overcome resistance to PARP inhibitors. Through this study, we will identify a novel treatment strategy for the majority of TNBC patients (BRCA1WT) who currently lack targeted therapies. Citation Format: Samyuktha Suresh, Lisa A. McPherson, James M. Ford. PRMT5 inhibitor synergizes with PARP inhibitors in triple-negative breast cancer cells [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 7595.

Abstract 6977: ETV6 is an unexplored partner of the Chr.8 q24.3 amplicon-embedded immortalizing oncogene TONSL in tumorigenesis

Abstract Approximately 20% of breast cancers exhibit recurring genomic amplification involving chromosome 8q24.3. In our previous study, we identified the Tonsoku-like DNA repair protein (TONSL) within this amplicon as an immortalizing oncogene of breast epithelial cells, that can promote Estrogen Receptor-positive (ER+) breast adenocarcinomas when combined with other breast cancer-associated oncogenes. Our findings indicated that TONSL-amplified breast cancer cells are dependent on TONSL for tumor growth and exhibited deregulated homologous recombination and cell cycle pathways. High levels of TONSL in primary breast cancers, particularly ER+ breast cancers, were associated with poor outcomes. Although TONSL itself is not druggable, owing to its lack of enzymatic activity, molecules that disrupt the TONSL interactome can be developed. Towards this goal, we performed immunoprecipitation (IP) with TONSL antibody using protein lysates from TONSL-immortalized primary breast epithelial cells and subjected the immunoprecipitates to mass spectrometry. We identified several proteins selectively enriched with the TONSL antibody, the most significant of which was ETS variant transcription factor 6 (ETV6). ETV6 is a modular protein consisting of a C-terminal DNA-binding ETS domain and N-terminal point (PNT) domain and is known to play a role as a transcriptional repressor during embryonic development and hematopoiesis. We confirmed these data by IP followed by western blot analysis of primary breast epithelial cells and TONSL amplified MDA-MB-436. Furthermore, we utilized ClusPro to model the TONSL-ETV6 interaction and predicted that the TONSL’s ankyrin repeats docks onto ETV6’s PNT in a favorable manner. To investigate the functional significance of this interaction, we evaluated ETV6-regulated genes using RNA-seq. Our analysis revealed that ETV6-regulated genes were downregulated following TONSL knockdown and vice versa. Most ETV6 target genes affected upon TONSL manipulation are involved in Epithelial-Mesenchymal Transition (EMT), particularly TWIST1, SNAI2 and ZEB1. Previous studies have demonstrated a role of ETV6 in limiting EMT by repressing the expression of EMT-associated transcription factors. Our findings revealed that TONSL overexpression in primary breast epithelial cells led to a significant increase in these EMT gene expression, whereas TONSL knockdown in MDA-MB-436 cells resulted in a moderate decrease. These findings suggest that TONSL binds to ETV6 and restricts its ability to bind to the regulatory regions of EMT-associated genes, leading to a transcriptional surge of EMT-associated genes leading to tumor progression. Further studies will contribute to our understanding of the role of the chr.8q24.3 amplicon, specifically the TONSL gene, in initiating/promoting breast tumorigenesis and the development of novel drugs that target the TONSL interactome. Citation Format: Aditi S. Khatpe, Emma Doud, Amber Mosely, Kathy D. Miller, Harikrishna Nakshatri. ETV6 is an unexplored partner of the Chr.8 q24.3 amplicon-embedded immortalizing oncogene TONSL in tumorigenesis [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 6977.

Abstract 7130: Dysregulated DNA repaired elicited by type 1 PRMT inhibitor in clear cell renal cell carcinoma enables development of novel combination therapies

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer, notorious for its resistance to treatment and dismal prognosis. Advanced or metastatic cases on average display an 8% 5-year survival rate. Given the considerable number of diagnoses in advanced stages, it is evident that we require innovative therapeutic approaches. Our lab has developed a methodology to establish cell lines from tumors and adjacent normal tissues sourced from patients. Consequently, we've amassed a collection of cell lines, including those from patient-derived ccRCC and normal kidney cells. Using these ccRCC models, we demonstrated that the type I protein arginine methyltransferase inhibitor, MS023, elicits a dysregulated DNA repair phenotype. RNA-Seq analysis of ccRCC cells treated with MS023 unveiled a marked downregulation of genes associated with the homologous recombination (HR) pathway for double-strand break repair and the Fanconi Anemia (FA) pathway for interstrand crosslink (ICL) repair. In a follow-up investigation using paired normal and cancerous cell lines from the same patient, we identified resistance to MS023 in normal kidney cells that is not mirrored in the corresponding ccRCC cell line. This hints at a potential therapeutic window and provides a strong rationale for clinical pursuit. Poly-ADP ribose polymerase (PARP) inhibitors are known to yield a synergistic effect when employed in HR-deficient diseases, such as BRCA-deficient breast cancers. Since MS023 induces an HR-deficient phenotype, we hypothesize that combining it with Talazoparib, a PARP inhibitor, will produce a similar synergistic effect. Additionally, mitomycin C (MMC) is a clinically approved compound that induces ICLs. As MS023 impedes ICL repair by downregulating crucial ICL-repairing genes, combining these two drugs should likewise generate a synergistic effect. Through a drug screen across two of our ccRCC cell lines, we discovered that the cells exhibit sensitivity to various PARP inhibitors. Employing synergy checkerboard assays and the highest single-agent model of synergy, we have found that the combination of MS023 and Talazoparib, as well as the combination of MS023 and MMC, demonstrate synergy, significantly reducing cellular viability at lower doses of the combination treatments compared to mono-treatment conditions at their respective doses. Preliminary in vivo studies using patient-derived xenograft models have revealed that both MS023 and Talazoparib are well-tolerated in mice, both when administered individually and in combination. Our current knowledge base indicates that MS023 holds promise for clinical investigation, both as a standalone treatment and in combination with Talazoparib or MMC. These results present an exciting opportunity to shed new light on the challenging therapeutic landscape faced by ccRCC patients today. Citation Format: Karen Arevalo, Joseph Walton, Jalna Meens, Angel Sau Ni Ng, Laurie Ailles. Dysregulated DNA repaired elicited by type 1 PRMT inhibitor in clear cell renal cell carcinoma enables development of novel combination therapies [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 7130.

Abstract 5605: Cutibacterium acnes invades prostate epithelial cells to induce BRCAness as a possible pathogen of prostate cancer

Abstract Abundant evidence suggests that chronic inflammation is linked to prostate cancer and that infection is a possible cause of prostate cancer. However, pathogen of prostate cancer remains to be found. To identify microbiota or pathogens associated with prostate cancer, we investigated the transcriptomes of 20 human prostate cancer tissues. We performed de novo assembly of non-human sequences from RNA-seq data and identified four bacteria as candidate microbiota in the prostate, including Moraxella osloensis, Uncultured chroococcidiopsis, Cutibacterium acnes, and Micrococcus luteus. Among these, C. acnes was detected in 19 of 20 prostate cancer tissue samples by immunohistochemistry. We then analyzed the gene expression profiles of prostate epithelial cells infected in vitro with C. acnes and found significant alterations in homologous recombination (HR) and Fanconi anemia pathway. Notably, electron microscopy showed that C. acnes invaded prostate epithelial cells and localized in perinuclear vesicles, whereas analysis of γH2AX foci and HR assay demonstrated impaired HR repair. In particular, BRCA2 was significantly down-regulated in C. acnes-infected cells. These findings suggest that C. acnes infection in the prostate could lead to HR deficiency and BRCAness which promotes DNA double-strand breaks, thereby increasing the risk of cancer development. Citation Format: Shingo Ashida, Chiaki Kawada, Hiroko Tanaka, Atsushi Kurabayashi, Ken-ichi Yagyu, Shuji Sakamoto, Kazuhiro Maejima, Satoru Miyano, Masanori Daibata, Hidewaki Nakagawa, Keiji Inoue. Cutibacterium acnes invades prostate epithelial cells to induce BRCAness as a possible pathogen of prostate 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 5605.

Abstract 6561: Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous ovarian cancer

Abstract Background: Ovarian Cancer (OC) is one of the leading causes of death among gynecological tumors with an insufficient understanding of OC disease evolution. Since tumor tissue is not always available, blood allows the investigation of single circulating tumor cells (sCTC) over time. We combined immunomagnetic enrichment and image-based sCTC sorting for phenotypic and genotypic analysis of sCTCs in the follow-up of OC to provide insights into sCTC heterogeneity and their malignant character. Patients and Methods: 10 ml blood of high-grade serous OC patients (pts) at the time of primary diagnosis (n=42), after chemotherapy (n=23) and after Avastin (n=11) was analyzed for sCTCs using density-gradient centrifugation to further apply MACS® using CD45 and CD235a antibodies coupled to MACS® MicroBeads. For image-based sCTC sorting (DEPArray®), an immunofluorescence protocol staining for Hoechst, cytokeratin (CK), the folate receptor alpha (FRa), Vimentin (Vim) and CD45 was applied. Whole genome amplification (WGA) was performed in recovered single cells, comprising cell lysis, DNA digestion, adaptor ligation and an amplification PCR for subsequent copy number alteration (CNA) analysis. Ampli1TM low-pass analysis was used to analyze single cell WGA products. 21 primary tumors (PT) were assessed for whole-genome CNA and the Ampli 1TM OncoSeek panel was used for deep sequencing of sCTCs. Results: Phenotypic assessment revealed FRa-positive cells in 28/42 (67%) pts, CK-positive cells in 32/42 (76%) pts and CK-FRa-double-positive cells in 15/42 (36%) pts, respectively, with a significant (p= 0.0205) reduction of cells after chemotherapy only documented for FRa-positive cells. Interestingly, cells with a high nuclear staining but no expression of target antigens were found in 37/42 pts (88%) and significantly (p=0.002) expanded during treatment. Genotyping revealed that only 7% (19/289) of single cells deriving from 13 different pts were aberrant. Whole genome CNA analysis in all pre-treatment sCTCs revealed enriched CNAs in chromosomes 2, 7 and 12. In post-treatment sCTCs, CNA dynamics highlighted the adaptive potential of the homologous recombination pathway and suggested further investigation of MAPK signaling in resistant OC. Low-pass CNA comparison between sCTCs and the PT was feasible in nine pts, 2/9 with persistent PT characteristics in sCTCs at different sampling times. TP53 variants were the most frequently detected ones, persistent CNA in the CDK4 oncogene and emerging CNA in the ALK oncogene were found in sCTCs from two pts. Conclusion: We identified sCTCs in the follow-up of OC and demonstrated interpatient as well as intrapatient heterogeneity of sCTCs. Matched sCTCs and PT data showed similar CNAs to a certain amount. Our low percentage of aberrant cells suggests that higher patient numbers are needed to confirm our findings to understand disease progression. Citation Format: Sabine Kasimir-Bauer, Nikolas H. Stoecklein, Rui P. Neves, Sven T. Liffers, Jan D. Kuhlmann, Pauline Wimberger, Paul Buderath, Rainer Kimmig, Carolin Salmon. Phenotypic and genotypic characterization of single circulating tumor cells in the follow-up of high-grade serous 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 6561.

Abstract 2524: Molecular and clinicopathologic landscape of HER2 positive breast cancer: A single institution study

Abstract Background: Human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase that is encoded by the ERBB2 gene, located on chromosome 17. Expression levels of HER2 are a key pathogenic and treatment-associated factor in breast carcinomas, with HER2 expression reported as per the ASCO/CAP scoring guidelines. Outside the intense focus on the HER2 overexpression few studies currently exist that look at the comprehensive molecular profile of these aggressive tumors. Design: Our institutional pathology database was searched for patients with breast carcinoma diagnosed between 2009 and 2013 and with HER2 scored as 3+ based on immunohistochemistry, or HER2 2+ with confirmatory positive FISH results. Comprehensive next-generation sequencing (NGS) was performed using the Illumina TruSight Oncology 523 gene panel, and clinicopathologic data were gathered from the institutional electronic health record system. A CoMutation plot was made using the CoMut python library. Results: The mean age of the patients at time of diagnosis was 56.1±12.7 years. The tumors were overwhelmingly high-grade, with 26/30 being grade 3 and the remaining 4/30 being grade 2 and a mean size of 2.6 cm. All were histologically invasive ductal carcinoma, and 26/30 were scored as HER2 3+ on IHC while 4/30 were HER2 2+ with positive FISH. ER was positive in 30% of cases (9/30), PR was positive in 23.3% (7/30), p53 was positive by IHC in 53.3% (16/30), and the mean Ki-67 proliferative index was 47.1%. 20 of the 30 (66%) cases meeting the inclusion criteria had pathogenic mutations detected by NGS testing. Several distinct molecular subgroups were seen on the CoMutation plot (figure 1). The mean tumor mutational burden (TMB) was 14.1 mutations per megabase. A significant subset of the tumors 70% (14/20) showed TP53 mutations, and 30% (6/20) show PIK3CA mutations. Of interest, only 1/14 tumors showed a co-occurring homologous recombination repair gene mutation and one tumor (1/20) showed mutations in 2 homologous recombination pathway genes (FANCA and BRCA1) as well as numerous additional mutations (tumor mutational burden of 65.3 mutations per megabase). Conclusion: Overall the mean TMB was 14.1 mutations/megabase in this HER2-positive breast cancer study set. Over 2/3 of our HER2-positive breast cancer cases also had TP53 mutations. In addition, 1/3 had PIK3CA mutations, suggesting that a subset of HER2-positive carcinomas also harbor other potentially actionable mutations which may increase the effectiveness of our existing interventions. Additional studies are needed to further subclassify genetically distinct subsets of HER2-positive breast cancers and to further understand clinical, pathologic and treatment-related differences in this aggressive breast cancer subset. Citation Format: Gokce Deniz Ardor, Zachary M. Coty-Fattal, Luis Z. Blanco, Kalliopi Siziopikou. Molecular and clinicopathologic landscape of HER2 positive breast cancer: A single institution study [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 2524.

Repression of the SUMO-conjugating enzyme UBC9 is associated with lowered double minutes and reduced tumor progression

ABSTRACT Double minutes (DMs), extrachromosomal gene fragments found within certain tumors, have been noted to carry onco- and drug resistance genes contributing to tumor pathogenesis and progression. After screening for SUMO-related molecule expression within various tumor sample and cell line databases, we found that SUMO-conjugating enzyme UBC9 has been associated with genome instability and tumor cell DM counts, which was confirmed both in vitro and in vivo. Karyotyping determined DM counts post-UBC9 knockdown or SUMOylation inhibitor 2-D08, while RT-qPCR and Western blot were used to measure DM-carried gene expression in vitro. In vivo, fluorescence in situ hybridization (FISH) identified micronucleus (MN) expulsion. Western blot and immunofluorescence staining were then used to determine DNA damage extent, and a reporter plasmid system was constructed to detect changes in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Our research has shown that UBC9 inhibition is able to attenuate DM formation and lower DM-carried gene expression, in turn reducing tumor growth and malignant phenotype, via MN efflux of DMs and lowering NHEJ activity to increase DNA damage. These findings thus reveal a relationship between heightened UBC9 activity, increased DM counts, and tumor progression, providing a potential approach for targeted therapies, via UBC9 inhibition.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Abstract A076: T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status

Abstract Epithelial ovarian cancer is the most lethal gynecologic malignancy, with 5-year survival rates of 46%. High grade serous carcinoma (HGSC) is the most frequent subtype of epithelial ovarian cancer. Approximately 50% of cases are homologous recombination deficient (HRD). Whereas maintenance treatment by PARP inhibitors has significantly improved the prognosis of patients with HRD tumors, clinical outcome in this malignancy is still poor. Despite the immunogenic nature of HGSC, in which T-cell infiltration correlates to improved clinical outcome following surgery and chemotherapy, the disease exhibits low response rates to immune checkpoint blockade (ICB) monotherapy in advanced line settings. We have previously identified epithelial malignancies including HGSC, CD4 and CD8 populations of tumor-infiltrating and antigen-specific T cells that display features of terminal exhaustion. These populations were key players in the response to anti-PD-1 in patients with epithelial tumors. Here, we set out to characterize tumor-infiltrating T cells in a cohort of 80 HGSC patients treated at our institution from whom we collected tumor samples prior to therapy in which HRD status was assessed. We used multiplex immunofluorescence of FFPE tumor samples to assess infiltration by CD4 and CD8 T cells and multiparametric flow cytometry of frozen viable cell suspensions to assess CD4 and CD8 T-cell exhaustion. The link between biological parameters and clinical outcome was assessed by univariable and multivariable analyses. Assessment of the T-cell infiltrate by immunofluorescence showed no difference in the absolute numbers, per tumor or stroma mm2, of CD8 or CD4 T cells between HRD and homologous recombination proficient (HRP) tumors. Instead, analysis of exhausted CD8 and CD4 T cells showed an increased frequency of terminally exhausted PD-1HighTIM-3+ CD8 T cells in HRD tumors. This population, which expresses the ectonucleotidase CD39, was significantly correlated to the presence of T regulatory cells (Treg), which agreed with previous studies showing comigration of effector and regulatory T cells in ovarian cancer. In addition, terminally exhausted CD4 T cells, expressing the immune checkpoints PD-1 and TIM-3 as well as CD39, were enriched in HRD tumors. Among HRD tumors, no difference was observed between the exhaustion profile in tumors presenting deleterious BRCA1/2 mutations compared to those with other mutations in the homologous recombination pathway. Univariable analyses showed exhaustion parameters to be significant predictors of progression-free survival (PFS). The frequencies of exhausted CD4 and CD8 T cells stood out as predictors of PFS in multivariable analyses. Altogether, these results provide the first evidence of a link between T-cell exhaustion and HRD status and reveal T-cell exhaustion as an independent predictor of disease outcome. They also underline the major role played by the antitumor immune response in HGSC and warrant the development of combination immunotherapies despite the lack of response to ICB monotherapy. Citation Format: Anna Salvioni, Mathilde Del, Marie Michelas, Pierre Vuattoux, Clara-Maria Scarlata, Carlos Martinez-Gomez, Nathalie Van Acker, François-Xavier Frenois, Guillaume Bataillon, Jean-Pierre Delord, Alejandra Martinez, Maha Ayyoub. T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A076.

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer

Abstract A035: Genome-wide CRISPRi screening reveals determinants of platinum sensitivity and resistance in high-grade serous ovarian cancer