DNA Damage Response Repair Research Articles

461 The effects of afamelanotide on UVR-induced DNA damage, acute inflammatory and DNA repair responses in human skin

461 The effects of afamelanotide on UVR-induced DNA damage, acute inflammatory and DNA repair responses in human skin

IMP2 drives chemoresistance by repressing cisplatin-induced apoptosis and ferroptosis via activation of IPO4 and SLC7A11 under hypoxia in bladder cancer

BackgroundCisplatin resistance is the leading cause of mortality in muscle-invasive bladder cancer (MIBC) cases. Previous evidence suggests that abnormal epitranscriptome modifications are associated with reduced chemotherapy responses. However, the exact underlying mechanism remains largely unknown.MethodsInsulin-like growth factor-2 mRNA-binding protein 2 (IMP2) was identified by clustered regularly interspaced short palindromic repeats (CRISPR) data screening, single-cell RNA-sequencing and sample analysis. To evaluate the regulatory role of IMP2, functional studies were conducted both in vitro and in vivo. To elucidate the underlying mechanisms, various techniques including immunofluorescence, fluorescent in situ hybridization, RNA pull-down, coimmunoprecipitation, and RNA immunoprecipitation were used.ResultsOur study revealed that IMP2 was overexpressed in chemoresistant MIBC and lung metastasis tissues. IMP2 inhibition markedly enhanced the sensitivity of BC cells to cisplatin both in vitro and in vivo. Mechanistically, IMP2 enhanced the mRNA stability of IPO4 and SLC7A11 in a m6A-dependent manner, augmenting the nuclear translocation of C/EBPδ to activate PRKDC-mediated DNA damage repair in response to cisplatin. Moreover, IMP2 upregulated SLC7A11 levels and suppressed cisplatin-induced ferroptosis. Combining ferroptosis and apoptosis inhibitors completely reversed cisplatin resistance caused by IMP2 overexpression. LINC00941, which was induced by HIF-1α-mediated transcriptional activation, specifically bound IMP2 and protects it from degradation.ConclusionsThis work demonstrated a novel mechanism involving the IMP2-IPO4/SLC7A11 pathway as a promising treatment target for cisplatin-resistant bladder cancer.

The aryl hydrocarbon receptor in environmentally induced cervical cancer: A Narrative review

The aryl hydrocarbon receptor (AhR) plays a crucial role in cellular responses to various environmental pollutants, including several known carcinogens. As a ligand-activated transcription factor, AhR activation modulates the expression of genes involved in critical cellular processes, including detoxification pathways, cell proliferation and differentiation, and immune system regulation. The AhR exhibits pleiotropic effects under normal physiological conditions, contributing to the development and function of various organ systems. AhR activity is important in angiogenesis, cardiomyocyte differentiation, oocyte maturation, oculomotor nerve formation, and hematopoietic stem cell maintenance. Additionally, AhR plays a role in regulating immune cell differentiation and function, maintaining the integrity of the intestinal epithelium and its associated immune system, and mediating UVB-induced DNA damage repair responses in the skin. It acts as a critical environmental sensor, mediating cellular responses to various exogenous ligands. Importantly, activation or inhibition of AhR affects distinct signaling pathways depending on the specific ligand and cellular context. Ligands for the AhR are divided into exogenous or endogenous and have agonistic or antagonistic activity. Recently, the AhR role was determined in cancer development. It can exert both tumor-promoting and tumor-suppressive effects depending on factors such as the specific ligand, cell type, and tissue microenvironment. Emerging evidence suggests that AhR may represent a promising target for immunotherapy and serve as a potential biomarker for cervical cancer. AhR interacts with apoptotic pathway, immune checkpoint system, steroid hormones, and immune cell regulation process in cervical cancer. Despite its potential significance, the precise role of AhR in cervical cancer development and progression is still unknown. In this review, we describe significant roles of AhR in gynecological cancers; e.g., in cervical cancer.

The eEF2 kinase coordinates the DNA damage response to cisplatin by supporting p53 activation

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) is a stress-responsive hub that inhibits the translation elongation factor eEF2, and consequently mRNA translation elongation, in response to hypoxia and nutrient deprivation. EEF2K is also involved in the response to DNA damage but its role in response to DNA crosslinks, as induced by cisplatin, is not known. Here we found that eEF2K is critical to mediate the cellular response to cisplatin. We uncovered that eEF2K deficient cells are more resistant to cisplatin treatment. Mechanistically, eEF2K deficiency blunts the activation of the DNA damage response associated ATM and ATR pathways, in turn preventing p53 activation and therefore compromising induction of cisplatin-induced apoptosis. We also report that loss of eEF2K delays the resolution of DNA damage triggered by cisplatin, suggesting that eEF2K contributes to DNA damage repair in response to cisplatin. In support of this, our data shows that eEF2K promotes the expression of the DNA repair protein ERCC1, critical for the repair of cisplatin-caused DNA damage. Finally, using Caenorhabditis elegans as an in vivo model, we find that deletion of efk-1, the worm eEF2K ortholog, mitigates the induction of germ cell death in response to cisplatin. Together, our data highlight that eEF2K represents an evolutionary conserved mediator of the DNA damage response to cisplatin which promotes p53 activation to induce cell death, or alternatively facilitates DNA repair, depending on the extent of DNA damage.

5-aminolevulinic acid photodynamic therapy protects against UVB-induced skin photoaging: A DNA-repairing mechanism involving the BER signalling pathway.

Low-dose 5-aminolevulinic acid photodynamic therapy (ALA-PDT) has been used to cope with skin photoaging, and is thought to involve DNA damage repair responses. However, it is still unknown how low-dose ALA-PDT regulates DNA damage repair to curb skin photoaging. We established a photoaging model using human dermal fibroblasts (HDFs) and rat skin. RNA-sequencing (RNA-seq) analysis was conducted to identify differentially expressed genes (DEGs) in HDFs before and after low-dose ALA-PDT treatment, followed by bioinformatics analysis. Senescence-associated β-galactosidase (SA-β-gal) staining was employed to assess skin aging-related manifestations and Western blotting to evaluate the expression of associated proteins. A comet assay was used to detect cellular DNA damage, while immunofluorescence to examine the expression of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in cells and skin tissues. In both invivo and invitro models, low-dose ALA-PDT alleviated the manifestations of ultraviolet B (UVB)-induced skin photoaging. Low-dose ALA-PDT significantly reduced DNA damage in photoaged HDFs. Furthermore, low-dose ALA-PDT accelerated the clearance of the photoproduct 8-oxo-dG in photoaged HDFs and superficial dermis of photoaged rat skin. RNA-seq analysis suggested that low-dose ALA-PDT upregulated the expression of key genes in the base excision repair (BER) pathway. Further functional validation showed that inhibition on BER expression by using UPF1069 significantly suppressed SA-β-gal activity, G2/M phase ratio, expression of aging-associated proteins P16, P21, P53, and MUTYH proteins, as well as clearance of the photoproduct 8-oxo-dG in photoaged HDFs. Low-dose ALA-PDT exerts anti-photoaging effects by activating the BER signalling pathway.

KDM7A-DT induces genotoxic stress, tumorigenesis, and progression of p53 missense mutation-associated invasive breast cancer.

Stress-induced promoter-associated and antisense lncRNAs (si-paancRNAs) originate from a reservoir of oxidative stress (OS)-specific promoters via RNAPII pausing-mediated divergent antisense transcription. Several studies have shown that the KDM7A divergent transcript gene (KDM7A-DT), which encodes a si-paancRNA, is overexpressed in some cancer types. However, the mechanisms of this overexpression and its corresponding roles in oncogenesis and cancer progression are poorly understood. We found that KDM7A-DT expression is correlated with highly aggressive cancer types and specific inherently determined subtypes (such as ductal invasive breast carcinoma (BRCA) basal subtype). Its regulation is determined by missense TP53 mutations in a subtype-specific context. KDM7A-DT transcribes several intermediate-sized ncRNAs and a full-length transcript, exhibiting distinct expression and localization patterns. Overexpression of KDM7A-DT upregulates TP53 protein expression and H2AX phosphorylation in nonmalignant fibroblasts, while in semi-transformed fibroblasts, OS superinduces KDM7A-DT expression in a TP53-dependent manner. KDM7A-DT knockdown and gene expression profiling in TP53-missense mutated luminal A BRCA variant, where it is abundantly expressed, indicate its significant role in cancer pathways. Endogenous over-expression of KDM7A-DT inhibits DNA damage response/repair (DDR/R) via the TP53BP1-mediated pathway, reducing apoptosis and promoting G2/M checkpoint arrest. Higher KDM7A-DT expression in BRCA is associated with KDM7A-DT locus gain/amplification, higher histologic grade, aneuploidy, hypoxia, immune modulation scores, and activation of the c-myc pathway. Higher KDM7A-DT expression is associated with relatively poor survival outcomes in patients with luminal A or Basal subtypes. In contrast, it is associated with favorable outcomes in patients with HER2+ER- or luminal B subtypes. KDM7A-DT levels are coregulated with critical transcripts and proteins aberrantly expressed in BRCA, including those involved in DNA repair via non-homologous end joining and epithelial-to-mesenchymal transition pathway. In summary, KDM7A-DT and its si-lncRNA exhibit several intrinsic biological and clinical characteristics that suggest important roles in invasive BRCA and its subtypes. KDM7A-DT-defined mRNA and protein subnetworks offer resources for identifying clinically relevant RNA-based signatures and prospective targets for therapeutic intervention.

Insights into the Dissociation Process and Binding Pattern of the BRCT7/8-PHF8 Complex.

DNA topoisomerase 2-binding protein 1 (Topbp1) plays a crucial role in activating the ataxia-telangiectasia mutated and rad3-related (ATR) complex to initiate DNA damage repair responses. For this process to occur, it is necessary for PHF8 to dissociate from Topbp1. Topbp1 binds to the acidic patch sequence (APS) of PHF8 through its C-terminal BRCT7/8 domain, and disrupting this interaction could be a promising strategy for cancer treatment. To investigate the dissociation process and binding pattern of BRCT7/8-PHF8, we employed enhanced sampling techniques, such as steered molecular dynamics (SMD) simulations and accelerated molecular dynamics (aMD) simulations, along with self-organizing maps (SOM) and time-resolved force distribution analysis (TRFDA) methodologies. Our results demonstrate that the dissociation of PHF8 from BRCT7/8 starts from the N-terminus, leading to the unfolding of the N-terminal helix. Additionally, we identified critical residues that play a pivotal role in this dissociation process. These findings provide valuable insights into the disassociation of PHF8 from BRCT7/8, which could potentially guide the development of novel drugs targeting Topbp1 for cancer therapy.

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge. The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

Abstract 689: BUB1 interferes with the repair of radiation-induced DNA damage to radiosensitize triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is associated with locoregional recurrence and low survival rates. Novel molecular targets are expected to improve the efficacy of radiation therapy (RT) in TNBC. BUB1, a mitotic checkpoint Serine/Threonine-protein kinase is known to impact chromosomal segregation during mitosis. However, its precise role in DNA damage repair in response to RT remains largely unknown. We demonstrated that BUB1 is overexpressed in BC (vs. normal breast) and considerably raised in TNBC with poor survival outcomes using differential gene expression analysis. In multiple TNBC cell lines, cell proliferation assays revealed that pharmacological (BAY1816032) or genomic (CRISPR) ablation of BUB1 was cytotoxic. Using clonogenic survival assays, we observed that BUB1 ablation radiosensitized TNBC cell lines (SUM159, MDA-MB-231, MDA-MB-468, BT-549) while its ablation did not radiosensitized breast epithelial cells (MCF10A). Furthermore, by depleting endogenous BUB1 (siRNAs and CRISPR) and reintroducing wild-type (WT) or kinase-dead (KD) BUB1, we demonstrated that inhibition of BUB1 kinase function is responsible for the radiosensitization phenotype. BUB1 ablation increased tumor doubling time in SUM159 xenografts when combined with radiation. Ki67 levels were also reduced in these tumors, confirming the efficacy of the combined treatment. BUB1 ablation led to persistent radiation-induced DNA DSB (gH2AX foci) indicating decreased DNA-DSB repair. BLRR assays confirmed that BUB1 impacts the non-homologous end joining (NHEJ) pathway. Biochemical analyses showed that BUB1 ablation stabilized total- and phospho- DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1i and DNAPKi prevented stabilization. To evaluate the recruitment of DDR proteins to the chromatin following RT, soluble nuclear and chromatin-enriched fractionations were performed which illustrated an increase in recruitment of phospho- and total-DNAPK, KAP1, and ATM to chromatin indicating that BUB1 is important in the activation and recruitment of critical NHEJ proteins to DSBs. In addition, BUB1 staining of TNBC TMAs demonstrated significant correlation between BUB1 protein expression with tumor grade and stage in TNBC patients. Our findings demonstrate that ablation of BUB1 sensitizes TNBC cell lines and xenograft to RT and BUB1 mediated radiosensitization may occur through NHEJ. Our results suggest that BUB1 may serve as a novel molecular target for radiosensitization in women with TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Pin Li, Stephen L. Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. BUB1 interferes with the repair of radiation-induced DNA damage to radiosensitize triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 689.

Abstract A007: Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair

Abstract Ser/Thr kinase BUB1 is known to regulate chromosomal segregation during mitosis. However, its exact role in DNA damage repair in response to radiotherapy remained elusive. Here, we demonstrate that inhibition of BUB1 kinase activity (BAY1816032) or genomic depletion (CRISPR) lead to radiation sensitization in triple negative breast cancer and lung cancer cell lines and in TNBC tumor xenograft models. Mechanistic studies demonstrated reduced DNA-DSB repair (γ-H2AX foci assay), and effect on the NHEJ pathway by the bioluminescent DNA repair reporter (BLRR). Gene expression analysis show that BUB1 expression correlates tightly with key members of the NHEJ machinery including H2AX, ATM, 53BP1, DNAPKcs, Ku70, Ku80, Lig4 among others. Cycloheximide-chase experiments demonstrate that BUB1 ablation (pharmacological or genomic) stabilizes DNAPKcs after irradiation. The half-lives (t1/2) of total-DNAPK, phospho-DNAPK could not be estimated in BUB1 ablated cells post radiotherapy. Conversely, we observed BUB1 stabilization following radiotherapy alone while pre-treatment with BUB1i or DNAPKi caused robust degradation of BUB1 (significantly shorter t1/2). Nuclear and chromatin fractionation assays revealed an increase in the phospho- and total- DNAPK, ATM, and KAP1 in nuclear fractions and in chromatin enriched fractions in BUB1 inhibited or depleted cells. There was no significant change in the levels of Ku70 and Ku80 in the nuclear fractions or in the chromatin enriched fractions. This data indicates that BUB1 may be involved in the activation and recruitment of key NHEJ proteins (ATM, DNAPKcs) to DSBs. Further experiments are underway to determine how BUB1 stabilizes DNAPKcs and regulate chromatin loading of key NHEJ factors following radiation. Additionally, we demonstrate that BUB1 mediate apoptotic cell death of cancer cells post-radiation. Our findings suggest that BUB1 may serve as a novel molecular target for radiosensitization in various solid cancers including TNBC. Citation Format: Sushmitha Sriramulu, Shivani Thoidingjam, Stephen Brown, Farzan Siddiqui, Benjamin Movsas, Michael Green, Corey Speers, Shyam Nyati. Ser/Thr kinase BUB1 stabilizes DNAPKcs in response to radiation induced DNA double strand break repair [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A007.

Abstract A028: Alcohol induces DNA damage and genomic instability in MCF-7 breast cancer cells and primary mammary tissues

Abstract Alcohol consumption has been associated with increased breast cancer risk. Previous studies indicated that alcohol-induced DNA damage plays a central role in carcinogenesis. However, the underlying molecular mechanisms remain unclear. This study aimed to examine the effect of alcohol exposure on key DNA damage/repair pathways in both in vitro and in vivo settings. When MCF-7 breast cancer cells were exposed to 0.1 ~ 0.8% alcohol, the treatment induced concentration-dependent DNA damage, as indicated by the increased 8-OHdG (8-hydroxy 2 deoxyguanosine) production and p-H2AX foci formation. Signal transduction analysis showed that alcohol induced DNA damage signals, including p53, p21, E2F-1 and CHK1, and the markers in DNA repair pathways, including XLF, Rad50 and DNA-PK. p53 knockdown in MCF-7 cells resulted in enhanced DNA damage and DNA repair responses, which underscores the role of p53 in alcohol-induced cellular regulation. We also demonstrated that alcohol induces sister chromatid exchanges (SCEs) in MCF-7 cells, and ALDH2 knockout enhanced alcohol induced SCEs, which suggests that alcohol-induced acetaldehyde (AA) is a key mediator in this process. Moreover, we examined the DNA damage markers in mammary tissues of FVB/N mice exposed to alcohol and showed that alcohol exposure in vivo induced similar patterns of DNA damage and repair responses. Taken together, our data highlights the contribution of alcohol-induced DNA damage in cellular injury and the consequent tumorigenesis, which provide fundamental support for further studies on alcohol-induced DNA damage and carcinogenesis. Citation Format: Xiaohe Yang. Alcohol induces DNA damage and genomic instability in MCF-7 breast cancer cells and primary mammary tissues [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A028.

Suppression of NBS1 Upregulates CyclinB to Induce Olaparib Sensitivity in Ovarian Cancer.

Background: Deficiencies in DNA damage repair responses promote chemotherapy sensitivity of tumor cells. The Nibrin homolog encoding gene Nijmegen Breakage Syndrome 1 (NBS1) is a crucial component of the MRE11-RAD50-NBN complex (MRN complex) and is involved in the response to DNA double-strand breaks (DSBs) repair that has emerged as an attractive strategy to overcome tumor drug resistance, but the functional relationship between NBS1 regulated DNA damage repair and cell cycle checkpoints has not been fully elucidated. Methods: In this study, lentivirus-mediated RNAi was used to construct NBS1-downregulated cells. Flow cytometry, qPCR, and immunohistochemistry were used to explore the regulatory relationship between NBS1 and CyclinB in vivo and in vitro. Results: Our findings suggest that NBS1 deficiency leads to defective homologous recombination repair. Inhibition of NBS1 expression activates CHK1 and CyclinB signaling pathways leading to cell cycle arrest and sensitizes ovarian cancer cells to Olaparib treatment in vitro and in vivo. NBS1-deficient ovarian cancer cells tend to maintain sensitivity to chemotherapeutic drugs through activation of cell cycle checkpoints. Conclusions: NBS1 may be a potential therapeutic target for epithelial ovarian cancer as it plays a role in the regulation of the DNA damage response and cell cycle checkpoints. Suppression of NBS1 upregulates CyclinB to induce Olaparib sensitivity in ovarian cancer.

Preclinical Studies Support the Clinical Development of LP-284 in Relapsed and Refractory B-Cell Lymphomas

Genomic instability, a hallmark of many cancers, often results from corrupted DNA damage response/repair (DDR) pathways. Targeting DDR deficiencies in cancer therapy has been successful in solid tumors with dysfunctional homologous recombination repair (HR). However, while less recognized, lymphomas also carry DDR defects such as HR deficiency (HRD) and can account for approximately 18% of all lymphomas. LP-284 is a fully synthetic DNA-damaging agent that induces double-stranded DNA breaks and displays elevated potency in cancer cells with HRD or transcription-coupled nucleotide excision repair defects, including multiple advanced B-cell lymphomas. In preclinical pharmacology studies, the mantle cell lymphoma (MCL) xenograft tumor mouse model derived from JeKo-1, which has mutated TP53 and checkpoint kinase 2 (CHEK2), an HRD score of 46, and was refractory to ibrutinib and bortezomib, showed near complete response after LP-284 treatment. It is noteworthy that 40~50% of MCL patients may be HRD due to the presence of mutations in the ataxia-telangiectasia mutated (ATM) gene. HRD is also likely to be prevalent in high-grade lymphomas, as IGH/MYC translocation or BCL2 expression has been reported to be associated with HR inhibition. Studies of LP-284 in the MYC/BCL2 dual-translocated OCILY1 tumor xenograft model have shown a robust efficacy profile with an average of 99% tumor growth inhibition. Key pharmacokinetic studies demonstrated that LP-284 is neither a substrate nor modulator of major CYP-450 enzymes, multi-drug resistance transporters, renal drug transporters, or hepatic drug transporters. Safety pharmacology studies indicated that LP-284 does not inhibit hERG potassium channel and has a low potential to affect cardiovascular functions. Toxicology repeat dose studies of LP-284 in Sprague Dawley rats and Beagle dogs, using intravenous infusion on Days 1, 8, and 15 in a 28-day cycle, were generally well tolerated with notable adverse effects in Beagle dogs consisting mainly of reversible hematologic toxicity and testicular toxicity. The highest non-severely toxic dose (HNSTD) in dogs was considered to be 0.4 mg/kg/dose. Overall, based on its impressive preclinical anti-tumor efficacy profile, low drug-drug interaction liability, and findings from toxicological studies, a Phase 1 study is currently planned with LP-284 to assess its safety, tolerability, pharmacokinetics, and clinical activity in patients with relapsed or refractory B-cell lymphoma. Clinical development will be informed by an emphasis on patient selection strategy based on DDR/HR biomarkers.

THU552 Understanding Radiation Therapy Sensitivity In Invasive Lobular Carcinoma Of The Breast

Abstract Disclosure: M.R. Boysen: None. M.J. Sikora: None. M. Shackleford: None. J. Sottnik: None. Invasive lobular carcinoma of the breast (ILC) affects ∼40,000 US women annually and is a top-ten most common cancer affecting women. Despite most ILC having biomarkers of “low risk” disease (e.g. ∼95% are estrogen receptor α /ER-positive), ILC is associated with poorer long-term outcomes and anti estrogen resistance. ILC are also notoriously non-responsive to chemotherapy, critically limiting treatment options for advanced or metastatic ILC. However, ILC are highly responsive to radiation therapy (XRT), which suppresses ILC recurrence and improves survival. We hypothesize that this sensitivity may be due to a dysfunction in DNA damage repair unique to ILC cells. We examined formation and resolution of DNA damage repair foci (namely RAD51, γH2AX and 53BP1 foci) by immunofluorescence with ILC and IDC cell lines treated with ionizing radiation +/- anti-estrogen therapy as surrogates for the initiation and resolution of DNA repair. We also assessed cell growth via 7-day growth assays with ILC and IDC cell lines treated with ionizing radiation. Imaging with immunofluorescence suggested a deficiency in γH2AX foci formation in ILC cells compared to IDC cells following treatment with XRT, despite robust formation of 53BP1 foci. This suggests that a deficiency in performing DNA damage repair in response to XRT is likely present in ILC cells. Additionally, our growth assays demonstrated an inability in ILC cells to recover from ionizing radiation compared to IDC cells. Taken together, the potential dysfunction in γH2AX foci formation in ILC cells suggests that ILC cells may not form the appropriate DNA repair structures to initiate the DNA damage response, suggesting that a form of DNA repair deficiency underlies radiation sensitivity in ILC cells. Presentation: Thursday, June 15, 2023

Inhibition of ATM-directed antiviral responses by HIV-1 Vif.

Emerging evidence indicates that HIV-1 hijacks host DNA damage repair (DDR) pathways to facilitate multiple facets of virus replication. Canonically, HIV-1 engages proviral DDR responses through the accessory protein Vpr, which induces constitutive activation of DDR kinases ATM and ATR. However, in response to prolonged DDR signaling, ATM directly induces pro-inflammatory NF-κB signaling and activates multiple members of the TRIM family of antiviral restriction factors, several of which have been previously implicated in antagonizing retroviral and lentiviral replication. Here, we demonstrate that the HIV-1 accessory protein Vif blocks ATM-directed DNA repair processes, activation of NF-κB signaling responses, and TRIM protein phosphorylation. Vif function in ATM antagonism occurs in clinical isolates and in common HIV-1 Group M subtypes/clades circulating globally. Pharmacologic and functional studies combine to suggest that Vif blocks Vpr-directed activation of ATM but not ATR, signifying that HIV-1 utilizes discrete strategies to fine-tune DDR responses that promote virus replication while simultaneously inhibiting immune activation.

ARTC1-mediated VAPB ADP-ribosylation regulates calcium homeostasis.

Mono-ADP-ribosylation (MARylation) is a post-translational modification that regulates a variety of biological processes, including DNA damage repair, cell proliferation, metabolism, and stress and immune responses. In mammals, MARylation is mainly catalyzed by ADP-ribosyltransferases (ARTs), which consist of two groups: ART cholera toxin-like (ARTCs) and ART diphtheria toxin-like (ARTDs, also known as PARPs). The human ARTC (hARTC) family is composed of four members: two active mono-ADP-ARTs (hARTC1 and hARTC5) and two enzymatically inactive enzymes (hARTC3 and hARTC4). In this study, we systematically examined the homology, expression, and localization pattern of the hARTC family, with a particular focus on hARTC1. Our results showed that hARTC3 interacted with hARTC1 and promoted the enzymatic activity of hARTC1 by stabilizing hARTC1. We also identified vesicle-associated membrane protein-associated protein B (VAPB) as a new target of hARTC1 and pinpointed Arg50 of VAPB as the ADP-ribosylation site. Furthermore, we demonstrated that knockdown of hARTC1 impaired intracellular calcium homeostasis, highlighting the functional importance of hARTC1-mediated VAPB Arg50 ADP-ribosylation in regulating calcium homeostasis. In summary, our study identified a new target of hARTC1 in the endoplasmic reticulum and suggested that ARTC1 plays a role in regulating calcium signaling.

Simultaneous Delivery of Dual Inhibitors of DNA Damage Repair Sensitizes Pancreatic Cancer Response to Irreversible Electroporation.

Pancreatic ductal adenocarcinoma (PDAC) is an abysmal disease refractory to most standard therapies. Irreversible electroporation (IRE) is a local ablative technique for the clinical treatment of solid tumors, including locally advanced and unresectable PDAC, by intratumorally delivering high-intensity electric pulses to permanently disrupt cell membranes and induce cell death. But the distribution of electric field is uneven within the tumor, and in some regions, tumor cells only experience temporary perturbation to their cell membrane, a phenomenon denoted as reversible electroporation (RE). These tumor cells may survive and therefore are the main culprit of tumor relapse after IRE. We herein showed that RE, although not killing tumor cells, induced DNA double-strand breaks and activated DNA damage repair (DDR) responses. Using reactive oxygen species-sensitive polymeric micelles coloaded with Olaparib, an inhibitor of poly(ADP-ribose) polymerase (PARP), and AZD0156, an inhibitor of ataxia telangiectasia mutated (ATM), the resultant nanoformulation (M-TK-OA) disrupted both homologous recombination and nonhomologous end joining signaling of the DDR response and impaired colony formation in pancreatic cancer cells after RE. The combination of IRE and M-TK-OA significantly prolonged animal survival in both subcutaneous and orthotopic murine PDAC models and elicited CD8+ T cell-mediated antitumor immunity with a sustained antitumor memory. The efficacy of combined IRE and M-TK-OA treatments was partially attributed to the activation of cyclic GMP-AMP synthase-stimulator of interferon genes innate immune responses. Our study suggests that dual inhibition of PARP and ATM with nanomedicine is a promising strategy to enhance the pancreatic cancer response to IRE.

EPIC-0307-mediated selective disruption of PRADX-EZH2 interaction and enhancement of temozolomide sensitivity to glioblastoma via inhibiting DNA repair and MGMT.

Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) has been limited by resistance. The level of O-6-methylguanine-DNA methyltransferase (MGMT) and intrinsic DNA damage repair factors are important for the TMZ response in patients. Here, we reported a novel compound, called EPIC-0307, that increased TMZ sensitivity by inhibiting specific DNA damage repair proteins and MGMT expression. EPIC-0307 was derived by molecular docking screening. RNA immunoprecipitation (RIP), and chromatin immunoprecipitation by RNA (ChIRP) assays were used to verify the blocking effect. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) assays were performed to explore the mechanism of EPIC-0307. A series of in vivo and in vitro experiments were designed to evaluate the efficacy of EPIC-0307 in sensitizing GBM cells to TMZ. EPIC-0307 selectively disrupted the binding of PRADX to EZH2 and upregulated the expression of P21 and PUMA, leading to cell cycle arrest and apoptosis in GBM cells. EPIC-0307 exhibited a synergistic inhibitory effect on GBM when combined with TMZ by downregulating TMZ-induced DNA damage repair responses and epigenetically silencing MGMT expression through modulating the recruitment of ATF3-pSTAT3-HDAC1 regulatory complex to the MGMT promoter. EPIC-0307 demonstrated significant efficacy in suppressing the tumorigenesis of GBM cells, restoring TMZ sensitivity. This study identified a potential small-molecule inhibitor (SMI) EPIC-0307 that selectively disrupted the PRADX-EZH2 interaction to upregulate expressions of tumor suppressor genes, thereby exerting its antitumor effects on GBM cells. EPIC-0307 treatment also increased the chemotherapeutic efficacy of TMZ by epigenetically downregulating DNA repair-associate genes and MGMT expression in GBM cells.

Abstract 6762: Tumor immune microenvironment &genomic features of non-small cell lung carcinomas in patients with HIV (PWH)

Abstract Introduction: Lung cancer is the leading cause of cancer-related death among PWH with a relative risk that is over 3-fold higher compared to the uninfected population. In addition, HIV-infected patients with non-small cell lung cancer (NSCLC) have a significantly worse prognosis. The determinants for these associations remain uncertain. Methods: We investigated the immune contexture of 18 primary NSCLCs from PWH and 19 clinicopathologically matched NSCLCs from HIV-negative cases represented in tissue microarrays using 37-plex Imaging Mass Cytometry (IMC) panels including cell phenotype markers (CK,CD4,CD8,CD20,CD45RO,CD25,etc), immunomodulatory targets (PD-L1,PD-L2,B7-H3,B7-H4,B2M,etc) and DNA damage response/repair (DDR) indicators (γH2AX, RAD51,BRCA1,53BP1,etc). The samples were analyzed using single-cell tissue segmentation and spatial analysis. A subset of cases was also studied using whole exome DNA sequencing (WES) and mRNA sequencing (RNAseq). Results: NSCLCs from PWH showed comparable levels of tumor infiltrating lymphocytes (TILs) relative to HIV-negative cases with a trend towards a lower CD4+/CD8+ cell ratio. However, CD4+ and CD8+ TILs in PWH showed higher levels of markers associated with T-cell activation and dysfunction including GZB, CD25, Ki-67, PD-1, LAG-3, and TIM-3 than non-HIV tumors. The epithelial tumor cells in HIV-associated cases showed higher levels of the immunomodulatory ligands PD-L1, CD47, B7-H4 and VISTA; and altered levels of the DDR markers γH2AX, BRCA1 and 53BP1 relative to the non-HIV controls. Differences were also seen in tumor-associated macrophages consistent with an immune regulatory environment. The spatial analysis of tumor and immune cells revealed a distinct topographic distribution of these cells in HIV-associated malignancies. WES analysis showed a higher nonsynonymous tumor mutational burden in HIV-associated tumors (400 vs 202 mean mutations/exome) with increased predicted HLA class-I neoantigens and a numerically higher immunoediting score than the non-HIV group. Exploratory analysis identified enrichment in RYR2 and KIAA1671 gene mutations in HIV-associated malignancies. RNAseq revealed distinct mRNA expression profiles of HIV-positive and negative NSCLCs with a fraction of transcripts showing differential expression associated with tumor suppressor functions, interleukin signaling and DDR. Conclusion: NSCLCs in PWH display profound immunological and genomic alterations suggesting defective immunosurveillance. Our study reveals novel characteristics of HIV-associated NSCLC with possible clinical implications for this growing and understudied population. Citation Format: Shruti Desai, Kishu Ranjan, Syim Salahuddin, Ramsey Yusuf, Jianlei Gu, Daiwei Tang, Yong Kong, Barani Kumar Rajendran, Hongyu Zhao, Yuval Kluger, Sarah Goldberg, Brinda Emu, Kurt Alex Schalper. Tumor immune microenvironment &genomic features of non-small cell lung carcinomas in patients with HIV (PWH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6762.

Abstract 2598: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, augments DNA damage repair and predicts therapy responses

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Methods: Analysis of DACH1 gene deletion and gene expression was conducted from public data bases and human prostate cancer samples. Transgenic mice were generated in which the Dach1 gene was deleted in the prostate of prostate Oncomice. Cells derived from Dach1 gene deletion mice and human prostate cancer cell lines with Dach1 knockdown were analyzed for DNA damage repair responses. Results: DACH1 gene deletion within the 13q21.31-q21.33 region, occurred in up to 18% of human PCa, and was associated with increased AR activity, and poor prognosis. DACH1 homozygous deletions more frequent in the metastatic site than in the primary tumors (Mich: 10% vs. 18%, N=59; FHCRC: 4% vs.11%, N= 54, SU2C: not profiled vs. 3.3, N=150). The prevalence of DACH1 heterozygous deletions was higher in the metastatic lesions than in primary tumors within a given cohort for three of six cohorts (Mich: 27.3% vs. 36%, N=59; FHCRC: 15% vs. 59%, N=54; SU2C: 0% vs. 26%, N=150, respectively). The patients with homozygous DACH1 deletions had reduced overall survival (medians of 84 vs. 120 months, N=667, log rank test P=9.3x10-3). Low DACH1 gene expression (expressed as a z-score with a z-score threshold of -1.25) was significantly correlated with earlier biochemical recurrence (BCR, log rank p value = 4.7x10-4, n=79). In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN) and was associated with increased DNA damage. Reduced Dach1 increased DNA damage in responses to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with resistance to TGFβ kinase and PARP inhibitors. Conclusions: Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies. Citation Format: Zhiping Li, Xuanmao Jiao, Gordon Robertson, Gabriele Di Sante, Anthony W. Ashton, Agnese DiRocco, Min Wang, Jun Zhao, Sankar Addya, Chenguang Wang, Peter A. McCue, Andrew P. South, Carlos Cordon-Cardo, Runzhi Liu, Kishan Patel, Rasha Hamid, Jorim Parmar, James B. DuHadaway, Nikolaus Schultz, Andrew Kossenkov, Lai Yee Phoon, Hao Chen, Li Lan, Yunguang Sun, Kenneth A. Iczkowski, Hallgeir Rui, Richard G. Pestell. The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, augments DNA damage repair and predicts therapy responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2598.