DNA Damage Response Biomarkers Research Articles

Mitigation of cisplatin-induced acute kidney injury through oral administration of FAAH Inhibitor PF-04457845.

Fatty acid amide hydrolase (FAAH) serves as the primary enzyme responsible for degrading the endocannabinoid anandamide (AEA). Inhibition of FAAH, either through pharmacological means or genetic manipulation, can effectively reduce inflammation in various organs, including the brain, colon, heart, and kidneys. Infusion of a FAAH inhibitor into the kidney medulla has been shown to induce diuretic and natriuretic effects. FAAH knockout mice have shown protection against both post-ischemia reperfusion injury and cisplatin-induced acute kidney injury (AKI), although through distinct mechanisms. The present study was based on the hypothesis that pharmacological inhibition of FAAH activity could mitigate cisplatin-induced AKI, exploring potential renoprotective mechanism. Male wild type C57BL/6 were administered an oral gavage of a FAAH inhibitor (PF-04457845, 5mg/kg) or vehicle (10% PEG200+5% Tween80+normal saline) at 72, 48, 24, and 2 hours before and 24 and 48 hours after a single intraperitoneal injection of cisplatin (Cis, 25 mg/kg). Mice were euthanatized 72 hours after cisplatin treatment. Compared to vehicle-treated mice, PF-04457845-treated mice showed a decrease of cisplatin-induced plasma creatinine, blood urea nitrogen levels, kidney injury biomarkers (NGAL and KIM-1) and renal tubular damage. The renal protection from oral gavage of PF-04457845 against cisplatin-induced nephrotoxicity was associated with an enhanced AEA tone and reduced levels of DNA damage response biomarkers p53 and p21. Our work demonstrates that PF-04457845 effectively alleviates cisplatin-induced nephrotoxicity in mice, underscoring the potential of orally targeting FAAH as a novel strategy to prevent cisplatin nephrotoxicity. Significance Statement Oral administration of FAAH inhibitor, can reduce cisplatin-induced DNA damage response, tubular damages, and kidney dysfunction. Inactivation of FAAH could be a potential strategy to prevent cisplatin-induced nephrotoxicity.

Abstract A004: Radiotherapy sensitizes preclinical models to DNA damage response agents

Abstract Introduction Radiotherapy is a key treatment for various cancers, with nearly 50% of all cancer patients receiving it as part of their therapeutic plan. It can activate cytotoxic signaling pathways to promote cancer cell death but also trigger cytoprotective mechanisms that respond to cellular damage. These cytoprotective mechanisms can be inhibited by targeted anticancer agents such as DNA damage response (DDR) agents, suggesting that radiotherapy could enhance the efficacy of these drugs. This study presents data showing radiotherapy enhances the potency of DDR agents in preclinical models. Methods and experimental procedures The MDA-MB-436 breast and A549 lung cancer cell lines were utilized to establish in vitro assays and in vivo models for the study. Agents targeting DNA-PK (AZD7648) and PARP (niraparib) were used as they work on pathways crucial for the repair of radiation-induced DNA double-strand breaks. The effects on cell viability and DDR biomarkers were assessed after treatment with irradiation (IR), compound or in combination both in vitro and in vivo. Targeted X-rays were delivered using an Xstrahl CIX3 cabinet irradiator, in vivo radiotherapy was delivered to the tumor only. Data An increased sensitivity to DDR agents when combined with radiotherapy was observed in vitro in both cell lines, compared to standalone treatments. A single dose of radiotherapy significantly reduced cell viability beyond the effects of radiotherapy or compound treatment alone. The induction of biomarkers by radiotherapy as monotherapy or in combination with DDR inhibitors was assessed. Changes in biomarker expression were consistent with those observed in the in vivo xenograft models. The administration of fractionated dosing regimens for radiotherapy was well-tolerated in vivo. Combination of radiotherapy with the DDR compounds led to tumor growth delay and enhanced survival outcomes. Conclusion In summary, in vitro assays and in vivo models have been established to investigate the synergistic effects of the combination of radiotherapy and DDR agents. Radiotherapy increased sensitivity of 2 human cancer cell lines to the DDR agents in vitro, and lead to tumor growth delay in vivo. These assays can be used as preclinical tools to identify novel compounds or mechanisms that can be successfully combined with radiotherapy. Citation Format: Pablo Binder, Theoni Katopodi, Lyndsey Hanson, Charlotte R. Bell, Eimear Flanagan, Paul Farrington, Nick Moore, Emily Wright, Yin Xin Ho, Tobias Bunday, Stewart Brown, Amy Cantrell, Yael Mamane, Jon Greenall, Shannon Sharman, John Woolley, Lorraine M. Mooney, Jane Kendrew. Radiotherapy sensitizes preclinical models to DNA damage response agents [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr A004.

Abstract A151: Establishing assays to investigate combinations of fractioned radiotherapy with DNA damage response agents in vitro and in vivo to enable investigation of radiosensitization and improved anti-tumour responses

Abstract Introduction: Radiotherapy is a main treatment modality for multiple cancer types, with approximately half of all cancer patients receiving radiotherapy as part of their treatment regimen. In addition to direct cytotoxic effects on cancer cells, it is now appreciated that radiosensitization using specific drugs inhibiting DNA damage response (DDR) pathways can enhance the sensitivity of tissues or cells to radiation therapy. Establishing assays to screen for potential radiosensitizing compounds is therefore valuable to inform drug discovery research. Methods: The MDA-MB-436 human breast cancer cell line was selected for assay optimization. DDR inhibitors of DNA-dependent protein kinase (DNA-PK) (AZD7648) and Poly-(ADP-Ribose)-Polymerase (PARP) (niraparib) were used as tool radiosensitizing compounds, as these proteins play key roles in the repair of radiation-induced DNA double strand breaks. Effects on cell viability, cell cycle progression and DDR biomarkers were determined under a variety of irradiation (IR) and compound conditions. In parallel, in vivo validation experiments were performed. Results and Conclusions: Both AZD7648 and niraparib radiosensitize MDA-MB-436 cells to IR treatment. A dose of 2 Gray (Gy) reduced the observed anti-proliferative compound IC50 compared to IR treatment or compound treatment alone. Furthermore, combined treatment of 2 Gy IR with either DDR inhibitor induces S-phase cell cycle arrest and increases cell death. Analysis of DDR biomarkers confirms their induction by radiotherapy in combination with DDR inhibitors. Similar changes to DDR biomarker expression were also observed in the in vivo xenograft mouse model. In summary, in vitro and in vivo assays have been established to investigate radiosensitization through the combination of IR and DDR inhibitors. Furthermore, these assays can identify novel compounds acting via a synergistic mechanism. Citation Format: Charlotte Bell, Stewart Brown, Amy Cantrell, Paul Farrington, Miguel Garcia, Ben Hodgson, Yin Xin Ho, Allan Jordan, Theoni Katopodi, Jane Kendrew, Wen Hao Neo, Stuart Thomson, Graeme E Walker, Emily Wright. Establishing assays to investigate combinations of fractioned radiotherapy with DNA damage response agents in vitro and in vivo to enable investigation of radiosensitization and improved anti-tumour responses [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A151.

Targeted DNA Damage Boost with Loncastuximab Tesirine in Combination with PARP Inhibitors in Diffuse Large B-Cell Lymphoma

MYC-over expressing lymphomas display inherent resistance to standard chemoimmunotherapy regimens and are characterised by genomic instability and constitutive activation of the DNA damage response (DDR) pathway. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate directed against CD19, a surface antigen broadly expressed in B-cell malignancies. Upon CD19 binding, Lonca induces DNA damage by delivering a pyrrolobenzodiazepine dimer-based, DNA crosslinking warhead into target cells. Here we hypothesised that DDR inhibition could enhance DNA damage induced by Lonca in CD19-positive lymphoma cells. Since MYC-overexpressing cells mainly rely on PARP activity to attenuate endogenous MYC-induced replicative stress, we combined Lonca with Talazoparib (an FDA-approved PARP inhibitor), in a panel of 12 DLBCL cell lines (7 with MYC-rearrangement), and in vivo in a MYC/BCL-2-rearranged DLBCL PDX model. Lonca and Talazoparib showed significant antiproliferative activity in a dose and time dependent manner in most cell lines at clinically achievable concentrations. The highest sensitivity to both compounds was observed in a BRCA2 mutant cell line (DOHH2). These data are in line with the known synthetic lethality between BRCA mutations and platinum derivatives and PARP inhibitors. The addition of Talazoparib significantly enhanced the antiproliferative effects of Lonca in DLBCL cell lines irrespective of MYC/BCL2 rearrangements and TP53 genomic alterations. Of note, the highest efficacy of the combination was observed in MYC-rearranged and MYC-overexpressing cell lines. The combination of Lonca and Talazo sinergistically increased DNA damage as evaluated by comet assay, induced enhanced cell death and caused specific changes in cell cycle dynamics. Enhanced apoptosis was confirmed by Annexin/PI staining and caspase 3/7 cleavage, assessed by caspase Glo assay and Western Blot analyses. As previously shown (Fusani et al., ASH 2021), while Lonca and Talazoparib affected cell cycle inducing G2/M phase arrest, the combination specifically increased the percentage of cells arrested in the BrdU negative S phase of cell cycle. Upon combined treatment, we observed increased DDR activation with the evaluation of several DDR biomarkers such as phospho(p)-ATM, pCHK2 Thr68, pCHK1 Ser345, gH2AX. To better understand the molecular mechanism underlying this synergistic interaction, we performed RNA-Seq analysis in two representative cell lines (OCI-LY18 and SUDHL5). Interestingly while Lonca did not cause significant changes in gene expression profiling (GEP) after 24 hours of incubation, the combination resulted in specific GEP changes such as down-regulation of histone H2B and aurora kinase A (AURKA), genes mainly involved in S and G2/M transition of the cell cycle. Similar results were obtained by combining Talazoparib and different PARP inhibitors with the alkylating agent cisplatin, indicating a class effect. The synergistic interaction of Lonca and Talazoparib was further confirmed in vivo in a MYC/BCL-2 double hit PDX model. As previously shown (Fusani et al., ASH 2021), Lonca/Talazoparib combination did not result in enhanced DNA damage induction in PBMC-derived B cells from healthy donors. Importantly, PBMC-derived T cells from healthy donors did not show any sign of DNA damage accumulation upon exposure to Lonca, Talazoparib and the combination. These data provide the rationale for future therapeutic strategies based on selective induction of DNA damage in neoplastic B cells in combination with DDR inhibition in aggressive MYC-positive B cell lymphoma.

Replicative stress in gastroesophageal cancer is associated with chromosomal instability and sensitivity to DNA damage response inhibitors

Gastroesophageal adenocarcinoma (GEA) is an aggressive malignancy with chromosomal instability (CIN). To understand adaptive responses enabling DNA damage response (DDR) and CIN, we analyzed matched normal, premalignant, and malignant gastric lesions from human specimens and a carcinogen-induced mouse model, observing activation of replication stress, DDR, and p21 in neoplastic progression. In GEA cell lines, expression of DDR markers correlated with ploidy abnormalities, such as number of high-level focal amplifications and whole-genome duplication (WGD). Integrating TP53 status, ploidy abnormalities, and DDR markers into a compositive score helped predict GEA cell lines with enhanced sensitivity to Chk1/2 and Wee1 inhibition, either alone or combined with irinotecan (SN38). We demonstrate that Chk1/2 or Wee1 inhibition combined with SN38/irinotecan shows greater anti-tumor activity in human gastric cancer organoids and an invivo xenograft mouse model. These findings indicate that specific DDR biomarkers and ploidy abnormalities may predict premalignant progression and response to DDR pathway inhibitors.

ITRAQ-based proteomic analysis reveals potential osteogenesis-promoted role of ATM in strontium-incorporated titanium implant.

The present study aims to reveal the osteogenic roles played by DNA damage response biomarkers through implementing isobaric tags for relative and absolute quantitation (iTRAQ) technique. First, sandblasted large-grit double acid-etched (SLA) titanium implant and strontium-incorporated (SLA-Sr) titanium implant were used for inserting in the tibiae of rats. iTRAQ technique was used to detect protein expression changes and identify differentially expressed proteins (DEPs). In total, 19,343 peptides and 4280 proteins were screened out. Among them, 91 and 138 DEPs were identified in the SLA-Sr group after implantation for 3 and 7 days, respectively. Ataxia-telangiectasia mutated (ATM) protein up-regulated on the 3rd day showed a trend of further up-regulation on the 7th day. Moreover, functional enrichment analyses were also conducted to explore the biological function of DEPs during the initial stage of osseointegration in vivo, which revealed that the biological functions of the DEPs on the 7th day were mainly related to "mismatch repair" and "mitotic G1 DNA damage checkpoint." Analysis of the Reactome signaling pathway showed that ATM was associated with TP53's regulation and activation. Finally, DNA damage repair related genes were selected for validation at mRNA and protein expression levels. Real-time reverse transcription-polymerase chain reaction and immunohistochemistry validation results demonstrated that mRNA expression level of ATM was higher in SLA-Sr group. In conclusion, SLA-Sr titanium implant could initiate DNA damage repair by activating expression levels of ATM. This study was striving to reveal new faces of better osseointegration and shedding light on the biological function and underlying mechanisms of this important procedure.

Biomarkers of DNA Damage Response Enable Flow Cytometry-Based Diagnostic to Identify Inborn DNA Repair Defects in Primary Immunodeficiencies

DNA damage is a constant event in every cell caused by exogenous factors such as ultraviolet and ionizing radiation (UVR/IR) and intercalating drugs, or endogenous metabolic and replicative stress. Proteins of the DNA damage response (DDR) network sense DNA lesions and induce cell cycle arrest, DNA repair, and apoptosis. Genetic defects of DDR or DNA repair proteins can be associated with immunodeficiency, bone marrow failure syndromes, and cancer susceptibility. Although various diagnostic tools are available to evaluate DNA damage, their quality to identify DNA repair deficiencies differs enormously and depends on affected pathways. In this study, we investigated the DDR biomarkers γH2AX (Ser139), p-ATM (Ser1981), and p-CHK2 (Thr68) using flow cytometry on peripheral blood cells obtained from patients with combined immunodeficiencies due to non-homologous end-joining (NHEJ) defects and ataxia telangiectasia (AT) in response to low-dose IR. Significantly reduced induction of all three markers was observed in AT patients compared to controls. However, delayed downregulation of γH2AX was found in patients with NHEJ defects. In contrast to previous reports of DDR in cellular models, these biomarkers were not sensitive enough to identify ARTEMIS deficiency with sufficient reliability. In summary, DDR biomarkers are suitable for diagnosing NHEJ defects and AT, which can be useful in neonates with abnormal TREC levels (T cell receptor excision circles) identified by newborn screening. We conclude that DDR biomarkers have benefits and some limitations depending on the underlying DNA repair deficiency.

Characterisation of Ovarian Cancer Cell Line NIH-OVCAR3 and Implications of Genomic, Transcriptomic, Proteomic and Functional DNA Damage Response Biomarkers for Therapeutic Targeting.

In order to be effective models to identify biomarkers of chemotherapy response, cancer cell lines require thorough characterization. In this study, we characterised the widely used high grade serous ovarian cancer (HGSOC) cell line NIH-OVCAR3 using bioinformatics, cytotoxicity assays and molecular/functional analyses of DNA damage response (DDR) pathways in comparison to an ovarian cancer cell line panel. Bioinformatic analysis confirmed the HGSOC-like features of NIH-OVCAR3, including low mutation frequency, TP53 loss and high copy number alteration frequency similar to 201 HGSOCs analysed (TCGA). Cytotoxicity assays were performed for the standard of care chemotherapy, carboplatin, and DDR targeting drugs: rucaparib (a PARP inhibitor) and VE-821 (an ATR inhibitor). Interestingly, NIH-OVCAR3 cells showed sensitivity to carboplatin and rucaparib which was explained by functional loss of homologous recombination repair (HRR) identified by plasmid re-joining assay, despite the ability to form RAD51 foci and absence of mutations in HRR genes. NIH-OVCAR3 cells also showed high non-homologous end joining activity, which may contribute to HRR loss and along with genomic amplification in ATR and TOPBP1, could explain the resistance to VE-821. In summary, NIH-OVCAR3 cells highlight the complexity of HGSOCs and that genomic or functional characterization alone might not be enough to predict/explain chemotherapy response.

0025 Circadian Dysregulation of DNA Repair and Increased Endogenous and Exogenous Sensitivity to DNA Damage Precipitate Elevated Cancer Risk Associated with Night Shift Work

Abstract Introduction The elevated cancer risk associated with night shift work is poorly understood. To investigate whether circadian disruption may play a role, we assessed the circadian transcriptome and its association with hallmark cancer pathways, as well as sensitivity to endogenous and exogenous sources of DNA damage, after in-laboratory simulated shift work. Methods N=14 healthy humans (aged 22-34y; 10m, 4f) were exposed to a simulated night shift schedule (daytime sleep: 10:00-18:00) or a simulated day shift schedule (nighttime sleep: 22:00-06:00) for 3 days (n=7 in each condition). After the simulated shift schedule, subjects underwent a 24h constant routine protocol, during which blood was collected every 3h. Lymphocytes were extracted from the blood samples and subjected to transcriptome analysis using a NanoString multiplex assay. We evaluated 726 mRNA cancer hallmark targets (NanoString PanCancer Pathway Panel) and 17 circadian clock genes, with 18 arrhythmic internal controls. Gene expression was analyzed for circadian rhythmicity using mixed-effects cosinor analysis. Further, lymphocytes were investigated for DNA damage using an alkaline comet assay and immunofluorescence assessment of DNA damage response biomarkers BRCA1 and γH2AX. Lymphocytes collected at 07:30 and 19:30 were also exposed to ionizing radiation (2.5Gy) and DNA damage response assessments were repeated. Results Simulated night shift caused widespread disruption of circadian rhythmicity, as measured under constant routine, for core clock genes and the transcriptome of cancer hallmark pathways. The DNA repair pathway showed significant enrichment of rhythmic genes (p<0.05) after the simulated day shift schedule only. Following simulated night shift, lymphocytes showed induction of endogenous DNA damage, with extended tail in the comet assay (p<0.001), and higher percentage of lymphocytes with BRCA1 and γH2AX foci (p<0.01). Lymphocytes collected at 19:30 showed enhanced impact of ionizing radiation as indicated by increased prevalence of cells with BRCA1 and γH2AX foci (p<0.05). Conclusion Circadian dysregulation of DNA repair mechanisms and increased sensitivity to DNA damage following night shift work may increase genomic instability and precipitate elevated cancer risk in night shift workers. Support NIH grants ES022640 and CA227381, CDMRP award W81XWH-18-1-0100, and Pacific Northwest National Laboratory BRAVE investment under DOE contract DE-AC05-76RL01830.

Development of an integrated assay in human TK6 cells to permit comprehensive genotoxicity analysis in vitro

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine10-H3-positive cell population within a small window of concentrations (0.157–0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G2M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.

The challenge of matching assays to biology in DNA damage response biomarkers for response to radiotherapy in bladder cancer

For the last 40 years of investigations into radiation (RT) for bladder preservation in muscle invasive bladder cancer (MIBC), the best predictor of response has remained early response. Specifically, the ability to achieve complete transurethral resection of bladder tumor (TURBT) prior to treatment (1) and complete or near-complete (Ta/Tis) response following a 4–5 week induction phase each are strongly associated with improved outcome (2).

Refinement of high-risk endometrial cancer classification using DNA damage response biomarkers: a TransPORTEC initiative

The TransPORTEC consortium previouslclassified high-risk endometrial cancer including poor-risk histologies such as clear cells, into four molecular subtypes “POLE mutated,” “microsatellite unstable,” “TP53 mutated,” and “no specific molecular profile.” We evaluated whether DNA damage response biomarkers could further refine this high-risk tumors classification, in particular the heterogeneous “no specific molecular profile” and “TP53 mutated” subsets recently qualified as poor prognosis in high-risk endometrial cancer. DNA damage response biomarkers including proteins involved in DNA damage (δ-H2AX), homologous recombination (RAD51), regulators of error-prone Non Homologous End-Joining (DNA-pk, FANCD2), and PARP-1 were evaluated in 116 high-risk tumors by immunohistochemistry. CD8 and PD-1 expression by immunochemistry and mutation analyses were performed previously. Survival outcome were calculated using Kaplan-Meier and Log-rank test. None of the DNA damage response biomarkers alone were prognostic. However markers were informative within molecular subsets. Among the “no specific molecular profile” subset, δ-H2AX+ was significantly predictive of poor disease free survival (Hazard Ratio = 2.56; p = 0.026), and among “TP53 mutated,” a DNA-pk+/FANCD2- profile (favouring error-prone Non Homologous End-Joining) predicted worst disease free survival (Hazard Ratio = 4.95; p = 0.009) resulting in five distinct prognostic subgroups from best to worst prognosis: group1 “POLE mutated/Microsatellite unstable” > group2 “no specific molecular profile with no DNA damage” > group3 “TP53 mutated/Non Homologous End-Joining negative” > group4 “no specific molecular profile with high DNA damage” > group5 “TP53 mutated/Non Homologous End-Joining positive”; p = 0.0002). Actionable targets were also different among subsets. Group3 had significantly higher infiltration of PD-1+ immune cells (p = 0.003), segregating with group1. Group2 had frequent PI3K pathway mutations and ER positivity. While group5, with the worst prognosis, had high DNA damage and PARP-1 expression providing a rationale for PARP inhibition. Our findings have refined the TransPORTEC prognostic classification of high-risk endometrial cancer into five distinct subgroups by integrating DNA damage response biomarkers and identified molecular subtype-specific therapeutic strategies.

Intra-patient and inter-patient comparisons of DNA damage response biomarkers in Nasopharynx Cancer (NPC): analysis of NCC0901 randomised controlled trial of induction chemotherapy in locally advanced NPC

BackgroundInter-patient heterogeneity in radiation-induced DNA damage responses is proposed to reflect intrinsic variations in tumour and normal tissue radiation sensitivity, but the prediction of phenotype by a molecular biomarker is influenced by clinical confounders and assay reproducibility. Here, we characterised the intrapatient and inter-patient heterogeneity in biomarkers of DNA damage and repair and radiation-induced apoptosis.MethodsWe enrolled 85 of 172 patients with locally advanced nasopharynx cancer from a randomised controlled phase II/III trial of induction chemotherapy added to chemo-radiotherapy. G0 blood lymphocytes were harvested from these patients, and irradiated with 1, 4, and 8 Gy ex vivo. DNA damage induction (1 Gy 0.5 h) and repair (4 Gy 24 h) were assessed by duplicate γH2AX foci assays in 50–100 cells. Duplicate FLICA assays performed at 48 h post-8 Gy were employed as surrogate of radiation-induced apoptosis; %FLICA-positive cells were quantified by flow cytometry.ResultsWe observed limited intrapatient variation in γH2AX foci and %FLICA readouts; median difference of duplicate foci scores was − 0.37 (IQR = − 1.256-0.800) for 1 Gy 0.5 h and 0.09 (IQR = − 0.685-0.792) for 4 Gy 24 h; ICC of ≥0.80 was observed for duplicate %FLICA0Gy and %FLICA8Gy assays of CD4+ and CD8+ T lymphocytes. As expected, we observed wide inter-patient heterogeneity in both assays that was independent of intrapatient variation and clinical covariates, with the exception of age, which was inversely correlated with %FLICAbackground-corrected (Spearman R = − 0.406, P < 0.001 [CD4+]; R = − 0.220, P = 0.04 [CD8+]). Lastly, an exploratory case-control analysis indicates increased levels of γH2AX foci at 4 Gy 24 h in patients with severe late radiotherapy-induced xerostomia (P = 0.05).ConclusionHere, we confirmed the technical reproducibility of DNA damage response assays for clinical implementation as biomarkers of clinical radiosensitivity in nasopharynx cancer patients.

Abstract A103: ATM inhibitor AZD0156 in combination with irinotecan: the use of mathematical modeling to predict efficacious doses and schedules from preclinical data

Abstract AZD0156 is a first- and only-in-class, potent, and selective ATM inhibitor. It is being investigated in clinical ascending dose studies in combination with olaparib or irinotecan. Preclinical data on the AZD0156 irinotecan combination have been used to determine which doses and schedules should be investigated to maximize efficacy. The effects of AZD0156 and irinotecan in combination on the distribution of cell cycle phases have been investigated in vitro by flow cytometry. Cell population growth and DNA damage response biomarkers such as pATM foci, gH2AX foci, and micronuclei were described in detail using high-throughput immunofluorescence imaging. In vivo, preclinical xenograft studies have been carried out to examine the effects on pharmacodynamics and tumor growth from different doses and schedules of both agents. These in vitro and in vivo data have been integrated into a mechanistic mathematical model that supports the combination hypothesis, and allows prediction of pharmacodynamic and tumor volume responses under different doses and schedules. In particular, the effects of clinical doses and schedules can be predicted by taking into account differences between mouse and human pharmacokinetics for the co-agents. It can be challenging to identify the optimum schedule during clinical ascending dose studies, especially in combination cases with multiple variables, such as the doses of each agent, time gaps between doses, and times without dosing between cycles. Preclinical information, together with mathematical modeling, can be used to guide the prioritization of clinical experiments. Citation Format: Elaine Cadogan. ATM inhibitor AZD0156 in combination with irinotecan: the use of mathematical modeling to predict efficacious doses and schedules from preclinical data [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A103.

Elevated APOBEC3B expression drives a kataegic-like mutation signature and replication stress-related therapeutic vulnerabilities in p53-defective cells

Background:Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system.Methods:We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities.Results:Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations.Conclusions:Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.

Abnormal levels of histone methylation in the retinas of diabetic rats are reversed by minocycline treatment

In this study we quantified the alterations of retinal histone post-translational modifications (PTMs) in diabetic rats using a liquid chromatography - tandem mass spectrometry (LC-MS/MS) approach. Some diabetic rats were subsequently treated with minocycline, a tetracycline antibiotic, which has been shown to inhibit the diabetes-induced chronic inflammation in the retinas of rodents. We quantified 266 differentially modified histone peptides, including 48 out of 83 methylation marks with significantly different abundancein retinas of diabetic rats as compared to non-diabetic controls. About 67% of these marks had their relative abundance restored to non-diabetic levels after minocycline treatment. Mono- and di-methylation states of histone H4 lysine 20 (H4K20me1/me2), markers related to DNA damage response, were found to be up-regulated in the retinas of diabetic rats and restored to control levels upon minocycline treatment. DNA damage response biomarkers showed the same pattern once quantified by western blotting. Collectively, this study indicates that alteration of some histone methylation levels is associated with the development of diabetic retinopathy in rodents, and the beneficial effect of minocycline on the retinas of diabetic rodents is partially through its ability to normalize the altered histone methylation levels.

Body mass index modifies the relationship between \u03b3-H2AX, a DNA damage biomarker, and pathological complete response in triple-negative breast cancer

BackgroundBody mass index (BMI) is largely investigated as a prognostic and predictive factor in triple-negative breast cancer (TNBC). Overweight and obesity are linked to a variety of pathways regulating tumor-promoting functions, including the DNA damage response (DDR). The DDR physiologically safeguards genome integrity but, in a neoplastic background, it is aberrantly engaged and protects cancer cells from chemotherapy. We herein verified the role of BMI on a previously assessed association between DDR biomarkers and pathological complete response (pCR) in TNBC patients treated with neoadjuvant chemotherapy (NACT).MethodsIn this retrospective analysis 54 TNBC patients treated with NACT were included. The relationship between DDR biomarkers, namely phosphorylated H2A Histone Family Member X (γ-H2AX) and phosphorylated checkpoint kinase 1 (pChk1), and pCR was reconsidered in light of BMI data. The Pearson’s Chi-squared test of independence (2-tailed) and the Fisher Exact test were employed to assess the relationship between clinical-molecular variables and pCR. Uni- and multivariate logistic regression models were used to identify variables impacting pCR. Internal validation was carried out.ResultsWe observed a significant association between elevated levels of the two DDR biomarkers and pCR in patients with BMI < 25 (p = 0.009 and p = 0.022 for γ-H2AX and pChk1, respectively), but not in their heavier counterpart. Results regarding γ-H2AX were confirmed in uni- and multivariate models and, again, for leaner patients only (γ-H2AXhigh vs γ-H2AXlow: OR 10.83, 95% CI: 1.79–65.55, p = 0.009). The consistency of this finding was confirmed upon internal validation.ConclusionsThe predictive significance of γ-H2AX varies according to BMI status. Indeed, elevated levels of γ-H2AX seemed associated with lower pCR rate only in leaner patients, whereas differences in pCR rate according to γ-H2AX levels were not appreciable in heavier patients. Larger investigations are warranted concerning the potential role of BMI as effect modifier of the relationship between DDR-related biomarkers and clinical outcomes in TNBC.

Clinical and biological significance of RAD51 expression in breast cancer: a key DNA damage response protein

Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n=1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group. The results were correlated with clinicopathological and molecular parameters and patients' outcome. RAD51 protein expression level was also assayed in a panel of cell lines using reverse phase protein array (RPPA). RAD51 was expressed in the nuclei (N) and cytoplasm (C) of malignant cells. Subcellular co-localisation phenotypes of RAD51 were significantly associated with clinicopathological features and patient outcome. Cytoplasmic expression (RAD51C(+)) and lack of nuclear expression (RAD51N(-)) were associated with features of aggressive behaviour, including larger tumour size, high grade, lymph nodal metastasis, basal-like, and triple-negative phenotypes, together with aberrant expression of key DDR biomarkers including BRCA1. All BRCA1-mutated tumours had RAD51C(+)/N(-) phenotype. RPPA confirmed IHC results and showed differential expression of RAD51 in cell lines based on ER expression and BRCA1 status. RAD51N(+) and RAD51C(+) tumours were associated with longer and shorter breast cancer-specific survival (BCSS), respectively. The RAD51N(+) was an independent predictor of longer BCSS (P<0.0001). Lack of RAD51 nuclear expression is associated with poor prognostic parameters and shorter survival in invasive BC patients. The significant associations between RAD51 subcellular localisation and clinicopathological features, molecular subtype and patients' outcome suggest that the trafficking of DDR proteins between the nucleus and cytoplasm might play a role in the development and progression of BC.

Abstract 5279: Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers

Abstract Robust PD assay results are valuable for informing decisions about continued preclinical and clinical development of new agents and for identifying effective combinations of targeted agents. The National Cancer Institute's Division of Cancer Treatment and Diagnosis (DCTD) develops and validates PD assays to obtain accurate information about drug effect on intended molecular targets in first-in-human clinical trials. Our group utilizes quantitative immunofluorescence assays (qIFA) of PD biomarkers in FFPE slides prepared from pre- and post-dose tumor biopsies collected from patients on early phase clinical trials. Paired biopsies are fixed and paraffin embedded, in parallel with tissue controls, such that all tissues are sectioned onto the same slide. Stringent methods to maintain the biopsy orientation are utilized during fixation, blocking and microtomy. A large number of sections are generated to increase the likelihood of finding optimal tumor regions of interest for biomarker analyses. Flanking H&amp;E slides are utilized to assess the quality of the tumor biopsies. Whole slide scans of the H&amp;E slides are shared with a clinical pathologist who determines whether the sections are sufficient or insufficient for the intended analysis. The pathology review allows the assay operator to select the optimal range of slides to stain and quantitatively analyze for the biomarker(s) of interest. Pathology-guided regions allow the operator to focus on tumor regions of interest and avoid normal tissue and/or confounding regions compromised by handling and processing artifacts. For nuclear biomarkers such as γH2AX and related DNA damage response biomarkers the pathologist annotates areas of sufficient tumor content and viability. For biomarker changes in tissue architecture such as that seen in Epithelial Mesenchymal Transition biomarkers, including E-Cadherin, Vimentin and β-catenin, the pathology annotation denotes areas of normal tissue and necrosis to eliminate from the qIFA analysis. All other areas of sufficient tumor cellularity from the entire slide are evaluated for the EMT biomarkers. Use of the pathology-annotated whole slide image as a tool for guidance of the operator performing the quantitative evaluation of the PD biomarker helps to ensure a non-subjective analysis. Finally, steps are taken to ensure proper storage of the paraffin slides, including paraffin dipping after microtomy, which is critical for preservation of labile epitopes such as phosphorylated proteins. We will present details of these optimized methods as well as key lessons learned during the preclinical and clinical implementation of qIFA measurements of PD biomarkers for various molecular targeted agents. Funded by NCI Contract No HHSN261200800001E. Citation Format: Katherine V. Ferry-Galow, Scott M. Lawrence, Tony Navas, Hala R. Makhlouf, Donna O. Butcher, Brad A. Gouker, William H. Yutzy, Jiuping Ji, Robert Kinders, Ralph E. Parchment, Shivaani Kummar, Joseph E. Tomaszewski, James H. Doroshow. Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2015-5279

Abstract 1731: Preclinical testing demonstrates strong activity of STA-12-8666, an HSP90 inhibitor-SN-38 conjugate, in small cell lung cancer (SCLC)

Abstract Purpose/Objectives: Small cell lung cancer (SCLC) is a highly aggressive disease representing 12-13% of all lung cancers, with 5 year survival rate of only 6%. While most patients respond initially to cytotoxic chemotherapies such as irinotecan, etoposide or carboplatin, resistance rapidly emerges and response to second line agents such as topotecan is limited, and in contrast to non-small cell lung cancer (NSCLC), few targetable oncogenes occur in SCLC. In this study, we tested new compound, STA-12-8666, which binds tumor-concentrated active form of shock protein 90 (HSP90) and has cleavable linker attached to SN-38, the active metabolite of irinotecan. Cleavage of the linker within the tumor provides time-release of SN-38 at high local concentration, while significantly limiting drug exposure and toxicity in non-transformed issue. The goal for this work was to evaluate STA-12-8666 for potential use as a new second line monotherapy, or as adjuvant in the frontline setting. Materials/Methods: Three dose levels of STA-12-8666 were evaluated in comparison to irinotecan, ganetespib, carboplatin, etoposide, and chemotherapy combinations in 4 independent SCLC xenograft models, including parental and cisplatin-resistant derivative cell lines (SCLC1, SR2), and a patient-derived xenograft (PDX). STA-12-8666 was also evaluated in drug combinations. Intratumoral responses were profiled using a mass spectrometry based approach to evaluate kinase pathway activation, and results confirmed by immunohistochemistry and western blot analysis. Pharmacokinetic analysis was performed to benchmark retention of STA-12-8666 to isomolar irinotecan in lung tumors. Results: In all three models, high dose (150 mg/kg) STA-12-8666 was well tolerated. In most cases, three doses administered at weekly intervals caused complete regression of established tumors, with response durable for &amp;gt; 2 months. Those tumors that regrew were responsive to re-dosing with STA-12-8666, and were subsequently eliminated. STA-12-8666 was also effective in limiting or eliminating tumors growth that had progressed following initial treatment on standard first and second line agents for SCLC. Low dose (50 mg/kg) STA-12-8666 controlled but did not eliminate tumors: however, it strongly enhanced the action of 30 mg/kg carboplatin, resulting in tumor elimination. Pharmacokinetic and proteomic analysis confirmed STA-12-8666 concentration in tumors, and identified a signature of DNA damage response biomarkers in STA-12-8666-treated tumors that strongly contrasted with the pattern induced by irinotecan. Conclusions: Together, these results indicate that STA-12-8666 may be a promising therapy in both frontline and second line settings for SCLC and strongly support the evaluation of this compound in Phase I/II clinical trials. Note: This abstract was not presented at the meeting. Citation Format: Anna Gaponova, AS Nikonova,A Deneka,BL Egleston,S Litwin,JS Duncan,K Duncan,H Borghaei,R Mehra,DA Proia,Y Boumber, Erica Golemis. Preclinical testing demonstrates strong activity of STA-12-8666, an HSP90 inhibitor-SN-38 conjugate, in small cell lung cancer (SCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1731. doi:10.1158/1538-7445.AM2015-1731