Repair-proficient Cells Research Articles

Machine learning prediction of prime editing efficiency across diverse chromatin contexts.

The success of prime editing depends on the prime editing guide RNA (pegRNA) design and target locus. Here, we developed machine learning models that reliably predict prime editing efficiency. PRIDICT2.0 assesses the performance of pegRNAs for all edit types up to 15 bp in length in mismatch repair-deficient and mismatch repair-proficient cell lines and in vivo in primary cells. With ePRIDICT, we further developed a model that quantifies how local chromatin environments impact prime editing rates.

Tumors cells with mismatch repair deficiency induce hyperactivation of pyroptosis resistant to cell membrane damage but are more sensitive to co-treatment of IFN-γ and TNF-α to PANoptosis

Hypermutated neoantigens in cancers with DNA mismatch repair deficiency (dMMR) are prerequisites for favorable clinical responses to immune-checkpoint blockade (ICB) therapy. However, TMB is not significantly associated with favorable prognosis from Preclinical and clinical studies. It implies that except for TMB, other mechanisms should be needed to contribute to successful cancer immunotherapy. We found that the hyperactivation of PANoptotic effective molecules in dMMR tumor cells caused cell membrane damage, induced ESCRT-mediated membrane repair, and protected tumor cells from the damage caused by Triton X-100, while DNA mismatch repair proficient (pMMR) tumor cells were sensitive to Triton X-100 mediating cell membrane damage due to the lack of ESCRT-mediated membrane repair. There was hyperactivation of GSDMD, GSDME, and p-MLKL in dMMR tumor cells. Co-treatment of IFN-γ and TNF-α induced rapid death of dMMR tumor cells by inducing PANoptosis including pyroptosis, apoptosis, and no necrosis. pMMR tumor cells had defects in the PANoptosis pathway and were resistant to co-treatment of IFN-γ and TNF-α. In conclusion, we can activate immune cells to release IFN-γ and TNF-α to overcome resistance to ICB treatment.

Genome-wide mapping of protein-DNA damage interaction by PADD-seq.

Protein-DNA damage interactions are critical for understanding the mechanism of DNA repair and damage response. However, due to the relatively random distributions of UV-induced damage and other DNA bulky adducts, it is challenging to measure the interactions between proteins and these lesions across the genome. To address this issue, we developed a new method named Protein-Associated DNA Damage Sequencing (PADD-seq) that uses Damage-seq to detect damage distribution in chromatin immunoprecipitation-enriched DNA fragments. It is possible to delineate genome-wide protein-DNA damage interactions at base resolution with this strategy. Using PADD-seq, we observed that RNA polymerase II (Pol II) was blocked by UV-induced damage on template strands, and the interaction declined within 2 h in transcription-coupled repair-proficient cells. On the other hand, Pol II was clearly restrained at damage sites in the absence of the transcription-repair coupling factor CSB during the same time course. Furthermore, we used PADD-seq to examine local changes in H3 acetylation at lysine 9 (H3K9ac) around cisplatin-induced damage, demonstrating the method's broad utility. In conclusion, this new method provides a powerful tool for monitoring the dynamics of protein-DNA damage interaction at the genomic level, and it encourages comprehensive research into DNA repair and damage response.

Preclinical Characterization of AZD5305, A Next-Generation, Highly Selective PARP1 Inhibitor and Trapper.

We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination. AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination. AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response. AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).

Abstract 2613: A modified Luria-Delbrück assay allows quantification of colorectal cancer persister cells’ mutation rate

Abstract When cancer cells are exposed to lethal doses of targeted therapies, the emergence of a subpopulation of drug-tolerant persister cells (DTPs) is often observed. We previously reported that colorectal cancer (CRC) cells exposed to targeted therapies activate an adaptive mutability stress response, involving DNA damage induction and a switch to low-fidelity DNA replication. Therefore, targeted treatment might lead to increased mutation rate in DTPs, but mutation rates of cancer cells during treatment have not been quantitatively assessed. Here, we combined biological experiments and mathematical modelling to characterize emergence and dynamics of DTPs. From this, we extrapolated parameters governing dynamics of cancer cells populations and designed a modified Luria-Delbrück assay on mammalian cells (MC-LD) to quantify mutations rates of CRC cells under standard growth conditions and during exposure to targeted therapy. We selected mismatch repair proficient CRC cell lines sensitive to different clinically used therapeutic agents, and derived clones to be used for experiments. By monitoring cell dynamics in drug-response growth assays, we found that CRC cells exposed to targeted therapy display a biphasic killing curve reaching a stable plateau, a pattern indicative of emergence of DTPs. By fitting model estimation to population growth assays, we predicted that, even if a subgroup of DTPs predated treatment, the majority of them emerged only upon exposure to targeted therapies. We also observed that DTPs slowly replicate under treatment, as shown by Carboxy fluorescein succinimidyl ester (CFSE) analysis and staining with 5-ethynyl-2’-deoxyuridine (EdU). We used these population dynamics parameters to design the MC-LD assay. CRC clones were plated in several 96-multiwell plates each, and after an expansion phase in standard culture conditions, treatment was added. After 3-4 weeks, a minority of wells showed growth of resistant colonies: based on the measured growth rates, we could predict that the resistant cells arose before treatment by spontaneous mutation. The remaining wells contained a homogenous population of DTPs. After several weeks of treatment, when pre-treatment resistant clones would have already emerged, late-emerging resistant colonies appeared in a subset of wells in which DTPs had previously been detected. Using the number of residual DTPs and resistant colonies to infer mutation rates, we found a 7- to 50-fold increase (depending on the cell line) in DTPs’ mutation rate compared to sensitive cells.In conclusion, we developed a new assay which allows quantitative comparisons of spontaneous and drug-induced mutation rates in cancer cells and showed that adaptive mutability in DTPs leads to increased mutation rates. This approach could be used to measure whether and how a wide range of environmental conditions affect DTP phenotype and mutation rates in mammalian cells. Citation Format: Alberto Sogari, Mariangela Russo, Simone Pompei, Mattia Corigliano, Giovanni Crisafulli, Andrea Bertotti, Marco Gherardi, Federica Di Nicolantonio, Marco Cosentino Lagomarsino, Alberto Bardelli. A modified Luria-Delbrück assay allows quantification of colorectal cancer persister cells’ mutation rate [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 2613.

Abstract 2597: Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a grim prognosis. Though the overall 5-year survival rates have recently improved to 10%, there is still an unmet need for better therapies. PARP inhibitors (PARPi)/platinum therapies have demonstrated clinical efficacy in BRCA1/2 mutated PDAC and has recently led to the FDA approval of PARPi (olaparib) in the maintenance setting, offering the best personalized treatment approach for such patients. Although promising, the benefits of these therapeutic strategies are currently limited to only a few patients. We have recently established that BARD1 (BRCA1- Associated-Ring-Domain-1), the main binding partner of BRCA1 in regulating homologous recombination repair is upregulated in PDAC cells exposed to PARPi/platinum compounds, by a post-transcriptional RNA binding mechanism. We therefore hypothesized that inhibition of BARD1 will sensitize cells to these agents. We found that inhibition of BARD1 (both by siRNA/shRNA and CRISPR-KO) not only sensitized the cells to olaparib or oxaliplatin but enhanced DNA damage in cells, as analyzed by comet assays and γ-H2AX immunofluorescence staining. BARD1 inhibition led to a decrease in homologous repair efficiency of cells as analyzed by the well established pDRGFP assay. To further understand BARD1 dependent downstream signaling, we performed comparative transcriptomic analysis of BARD1 silenced cells compared to scramble control. RNA seq analysis revealed modulation of several cell cycle checkpoints and DNA damage response pathways. Notably, through gene set enrichment analysis (GSEA), we found that silencing BARD1 causes de-enrichment of pathway regulating Polo-Like-Kinase-1 (PLK1, a serine/threonine kinase) activity (p-val=0.05, FDR=0.07). PLK1 is frequently upregulated in PDAC cells and promotes progression of cells through mitosis. Based on these results, we found that a PLK1 specific inhibitor (onvansertib) synergized with olaparib to reduce cell survival of homologous repair proficient (HR-P) PDAC cells. Importantly, the mechanism of drug synergy involved induction of apoptosis and DNA damage, as assessed by cleaved caspase-3 and γ-H2AX protein analysis. These results are suggestive of targeting the BARD1-PLK1 axis in order to enhance sensitivity of PARPi/platinums in HR proficient cells. Our ongoing and future studies are aimed at understanding the interaction of BARD1 and PLK1 in PDAC cells and analyze the effects of onvansertib and olaparib on tumor growth in vivo. Accomplishment of these studies will establish novel therapeutic targets and synthetic lethal combinations that have not been explored yet for deadly pancreatic cancers, ultimately benefiting patient survival in the long run. Citation Format: Carolyn Coats, Anoohya Arkala, Eric Londin, Avinoam Nevler, Jonathan R. Brody, Charles J. Yeo, Aditi Jain. Inhibition of BARD1-PLK1 axis enhances PARP inhibitor/platinum sensitivity in homologous repair proficient pancreatic ductal adenocarcinoma [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 2597.

Abstract 1593: Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer

Abstract Patients affected by colorectal cancer (CRC) with microsatellite instability (MSI), which is caused by DNA mismatch repair deficiency (MMRd), are eligible for therapies based on immune checkpoint inhibitors (CPI), while microsatellite stable (MSS) tumors are not. However, a subset of MSS CRCs contains variable fractions of MMRd cells. How the presence of MMRd cells in tumors classified as MSS impacts cancer immune surveillance is largely unknown. It is also unclear whether the pharmacological modulation of MMRd cells percentage in MMR heterogeneous tumor can occur and if this might result in the improvement of tumor immune control.To shed light on these aspects we studied isogenic mismatch repair proficient and deficient mouse tumor cells, generated by genetic inactivation of MLH1, a key component of the MMR machinery. We mixed MLH1+/+and MLH1-/-cells at different ratios, injecting the resulting heterogeneous populations in mice. In the presence of a competent immune system, the tumorigenic potential and immune surveillance of MSS/MSI (MLH1+/+/MLH1-/-) heterogeneous tumors was dependent upon the MMRd fraction. Tumor rejection was observed when at least 50% of the cells were MMRd, but tumor growth delay was also evident when as low as 20% of MMRd cells were present in the mixed population and this was paralleled by immune infiltration of the tumor. Molecular profiles of samples from MSS/MSI heterogeneous tumors that evaded immune control, showed enrichment of the MSS fraction. Treatment of MSS/MSI mixed populations with the antimetabolite 6-Thioguanine (6TG) greatly enriched the MMRd fraction and improved immune response. Overall, these results suggest that genetic and/or pharmacological modulation of the DNA mismatch repair machinery can foster immune surveillance of MMR heterogeneous tumors and modulate the cancer immune environment. Citation Format: Vito Amodio, Giovanni Germano, Benedetta Mussolin, Simona Lamba, Rosaria Chilà, Giuseppe Rospo, Caterina Marchiò, Silvia Marsoni, Gianluca Mauri, Federica Di Nicolantonio, Alberto Bardelli. Genetic and pharmacological modulation of DNA mismatch repair promotes immune surveillance in murine colorectal cancer [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 1593.

Abstract PO-131: RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer

Abstract Breast cancer (BC) is the second most diagnosed malignant disease in women, and one of the leading causes of cancer-related deaths. Triple-negative breast cancer (TNBC) is the most aggressive and difficult to treat subtype of BC because it is highly metastatic and lacks targeted therapies. African American (AA) women have a higher death rate from BC than women of other races and ethnicities. The higher incidences of TNBCs and their aggressive growth in young AA women contributing to higher death rates indicate a biological basis for this difference. Thus, it is imperative to understand the molecular mechanisms that contribute to aggressive tumor growth in AA women, identify biomarkers to select patients who will respond to existing therapies, and develop effective therapeutics to reduce this disparity. Our studies identified that multiple TNBC cells derived from AA women are inherently chemoresistant and exhibit aggressive growth behavior compared to TNBC cells derived from European American (EA) patients. Our preliminary screenings showed that the DNA repair protein, RAD51, is overexpressed in AA TNBC patients and correlates a poor prognosis relative to EA TNBC patients. Analysis of AA and EA TNBC tumor specimens indicated the epigenetic regulation of RAD51 by promoter methylations and microRNAs. Furthermore, AA women diagnosed with TNBC, have a considerably lower incidence of germline BRCA1 mutations than women of other racial or ethnic groups. This indicates most TNBC tumors in AA patients are DNA repair proficient and have intact cell cycle checkpoint mechanisms that protect them from chemotherapy-induced DNA damage and promote therapeutic resistance. Our drug screenings identified CHK1 inhibitor, Prexasertib caused DNA repair deficiency in BRCA wild-type TNBC cells by promoting proteasome-mediated degradation of BRCA1 and RAD51 proteins. Therefore, we designed a synthetic lethality-based drug combination of Prexasertib with PARP inhibitors (PARPi) in DNA repair proficient TNBC cells. Data from our preclinical evaluations show Prexasertib and Olaparib cause increased DNA strand breaks, mitotic catastrophe, and synergistic TNBC cell lethality compared to individual drug treatments. Additionally, computational analysis of TCGA data revealed a RAD51 upregulation in TNBC tumors compared to normal breast tissues and other subtypes of BC which renders as a poor prognostic marker for these patients. Remarkably, there was an interesting discrepancy in RAD51 expression levels between different racial groupings, with AA and Asian BC patients having higher levels of RAD51 expression than Caucasian BC patients. Consistent with these observations, AA and Asian TNBC patients showed decreased survival probability. Together, our data indicate that RAD51 and its epigenetic regulators could be biomarkers for aggressive TNBC and racial disparity in BC therapeutic outcomes and suggests a novel combination therapy involving Prexasertib and Olaparib may improve prognosis and reduce racial disparity in TNBC. Citation Format: Ganesh N. Acharya, Chinnadurai Mani, Upender Manne, Komaraiah Palle. RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-131.

Kinetics of DNA Repair Under Chronic Irradiation at Low and Medium Dose Rates in Repair Proficient and Repair Compromised Normal Fibroblasts.

We present time and dose dependencies for the formation of 53BP1 and γH2AX DNA damage repair foci after chronic radiation exposure at dose rates of 140, 250 and 450 mGy/day from 3 to 96 h, in human and mouse repair proficient and ATM or DNA-PK deficient repair compromised cell models. We describe the time/dose-response curves using a mathematical equation which contains a linear component for the induction of DNA damage repair foci after irradiation, and an exponential component for their resolution. We show that under conditions of chronic irradiation at low and medium dose rates, the processes of DNA double-strand breaks (DSBs) induction and repair establish an equilibrium, which in repair proficient cells manifests as a plateau-shaped dose-response where the plateau is reached within the first 24 h postirradiation, and its height is proportionate to the radiation dose rate. In contrast, in repair compromised cells, where the rate of repair may be exceeded by the DSB induction rate, DNA damage accumulates with time of exposure and total absorbed dose. In addition, we discuss the biological meaning of the observed dependencies by presenting the frequency of micronuclei formation under the same irradiation conditions as a marker of radiation-induced genomic instability. We believe that the data and analysis presented here shed light on the kinetics of DNA repair under chronic radiation and are useful for future studies in the low-to-medium dose rate range.

Abstract ND05: Discovery and first structural disclosure of AZD5305: A next generation, highly selective PARP1 inhibitor and trapper

Abstract Poly ADP-ribose polymerase (PARP) inhibitors have demonstrated clinical effectiveness in the treatment of homologous recombination deficient tumors; however, hematological and other toxicities have limited their use as a class of agents, both as monotherapy and as combination with already poorly tolerated chemotherapies. Recent literature suggests that inhibition of PARP2, a feature shared by all the current clinical PARP inhibitors, is not required for efficacy and may in fact be associated with hematological effects. These observations led to our hypothesis that a highly selective and potent PARP1 inhibitor and trapper would retain efficacy while improving therapeutic index, thus expanding clinical combinations options. A high throughput screen against PARP1 and PARP2 identified a compound series with selectivity for binding to PARP1 over PARP2. This set of compounds was refined further by screening for the ability to trap PARP1 to DNA and selectively inhibit growth of BRCA mutant cell lines. Further lead optimization efforts utilizing PARP1 enzyme structural information, lead to the discovery of to AZD5305. AZD5305 is a highly potent inhibitor of PARP1, with significant PARP1-DNA trapping activity, no PARP2-activity, nor binding activity to any other members of the PARP family. AZD5305 has excellent secondary pharmacology and physicochemical properties as well as high oral bioavailability in preclinical species. AZD5305 has shown unique properties in preclinical toxicology models. When dosed daily for 14 days in rats at clinically equivalent doses, olaparib (a PARP1/2 inhibitor), causes up to 50% reduction in hemoglobin, in line with clinically observed anemia. Under the same experimental conditions, AZD5305 had no effects on any hematological parameters measured, when dosed at predicted clinically efficacious doses. In vitro, AZD5305 inhibits growth selectively in cell lines with deficiencies in DNA repair pathways, with IC50s in the single-digit nM range, while having no or minimal growth inhibitory effects in other cells (IC50s >10μM). The difference observed between the effects on DNA repair deficient and proficient cell lines is unprecedented with PARP inhibitors and indicates the great potential for AZD5305 to demonstrate an improved therapeutic index in the clinic. Treatments of xenografts and PDX models with AZD5305 at doses ≥ 0.1 mg/kg QD, compared to olaparib 100 mg/kg QD, resulted in greater depth of tumor regressions, as well as significantly longer duration of regressions following cessation of dosing. Overall, AZD5305 is a next generation PARP inhibitor and trapper, with enhanced anticancer efficacy and an improved safety profile in preclinical models compared to other PARP inhibitors. These features make AZD5305 an exciting and unique clinical candidate, with multiple clinical options as monotherapy and in combination. AZD5305 is in Ph1 clinical trials. Citation Format: Elisabetta Leo, Jeffrey Johannes. Discovery and first structural disclosure of AZD5305: A next generation, highly selective PARP1 inhibitor and trapper [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 ND05.

Nucleoside Analogs Radiosensitize G0 Cells by Activating DNA End Resection and Alternative End-Joining

Alternative end-joining (alt-EJ) is a DNA end resection-dependent, error-prone pathway utilized by vertebrate cells to repair DNA double-strand breaks (DSBs), but its engagement is linked to chromosomal translocations and genomic instability. Here, we report that when proliferating cells are exposed to ionizing radiation, treatment with nucleoside analogs (NAs) causes strong radiosensitization by increasing engagement of alt-EJ, while at the same time suppressing homologous recombination (HR) in S- and G2phase cells. This NA-mediated pathway shift may reflect a passive compensatory engagement of alt-EJ following HR suppression that is specific for S- and G2-phase cells, and/or the direct activation of alt-EJ throughout the cell cycle. To distinguish between these possibilities, we utilize here a cell culture model that exploits genetic and cell cycle-dependent inactivation of DSB repair pathways, to exclusively study alt-EJ and its modulation by NAs in murine and human cell lines. To this end, we allow LIG4-/--deficient cells to accumulate in G1/G0 phase by transfer to serum-deprived media and obtain cells deficient in c-NHEJ owing to the genetic LIG4 knockout, deficient in HR owing to the absence of S- or G2-phase cells, and compromised in their ability to carry out alt-EJ owing to their accumulation in G0. We find that in these cells irradiation and treatment with the NA, β-arabinofuranosyladenine (araA), and to a lesser degree with other NAs, promptly activates suppressed alt-EJ that now functions at levels approximating those of c-NHEJ in wild-type cells. Results at high dose (20 Gy) generated using pulsed-field gel electrophoresis (PFGE) are corroborated by results at low dose (1 Gy) generated by scoring 53BP1 foci. Strikingly, araA treatment activates a normally undetectable DNA-end-resection at DSBs, which requires ATR activity, but proceeds unimpeded after CtIP knockdown. Treatment with araA increases the formation of chromosomal aberrations and enhances radiation-induced cell killing. The results support direct stimulation of resection by NAs and alt-EJ as a mechanism of their documented radiosensitizing potential. We propose that this stimulation also occurs in repair-proficient cells and that it occurs throughout the cell cycle. It may therefore be harnessed to develop protocols combining NAs with radiation to treat human cancer.

Radiation-induced DNA damage and repair effects on 3D genome organization

The three-dimensional structure of chromosomes plays an important role in gene expression regulation and also influences the repair of radiation-induced DNA damage. Genomic aberrations that disrupt chromosome spatial domains can lead to diseases including cancer, but how the 3D genome structure responds to DNA damage is poorly understood. Here, we investigate the impact of DNA damage response and repair on 3D genome folding using Hi-C experiments on wild type cells and ataxia telangiectasia mutated (ATM) patient cells. We irradiate fibroblasts, lymphoblasts, and ATM-deficient fibroblasts with 5 Gy X-rays and perform Hi-C at 30 minutes, 24 hours, or 5 days after irradiation. We observe that 3D genome changes after irradiation are cell type-specific, with lymphoblastoid cells generally showing more contact changes than irradiated fibroblasts. However, all tested repair-proficient cell types exhibit an increased segregation of topologically associating domains (TADs). This TAD boundary strengthening after irradiation is not observed in ATM deficient fibroblasts and may indicate the presence of a mechanism to protect 3D genome structure integrity during DNA damage repair.

Abstract A120: Adaptive mutability of colorectal cancers in response to targeted therapies

Abstract Success in eradicating human cancer with targeted therapies is limited by the emergence of secondary resistance. The prevalent view is that resistance is a fait accompli: when treatment is initiated, tumors already contain drug-resistant mutant cells. However, when cancer cells are challenged with targeted agents, the emergence of drug tolerant ‘persister’ cell population is often observed. Persisters survive exposure to targeted therapies through non-genetic, poorly understood mechanisms, and constitute a reservoir from which genetically divergent, drug-resistant derivatives eventually emerge. Analogously, when bacteria are exposed to stress, such as antibiotic treatment, persister cells can survive and, by switching from high-fidelity to low-fidelity DNA replication process, increase transiently their mutation rate (adaptive mutability), thus improving chances of survival. We used colorectal cancer (CRC) as model system to explore the hypothesis that, in addition to pre-existing drug resistant cells, resistance to targeted therapies could be fostered by a transient increase in genomic instability during treatment, leading to de novo genetic alterations. We found that, when exposed to EGFR and/or BRAF inhibition, mismatch repair proficient (MMRp) CRC cell lines exhibited a down-modulation of MMR and Homologous Recombination (HR) DNA repair genes, and a concomitant up-regulation of error-prone polymerases, resulting in reduced MMR and HR proficiency. Therapy-induced modulation of DNA repair genes was transient and returned to initial levels upon removal of the drugs, or when the cells developed permanent resistance to targeted agents. Notably, MLH1 and MSH2 MMR genes were down-regulated in patients-derived xenografts and tissue samples obtained at clinical response (minimal residual disease) compared to pre-treatment samples, confirming the clinical relevance of our findings. Activation of error-prone polymerases was associated with increased ROS production and accumulation of DNA damage marker γ-H2AX, in a time- a dose-dependent manner upon drug administration. The combination of DNA repair down-modulation and error-prone polymerases upregulation increases mutagenic ability and triggers microsatellite instability under drug-induced stress in CRC cells. Our results demonstrate that cancer cells, like unicellular organisms, evade therapeutic pressures by enhancing mutability. The notion that cancer cells exposed to targeted therapies activate a stress-induced adaptive mutability process may prompt the design of novel therapeutic strategies aimed at interfering with clonal evolution, reducing the generation of new variants during therapeutic treatment. Citation Format: MARIANGELA RUSSO, Giovanni Crisafulli, Alberto Sogari, Nicole Megan Reilly, Sabrina Arena, Simona Lamba, Alice Bartolini, Vito Amodio, Alessandro Magrì, Luca Novara, Ivana Sarotto, Zachary Nagel, Cortt Piett, Alessio Amatu, Andrea Sartore-Bianchi, Salvatore Siena, Andrea Bertotti, Livio Trusolino, Federica Di Nicolantonio, Alberto Bardelli. Adaptive mutability of colorectal cancers in response to targeted therapies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A120. doi:10.1158/1535-7163.TARG-19-A120

Poly (ADP) Ribose Glycohydrolase Can Be Effectively Targeted in Pancreatic Cancer.

Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have an average survival of less than 1 year, underscoring the importance of evaluating novel targets with matched targeted agents. We recently identified that poly (ADP) ribose glycohydrolase (PARG) is a strong candidate target due to its dependence on the pro-oncogenic mRNA stability factor HuR (ELAVL1). Here, we evaluated PARG as a target in PDAC models using both genetic silencing of PARG and established small-molecule PARG inhibitors (PARGi), PDDX-01/04. Homologous repair-deficient cells compared with homologous repair-proficient cells were more sensitive to PARGi in vitro. In vivo, silencing of PARG significantly decreased tumor growth. PARGi synergized with DNA-damaging agents (i.e., oxaliplatin and 5-fluorouracil), but not with PARPi therapy. Mechanistically, combined PARGi and oxaliplatin treatment led to persistence of detrimental PARylation, increased expression of cleaved caspase-3, and increased γH2AX foci. In summary, these data validate PARG as a relevant target in PDAC and establish current therapies that synergize with PARGi. SIGNIFICANCE: PARG is a potential target in pancreatic cancer as a single-agent anticancer therapy or in combination with current standard of care.

P12 - 8-OxoG Formation: Impact in Gene Promoters on Transcription and Mapping Studies

Studies with the regulatory VEGF promoter G-quadruplex (G4)-forming sequence have identified this G-rich region is prone to oxidation yielding 8-oxoguanine (OG),1 and we postulated that oxidation of the regulatory sequence could alter gene expression. We used synthetic chemistry to install the VEGF G4 with OG at oxidation-prone sites in the promoter of a luciferase gene that was transfected in mammalian cells to understand how gene expression changed. The modified sequences were installed in either the native coding strand or the non-native template strand of the promoter.1,2 Monitoring the time dependency in gene expression found a nearly 300% increase in expression when OG was in the coding strand and a nearly 400% decrease in expression for OG in the template strand. Studies in base excision repair (BER) proficient or deficient cell lines support BER as the process for gene activation for OG in the coding strand, and template strand OG turned gene expression off in a BER-independent process. Furthermore, we found that human DNA repair gene promoters are favorably biased with potential G4-forming sequences that can alter gene expression when OG is present. Thus, we hypothesize that certain potential G4-forming sequences are switches for gene expression during oxidative stress.3 To determine whether OG is prevalent in gene regulatory regions we developed a sequencing protocol and found enrichment of OG in gene promoters and 5'-UTRs.4

Abstract 4852: Induction of HRD by targeting G9a and MTDH in DNA repair proficient endometrial cancer cells

Abstract The homologous recombination repair (HRR) pathway is required for high-fidelity repair of double-strand DNA breaks. Cancer cells with HRR deficiency due to somatic BRCA1 or BRCA2 mutations are sensitive to platinum agents and targeted therapy with PARP inhibitors. Endometrial cancer is the most common cancer of the female reproductive system. DNA damage agents are commonly used in treatment but tumors often develop resistance. Metadherin (MTDH) has been shown to be amplified in a wide range of solid tumors as well as in leukemia and lymphoma. Overexpression of MTDH is correlated with angiogenesis, metastasis, and chemoresistance. Histone lysine N-methyl-transferase G9a catalyzes the demethylation of histone H3 at lysine residue 9 (H3K9) resulting in a repressive histone modification. The objective of this study is to validate the function of MTDH and G9a in HRR and test the potential to target MTDH or G9a to increase drug sensitivity. p21 expression was induced when G9a was depleted. DNA damage induced MTDH expression at 4 hours after 8Gy γ-irradiation in the well validated endometrial adenocarcinoma cell line Ishikawa H. However, induction of MTDH protein expression by the same radiation dosage was reduced when G9a was depleted. The G9a inhibitor UNC0642 itself can also induce the expression of MTDH after 24 hours treatment. Ishikawa parental cells and two Ishikawa sub-cell lines in which G9a was depleted by CRISPR were tested for the DNA damage biomarker γH2Ax and Rad51 foci formation at 4 hours following 8Gy irradiation. G9a depletion was shown to decrease Rad51 foci formation and to increase the formation of γH2Ax foci. Two natural products, Celastrol and Pristimerim were found to inhibit MTDH, FANCD2 and FANCI protein levels, which may be used to induce BRCA-ness in homologous recombination repair proficient cancer cells. G9a depletion results in the reduction of irradiation-induced Rad51 foci formation and an increase of irradiation-induced γH2Ax foci formation. We suggest that G9a depletion can increase cancer cell sensitivity to DNA damage agents. MTDH is induced by DNA damage and is a G9a inhibitor. MTDH depletion causes reduction of irradiation-induced Rad51 foci. This study is supported by the University of Iowa Department of Obstetrics and Gynecology Research Development Fund, NIH grant R01CA99908 and R01CA184101. Citation Format: Xiangbing Meng, Shujie Yang, Jianling Bi, Yiyang Li, Yuping Zhang, Sudartip Areecheewaku, Mary Li, Zihan Wang, Aliasger K. Salem, Kimberly K. Leslie. Induction of HRD by targeting G9a and MTDH in DNA repair proficient endometrial cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4852.

Abstract 1853: The Chk1 inhibitor, SRA737, synergizes with niraparib to kill cancer cells via multiple cell death pathways

Abstract Targeting the DNA damage response (DDR) network is a promising strategy for the development of new cancer therapies. Checkpoint kinase 1, Chk1, is a central mediator of the DDR network and the potent, selective oral Chk1 inhibitor, SRA737, is being investigated in clinical trials. A distinct class of DDR inhibitors targeting PARP (PARPi) are approved for the treatment of ovarian cancers; however, tumors with functional homologous recombination (HR) repair are less sensitive to their effects, thereby limiting the clinical potential of these agents. Several reports have described the synergistic combination of Chk1i and PARPi, although the mechanism of anti-tumor activity has not been well defined. We explored the efficacy and mechanism of cytotoxicity of SRA737 in combination with the PARPi, niraparib, in HR repair proficient tumor cell lines. In short-term cell viability assays, the combination of SRA737 and niraparib elicited greater tumor cell death than either agent alone, as early as 12 hours after exposure to drug. Combination indices determined from colony forming assays indicated synergistic activity (CI < 0.7) using clinically achievable concentrations of each agent. Quantitative immunofluorescence studies revealed activation of ATM and phosphorylation of H2AX within 4 hours of treatment, indicating induction of DNA double strand breaks and activation of DDR signaling. Concurrent changes in the phosphorylation of mTOR, AMPK and the downstream target ULK1 suggested an induction of autophagy. Consistent with this hypothesis, the single agents, as well as the combination, led to decreases in p62 and LAMP2 levels and simultaneous increases in ATG5 and Beclin1 expression, and ATG13 phosphorylation. Autophagic flux was confirmed in cells expressing an LC3-GFP-RFP reporter plasmid. Genetic knockdown of autophagy components resulted in partial rescue of cell viability, suggesting that autophagy-dependent cell death may represent a mechanism of cytotoxicity for this DDR combination. Given that rescue of cell death was incomplete following abrogation of autophagy, we additionally examined the involvement of apoptotic pathways. SRA737 and niraparib treatment resulted in reduced levels of anti-apoptotic proteins, BCL-XL and MCL-1, and increased levels of the pro-apoptotic protein, BIM. Moreover, knock down of pro-apoptotic proteins or over-expression of anti-apoptotic proteins partially rescued combination-induced lethality. Collectively these results argue that toxic autophagy, as well as the intrinsic and extrinsic apoptosis pathways, contribute to SRA737 and niraparib-induced tumor cell killing. The involvement of multiple mechanisms of cell death may decrease the likelihood of cancer cells to acquire resistance to these agents. These findings support further investigation of SRA737 in combination with PARPi, including niraparib, in HR repair proficient cancers. Citation Format: Laurence Booth, Jane Roberts, Andrew Poklepovic, Ryan J. Hansen, Bryan Strouse, Snezana Milutinovic, Christian Hassig, Paul Dent. The Chk1 inhibitor, SRA737, synergizes with niraparib to kill cancer cells via multiple cell death pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1853.

25IN - The relationship between DNA damage and immune checkpoint activation in cancer

Background: In cancer the presence of T cell immune infiltration has been recognised as a prognostic factor, however the mechanisms underpinning this response are not clearly defined. Our group and others have identified a relationship between type I Interferon immune signalling and loss of DNA repair, particularly the Fanconi Anemia pathway, in several different types of cancer. We therefore investigated the mechanism activating this immune response in the context of abnormal DNA repair. Methods: Preclinical isogenic cell line systems and human tumours were used to identify the relationship between loss of function of DNA repair genes and activation of immune signalling. A panel of Chemotherapeutic agents were studied for their effect on immune activation. Results: IHC analysis demonstrated that both intra-tumoral and stromal CD8+ and CD4+ T cell infiltration were associated with DNA repair deficient breast tumors. The CXCL10 and CCL5 cytokines as well as the immune checkpoint target PD-L1 were shown to be significantly up-regulated in DNA repair deficient tumours and in tissue culture models when compared to DNA proficent tumours. Furthermore, conditioned media from DNA repair deficient cell lines stimulated inward migration of peripheral blood mononuclear cells, when compared to media from proficient cells, indicating the presence of active cytokines. We identified constitutive activation of the innate immune pathway STING/TBK1/IRF3 specifically in DNA repair deficient tumour cells when compared to proficient cells and found that binding of the DNA sensor cGAS to cytosolic DNA fragments was required for this immune response. In addition, we identified several chemotherapeutic agents that were able to activate the immune response in DNA repair proficient cell lines through DNA damage, the development of cytoplasmic DNA fragments and the consequent activation of the STING/TBK1/IRF3 immune pathway. Conclusions: We have identified that the STING/TBK1/IRF3 immune pathway is constitutively activated by cytoplasmic DNA in DNA repair deficient cancers and may explain lymphocytic infiltration and the response to immune checkpoint based therapy. Some conventional chemotherapy agents such as doxorubicin are able to active this pathway in DNA repair proficient cancers and may represent a logical combination with immune checkpoint targeted drugs in clinical trials. Legal entity responsible for the study: Queen's University of Belfast Funding: McClay Foundation Disclosure: R. Kennedy: I receive payment as the Global VP and Medical Director for Almac Diagnostics.

MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia.

Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous deletions in MSH6, a crucial part of DNA mismatch repair, are frequently detected. Our aim was to determine whether MSH6 deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down MSH6 in mismatch repair proficient cell lines (697 and UOCB1) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold) in vitro, as well as increased resistance to 6-mercaptopurine treatment in vivo. No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070 vs. 1722 fmol/μg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure. MSH6 knockdown cells showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant MSH6 adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.

Reactive oxygen species-induced toxicity and mutants in chinese hamster ovary cells

The chinese hámster ovary (CHO) cell system is widely used system to study spontaneous and induced-toxicity and mutagenicity in mammalian cells. By comparing toxicity and mutation spectrum of identically exposed repair proficient and repair-deficient cell lines, the possible roles of DNA repair in toxicity may be defined. In this study we propose that parental CHO-K1-BH4 is more resistant to oxidative damage caused by hydrogen peroxide (H3O2), added directly to the medium or generated steadily by glucose oxidase (Go), paraquat and xanthine oxidase (XO) than its derivatives AS52 and XRS-5. We hypothesize that the various CHO cell lines differ in sensitivity to oxidative damage and that the observed toxicity may be due to accumulation of DNA breaks and impairment of the mitochondrial functions. High toxicity was observed (low clonogenic survival) for all tested compounds in all cell lines. Line XRS-5 seemed to be more sensitive to reactive oxygen species (ROS)-induced toxicity. CHO-K1-BH4 parental cell line appeared to be more resistant to the compounds studied when compared to the radiosensitive XRS-5 cell line. Glucose oxidase (4mU/ml) resulted equitoxic to parental and derivatives cell lines, 500 mM of H3O2 Increased ~4 fold the mutant frequency above the background in AS52. Xanthine oxidase (100 mU/ml) increased ~4 fold the mutagenicity frequency in XRS-5. None of the other tested compounds induced 6-TG resistance as mutagenic response. This difference may be due to different ability to handle oxidative damage associated with genetic composition and repair enzymatic responses.