Increased DNA Double-strand Breaks Research Articles

Loss of O6-methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme. The roles of this enzyme in immune cells remain unclear. In this study, we explored the roles of MGMT in bone marrow-derived murine macrophages (BMMs) via the use of MGMT knockout (KO) mice. Loss of MGMT altered the response to TLR3 agonists (poly (I:C)), such as dampening the production of TNFα and IL-6. Increased DNA double-strand breaks (DSBs) were observed in MGMT-KO macrophages but did not result in increased cell death. MGMT localized to both nuclei and mitochondria at increasing levels during poly (I:C) stimulation. MGMT deficiency increased the production of mitochondrial reactive oxygen species (mtROS), which was correlated with increased mitophagy. The underlying mechanism involves mediation through activation of the AMPKα pathway. Taken together, our findings reveal the roles of MGMT in macrophages in regulating the response to TLR3, which links DSBs to mtROS and mitophagy via the AMPKα pathway. These roles may have consequences for the inflammatory response and chronic inflammation.

Enhancing transcription–replication conflict targets ecDNA-positive cancers

Extrachromosomal DNA (ecDNA) presents a major challenge for cancer patients. ecDNA renders tumours treatment resistant by facilitating massive oncogene transcription and rapid genome evolution, contributing to poor patient survival1–7. At present, there are no ecDNA-specific treatments. Here we show that enhancing transcription–replication conflict enables targeted elimination of ecDNA-containing cancers. Stepwise analyses of ecDNA transcription reveal pervasive RNA transcription and associated single-stranded DNA, leading to excessive transcription–replication conflicts and replication stress compared with chromosomal loci. Nucleotide incorporation on ecDNA is markedly slower, and replication stress is significantly higher in ecDNA-containing tumours regardless of cancer type or oncogene cargo. pRPA2-S33, a mediator of DNA damage repair that binds single-stranded DNA, shows elevated localization on ecDNA in a transcription-dependent manner, along with increased DNA double strand breaks, and activation of the S-phase checkpoint kinase, CHK1. Genetic or pharmacological CHK1 inhibition causes extensive and preferential tumour cell death in ecDNA-containing tumours. We advance a highly selective, potent and bioavailable oral CHK1 inhibitor, BBI-2779, that preferentially kills ecDNA-containing tumour cells. In a gastric cancer model containing FGFR2 amplified on ecDNA, BBI-2779 suppresses tumour growth and prevents ecDNA-mediated acquired resistance to the pan-FGFR inhibitor infigratinib, resulting in potent and sustained tumour regression in mice. Transcription–replication conflict emerges as a target for ecDNA-directed therapy, exploiting a synthetic lethality of excess to treat cancer.

Characterization of macrophages associated with human skin models exposed to UV radiation.

Skin macrophages play important roles in the response to external stimuli. Human skin equivalents (HSEs) incorporating the human monocytic cell line THP-1 were fabricated to generate immunocompetent human skin models. These HSEs were used to investigate the influence of the skin microenvironment and ultraviolet A (UVA) on macrophages. Transcriptomic analysis revealed that THP-1 cells in HSEs were enriched in extracellular matrix interaction hallmark but downregulated in DNA replication hallmark. Upon UVA exposure, immunocompetent HSEs presented epidermal distortion and increased DNA double-strand breaks (DSBs). The genes associated with oxidative stress and the inflammatory response were significantly upregulated in THP-1 cells. When the photoprotective agent mycosporine-2-glycine from cyanobacteria was applied to HSEs, the incidence of UVA-induced DSBs was significantly lower, and inflammatory and UV response hallmarks were downregulated in THP-1 cells. Taken together, these results suggest that immunocompetent HSEs can be used to investigate the responses of skin-resident macrophages to external stimuli.

Aurora Kinase A Inhibition Potentiates Platinum and Radiation Cytotoxicity in Non-Small-Cell Lung Cancer Cells and Induces Expression of Alternative Immune Checkpoints.

Despite major advances in non-small-cell lung cancer (NSCLC) treatment, the five-year survival rates for patients with non-oncogene-driven tumors remain low, necessitating combinatory approaches to improve outcomes. Our prior high-throughput RNAi screening identified Aurora kinase A (AURKA) as a potential key player in cisplatin resistance. In this study, we investigated AURKA's role in platinum and radiation sensitivity in multiple NSCLC cell lines and xenograft mouse models, as well as its effect on immune checkpoints, including PD-L1, B7x, B7-H3, and HHLA2. Of 94 NSCLC patient tumor specimens, 91.5% tested positive for AURKA expression, with 34% showing moderate-to-high levels. AURKA expression was upregulated following cisplatin treatment in NSCLC cell lines PC9 and A549. Both AURKA inhibition by alisertib and inducible AURKA knockdown potentiated the cytotoxic effects of cisplatin and radiation, leading to tumor regression in doxycycline-inducible xenograft mice. Co-treated cells exhibited increased DNA double-strand breaks, apoptosis, and senescence. Additionally, AURKA inhibition alone by alisertib increased PD-L1 and B7-H3 expression. In conclusion, our study demonstrates that AURKA inhibition enhances the efficacy of platinum-based chemotherapy in NSCLC cells and modulates the expression of multiple immune checkpoints. Therefore, combinatory regimens with AURKA inhibitors should be strategically designed and further studied within the evolving landscape of chemo-immunotherapy.

PRAME induces genomic instability in uveal melanoma

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer.

225Ac-iPSMA-RGD for Alpha-Therapy Dual Targeting of Stromal/Tumor Cell PSMA and Integrins

Prostate-specific membrane antigens (PSMAs) are frequently overexpressed in both tumor stromal endothelial cells and malignant cells (stromal/tumor cells) of various cancers. The RGD (Arg-Gly-Asp) peptide sequence can specifically detect integrins involved in tumor angiogenesis. This study aimed to preclinically evaluate the cytotoxicity, biokinetics, dosimetry, and therapeutic efficacy of 225Ac-iPSMA-RGD to determine its potential as an improved radiopharmaceutical for alpha therapy compared with the 225Ac-iPSMA and 225Ac-RGD monomers. HEHA-HYNIC-iPSMA-RGD (iPSMA-RGD) was synthesized and characterized by FT-IR, UV-vis, and UPLC mass spectroscopy. The cytotoxicity of 225Ac-iPSMA-RGD was assessed in HCT116 colorectal cancer cells. Biodistribution, biokinetics, and therapeutic efficacy were evaluated in nude mice with induced HCT116 tumors. In vitro results showed increased DNA double-strand breaks through ROS generation, cell apoptosis, and death in HCT116 cells treated with 225Ac-iPSMA-RGD. The results also demonstrated in vivo cytotoxicity in cancer cells after treatment with 225Ac-iPSMA-RGD and biokinetic and dosimetric properties suitable for alpha therapy, delivering ablative radiation doses up to 237 Gy/3.7 kBq to HCT116 tumors in mice. Given the phenotype of HCT116 cancer cells, the results of this study warrant further dosimetric and clinical studies to determine the potential of 225Ac-iPSMA-RGD in the treatment of colorectal cancer.

Targeting the COMMD4–H2B protein complex in lung cancer

BackgroundLung cancer is the biggest cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 85–90% of all lung cancers. Identification of novel therapeutic targets are required as drug resistance impairs chemotherapy effectiveness. COMMD4 is a potential NSCLC therapeutic target. The aims of this study were to investigate the COMMD4-H2B binding pose and develop a short H2B peptide that disrupts the COMMD4-H2B interaction and mimics COMMD4 siRNA depletion.MethodsMolecular modelling, in vitro binding and site-directed mutagenesis were used to identify the COMMD4-H2B binding pose and develop a H2B peptide to inhibit the COMMD4-H2B interaction. Cell viability, DNA repair and mitotic catastrophe assays were performed to determine whether this peptide can specially kill NSCLC cells.ResultsBased on the COMMD4-H2B binding pose, we have identified a H2B peptide that inhibits COMMD4-H2B by directly binding to COMMD4 on its H2B binding binding site, both in vitro and in vivo. Treatment of NSCLC cell lines with this peptide resulted in increased sensitivity to ionising radiation, increased DNA double-strand breaks and induction of mitotic catastrophe in NSCLC cell lines.ConclusionsOur data shows that COMMD4-H2B represents a novel potential NSCLC therapeutic target.

Hexyl-pentynoic acid serves as a novel radiosensitizer for breast cancer by inhibiting UCHL3-dependent Rad51 deubiquitination

ObjectiveTo investigate the effects and underlying mechanism of 2-hexyl-4-pentynoic acid (HPTA), a derivative of valproic acid (VPA), on radiotherapy in breast cancer. MethodsMCF7 cells and 7,12-dimethylbenz-[α]-anthracene (DMBA)-induced transformed human normal breast cells (MCF10A–DMBA cells) were irradiated with 8 ​Gy X-rays. For both cells there were four groups: control, valproic acid (VPA)/HPTA, IR, and VPA/HPTA+IR groups. MTT and clonogenic survival assays were performed to assess cell proliferation, and comet assay was performed to evaluate DNA damage. Protein expression of γH2AX, 53BP1, Rad51, and BRCA1 was examined via immunofluorescence and immunoblotting. Cycloheximide chase and ubiquitination experiments were conducted to determine Rad51 ubiquitination. In vivo experiments involved a rat model of DMBA-induced breast cancer, with four fractionated doses of 2 ​Gy. Tumor tissue pathological changes and γH2AX, Rad51, and UCHL3 expression levels were measured by hematoxylin-eosin staining, immunohistochemistry, and immunoblotting. ResultsCompared with the IR group, 15 ​μmol/L HPTA reduced the cell proliferation ability of irradiated MCF7 cells (t=2.16, P<0.05). The VPA/HPTA+IR group exhibited significantly increased DNA double-strand breaks relative to those in the IR group (VPA+IR vs. IR, t=13.37, P<0.05; HPTA+IR vs. IR, t=8.48, P<0.05). Immunofluorescence and immunoblotting experiments demonstrated that the VPA/HPTA+IR group displayed significantly increased cell foci formation, γH2AX and 53BP1 protein expression levels compared to the IR group [(γH2AX: VPA+IR vs. IR, t=8.88, P< 0.05; HPTA+IR vs. IR, t=8.90, P< 0.05), (53BP1, VPA+IR vs. IR, t=5.73, P< 0.05; HPTA+IR vs. IR, t=6.40, P< 0.05)]. Further, Rad51 expression was downregulated (VPA+IR vs. IR, t=3.12, P< 0.05; HPTA+IR vs. IR, t ​= ​2.70, P<0.05), and Rad51 inhibition effectively counteracted HPTA-induced radiosensitization. Ubiquitination detection further verified that HPTA inhibits Rad51 expression via UCHL3-dependent Rad51 deubiquitination. In vivo study results showed that 20 ​mg/kg HPTA significantly enhanced the radiosensitivity of breast tumors in rats by inhibiting Rad51 expression. ConclusionsHPTA is a highly effective radiosensitizer that enhances the radiotherapeutic efficacy of breast cancer treatment through UCHL3-dependent deubiquitination of Rad51.

Radiobiological Characterization of Pancreatic Cancer Patient-Derived Organoids

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive and lethal tumors with a 5-year survival rate of less than 10%. Neoadjuvant radio(chemo)therapy aiming tumor downsizing fails in about 70% due to high heterogeneity, strong desmoplastic stroma and intrinsic radioresistance. In this project, pancreatic cancer patient-derived organoids (PDOs) are characterized regarding radioresponse, DNA-damage, proliferation and hypoxia, and clinical patients' outcome is correlated with the preclinical radiobiological data. In contrast to 2D monolayer cultures, PDOs maintain similar appearance, organization and functionality as the original tissue and therefore might have the potential as advanced preclinical model in radiation oncology. The radiation response of nine different pancreatic cancer PDO lines was determined by 3D cell viability assay. PDOs were irradiated with 0, 2, 4, 6, and 8 Gy (CellRad, Precision, USA) 24h after seeding and the ATP-dependent viability assay was performed 72h and 7d after irradiation (RT). Changes in morphology, number, and size were investigated by microscopy at different time points after RT. PDOs were characterized immunohistochemically by y-H2AX (DNA damage), and Ki-67 (proliferation) staining. RNA sequencing data of treatment-naive PDOs were analyzed by gene set enrichment analyses (GSEA) regarding radioresistance. Preclinical results were correlated with corresponding clinical data of PDAC patients. After optimization of the experimental set-up, PDOs showed a dose-dependent decrease in viability 7d after RT and heterogeneity in radioresponse. PDO lines were classified into radiosensitive, -intermediate, and -resistant subclasses. Immunohisto-chemical staining showed a significant increase in DNA double-strand breaks after RT. A correlation between radiosensitivity and enhanced proliferation index Ki67 was observed. Based on RNA sequencing data, OXPHOS- and hypoxia-dependent genes, amongst others, were identified as pathways significantly differentially regulated between the subclasses by GSEA. Preclinical radioresistance was associated with worse survival and poor clinical outcome. The results of the preclinical experiments demonstrate the heterogeneity among PDOs in response to RT reflecting the clinical situation of patients with PDAC. The findings from the GSEA show promising aspects for further experiments to understand the role of hypoxia in PDAC and its effect on radioresistance. PDOs have the potential as a novel translational research platform in radiation oncology. Prospectively, we aim to implement the screening of the radiosensitivity of PDOs in clinical practice for the realization of truly personalized radiotherapy in PDAC patients.

Dual enhancement in the radiosensitivity of prostate cancer through nanoparticles and chemotherapeutics

BackgroundRadiotherapy (RT) is an essential component in the treatment regimens for many cancer patients. However, the dose escalation required to improve curative results is hindered due to the normal tissue toxicity that is induced. The introduction of radiosensitizers to RT treatment is an avenue that is currently being explored to overcome this issue. By introducing radiosensitizers into tumor sites, it is possible to preferentially enhance the local dose deposited. Gold nanoparticles (GNPs) are a potential candidate that have shown great promise in increasing the radiosensitivity of cancer cells through an enhancement in DNA damage. Furthermore, docetaxel (DTX) is a chemotherapeutic agent that arrests cells in the G2/M phase of the cell cycle, the phase most sensitive to radiation damage. We hypothesized that by incorporating DTX to GNP-enhanced radiotherapy treatment, we could further improve the radiosensitization experienced by cancer cells. To assess this strategy, we analyzed the radiotherapeutic effects on monolayer cell cultures in vitro, as well as on a mice prostate xenograft model in vivo while using clinically feasible concentrations for both GNPs and DTX.ResultsThe introduction of DTX to GNP-enhanced radiotherapy further increased the radiotherapeutic effects experienced by cancer cells. A 38% increase in DNA double-strand breaks was observed with the combination of GNP/DTX vs GNP alone after a dose of 2 Gy was administered. In vivo results displayed significant reduction in tumor growth over a 30-day observation period with the treatment of GNP/DTX/RT when compared to GNP/RT after a single 5 Gy dose was given to mice. The treatment strategy also resulted in 100% mice survival, which was not observed for other treatment conditions.ConclusionsIncorporating DTX to work in unison with GNPs and RT can increase the efficacy of RT treatment. Our study suggests that the treatment strategy could improve tumor control through local dose enhancement. As the concentrations used in this study are clinically feasible, there is potential for this strategy to be translated into clinical settings.

DNA damage-induced senescence is associated with metabolomic reprogramming in breast cancer cells

Senescence due to exogenous and endogenous stresses triggers metabolic reprogramming and is associated with many pathologies, including cancer. In solid tumors, senescence promotes tumorigenesis, facilitates relapse, and changes the outcomes of anti-cancer therapies. Hence, cellular and molecular mechanisms regulating senescent pathways make attractive therapeutic targets. Cancer cells undergo metabolic reprogramming to sustain the growth-arrested state of senescence. In the present study, we aimed to understand the metabolic reprogramming in MCF-7 breast tumor cells in response to two independent inducers of DNA damage-mediated senescence, including ionizing radiation and doxorubicin. Increased DNA double-strand breaks, as demonstrated by γH2AX staining, showed a senescence phenotype, with expression of senescence-associated β-galactosidase accompanied by the upregulation of p21 and p16 in both groups. Further, untargeted analysis of the senescence-related extracellular metabolome profile of MCF-7 cells showed significantly reduced concentrations of carnitine and pantothenic acid and increased levels of S-adenosylhomocysteine in doxorubicin-treated cells, indicating the accumulation of ROS mediated DNA damage and impaired mitochondrial membrane potential. Similarly, a significant decline in the creatine level was observed in radiation-exposed cells, suggesting an increase in oxidative stress-mediated DNA damage. Our study, therefore, provides key effectors of the metabolic changes in doxorubicin and radiation-induced early senescence in MCF-7 breast cancer cells.

Enzymatic depletion of l-Met using an engineered human enzyme as a novel therapeutic strategy for melanoma.

Many cancers, including melanoma, have a higher requirement for l-methionine in comparison with noncancerous cells. In this study, we show that administration of an engineered human methionine-γ-lyase (hMGL) significantly reduced the survival of both human and mouse melanoma cells in vitro. A multiomics approach was utilized to identify global changes in gene expression and in metabolite levels with hMGL treatment in melanoma cells. There was considerable overlap in the perturbed pathways identified in the two data sets. Common pathways were flagged for further investigation to understand their mechanistic importance. In this regard, hMGL treatment induced S and G2 phase cell cycle arrest, decreased nucleotide levels, and increased DNA double-strand breaks suggesting an important role for replication stress in the mechanism of hMGL effects on melanoma cells. Further, hMGL treatment resulted in increased cellular reactive oxygen specieslevels and increased apoptosis as well as uncharged transfer RNApathway upregulation. Finally, treatment with hMGL significantly inhibited the growth of both mouse and human melanoma cells in orthotopic tumor models in vivo. Overall, the results of this study provide a strong rationale for further mechanistic evaluation and clinical development of hMGL for the treatment of melanoma skin cancer and other cancers.

Hexavalent Chromium Disrupts Oocyte Development in Rats by Elevating Oxidative Stress, DNA Double-Strand Breaks, Microtubule Disruption, and Aberrant Segregation of Chromosomes.

Environmental and occupational exposure to hexavalent chromium, Cr(VI), causes female reproductive failures and infertility. Cr(VI) is used in more than 50 industries and is a group A carcinogen, mutagenic and teratogenic, and a male and female reproductive toxicant. Our previous findings indicate that Cr(VI) causes follicular atresia, trophoblast cell apoptosis, and mitochondrial dysfunction in metaphase II (MII) oocytes. However, the integrated molecular mechanism of Cr(VI)-induced oocyte defects is not understood. The current study investigates the mechanism of Cr(VI) in causing meiotic disruption of MII oocytes, leading to oocyte incompetence in superovulated rats. Postnatal day (PND) 22 rats were treated with potassium dichromate (1 and 5 ppm) in drinking water from PND 22-29 and superovulated. MII oocytes were analyzed by immunofluorescence, and images were captured by confocal microscopy and quantified by Image-Pro Plus software, Version 10.0.5. Our data showed that Cr(VI) increased microtubule misalignment (~9 fold), led to missegregation of chromosomes and bulged and folded actin caps, increased oxidative DNA (~3 fold) and protein (~9-12 fold) damage, and increased DNA double-strand breaks (~5-10 fold) and DNA repair protein RAD51 (~3-6 fold). Cr(VI) also induced incomplete cytokinesis and delayed polar body extrusion. Our study indicates that exposure to environmentally relevant doses of Cr(VI) caused severe DNA damage, distorted oocyte cytoskeletal proteins, and caused oxidative DNA and protein damage, resulting in developmental arrest in MII oocytes.

Abstract 2667: PARG inhibition augments CHK1 inhibitor-induced replication stress and synergistically kills ovarian cancer cells

Abstract Ovarian cancer (OC) is one of the leading causes of cancer-related deaths in women in the United States. PARP inhibitors showed promising clinical responses; however, most of the patients eventually relapse and succumb to the resistant disease. Currently, it lacks effective therapies to prevent and treat OC, which warrants novel drugs and combination therapies to improve patients' prognoses. Epithelial OCs are heavily dependent on ATR-CHK1-mediated DNA damage checkpoint signaling for survival, which is also regulated by hedgehog/GLI1 (Hh) signaling in cancer cells. However, both Hh/GLI1 and CHK1 inhibitors failed to show promising results in clinical trials as single agents. Poly(ADP-ribose) glycohydrolase (PARG) is the critical enzyme that removes Poly-ADP ribosylated (PARylated) proteins upon replication stress. We hypothesized that inhibition of PARG could amplify CHK1 inhibitor-induced replication stress by trapping PARylated proteins on the chromatin and compromising DNA repair and causing a metabolic and mitotic catastrophe. We evaluated CHK1 inhibitor prexasertib in combination with PARG inhibitor (PARGi) to target metabolic and DNA repair crosstalk to effectively kill OC cells and to overcome resistance. Our studies demonstrate that prexasertib treatment induces PARylation by inducing replication stress. Similarly, PARG inhibition by (PDD00017273) fails to remove PARylation and activates checkpoint kinase 1 (CHK1) by phosphorylating at S296, S317, and S345 in OC cells. Based on these observations, we hypothesized that a combination of CHK1 and PARG inhibition would augment oncogenic replication stress in OC cells and cause increased DNA damage and cell death. Interestingly, our evaluation of prexasertib and PARGi combination treatment by clonogenic survival and MTT assays showed synergistic killing in a panel of OC cells, including in isogenic chemoresistant models as well as 3D organoid models of primary OC cells compared to individual drugs. As predicted, combined treatment increased DNA double-strand breaks as demonstrated by COMET assays, and increased γH2AX foci, pRPA32 foci, perturbed S/G2 phase arrest, and nuclear disintegration relative to individual-drug treatment. Furthermore, significant depletion of NAD+ levels was seen in PARGi-treated OC cells compared to untreated cells. Together, these results indicate that this combination treatment cause persistence of heavy PARylation at DNA damage sites abrogates cell cycle checkpoint mechanisms, exhausts cellular NAD+ levels, and inhibits DNA repair leading to the metabolic and mitotic collapse of cancer cells and synergistic lethality in OC cells. Currently, prexasertib is under clinical trials and our studies will provide novel mechanistic insight into the therapeutic potential of this combination and provides preclinical evidence to develop further this combination therapy in treating OC. Citation Format: Ganesh Acharya, Chinnadurai Mani, Mark B. Reedy, Komaraiah Palle. PARG inhibition augments CHK1 inhibitor-induced replication stress and synergistically kills ovarian cancer cells [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 2667.

Therapeutic efficacy of cyclin-dependent kinase inhibition in combination with ionizing radiation for lung cancer

Purpose To evaluate the therapeutic efficacy of cyclin-dependent kinase (CDK) inhibition in combination with ionizing radiation for lung cancer. Materials and methods Human lung adenocarcinoma (A549) and squamous cell carcinoma (H520) cells were used to evaluate the therapeutic efficacy of CDK inhibition in combination with ionizing radiation in vitro using colony formation assay, γH2AX immunofluorescence staining, western blotting, and cell cycle phase analysis. We also performed in vivo evaluations of ectopic tumor growth. Results In vitro pretreatment with the CDK inhibitor, seliciclib, before irradiation significantly decreased the survival of A549 and H520 cells in a dose-dependent manner. Although CDK inhibition alone did not increase the intensity of γH2AX foci, its combination with ionizing radiation increased DNA double-strand breaks, as shown by γH2AX immunofluorescence staining and western blotting. The combination of CDK inhibition and ionizing radiation-induced G2/M arrest and increased apoptosis, as evidenced by the increased proportion of cells in G2/M arrest, subG1 apoptotic population, and expression of apoptotic markers (cleaved PARP-1 and cleaved caspase-3). Mechanistic studies showed reduced expression of cyclin A with combined treatment, indicating cell cycle shifting effects. An in vivo xenograft model showed that the combination of CDK inhibition and ionizing radiation delayed xenograft tumor growth, and increased the proportion of cleaved PARP-1- and cleaved caspase-3-positive cells, compared to either treatment alone. Conclusions We provide preclinical tumoricidal evidence that the combination of CDK inhibition and ionizing radiation is an efficacious treatment for lung cancer.

Bortezomib abrogates temozolomide-induced autophagic flux through an ATG5 dependent pathway.

Introduction: Glioblastoma (GBM) is invariably resistant to temozolomide (TMZ) chemotherapy. Inhibiting the proteasomal pathway is an emerging strategy to accumulate damaged proteins and inhibit their lysosomal degradation. We hypothesized that pre-treatment of glioblastoma with bortezomib (BTZ) might sensitize glioblastoma to temozolomide by abolishing autophagy survival signals to augment DNA damage and apoptosis. Methods: P3 patient-derived glioblastoma cells, as well as the tumour cell lines U87, HF66, A172, and T98G were investigated for clonogenic survival after single or combined treatment with temozolomide and bortezomib in vitro. We investigated the requirement of functional autophagy machinery by utilizing pharmacological inhibitors or CRISPR-Cas9 knockout (KO) of autophagy-related genes -5 and -7 (ATG5 and ATG7) in glioblastoma cells and monitored changes in autophagic flux after temozolomide and/or bortezomib treatments. P3 wild-type and P3 ATG5-/- (ATG5 KO) cells were implanted orthotopically into NOD-SCID mice to assess the efficacy of bortezomib and temozolomide combination therapy with and without functional autophagy machinery. Results: The chemo-resistant glioblastoma cells increased autophagic flux during temozolomide treatment as indicated by increased degradation of long-lived proteins, diminished expression of autophagy markers LC3A/B-II and p62 (SQSTM1), increased co-localisation of LC3A/B-II with STX17, augmented and no induction of apoptosis. In contrast, bortezomib treatment abrogated autophagic flux indicated by the accumulation of LC3A/B-II and p62 (SQSTM1) positive autophagosomes that did not fuse with lysosomes and thus reduced the degradation of long-lived proteins. Bortezomib synergistically enhanced temozolomide efficacy by attenuating cell proliferation, increased DNA double-strand breaks, and apoptosis in an autophagy-dependent manner. Abolishing autophagy in ATG5 KOs reversed the bortezomib-induced toxicity, rescued glioblastoma cell death and reduced animal survival. Discussion: We conclude that bortezomib abrogates temozolomide induced autophagy flux through an ATG5 dependent pathway.

Investigation of the antioxidant status and the number of double-stranded DNA breaks in models of brain tumor lesion by metastases of non-small cell lung cancer in vivo

Purpose of the study. Evaluation of the antioxidant status and DNA damage in tissues of subcutaneous xenografts of non-small cell lung cancer and in peritumoral tissues created using the A549 and H1299 cell cultures.Materials and methods. The study included 35 intact male Balb/c Nude immunodeficient mice. Cell lines A549 and H1299 were used as transplantable tumor biomaterial. A CDX model was created in accordance with the protocol for supratentorial injections (Ozawa T., James C. D., 2010) adapted for this experiment. Growth rates were controlled and intracranial xenografts were visualized using a high-resolution micro-CT system. The activity of catalase and superoxide dismutase was determined with non-denaturing electrophoresis in 8 % and 12 % polyacrylamide gel. The concentrations of sulfhydryl groups were determined according to Ellman. The DNA damage in lymphocytes was determined by the comet assay.Results. The experiment resulted in the creation of models of brain tumors characterized by intracranial growth pattern in 100 %. The activity of catalase in the studied lysates of intracranial xenografts, peritumoral tissue and healthy tissues of tumor-bearing animals in all experimental groups increased statistically significantly relative to the healthy tissue of intact animals, and the greatest differences from the control were recorded in the group of animals with implanted H1299 culture at a concentration of 1 × 106 . Superoxide dismutase activity in the studied lysates of intracranial xenografts and peritumoral tissues statistically significantly increased compared to the control sample in all experimental groups. The highest increase in the SOD activity was observed in the tissues of intracranial xenografts with the highest tumor load, which amounted to 28.8 % and 32.9 % of the changes relative to the control sample. A statistically significant increase in the concentration of SH-groups relative to the control sample in tumor tissue lysates was revealed in all experimental groups, and the highest concentration (36.2 ± 0.47) was observed in the group of experimental animals with the highest tumor load. Percentage change in tail moment (DNA damage indicator) in groups O1, O2, O3 and O4 increased statistically significantly compared to the control sample by 55.8 %, 111.8 %, 97.3 % and 170 %, respectively.Conclusions. The observed increase in the activity of the antioxidant defense system, accumulation of oxidative modifications of proteins, and an increase in DNA double-strand breaks in the tissues of intracranial xenografts of non-small cell lung cancer in vivo suggest that the created models reflect processes similar to those in tumors of human non-small cell lung cancer.

Dnmt3a Loss-of-Function Enhances DNA Repair in Proliferative HSCs Under Inflammatory Stress

Somatic mutations in DNMT3A can be enriched during clonal hematopoiesis by conditions with elevated type II interferon signaling. We have recently demonstrated that loss of functional Dnmt3a endows HSCs with a fitness advantage during chronic IFNg-driven inflammation. The increased fitness of Dnmt3a-mutant HSCs in these conditions can be partly attributed to the insensitivity to IFNg-primed apoptosis upon proliferative stress and delayed cell cycle activation from quiescence. However, it is unknown if these two mechanisms are related; if not, what is the underlying mechanism of inflammation-induced apoptotic resistance by Dnmt3a loss in HSCs? If the apoptotic resistance was merely due to delayed cell cycle activation from quiescence, proliferative activation should normalize the apoptotic levels of IFNg-exposed Dnmt3a-mutant HSCs. To test this, we induced HSCs into cell cycle by injecting 5-fluorouracil (5FU) into control (Vav-cre-; Dnmt3af/f) and mice lacking Dnmt3a in hematopoietic cells (3aKO, Vav-cre+; Dnmt3af/f). Kinetic analysis by BrdU labeling revealed that HSCs reached proliferation peak 8-days post-5FU treatment (>90% BrdU+). To capture the maximal number of proliferative HSCs, we injected PBS or recombinant mouse IFNg into 5FU-treated control and 3aKO mice on day 7 and 8. Phenotypic HSCs (Lin-cKit+EPCR+CD150+CD48-) were purified 2h after the second injection and analyzed for apoptosis. Cycling control HSCs were significantly more apoptotic upon IFNg exposure, while cell-cycle activated 3aKO HSCs remained insensitive to IFNg-primed apoptosis. Functional validation by colony formation assay showed IFNg-exposed cycling control HSCs formed significantly fewer colonies, while 3aKO HSCs gave rise to significantly more colonies regardless of exposure. These data suggest that Dnmt3a mutation may render HSCs resistant to IFNg-primed apoptosis via cell-cycle independent mechanisms. It has been documented that inflammation causes HSC apoptosis by inducing DNA damage through various mechanisms. Thus, the apoptotic resistance observed in Dnmt3a mutant HSCs could be due to 1) resistance to inflammation-induced DNA damage or 2) enhanced resolution of DNA damage. To test if Dnmt3a mutation renders HSCs resistant to IFNg-primed DNA damage, neutral comet assay was performed using PBS or IFNg-exposed quiescent HSCs. Interestingly, both genotypes showed increased DNA double-strand breaks (DSBs) upon IFNg exposure, suggesting that quiescent Dnmt3a-mutant HSCs are susceptible to IFNg-primed DSBs. DNA repair is one mechanism enabling DSB resolution, and is functionally active in proliferative HSCs. Thus, we performed neutral comet assay on cycling HSCs to quantify DSBs following PBS/IFNg exposure. Expectedly, IFNg significantly increased DSBs in cycling control but not in 3aKO HSCs, suggesting Dnmt3a loss may endow HSCs with augmented DNA repair. Similarly, when DNA damage was determined in control and Dnmt3a-knockout (3aKO) murine myeloblast cells (32D) 4h and 24h post-IFNg exposure, a comparable increase in DNA damage was observed 4h post-exposure in both genotypes. Interestingly, DNA damage levels were significantly reduced 24h later only in the 3aKO cells. This data paralleled HSC data, implying that Dnmt3a ablation facilitates augmented DNA repair. DSBs are repaired primarily via three universal yet distinct pathways, homologous recombination (HR), non-homologous end joining (NHEJ), and alternative NHEJ. To investigate how Dnmt3a loss facilitates enhanced DNA repair in response to IFNg exposure, we performed a DNA repair reporter assay in 32D genotypes 24h after IFNg exposure. The plasmid repair reporter assay was built on the detection of a green fluorescent protein (GFP) expression, which is otherwise disrupted by the insertion of I-SceI restriction enzyme recognition sites. After cutting with I-SceI, GFP can only be expressed after specific repair by one of the three pathways. Results showed Dnmt3a loss specifically enhanced the NHEJ pathways upon IFNg exposure, while other pathways were comparable between genotypes and treatments. In summary, our results demonstrated that Dnmt3a loss protects cell-cycle activated HSCs from inflammation-primed DSBs and apoptosis via augmented NHEJ as one mechanism. Future studies will investigate the molecular mechanisms underlying the enhanced NHEJ in Dnmt3a-mutant HSCs in response to chronic inflammation.

HDAC8 Disrupts DNA Damage Response By Deacetylation of U2AF1 in Inv(16) Acute Myeloid Leukemia

Genomic instability due to defective DNA damage response (DDR) and DNA repair increases the probability of acquiring mutations, which may contribute to the pathogenesis of acute myeloid leukemia (AML). Inv(16) AML [inv(16)(p13q22) or t(16;16)(p13.1;q22)] creates a fusion gene CBFb-MYH11 (CM) by fusing core binding factor b (CBFb) with MYH11 which encodes smooth muscle myosin heavy chain (SMMHC). Our previous studies using a conditional Cbfb-MYH11 knock-in (KI) mouse model showed that preleukemic progenitors are prone to leukemia initiation, however, the underlying mechanism is not clear. Here, we show increased DNA double-strand breaks measured by comet assay and γH2AX staining in CM preleukemic progenitors compared to control counterparts. RNA-seq using sorted LSK (Lin-Sca1+Kit+) cells isolated from BM of CM KI mice (n = 5) and control (n = 4) identified a total of 1019 genes differentially expressed (526 up; 493 down; > 1.5-fold change; p < 0.01). Gene set enrichment analysis revealed that G2/M checkpoint, E2F targets, and mitotic spindle pathways were differentially downregulated in CM LSK, suggesting a possible contribution of cell cycle checkpoint dysregulation. To further explore the mechanism underlying CM-induced DNA damage, we expressed CM in 32D cells and subjected them to irradiation (IR; 3.5 Gy) followed by γH2AX and RAD51 staining. Significantly more γH2AX foci per nucleus were observed in CM cells (CM 35.03 vs Ctrl 24.71 at 0.5h, p<0.0001; CM 14.79 vs Ctrl 11.67 at 3h, p=0.0186), while fewer RAD51 foci per nucleus were seen in CM cells (CM 4.932 vs Ctrl 9.6 at 3h, p<0.0001), suggesting that CM may impair homologous recombination (HR)-mediated DNA repair. We reported that CM interacts with HDAC8 and enhances its activity, thus we examined whether HDAC8 may mediate the impact on DDR and HR activity. HDAC8 overexpression (OE) in 32D cells resulted in significantly more γH2AX foci per nucleus after IR (3.5 Gy) (HDAC8-OE 42.20 vs Ctrl 32.17 at 0.5h, p<0.0001; HDAC8-OE 15.45 vs Ctrl 10.63 at 3h, p<0.0001). Similar to CM cells, fewer RAD51 foci per nucleus were seen in HDAC8-OE cells (HDAC8-OE 11.63 vs Ctrl 17.39 at 3h, p<0.0001). To verify HDAC8 function in HR repair, we used the I-SceI and DR-GFP (homology-directed repair) or EJ5-GFP (end-joining-directed repair) DNA repair reporter (Gunn A et al, Methods Mol Biol, 2012). The frequency of repaired GFP+ population in the HR-directed repair reporter was reduced in HDAC8-OE cells compared to control (HDAC8-OE 7.807% vs Ctrl 11.34%, p=0.0169), while no difference was observed in the NHEJ-directed repair reporter. These results indicate that high HDAC8 expression disturbs HR DNA repair. The U2 small nuclear RNA auxiliary factor 1 (U2AF1) has been reported as a component of BRCA1-mRNA splicing machinery that regulates pre-mRNA splicing of critical genes involved in HR DNA repair. Here, we show that U2AF1 is subjected to post-translational acetylation and that U2AF1 interacts with HDAC8. We show that U2AF1 acetylation and its interaction with HDAC8 were increased following DNA damage and that HDAC8 is critical for deacetylating U2AF1. Site-directed mutagenesis for reported putative acetylation lysine sites (K15, K23, K175) (Elia AE et al, Mol Cell, 2015) revealed that the acetylation level of U2AF1-K23R decreased after IR and corresponded to reduced interaction with HDAC8 and the BRCA1/BCLAF1 complex. Notably, the interaction with BRCA1/BCLAF1 complex was rescued by mutating lysine to the acetylation mimic glutamine (Q). These results indicate that the acetylation state of U2AF1 is essential for the assembly of BRCA1-mRNA splicing complex and suggest that HDAC8 may regulate HR DNA repair by modulating the U2AF1 acetylation state. Similar to HDAC8 OE, CM expression reduced U2AF1 acetylation and impaired its interaction with BRCA1/BCLAF1 complex. Collectively, our results suggest that the increased DNA damage induced by CM may be due to the reduced acetylation of U2AF1 resulting from high HDAC8 activity. These results highlight a direct impact of inv(16) fusion protein and high HDAC8 activity on increased genomic instability in AML.

DNAR-08. THE ROLE OF SMARCAL1 AS A SYNTHETIC LETHAL VULNERABILITY IN ATRX-DEFICIENT GLIOMAS

Abstract Mechanisms to maintain telomere length over successive cell divisions are a requirement for cancer cell immortalization. Although many cancers maintain telomere length through the activation of telomerase, ~10-15% of human cancers use telomerase-independent mechanisms of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). In gliomas, the ALT phenotype is associated with loss-of-function mutations in the ATRX gene in IDH-mutant astrocytomas, adult and pediatric glioblastomas (GBM), high-grade astrocytomas with piloid features, and pleomorphic xanthoastrocytomas. In adult GBM, we previously identified a relatively rare subset of ALT-positive ATRX wildtype tumors that harbored loss-of-function mutations in the SMARCAL1 gene, and we found that loss of SMARCAL1 activity plays a causative role in the onset of ALT in these tumors. SMARCAL1 is an annealing helicase that localizes to sites of DNA damage and replication stress and catalyzes the reversal of stalled replication forks. To better understand the relationship between ATRX mutations, SMARCAL1 activity, and the ALT phenotype, we investigated the localization and function of SMARCAL1 in ALT-positive glioma cells and xenografts with native ATRX deficiency. We found that SMARCAL1 localizes to ALT-associated PML bodies in astrocytoma cell lines with concurrent ATRX and IDH1 mutations (derived from grade 3 and 4 tumors). Our data show that inducible suppression of SMARCAL1 via doxycycline-induced RNAi leads to increased DNA double-strand breaks, increased abundance of extrachromosomal telomeric repeats (c-circles), and increased sensitivity to irinotecan, a topoisomerase I inhibitor. In an orthotopic mouse model, SMARCAL1 depletion exhibited a chemosensitizing effect in combination with irinotecan against a patient-derived xenograft (astrocytoma, IDH-mutant Grade 4). Based on these data, we hypothesize that SMARCAL1 activity is critical for resolving ALT-associated replication stress in ATRX-deficient malignant gliomas. We therefore propose that SMARCAL1 functions as a synthetic lethal vulnerability in ATRX-deficient ALT-positive gliomas and that SMARCAL1 inhibition is a viable therapeutic strategy in these tumors.