Expression Of DNA Damage Repair Genes Research Articles

IGFBP3 regulates airway basal cell stemness and airway regeneration

Basal Cells (BCs) are multipotent progenitor cells of the upper airway and critical in airway epithelial regeneration. Impaired self-renewal and differentiation capacity of BCs is a significant cause of chronic respiratory diseases. However, mechanisms mediating these changes remain largely unexplored. We, and others, have observed attenuated growth and differentiation of BCs after repeated cell divisions in vitro. To investigate mechanisms regulating BC stemness, we performed bulk RNA-sequencing to analyze transcriptomes of early and late passage BCs maintained in culture. Our analyses reveal significant differences in the expression of genes involved in IGF- and IGFBP3-dependent pathways that are known to modulate cellular growth and ROS generation. The role of these pathways in regulating BC stemness have not been systematically investigated. We hypothesized that IGFBP3 regulates BC growth and differentiation by modulating cellular redox status and DNA damage repair. Herein, we demonstrate that passage of non-diseased human BCs over 5 passages increases IGFBP3 transcript in 3 independent growth conditions. Interestingly, continued passage decreased IGFBP3 expression and significantly altered its intracellular localization. In contrast to low passage cells where IGFBP3 is largely cytosolic, high passage cells almost exclusively express IGFBP3 in their nuclei where it is known to participate in DNA repair. Consistent with this, we observed increased DNA repair gene expression in high passage cells coupled with significant reduction in thioredoxin levels. Together, these results suggest that serial passage of BCs causes oxidative stress-mediated BC exhaustion and altered IGFBP3 expression and localization. To investigate the role of IGFBP3 in BC growth and differentiation, we knocked down IGFBP3 expression and, as hypothesized, IGFBP3 knock down of low passage cells attenuated growth, BC markers, and DNA damage repair gene expression. In addition, differentiation to secretory, ciliated and club cells at the air-liquid interphase was significantly attenuated. In conclusion, our data support an IGFBP3-dependent regulation of BC stemness by preserving growth and differentiation. This study was supported by the NIH R01HL139828 (ALR) and Cystic Fibrosis Foundation grants FIRTH17XX0 (ALR), RYAN21XX0, FIRTH21XX0 (ALR, CNM). BCs for this study were provided though the Center for Gene Therapy Cell Core funded by the NIH:NIDDK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PRMT5 Inhibitors Regulate DNA Damage Repair Pathways in Cancer Cells and Improve Response to PARP Inhibition and Chemotherapies.

Expression of PRMT5 is highly positively correlated to DNA damage repair (DDR) and DNA replication pathway genes in many types of cancer cells, including ovarian and breast cancer. In the present study, we investigated whether pharmacological inhibition of PRMT5 downregulates DDR/DNA replication pathway genes and sensitizes cancer cells to chemotherapy and PARP inhibition. Potent and selective PRMT5 inhibitors significantly downregulate expression of multiple DDR and DNA replication genes in cancer cells. Mechanistically, PRMT5 inhibition reduces the presence of PRMT5 and H4R3me2s on promoter regions of DDR genes such as BRCA1/2, RAD51 and ATM. PRMT5 inhibition also promotes global alternative splicing changes. Our data suggests that PRMT5 inhibition regulates expression of FANCA, PNKP and ATM by promoting exon skipping and intron retention. Combining C220 or PRT543 with Olaparib or chemotherapeutic agents such as cisplatin demonstrates a potent synergistic interaction in breast and ovarian cancer cells in vitro. Moreover, combination of PRT543 with Olaparib effectively inhibits the growth of patient-derived breast and ovarian cancer xenografts. Furthermore, PRT543 treatment significantly inhibits growth of Olaparib resistant tumors in vivo. These studies reveal a novel mechanism of PRMT5 inhibition and suggest beneficial combinatorial effects with other therapies, particularly in patients with tumors that are resistant to therapies dependent on DNA damage as their mechanism of action.

8-Oxoguanine DNA Glycosylase 1 Upregulation as a Risk Factor for Obesity and Colorectal Cancer.

DNA damage has been extensively studied as a potentially helpful tool in assessing and preventing cancer, having been widely associated with the deregulation of DNA damage repair (DDR) genes and with an increased risk of cancer. Adipose tissue and tumoral cells engage in a reciprocal interaction to establish an inflammatory microenvironment that enhances cancer growth by modifying epigenetic and gene expression patterns. Here, we hypothesize that 8-oxoguanine DNA glycosylase 1 (OGG1)-a DNA repair enzyme-may represent an attractive target that connects colorectal cancer (CRC) and obesity. In order to understand the mechanisms underlying the development of CRC and obesity, the expression and methylation of DDR genes were analyzed in visceral adipose tissue from CRC and healthy participants. Gene expression analysis revealed an upregulation of OGG1 expression in CRC participants (p < 0.005) and a downregulation of OGG1 in normal-weight healthy patients (p < 0.05). Interestingly, the methylation analysis showed the hypermethylation of OGG1 in CRC patients (p < 0.05). Moreover, expression patterns of OGG1 were found to be regulated by vitamin D and inflammatory genes. In general, our results showed evidence that OGG1 can regulate CRC risk through obesity and may act as a biomarker for CRC.

JNJ-64619178 radiosensitizes and suppresses fractionated ionizing radiation-induced neuroendocrine differentiation (NED) in prostate cancer.

Radiation therapy (RT) is a standard treatment regimen for locally advanced prostate cancer; however, its failure results in tumor recurrence, metastasis, and cancer-related death. The recurrence of cancer after radiotherapy is one of the major challenges in prostate cancer treatment. Despite overall cure rate of 93.3% initially, prostate cancer relapse in 20-30% patients after radiation therapy. Cancer cells acquire radioresistance upon fractionated ionizing radiation (FIR) treatment, eventually undergo neuroendocrine differentiation (NED) and transform into neuroendocrine-like cells, a mechanism involved in acquiring resistance to radiation therapy. Radiosensitizers are agents that inhibit the repair of radiation-induced DNA damage. Protein arginine methyltransferase 5 (PRMT5) gets upregulated upon ionizing radiation treatment and epigenetically activates DNA damage repair genes in prostate cancer cells. In this study, we targeted PRMT5 with JNJ-64619178 and assessed its effect on DNA damage repair gene activation, radiosensitization, and FIR-induced NED in prostate cancer. γH2AX foci analysis was performed to evaluate the DNA damage repair after radiation therapy. RT-qPCR and western blot were carried out to analyze the expression of DNA damage repair genes. Clonogenic assay was conducted to find out the surviving fraction after radiation therapy. NED was targeted with JNJ-64619178 in androgen receptor (AR) positive and negative prostate cancer cells undergoing FIR treatment. JNJ-64619178 inhibits DNA damage repair in prostate cancer cells independent of their AR status. JNJ-64619178 impairs the repair of ionizing radiation-induced damaged DNA by transcriptionally inhibiting the DNA damage repair gene expression and radiosensitizes prostate, glioblastoma and lung cancer cell line. It targets NED induced by FIR in prostate cancer cells. JNJ-64619178 can radiosensitize and suppress NED induced by FIR in prostate cancer cells and can be a potential radiosensitizer for prostate cancer treatment.

Metabolic modulation of CtBP dimeric status impacts the repression of DNA damage repair genes and the platinum sensitivity of ovarian cancer.

Platinum drug-based chemotherapy plays a dominant role in OC (ovarian cancer) treatment. The expression of DNA damage repair (DDR) genes is critical in distinguishing drug-sensitive and drug-refractory patients, as well as in the development of drug resistance in long-term treated patients. CtBP is a highly expressed oncogene in OC and was found to repress DDR genes expression in our previous study. In the present study, the formation of CtBP dimers in live cells was studied, and the functional differences between monomeric and oligomeric CtBP were explored by CHIP-seq and RNA-seq. Besides, the dynamics of CtBP dimer formation in response to the metabolic modulation were investigated by the protein fragment complementation (PCA) assays. We show that dimerized CtBP, but not the dimerization-defective mutant, binds to and represses DDR gene expression in OC cells. Treatment of the mice tumors grown from engrafted OC cells by cisplatin disclosed that high-level CtBP expression promotes the CtBP dimerization and increases the therapeutic effect of cisplatin. Moreover, the CtBP dimerization is responsive to the intracellular metabolic status as represented by the free NADH abundance. Metformin was found to increase the dimerization of CtBP and potentiate the therapeutic effect of cisplatin in a CtBP dimerization-dependent manner. Our data suggest that the CtBP dimerization status is a potential biomarker to predict platinum drug sensitivity in patients with ovarian cancer and a target of metformin to improve the therapeutic effect of platinum drugs in OC treatment.

A novel DNA damage repair-related signature for predicting prognositc and treatment response in non-small lung cancer.

DNAdamage repair (DDR) is essential for maintaining genome integrity and modulating cancer risk, progression, and therapeutic response. DDR defects are common among non-small lung cancer (NSCLC), resulting in new challenge and promise for NSCLC treatment. Thus, a thorough understanding of the molecular characteristics of DDR in NSCLC is helpful for NSCLC treatment and management. Here, we systematically analyzed the relationship between DDR alterations and NSCLC prognosis, and successfully established and validated a six-DDR gene prognostic model via LASSO Cox regression analysis based on the expression of prognostic related DDR genes, CDC25C, NEIL3, H2AFX, NBN, XRCC5, RAD1. According to this model, NSCLC patients were classified into high-risk subtype and low-risk subtype, each of which has significant differences between the two subtypes in clinical features, molecular features, immune cell components, gene mutations, DDR pathway activation status and clinical outcomes. The high-risk patients was characterized with worse prognosis, lower proportion and number of DDR mutations, unique immune profile and responsive to immunetherapy. And the low-risk patients tend to have superior survival, while being less responsive to immunotherapy and more sensitive to treatment with DNA-damaging chemotherapy drugs. Overall, this molecular classification based on DDR expression profile enables hierarchical management of patients and personalized clinical treatment, and provides potential therapeutic targets for NSCLC.

DNA damage repair gene signature model for predicting prognosis and chemotherapy outcomes in lung squamous cell carcinoma

BackgroundLung squamous cell carcinoma (LUSC) is prone to metastasis and likely to develop resistance to chemotherapeutic drugs. DNA repair has been reported to be involved in the progression and chemoresistance of LUSC. However, the relationship between LUSC patient prognosis and DNA damage repair genes is still unclear.MethodsThe clinical information of LUSC patients and tumour gene expression level data were downloaded from the TCGA database. Unsupervised clustering and Cox regression were performed to obtain molecular subtypes and prognosis-related significant genes based on a list including 150 DNA damage repair genes downloaded from the GSEA database. The coefficients determined by the multivariate Cox regression analysis and the expression level of prognosis-related DNA damage repair genes were employed to calculate the risk score, which divided LUSC patients into two groups: the high-risk group and the low-risk group. Immune viability, overall survival, and anticarcinogen sensitivity analyses of the two groups of LUSC patients were performed by Kaplan–Meier analysis with the log rank test, ssGSEA and the pRRophetic package in R software. A time-dependent ROC curve was applied to compare the survival prediction ability of the risk score, which was used to construct a survival prediction model by multivariate Cox regression. The prediction model was used to build a nomogram, the discriminative ability of which was confirmed by C-index assessment, and its calibration was validated by calibration curve analysis. Differentially expressed DNA damage repair genes in LUSC patient tissues were retrieved by the Wilcoxon test and validated by qRT–PCR and IHC.ResultLUSC patients were separated into two clusters based on molecular subtypes, of which Cluster 2 was associated with worse overall survival. A prognostic prediction model for LUSC patients was constructed and validated, and a risk score calculated based on the expression levels of ten DNA damage repair genes was employed. The clinical utility was evaluated by drug sensitivity and immune filtration analyses. Thirteen-one genes were upregulated in LUSC patient samples, and we selected the top four genes that were validated by RT–PCR and IHC.ConclusionWe established a novel prognostic model based on DNA damage repair gene expression that can be used to predict therapeutic efficacy in LUSC patients.

Inhibition of PRMT5 Disrupts Cell Cycle Progression and DNA Damage Signaling, Revealing a Potential Novel Combination Therapy for Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and remains a therapeutic challenge. There is an urgent need for novel therapeutic strategies for PDAC, and the targeting of pathways highly relevant to its pathobiology is critical. PDAC cells often possess defects in DNA damage repair (DDR) pathways that are not present in healthy cells, which can be exploited for therapeutic strategies. Protein arginine methyltransferase 5 (PRMT5) is a type II histone arginine methyltransferase involved in various cellular processes, such as cell cycle progression and DDR. In PDAC tumors, PRMT5 expression is elevated and predicts shorter overall survival and disease‐free patient survival than those with lower PRMT5 expression. Here, we examined the effect of PRMT5 inhibition on PDAC in vitro, the impact of its inhibition on cell cycle and DDR, as well as potential synergy between PRMT5 inhibition and DNA damage‐inducing ionizing radiation (IR) as a novel therapeutic strategy for PDAC. Treatment of PDAC cells withthe PRMT5 inhibitor (PRMT5i), EPZ015938, resulted in a dose‐dependent decrease of cell growth by time‐lapse live‐cell imaging and clonogenic survival assays. Flow cytometry‐based cell cycle analysis showed that PDAC cells treated with EPZ015938 had increased numbers in G2/M, suggesting that PRMT5 inhibition triggers a G2/M arrest. Using a phospho‐protein array of cell cycle control proteins, we found higher levels of WEE1, P‐S216‐CDC25C, and P‐Y15‐CDC2, supporting G2/M arrest following treatment with EPZ015938. The phospho‐protein array also revealed an increase in P‐S15‐TP53, a signal of DNA damage mediated by the DNA damage sensors, ATM/ATR, along with a decrease in ATM. We confirmed the ATM downregulation at the transcript level by quantitative RT‐PCR. Pathway‐specific qPCR array analysis displayed differential expression of DDR genes related to ATM and G2/M DNA damage checkpoint regulation, further implicating a mechanistic role for these pathways in response to PRMT5i. The downregulation of ATM combined with the presence of G2/M arrest and signals of DNA damage led us to examine the related ATR pathway. In protein lysates from PDAC cells treated with PRMT5i followed by western blot, we found increased phosphorylation of ATR at S1981 and H2A.X at S139. This suggests that PRMT5 inhibition activates DDR in PDAC cells via ATR. To harness the potential vulnerability presented by PRMT5i‐mediated G2/M arrest, we combined PRMT5i treatment with ionizing radiation (IR) exposure to find that the combination of EPZ015938 and IR displayed a greater reduction in PDAC cell growth in vitro over individual treatments. In conclusion, we find that PRMT5 is a critical player in DNA damage via modulation of the ATM/ATR pathways, and combined PRMT5i and IR have beneficial anti‐tumor effects in vitro, revealing an exciting avenue for further exploration as a potential new therapeutic strategy in PDAC.

Abstract P3-14-03: The DNA damage repair landscape in Black women with breast cancer

Abstract Black women have 42% higher mortality rate from estrogen receptor positive (ER+) breast cancer than white women. Molecular mechanisms underlying worse outcome in black women are understudied. Recently, downregulation of specific DNA damage repair (DDR) genes was shown to causally induce resistance to standard care endocrine therapy by dysregulating cell cycle regulation, and to associate with poor outcome in white women with ER+ breast cancer. However, frequency and patterns of DDR dysregulation in Black women with ER+ breast cancer and impact on survival outcomes remains untested. By assessing RNA expression of 104 DDR genes across three tumor, and two normal breast, datasets, here, we map for the first time, global patterns of DDR gene expression regulation specific to Black women, both in tumors and in the normal breast. We identify a specific subset of 8 candidate DDR genes that are dysregulated at the RNA level in tumors from Black women. Of note, a novel DDR regulation signature where genes from the homologous recombination pathway are upregulated and genes from SSBR pathways (mainly base excision repair) are coincidently downregulated is almost uniquely detectable in tumors from Black women (8% incidence relative to 1% in tumors from white women, p=0.01). Moreover, this coincident DDR signature associates with dysregulated cell cycle gene expression (p&amp;lt;0.001). In accordance, patients whose tumors demonstrate this coincident DDR signature also have significantly worse survival outcomes across datasets (hazard ratio of 9.5, p&amp;lt;0.001). Overall, these results constitute the first systematic analysis of differences in DDR gene expression regulation between Black and white women and identify a specific DDR signature associated with poor outcome in tumors from Black women. These results provide new grounds for refining biomarker profiles and improving precision medicine for underserved populations. Citation Format: Aloran Mazumder, Athena Jimenez, Rachel E Ellsworth, Sophia George, Stephen J Freedland, Matthew N Bainbridge, Svasti Haricharan. The DNA damage repair landscape in Black women with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-03.

Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9-mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

CD137 (4-1BB) costimulation of CD8+ T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

CD137 (4-1BB; TNFSR9) is an activation-induced surface receptor that through costimulation effects provide antigen-primed T cells with augmented survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being utilised for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cell is superior to that provided in-trans in terms of T cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mouse and human. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell cycle and DNA damage repair gene expression programs. Here we report that the superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which may provide costimulation either in cis or in trans.

KDM6 Demethylases Integrate DNA Repair Gene Regulation: Loss of KDM6A Sensitizes AML to PARP Inhibition and Potentiates with BCL2 Blockade

Acute myeloid leukemia (AML) is a heterogeneous, aggressive hematological malignancy with dismal prognosis where limited targeted therapies are currently available. Poly-(ADP-ribose)-polymerase (PARP) inhibition has emerged as an important therapeutic arsenal to target homologous recombination-deficient tumors. However, molecular understanding of PARP blockade in the context of epigenetic derangements and transcriptional plasticity in human elderly AML pathogenesis remains unexplored. KDM6 proteins are H3K27 demethylases that critically regulate chromatin architecture in multi-cellularity and tumorigenesis (Tran, Mol Cell Biol 2020). KDM6A escapes X-chr inactivation, and Utx-/- female mice spontaneously develop aging associated myeloid leukemia (Gozdecka, Nat Genet 2018; Sera, Blood 2021). In addition, KDM6A loss of function mutation is implicated in AML relapse (Stief, Leukemia 2020). In contrast, KDM6B primarily exerts an oncogenic function in heme-malignancies. Together, KDM6A and KDM6B play cell type-specific function in leukemia, and KDM6 proteins and associated signaling emerge as important focal point for developing molecular targeted therapy.We identify that KDM6 demethylase activity critically regulates DNA damage repair (DDR) gene expression program in AML. Transcriptome analysis indicated a significant downregulation of expression of DDR genesets in both KDM6A deficient human AML and Utx -/- pre-leukemic cells. Lentiviral shRNA screening performed in response to low-dose γ-irradiation in AML stem cells, revealed a radioprotective function of KDM6A. Expression of KDM6s is regulated by genotoxic stress in a time-dependent manner, and deficiency of JmjC catalytic function impaired DDR transcriptional activation and compromised repair potential. Mechanistically, quantitative ChIP experiments also revealed co-operation between KDM6A and SWI/SNF facilitating dynamic chromatin remodeling at TSS/promoter to induce DDR gene expression. To interrogate changes in chromatin accessibility we performed ATAC-seq analysis in KDM6 deficient AML. Motif enrichment highlighted that while KDM6A depletion led to reduced chromatin access to 140 transcription factors (TFs), only 56 TF binding sites showed increased accessibility. Overall, changes in chromatin accessibility, associated with a reduced binding of DDR regulatory TFs in KDM6 deficient AML, account for a compromised DDR function.In agreement with these findings an array of KDM6 deficient AML cells were more sensitive to PARP inhibition, and pre-clinical mice models xenotransplanted with KDM6A loss of function AML line showed an increased susceptibility to PARP blockade in vivo. FLT3-ITD positive AML with a lower KDM6A expression was more sensitive to olaparib. In addition, olaparib administration significantly reduced bone marrow engraftment of patient-derived xenografts of KDM6A-mutant primary AML. Interestingly, KDM6A expression is upregulated in venetoclax-resistant monocytic-AML compared to venet-sensitive primitive-AML. Using venet responsive isogenic lines we demonstrated that attenuation of KDM6 function increased mitochondrial activity, intracellular ROS levels, de-repressed BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6 loss resulted in transcriptional repression of BCL2A1, commonly associated with venet resistance (Zhang, Nat Cancer 2020). Corroborating these results, dual targeting of PARP with BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing primary AML apoptosis, and KDM6A loss further enhanced this synergism.In sum, our study illustrates a molecular mechanistic rationale in support for a novel combination targeted therapy for AML, and posit KDM6A as a molecular determinant for therapeutic efficacy. Intriguingly, KDM6A functions as a gatekeeper of BCL2 and BCL2A1 expression. Similar to TET2 although bi-allelic Utx loss causes evolution to myeloid neoplasms, minimal KDM6 activity is important for survival of human AML cells. KDM6s have been implicated in solid tumors, and both PARP and BCL2 inhibitors are being tested in cancer patients, underscoring a wider scope of application. To conclude, KDM6A unfolds to be a central regulator for susceptibility of AML to both PARP and BCL2 inhibition, expanding the possibility to characterize effective combination targeted therapy for AML in clinical settings. DisclosuresMinden: Astellas: Consultancy. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 1691: CD137 (4-1BB) costimulation of CD8 T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation

Abstract CD137 (4-1BB, TNFSFR9) costimulation provides antigen-primed T cells with increased survival, proliferation and effector functions as well as metabolic advantages. These immunobiological mechanisms are being harnessed for cancer immunotherapy with agonist CD137-binding and crosslinking-inducing agents that elicit CD137 intracellular signaling. In this study, side-by-side comparisons show that provision of CD137 costimulation in-cis with regard to the TCR-CD3-ligating cells is far superior to that provided in-trans in terms of T-cell activation, proliferation, survival, cytokine secretion and mitochondrial fitness in mice and humans. Cis ligation of CD137 relative to the TCR-CD3 complex results in more intense canonical and non-canonical NF-κB signaling and provides a more robust induction of cell-cycle and DNA damage repair gene expression programs. Superiority of cis versus trans CD137-costimulation is readily observed in vivo and is relevant for understanding the immunotherapeutic effects of CAR T cells and CD137 agonistic therapies currently undergoing clinical trials, which provide costimulation either in cis or in trans. Citation Format: Itziar Otano, Arantza Azpilikueta, Javier Glez-Vaz, Peter Ellmark, Sara Fritzell, Gabriela Fernandez-Hoyos, Michelle Hase Nelson, Maite Alvarez, María del Carmen Ochoa, Elixabet Bolaños, Doina Cuculescu, Patricia Jauregui, Sandra Sanchez, Iñaki Etxeberría, María E. Rodriguez-Ruiz, Miguel F. Sanmamed, Alvaro Teijeira, Pedro Berraondo, Igancio Melero. CD137 (4-1BB) costimulation of CD8 T cells is more potent when provided in cis than in trans with respect to CD3-TCR stimulation [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 1691.

Comprehensive analysis of DNA damage repair in squamous cell carcinoma subtypes

AimsDefective components resulting from DNA damage and repair mechanisms have been found to be underlying causes that affect the development and progression of different types of cancers, including squamous cell carcinoma (SCC). A more detailed classification of SCC is necessary for better application of DNA damage repair therapies. Materials and methodsWe aimed to characterize the molecular profile of SCC by developing a classification system based on DNA damage repair gene expression profiles. An integrative analysis was performed using a metadata set of 1374 SCC human samples from the UCSC Genome Browser. We then analyzed genomic alterations and mutations, and genes-TF-microRNA regulatory relationships and conducted enrichment, survival, and immune infiltration analyses. Key findingsThis study was conducted on a total of 1374 SCC patients and 402 DNA damage repair genes. Two subtypes were established using consensus clustering, with 1143 patients being of the Non DDR subtype and 231 patients being of the DDR subtype. MATH, mutation burden, and heterogeneity were significantly higher in Non-DDR subtype than in DDR subtype. Next, a total of 1081 differentially expressed genes and 21 microRNAs were identified between the two subtypes and a genes-TF-microRNA regulatory network was constructed. In addition, stromal score, immune score and ESTIMATE score were significantly lower for the Non-DDR subtype, while tumor purity was significantly lower for the DDR subtype. In addition, five pathways associated with DNA damage repair were all enriched in the DDR subtype. SignificanceOur study established two subtypes of SCC based on DNA damage repair, which may help to predict prognosis and determine the most suitable treatment for SCC patients.

Discovery of MFH290: A Potent and Highly Selective Covalent Inhibitor for Cyclin-Dependent Kinase 12/13.

Genetic depletion of cyclin-dependent kinase 12 (CDK12) or selective inhibition of an analog-sensitive CDK12 reduces DNA damage repair gene expression, but selective inhibition of endogenous CDK12 is difficult. Here, we report the development of MFH290, a novel cysteine (Cys)-directed covalent inhibitor of CDK12/13. MFH290 forms a covalent bond with Cys-1039 of CDK12, exhibits excellent kinome selectivity, inhibits the phosphorylation of serine-2 in the C-terminal domain (CTD) of RNA-polymerase II (Pol II), and reduces the expression of key DNA damage repair genes. Importantly, these effects were demonstrated to be CDK12-dependent as mutation of Cys-1039 rendered the kinase refractory to MFH290 and restored Pol II CTD phosphorylation and DNA damage repair gene expression. Consistent with its effect on DNA damage repair gene expression, MFH290 augments the antiproliferative effect of the PARP inhibitor olaparib.

Differential microRNA expression profiles associated with microsatellite status reveal possible epigenetic regulation of microsatellite instability in gastric adenocarcinoma.

BackgroundAlthough microsatellite instability (MSI) is a powerful predictive biomarker for the efficacy of immunotherapy, the mechanism of MSI in sporadic gastrointestinal cancer is not fully understood. However, epigenetics, particularly microRNAs, has been suggested as one of the main regulators that contribute to the MSI formation.MethodsWe used microRNA expression data of 386 gastric adenocarcinoma samples from The Cancer Genome Atlas (TCGA) database to identify differential microRNA expression profiles by different MSI status. We also obtained putative common target genes of the top differential microRNAs with miRanda online tools, and we analyzed these data by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment (KEGG).ResultsWe found that 56 and 67 gastric adenocarcinoma samples were positive for low and high MSI, respectively, and that a high MSI status was associated with age, sex and subregion (P=0.049, 0.014 and 0.007, respectively). In the 67 samples with a high MSI status, expression levels of 14 microRNAs were upregulated but five microRNAs were downregulated as assessed by the fold change (FC), compared with that of the 56 samples with a low MSI status (P<0.05, |FC| >2). Further analysis suggested that the expression of miR-210-3p, miR-582-3p, miR-30a-3p and miR-105-5p predicted a high MSI status (P=4.93×10−10, 5.63×10−10, 3.23×10−9 and 7.64×10−4, respectively). Regulation of the transcription pathways ranked the top of lists from both GO and KEGG analyses, and these microRNAs might regulate DNA damage-repair genes that were also associated with a high MSI status.ConclusionsMiR-30a-3p and miR-105-5p are potential biomarkers for the MSI-H gastric adenocarcinoma, possibly by altering expression of DNA damage-repair genes.

Integrative analyses identify a DNA damage repair gene signature for prognosis prediction in lower grade gliomas.

Background: The DNA damage repair (DDR) pathways play important roles for regulating cancer progression and therapeutic response. IDH mutations, well-known prognosis biomarkers for glioma, lead to hypermethylation of tumor cells and affect genes' expression. Whether IDH mutations affect glioma prognosis through influencing the expression of DDR genes remains unclear. Methods: A total of 272 DDR genes were selected for differential expression and survival analysis. The identified genes were then utilized to construct the prognosis predicting model. Results: PARPBP, PLK3, POLL and WEE1 were found differential expressed between IDH mutations carriers and wild-type carriers, and were associated with survival of low grade glioma (LGG) patients. The predicting algorithm can predicts the prognosis of LGG patients. Conclusion: IDH mutations may affect LGG prognosis through regulation of DDR pathways.

Environmentally relevant exposure to dibutyl phthalate disrupts DNA damage repair gene expression in the mouse ovary†.

Phthalates have a history of reproductive toxicity in animal models and associations with adverse reproductive outcomes in women. Human exposure to dibutyl phthalate (DBP) occurs via consumer products (7-10μg/kg/day) and medications (1-233μg/kg/day). Most DBP toxicity studies have focused on high supraphysiological exposure levels; thus, very little is known about exposures occurring at environmentally relevant levels. CD-1 female mice (80days old) were treated with tocopherol-stripped corn oil (vehicle control) or DBP dissolved in oil at environmentally relevant (10 and 100μg/kg/day) or higher (1000μg/kg/day) levels for 30days to evaluate effects on DNA damage response (DDR) pathway genes and folliculogenesis. DBP exposure caused dose-dependent effects on folliculogenesis and gene expression. Specifically, animals exposed to the high dose of DBP had more atretic follicles in their ovaries, while in those treated with environmentally relevant doses, follicle numbers were no different from vehicle-treated controls. DBP exposure significantly reduced the expression of DDR genes including those involved in homologous recombination (Atm, Brca1, Mre11a, Rad50), mismatch repair (Msh3, Msh6), and nucleotide excision repair (Xpc, Pcna) in a dose-specific manner. Interestingly, staining for the DNA damage marker, γH2AX, was similar between treatments. DBP exposure did not result in differential DNA methylation in the Brca1 promoter but significantly reduced transcript levels for the maintenance DNA methyltransferase, Dnmt1, in the ovary. Collectively, these findings show that oral exposure to environmentally relevant levels of DBP for 30days does not significantly impact folliculogenesis in adult mice but leads to aberrant ovarian expression of DDR genes.

Comprehensive Profiling of DNA Repair Defects in Breast Cancer Identifies a Novel Class of Endocrine Therapy Resistance Drivers.

Purpose: This study was undertaken to conduct a comprehensive investigation of the role of DNA damage repair (DDR) defects in poor outcome ER+ disease.Experimental Design: Expression and mutational status of DDR genes in ER+ breast tumors were correlated with proliferative response in neoadjuvant aromatase inhibitor therapy trials (discovery dataset), with outcomes in METABRIC, TCGA, and Loi datasets (validation datasets), and in patient-derived xenografts. A causal relationship between candidate DDR genes and endocrine treatment response, and the underlying mechanism, was then tested in ER+ breast cancer cell lines.Results: Correlations between loss of expression of three genes: CETN2 (P < 0.001) and ERCC1 (P = 0.01) from the nucleotide excision repair (NER) and NEIL2 (P = 0.04) from the base excision repair (BER) pathways were associated with endocrine treatment resistance in discovery dataset, and subsequently validated in independent patient cohorts. Complementary mutation analysis supported associations between mutations in NER and BER genes and reduced endocrine treatment response. A causal role for CETN2, NEIL2, and ERCC1 loss in intrinsic endocrine resistance was experimentally validated in ER+ breast cancer cell lines, and in ER+ patient-derived xenograft models. Loss of CETN2, NEIL2, or ERCC1 induced endocrine treatment resistance by dysregulating G1-S transition, and therefore, increased sensitivity to CDK4/6 inhibitors. A combined DDR signature score was developed that predicted poor outcome in multiple patient cohorts.Conclusions: This report identifies DDR defects as a new class of endocrine treatment resistance drivers and indicates new avenues for predicting efficacy of CDK4/6 inhibition in the adjuvant treatment setting. Clin Cancer Res; 24(19); 4887-99. ©2018 AACR.

93P DNA damage repair protein expression and EGFR gene status in NSCLC

Background: Epidermal growth factor receptor (EGFR) is considered to be one of the key driver genes in non-small cell lung cancer (NSCLC). This study explored the potential association between EGFR and DNA damage repair gene expression (Ku70 and Rad51), and their relationship with prognosis in patients with NSCLC. Methods: We reviewed the clinical information of 79 patients. The expression of Ku70 and Rad51 were determined via immunohistochemistry in surgically resected of lung adenocarcinoma and densitometry analysis using image-pro plus 6.0 software. The clinical prognostic value of protein expression was investigated with univariate and multivariate survival analysis. Results: Compared with EGFR mutant patients, mean integral optical density (IOD) of Rad51 and Ku70 had no statistically significant difference in the wild type. Separating patients into low and high expression group according to the median value of IOD, Spearman's test showed no significant correlation between Ku70 and Rad51 expression level and EGFR gene status, while tumor tissue with T1-T2 staging had a lower Ku70 protein expression than T3-T4 (p < 0.05) according to the IOD. No statistical significant difference was found between in mean IOD of Ku70 and Rad51 between recurrence and non-recurrence. Kaplan-Meier analysis revealed that patients with Ku70 low expression tended to have poorer DFS than high expression group (20.6 vs. 22.7 months, p > 0.05). PFS of both Ku70 and Rad51 low expression group had an improving tendency comparing to high expression group (Ku70: 9.0 vs. 6.0 months, and Rad51: 10.0 vs. 7.0 months, both p > 0.05). Multivariate analysis showed, (negative vs. positive) was an independent impact factor of DFS; gender (female vs. male), EGFR status (mutant vs. wild type) and Ku70 expression level (low vs. high) were independent impact factors of PFS. Tabled 1Logistic regression between different proteins and possible impact factorProteinVariablesSEExp(B)pKu70EGFR0.4880.6840.688T-stage0.8330.0440.044Rad51EGFR0.4780.9250.925T-stage0.7320.1640.164 Open table in a new tab Conclusions: We provide clinical evidence that Ku70 protein expression level is lower in higher T-stage tumor tissue than the lower. DNA damage repair protein expression level has not been detected to have significant correlation with EGFR gene status and disease recurrence, while tumor high expression level of Ku70 protein might have worse response to systemic therapy. However, further study is required. Legal entity responsible for the study: Thoracic Oncology Center, West China Hospital Funding: State Key Laboratory of Biotherapy and Cancer Center of Sichuan province in China Disclosure: All authors have declared no conflicts of interest.