Inhibition Of Homologous Recombination Research Articles

Dual activities of a silencing information regulator complex in yeast transcriptional regulation and DNA‐damage response

AbstractThe Saccharomyces cerevisiae silencing information regulator (SIR) complex contains up to four proteins, namely Sir1, Sir2, Sir3, and Sir4. While Sir2 encodes a NAD‐dependent histone deacetylase, other SIR proteins mainly function as structural and scaffold components through physical interaction with various proteins. The SIR complex displays different conformation and composition, including Sir2 homotrimer, Sir1‐4 heterotetramer, Sir2‐4 heterotrimer, and their derivatives, which recycle and relocate to different chromosomal regions. Major activities of the SIR complex are transcriptional silencing through chromosomal remodeling and modulation of DNA double‐strand‐break repair pathways. These activities allow the SIR complex to be involved in mating‐type maintenance and switching, telomere and subtelomere gene silencing, promotion of nonhomologous end joining, and inhibition of homologous recombination, as well as control of cell aging. This review explores the potential link between epigenetic regulation and DNA damage response conferred by the SIR complex under various conditions aiming at understanding its roles in balancing cell survival and genomic stability in response to internal and environmental stresses. As core activities of the SIR complex are highly conserved in eukaryotes from yeast to humans, knowledge obtained in the yeast may apply to mammalian Sirtuin homologs and related diseases.

Telomere Checkpoint in Development and Aging

The maintenance of genome integrity through generations is largely determined by the stability of telomeres. Increasing evidence suggests that telomere dysfunction may trigger changes in cell fate, independently of telomere length. Telomeric multiple tandem repeats are potentially highly recombinogenic. Heterochromatin formation, transcriptional repression, the suppression of homologous recombination and chromosome end protection are all required for telomere stability. Genetic and epigenetic defects affecting telomere homeostasis may cause length-independent internal telomeric DNA damage. Growing evidence, including that based on Drosophila research, points to a telomere checkpoint mechanism that coordinates cell fate with telomere state. According to this scenario, telomeres, irrespective of their length, serve as a primary sensor of genome instability that is capable of triggering cell death or developmental arrest. Telomeric factors released from shortened or dysfunctional telomeres are thought to mediate these processes. Here, we discuss a novel signaling role for telomeric RNAs in cell fate and early development. Telomere checkpoint ensures genome stability in multicellular organisms but aggravates the aging process, promoting the accumulation of damaged and senescent cells.

Inhibition of p53 and ATRX increases telomeric recombination in primary fibroblasts.

Telomere length can be maintained either by the telomerase enzyme or by alternative lengthening of telomeres (ALT), which is based on telomeric recombination. However, both mechanisms are inactive in most human somatic cells. ATRX has been previously identified as an ALT repressor gene. Nonetheless, TP53 is also deficient in most ALT cell lines, and previous works showed that it is an inhibitor of homologous recombination (HR). Despite this, the role of p53 as an ALT repressor has not been previously examined. Therefore, we investigated the effects of p53 and ATRX inhibition on normal human fibroblasts (devoid of any mutation), in the presence or absence of X-ray-induced telomeric damage. Performing immunofluorescence with antibodies for RAD51, H2AX, and TRF1 (for studying HR-mediated DNA damage repair) and CO-FISH (for telomeric sister chromatid exchanges), we observed that HR is a normal mechanism for the repair of telomeric damage, present also in noncancer cells. Moreover, we discovered that telomeric HR, as for HR in general, is significantly inhibited by p53. Indeed, we observed that inhibition of p53 drastically increases telomeric sister chromatid exchanges. We also confirmed that ATRX inhibition increases telomeric recombination. In particular, we observed an increase in crossover products, but a much higher increase in noncrossover products.

Elevated APE1 Dysregulates Homologous Recombination and Cell Cycle Driving Genomic Evolution, Tumorigenesis, and Chemoresistance in Esophageal Adenocarcinoma

The purpose of this study was to identify drivers of genomic evolution in esophageal adenocarcinoma (EAC) and other solid tumors. An integrated genomics strategy was used to identify deoxyribonucleases correlating with genomic instability (as assessed from total copy number events in each patient) in 6 cancers. Apurinic/apyrimidinic nuclease 1 (APE1), identified as the top gene in functional screens, was either suppressed in cancer cell lines or overexpressed in normal esophageal cells and the impact on genome stability and growth was monitored invitro and invivo. The impact on DNA and chromosomal instability was monitored using multiple approaches, including investigation of micronuclei, acquisition of single nucleotide polymorphisms, whole genome sequencing, and/or multicolor fluorescence in situ hybridization. Expression of 4 deoxyribonucleases correlated with genomic instability in 6 human cancers. Functional screens of these genes identified APE1 as the top candidate for further evaluation. APE1 suppression in EAC, breast, lung, and prostate cancer cell lines caused cell cycle arrest; impaired growth and increased cytotoxicity of cisplatin in all cell lines and types and in a mouse model of EAC; and inhibition of homologous recombination and spontaneous and chemotherapy-induced genomic instability. APE1 overexpression in normal cells caused a massive chromosomal instability, leading to their oncogenic transformation. Evaluation of these cells by means of whole genome sequencing demonstrated the acquisition of changes throughout the genome and identified homologous recombination as the top mutational process. Elevated APE1 dysregulates homologous recombination and cell cycle, contributing to genomic instability, tumorigenesis, and chemoresistance, and its inhibitors have the potential to target these processes in EAC and possibly other cancers.

PilE G-Quadruplex Is Recognized and Preferentially Bound but Not Processed by the MutL Endonuclease from Neisseria gonorrhoeae Mismatch Repair Pathway

The human pathogen Neisseria gonorrhoeae uses a homologous recombination to undergo antigenic variation and avoid an immune response. The surface protein pilin (PilE) is one of the targets for antigenic variation that can be regulated by N. gonorrhoeae mismatch repair (MMR) and a G-quadruplex (G4) located upstream of the pilE promoter. Using bioinformatics tools, we found a correlation between pilE variability and deletion of DNA regions encoding ngMutS or ngMutL proteins, the main participants in N. gonorrhoeae methyl-independent MMR. To understand whether the G4 structure could affect the ngMutL-mediated regulation of pilin antigenic variation, we designed several synthetic pilE G4-containing oligonucleotides, differing in length, and related DNA duplexes. Using CD measurements and biochemical approaches, we have showed that (i) ngMutL preferentially binds to pilE G4 compared to DNA duplex, although the latter is a cognate substrate for ngMutL endonuclease, (ii) protein binding affinity decreases with shortening of quadruplex-containing and duplex ligands, (iii) the G4 structure inhibits ngMutL-induced DNA nicking and modulates cleavage positions; the enzyme does not cleave DNA within G4, but is able to bypass this noncanonical structure. Thus, pilE G4 may regulate the efficiency of pilin antigenic variation by quadruplex binding to ngMutL and suppression of homologous recombination.

EXTH-63. COMBINED INHIBITION OF TOP1 AND PARP: A NOVEL THERAPEUTIC STRATEGY FOR GBM WITH PTEN DEFICIENCY

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive and lethal type of brain tumor. Activation of PI3K/mTOR pathway along with the loss of its primary negative regulator, phosphatase and tensin homolog (PTEN), occurs in nearly 50% of GBM patients. As PTEN is known to promote DNA damage repair deficiency, here we investigated whether PTEN deficiency presents a vulnerability to a simultaneous induction of DNA damage and suppression of repair mechanisms by combining topoisomerase I (TOP1) and PARP inhibitors. METHODS We used patient-derived GBM cells and stem-like cells to determine response to LMP400 (Indotecan), a novel non-camptothecin TOP1 inhibitor, and the PARP inhibitors Olaparib or Niraparib. Treatment efficacy was also determined using cell viability, cell cycle, DNA damage, repair, and apoptosis assays in a pair of isogenic PTEN-null and PTEN-WT glioma cell lines derived from a genetically engineered mouse GBM model. RNAseq analysis was performed to identify treatment-induced dysregulated pathways. RESULTS PTEN-deficient cells are highly sensitive to LMP400 and PTEN rescue lessens sensitivity to the treatment. Combining LMP400 with PARP inhibitors, Olaparib or Niraparib, leads to synergistic cytotoxicity. LMP400/Niraparib combination induces G2/M cell cycle arrest, DNA damage, suppression of homologous recombination (HR)-related proteins and activation of caspase 3/7 activity significantly more in PTEN-null cells compared to isogenic PTEN-WT cells. Gene set enrichment analysis revealed suppression of cell cycle and DNA damage repair as well as activation of cell death pathways. Finally, CRISPR-Cas9 KO screening suggests that LMP400 is not likely to be a substrate for ABC transporters, suggesting the brain penetration and supporting the use in brain tumor patients. CONCLUSION Combined inhibition of TOP1 and PARP induces synergistic antiglioma effects selectively in PTEN-null glioblastoma cells, providing a strong scientific premise for a clinical trial of combined treatment with LMP400 and Niraparib in a subset of GBM with PTEN deficiency.

Abstract 216: Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models

Abstract Purpose: CDK4/6 inhibitors (CDK4/6i) are standard of care for the treatment of locally advanced and metastatic estrogen receptor-positive (ER+), HER2-negative metastatic breast cancer (BC). CDK4/6 inhibition + radiation therapy (RT) is synergistic in both ER+ and triple negative breast cancers (TNBC), but the underlying mechanism is not entirely understood. In this study, we evaluated how pre-existing or genetically engineered deficits in DNA damage response genes (BRCA1/2, RAD51, RB1, XRCC6, TP53) influence radiosensitization. We hypothesized that inhibition of homologous recombination (HR) would prevent CDK4/6i-mediated radiosensitization and blocking non-homologous end joining (NHEJ) would be synergistic. Methods: Cellular proliferation assays determined the half-maximal inhibitory concentrations (IC50) of the 3 approved CDK4/6i palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays determined the radiation enhancement ratios (rERs) and evaluated the efficacy of CDK4/6i + RT. Immunofluorescence assays measured RAD51 foci formation and quantified micronuclei formation following RT and/or CDK4/6 inhibition. Immunoprecipitation with myc-RAD51 and GFP-RB assessed potential protein-protein interactions. Results: While ER+ and TNBC cell lines with wild type BRCA1 expression are radiosensitized by CDK4/6i, BRCA1-deficient SUM-149 cells are not radiosensitized by CDK4/6i at concentrations up to 1µM (rER: 0.92-1.01). In an MCF-7 isogenic model of BRCA2 knockout, CDK4/6i-mediated radiosensitization was abolished compared to Cas9 control or parental cell lines. In ER+ BC cell lines (MCF-7-p53 wt, T47D-p53 mutant), transient or genetic knockdown of RAD51 prevented CDK4/6i-induced radiosensitization. The total quantity of RT-induced RAD51 foci increased in vitro following overexpression of RB-a tumor suppressor and downstream target of CDK4/6. RB overexpression also rescued CDK4/6i-mediated radiosensitization in RB-deficient cell lines through changes in HR efficiency but not via NHEJ or altered micronuclei formation. Moreover, immunoprecipitation of RAD51 in ER+ (MCF-7) and TNBC (MDA-MB-231) cells exhibited an interaction with RB. Conversely, loss of the NHEJ-associated protein Ku70 (XRCC6) was synergistic with palbociclib + RT in MCF7 (rER: 1.76-2.44) and T47D (rER: 1.61-3.88) cells. Finally, CRISPR Cas9-mediated loss of the tumor suppressor p53 (TP53) did not affect radiosensitization induced by CDK4/6i in isogenic p53 wt ER+ (MCF-7, rER: 1.19-1.33) and p53 wt TNBC (CAL-51, rER: 1.23-1.52) cell lines with TP53 loss. Conclusions: Taken together, our results in multiple non-overlapping isogenic models of ER+ and TNBC suggest that CDK4/6i-mediated radiosensitization of BC cell lines occurs through impaired HR activity and RB signaling, and not through the actions of p53 or NHEJ-mediated DNA repair. Citation Format: Kassidy M. Jungles, Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Meilan Liu, Lynn Lerner, Lori J. Pierce, James M. Rae, Corey W. Speers. Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 216.

Activation of homologous recombination in G1 preserves centromeric integrity.

Centromeric integrity is key for proper chromosome segregation during cell division1. Centromeres have unique chromatin features that are essential for centromere maintenance2. Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements3, little is known about how centromere integrity in response to DNA damage is preserved. DNA repair by homologous recombination requires the presence of the sister chromatid and is suppressed in the G1 phase of the cell cycle4. Here we demonstrate that DNA breaks that occur at centromeres in G1 recruit the homologous recombination machinery, despite the absence of a sister chromatid. Mechanistically, we show that the centromere-specific histone H3 variant CENP-A and its chaperone HJURP, together with dimethylation of lysine 4 in histone 3 (H3K4me2), enable a succession of events leading to the licensing of homologous recombination in G1. H3K4me2 promotes DNA-end resection by allowing DNA damage-induced centromeric transcription and increased formation of DNA-RNA hybrids. CENP-A and HJURP interact with the deubiquitinase USP11, enabling formation of theRAD51-BRCA1-BRCA2 complex5 and rendering the centromeres accessible to RAD51 recruitment and homologous recombination in G1. Finally, we show that inhibition of homologous recombination in G1 leads to centromeric instability and chromosomal translocations. Our results support a model in which licensing of homologous recombination at centromeric breaks occurs throughout the cell cycle to prevent the activation of mutagenic DNA repair pathways and preserve centromeric integrity.

Depletion of NK6 Homeobox 3 (NKX6.3) causes gastric carcinogenesis through copy number alterations by inducing impairment of DNA replication and repair regulation

Genomic stability maintenance requires correct DNA replication, chromosome segregation, and DNA repair, while defects of these processes result in tumor development or cell death. Although abnormalities in DNA replication and repair regulation are proposed as underlying causes for genomic instability, the detailed mechanism remains unclear. Here, we investigated whether NKX6.3 plays a role in the maintenance of genomic stability in gastric epithelial cells. NKX6.3 functioned as a transcription factor for CDT1 and RPA1, and its depletion increased replication fork rate, and fork asymmetry. Notably, we showed that abnormal DNA replication by the depletion of NKX6.3 caused DNA damage and induced homologous recombination inhibition. Depletion of NKX6.3 also caused copy number alterations of various genes in the vast chromosomal region. Hence, our findings underscore NKX6.3 might be a crucial factor of DNA replication and repair regulation from genomic instability in gastric epithelial cells.

Abstract SY37-01: Genomic instability and hypoxia as co-drivers of prostate cancer aggression

Abstract Understanding the mechanistic basis for transition to late stage aggressive disease is vital for both assigning patient risk status in the localised setting and also identifying novel treatment strategies to prevent progression. Sub-regions of intratumoral hypoxia are found in all solid tumours and are associated with many biologic drivers of tumour progression. Crucially, more recent findings show the co-presence of hypoxia and genomic instability can confer a uniquely adverse prognosis in prostate cancer. In-depth informatic and functional studies suggests a role for hypoxia in co-operating with oncogenic drivers (e.g. loss of PTEN) and suppressing DNA repair capacity. To delineate a direct relationship between hypoxia and PTEN loss, we interrogated the impact of chronic hypoxia (1% O2, 0.2% O2; 72 h) on PTEN oncogenic signalling and chromosome instability. Quantitative immunofluorescence of centrosome aberrations, using antibodies to phospho-Histone H3 and Centrin-1, show significant increases in centriole numbers (p < 0.05) in mitotic cells under chronic hypoxic conditions and an increase in the incidence of micronuclei (p < 0.005) specifically in chronic hypoxic cells with PTEN loss. Hypoxic suppression of homologous recombination represents a "contextual lethal" vulnerability in hypoxic prostate tumours which could extend the application of existing DNA repair targeting agents such as poly-ADP ribose polymerase inhibitors. Additional mechanistic studies using hTERT-immortalized, primary prostate epithelial cells isogenic for c-MYC, BRCA2, or TP53/RB (SV40-transduced) were studied as to the relationship between hypoxia, inherent versus acquired genetic instability and DNA repair capacity in-vitro and in-situ. Chronic hypoxia (20 generation doublings at 0.2% O2) was sufficient to drive increased copy number alterations (CNAs) in selected clones by up to 30% in TP53/RB null cells. This finding is now being quantitated with a clonal evolution model and genotype-phenotype associations are being validated using functional transformation and invasion assays. Further investigation is now required to assess this relationship on the background of existing genomic alterations relevant to prostate and other cancers and also characterise the role of hypoxia in driving early metastatic spread. Supported by core funding from Cancer Research UK Manchester Institute and grants within the Cancer Research UK Manchester RadNet Unit, NIHR Manchester Biomedical Research Centre and a Prostate Cancer UK-Movember Centre of Excellence award. Citation Format: Robert G. Bristow. Genomic instability and hypoxia as co-drivers of prostate cancer aggression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY37-01.

Abstract IA-003: Oxygen dependent resistance to PARP inhibitors

Abstract Severe hypoxia causes resistance to conventional chemotherapy and has been reported to synergize with PARP inhibitors (PARPi) through suppression of homologous recombination (HR). While this synergistic killing is true at oxygen levels less than 0.5%, our study shows that less severe hypoxia (e.g. 2% oxygen) is instead associated with resistance to PARPi in HR proficient cells. Interestingly, we demonstrate that HR deficient hypoxic tumors are significantly less responsive to PARPi, due to limited ROS-induced intrinsic DNA damage. To determine the contribution of hypoxic cells to PARPi, we used the hypoxic cytotoxin Tirapazamine to target hypoxic tumor cells. We found that the elimination of hypoxic tumor cells by Tirapazamine led to a substantial antitumor response with PARPi compared to PARPi treated tumors alone, without enhancing normal tissue toxicity. These studies indicate that tumor hypoxia reduces the efficacy of PARPi to tumor cells and that eliminating hypoxic tumor cells will enhance the efficacy of PARPi therapy. Citation Format: Manal Mehibel, Jimmy Xu, Grace Li, Jung Moon, Kaushik Thakkar, Anh Diep, Ryan Kim, Joshua Bloomstein, Siren Xiao, Julien Bacal, Joshua Saldivar, Quynh Le, Karlene Cimprich, Erinn Rankin, Amato Giaccia. Oxygen dependent resistance to PARP inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-003.

Abstract PO-032: Mechanistic studies of hypoxia as a driver of genomic instability in prostate cancer

Abstract Prostate cancer (PCa) is the 2nd most prevalent cancer in men and has poor patient outcome upon transition from localized disease to the castration-resistant and metastatic state. Numerous studies have indicated intratumoral hypoxia as a key driver in disease progression across multiple cancers and in prostate cancer. Hypoxia is associated with local recurrence, metastatic capacity, androgen independence and neuroendocrine differentiation (Ashton and Bristow, Brit.J.Rad, 2020). Crucially, among localized PCa patients, hypoxia and genomic instability have an independent and additive effect on biochemical relapse and bioinformatic studies suggest that hypoxia co-segregates mutation signatures representing defects in homologous recombination (HR) and mismatch repair (MMR) DNA repair pathways (Bhandari et al., Nat Comms, 2020). In order to investigate whether hypoxia directly drives genome instability in prostate cancer, mechanistic studies using hTERT-immortalized, primary prostate epithelial cells isogenic for c-MYC, BRCA2, or TP53/RB (SV40-transduced as PNT2 cells) were studied as to the relationship between hypoxia, inherent versus acquired genetic instability, and DNA repair capacity in-vitro and in-situ. Chronic hypoxia (20 generation doublings at 0.2% O2) was sufficient to drive increased copy number alterations (CNAs; based on low-pass whole genome sequencing - WGS) in selected clones by up to 30% in TP53/RB null cells, but not hTERT immortalized models. This finding is now being quantitated within a clonal evolution model and genotype-phenotype associations are being validated using functional transformation and invasion assays. Associated mechanistic studies have revealed that the hypoxic suppression of homologous recombination (HR) factor transcript and protein abundance (e.g up to ~60% reduction of RAD51/BRCA2 in PC3, DU145 and PNT2 cells) and associated with reduced RAD51 nuclear foci induction or defective DR-GFP reconstitution, is highly-dependent on cell density. This suggests that reduced HR and MMR repair capacity may exist in situ within hypoxic tumor oxygen gradients with close cell-cell interactions; in situ studies of RAD51 and other DNA repair proteins across hypoxic gradients in primary human tumors will be presented. These translational studies will accompany the HYPROGEN trial in Manchester in which patients who present with treatment-naïve M1 bone metastatic disease are given oral pimonidazole prior to prostate and bone metastasis biopsies to directly study genomic instability (using WGS) and hypoxia (pimonidazole staining and RNA hypoxic signatures) to understand these relationships in paired primary-metastatic samples. This data will provide a mechanistic basis for the adverse prognosis seen in patients whose tumors have both hypoxia and genome instability, as well as fresh evidence for stratification and treatment intensification of localized PCa patients with hypoxic tumors. (Supported by a core grant from Cancer Research UK to RGB) Citation Format: Jack Ashton, Richard Rebello, Stephen Lyons, Robert G. Bristow. Mechanistic studies of hypoxia as a driver of genomic instability in prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-032.

Abstract IA-001: Genomic architecture and targets in hypoxic tumors

Abstract Understanding the mechanistic basis for transition to late stage aggressive disease is vital for both assigning patient risk status in the localized setting and also identifying novel treatment strategies to prevent progression. Subregions of intratumoral hypoxia are found in all solid tumors and are associated with many biologic drivers of tumor progression. Crucially, more recent findings show the co-presence of hypoxia and genomic instability can confer a uniquely adverse prognosis. In-depth informatic and functional studies suggests a role for hypoxia in co-operating with oncogenic drivers (e.g. loss of PTEN) and suppressing DNA repair capacity to alter clonal adaption and evolution and driving an aggressive mutator phenotype. More specifically, hypoxic suppression of homologous recombination represents a "contextual lethal" vulnerability in hypoxic prostate tumors which could extend the application of existing DNA repair targeting agents such as poly-ADP ribose polymerase inhibitors. Further investigation is now required to assess this relationship on the background of existing genomic alterations relevant to prostate and other cancers, and also characterize the role of hypoxia in driving early metastatic spread. Supported by core funding from Cancer Research UK Manchester Institute and grants within the Cancer Research UK Manchester RadNet Unit, NIHR Manchester Biomedical Research Centre and a Prostate Cancer UK-Movember Centre of Excellence award. Citation Format: Robert G. Bristow. Genomic architecture and targets in hypoxic tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-001.

Ongoing Spontaneous DNA Damage Creates Synthetic Lethality Targeted By Novel RAD51 Inhibitors in Multiple Myeloma

Unrepaired DNA double-strand breaks (DSBs) pose a serious threat to genomic stability, potentially leading to the formation of oncogenic mutations, as well as translocations, deletions and amplifications. Almost all multiple myeloma (MM) patients are characterized by such copy number alterations and structural variations. Therefore, we evaluated and report that MM cells have increased ongoing spontaneous DNA damage which could be exploited to create synthetic lethality in combination with inhibitors of homologous recombination (HR). We evaluated various types of DNA damage in peripheral blood mononuclear cells (PBMCs)from 15healthy controls, 4 MM cell lines and CD138+ bone marrow plasma cells (BMPCs) from 16 patients with monoclonal gammopathy of undetermined significance, 18 with smoldering myeloma (SMM) and 15 with MM. Relative to normal PBMCs, MGUS and MM cells showed significantly higher levels of endogenous DNA breaks (measured as olive tail moments with single-cell gel electrophoresis), abasic sites (Elisa-based detection) as well as DNA break repair efficiency (all P<0.001). Interestingly, a gradual significant increase of all the DNA damage parameters was observed in BMPCs duringthe progression from MGUS to MM, with PBMCs from healthy controls showing the least levels (all P<0.05).Thus, MM cells have both the increased levels of endogenously generated DNA damage as well as DNA repair. A functioning HR is thus a dependency for MM cell survival. Our data confirms elevated HR activity in MM contributing to both genomic evolution as well as tumor growth. We have further exploited this molecular and functional dependency by targeting RAD51, a recombinase involved in the recognition of sequence homology and exchange of DNA strands between homologous sequences, playing a key role in the HR. We evaluated antitumor activity of novel RAD51 inhibitors (CYT-0178 and CYT-0851) in MM. CYT-0851 is currently in clinical testing in a range of cancers. First, we evaluated HR using 3 different functional HR assays in MM as well as other cancer cell lines. Treatment of MM1S and FLO-1 cancer cell lines with CYT-0178 at 5 mM demonstrated > 70% inhibition of HR activity measured using plasmid-based assay. Inhibition of HR by CYT-0178 was also demonstrated in U2OS cell using DRGFP (Addgene) assay. Moreover, treatment of RPMI-8226, MM1S, H929 MM cell lines with CYT-0851caused a dose-dependent inhibition of HR activity as assessed by homologous strand exchange assay. To evaluate the impact of these drugs on cell viability, MM cell lines (U266, RPMI8226 and MM1S) and normal cells were incubated with CYT-0851 and CYT-0178 at various concentrations and durations. Treatment with CYT-0851 induced dose- and time-dependent cytotoxicity in all myeloma cell lines tested with minimal impact on normal cells. For CYT-0178, the treatment at 10 𝛍M for seven days resulted in 50% to ~80% cell death in MM cell lines tested, although lower (5 𝛍M) dose had no significant impact. We further evaluated combination of these inhibitors with conventional MM drugs melphalan, cyclophosphamide and lenalidomide,for impact on cytotoxicity. Treatment with CYT-0851 led to a synergistic anticancer effect when combined with these chemotherapeutic agents in both the MM cell lines tested. Increased cytotoxicity of lenalidomide and melphalan was also observed by CYT-0178 in MM1S cells. In summary, we demonstrate that MM cells have ongoing endogenous DNA damage which provides basis for synthetic lethality in combination with HR inhibitors while sparing normal cells. Moreover, novel RAD51 inhibitors CYT-0178 and CYT-0851 inhibit HR activity, impair cell growth and increase efficacy of existing drugs in multiple myeloma. Disclosures Day: Cyteir Therapeutics: Employment. Munshi:Takeda: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy.

IDamage: a method to integrate modified DNA into the yeast genome.

In order to explore the mechanisms employed by living cells to deal with DNA alterations, we have developed a method by which we insert a modified DNA into a specific site of the yeast genome. This is achieved by the site-specific integration of a modified plasmid at a chosen locus of the genome of Saccharomyces cerevisiae, through the use of the Cre/lox recombination system. In the present work, we have used our method to insert a single UV lesion into the yeast genome, and studied how the balance between error-free and error-prone lesion bypass is regulated. We show that the inhibition of homologous recombination, either directly (by the inactivation of Rad51 recombinase) or through its control by preventing the polyubiquitination of PCNA (ubc13 mutant), leads to a strong increase in the use of Trans Lesion Synthesis (TLS). Such regulatory aspects of DNA damage tolerance could not have been observed with previous strategies using plasmid or randomly distributed DNA lesions, which shows the advantage of our new method. The very robust and precise integration of any modified DNA at any chosen locus of the yeast genome that we describe here is a powerful tool that will enable the exploration of many biological processes related to replication and repair of modified DNA.

Abstract 3823: Inhibition of homologous recombination, PARP inhibitor, or dianhydrodulcitol overcomes temozolomide-resistance in glioma cells

Abstract Glioblastoma is one of the most aggressive tumors in the central nervous system tumors. The standard therapy for glioblastomas is maximal safe resection, followed by radiation therapy and chemotherapy with temozolomide (TMZ). The acquisition of resistance to TMZ is big problem. TMZ is a DNA-methylating agent, delivering a methyl group to DNA (O6-guanine, N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine, mispairs with thymine, leading to futile DNA mismatch repair (MMR), formation of double strand breaks and eventual cell death, when O6-methylguanine DNA methyltransferase (MGMT) is absent. N7-methylguanine and N3-methyladenine are repaired by base excision repair. The object of the study was to reveal the mechanisms of resistance to TMZ and to find the way to overcome the resistance in glioma cells. Several clones of TMZ-resistant U251 were obtained and analyzed. #3 clone showed G2 arrest after TMZ exposure and this arrest was abrogated sooner compared to parental U251. TMZ did not induce G2 arrest in #8 clone. The expression of MGMT was not found in U251 parental cells, #3 cells nor #8 cells. The ability of homologous recombination (HR) was increased in #3 clone, and suppression of HR resensitized #3 clone to TMZ. The protein levels of MSH6, which was one of the main proteins associated with MMR, was reduced in #8 clone. Inhibition of base excision repair by PARP inhibitor resensitized #8 clone to TMZ. Dianhydrodulcitol, alkylating agent, suppressed the proliferation in U251 and even U251-derived resistant clones. These effects were shown in U87MG-dereived resistant clones and MGMT-proficient cell lines. To select the way according to the mechanisms of resistance to TMZ could overcome the resistance in glioma cells. Citation Format: SHIGEO OHBA, Yuichi Hirose. Inhibition of homologous recombination, PARP inhibitor, or dianhydrodulcitol overcomes temozolomide-resistance in glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3823.

Abstract 2566: CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage

Abstract Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe. Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2566.

Abstract 2566: CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage

Activation Induced Cytidine Deaminase (AID) is a DNA-directed cytidine deaminase that plays a critical role in somatic hypermutation and immunoglobulin class switching in activated B- lymphocytes. AID is often ectopically expressed and promotes genomic hypermutation in a range of lymphoid malignancies. Deamination of genomic cytidines results in point mutations, single strand DNA breaks (SSB), and double strand DNA breaks (DSB). The resulting DNA damage stress leads to an obligate dependency on homologous recombination for cellular survival. RAD51 is a critical component of the homologous recombination pathway, forming nucleoprotein filaments at sites of DNA damage or replication fork stalls. RAD51 mediates homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We have previously shown that RAD51 depletion leads to accumulation of DNA breaks, and ultimately cell death, in AID expressing cells. We now describe the mechanism of action for a novel small molecule, CYT01B, that targets RAD51 mediated recombination. Treatment of AID-expressing cells with CYT01B, attenuates RAD51 focus formation, reduces the nuclear concentration of RAD51, and promotes RAD51 protein degradation. CYT01B reduces homologous recombination activity in cells, culminating in cytologically visible chromosome fragmentation following DNA damage. Finally, CYT01B treated cells undergo replication catastrophe associated with caspase 3/7 activation and persistent PCNA nuclear signal. We conclude that CYT01B is a novel homologous recombination inhibitor, and propose a new synthetic lethality mechanism, induced by CYT01B, mediated by DNA replication stress and replication catastrophe. Citation Format: Melinda Day, Amber Cyr, Tyler Maclay, Kevin Mills. CYT01B suppresses RAD51 focus formation and induces replication catastrophe following AID induced DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2566.

OC-0264 CDK4/CDK6 inhibition radiosensitises HPV-neg HNSCC through inhibition of homologous recombination

OC-0264 CDK4/CDK6 inhibition radiosensitises HPV-neg HNSCC through inhibition of homologous recombination

Neutrophil‐induced genomic instability impedes resolution of inflammation and wound healing

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of tissue injury associated with inflammatory bowel diseases (IBD). The pathological effects of PMNs are largely attributed to the release of soluble mediators and reactive oxygen species (ROS). We identified a new, ROS‐independent mechanism, whereby activated, tissue‐infiltrating PMNs release microparticles armed with pro‐inflammatory microRNAs (miR‐23a and miR‐155). Using IBD clinical samples, in vitro and in vivo injury models, we show that PMN‐derived miR‐23a/155 promote accumulation of double‐strand breaks (DSBs) by inducing Lamin B1‐dependent replication fork collapse, and inhibition of homologous recombination (HR) by targeting HR‐regulator RAD51. DSB accumulation in injured epithelium led to impaired colonic healing and genomic instability. Targeted inhibition of miR‐23a/155 in cultured intestinal epithelial cells and in acutely injured mucosa decreased the detrimental effects of PMNs and enhanced tissue healing responses, suggesting that this approach can be used in therapies aimed at resolution of inflammation, wound healing and potentially prevent neoplasia.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (NIH) DK101675, Digestive Health Foundation, Chicago and by the Robert H. Lurie Comprehensive Cancer Center(Eisenberg Scholar grant).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.