Increased DNA Damage Repair Research Articles

Deciphering the Molecular Mechanisms behind Drug Resistance in Ovarian Cancer to Unlock Efficient Treatment Options.

Ovarian cancer is a highly lethal form of gynecological cancer. This disease often goes undetected until advanced stages, resulting in high morbidity and mortality rates. Unfortunately, many patients experience relapse and succumb to the disease due to the emergence of drug resistance that significantly limits the effectiveness of currently available oncological treatments. Here, we discuss the molecular mechanisms responsible for resistance to carboplatin, paclitaxel, polyadenosine diphosphate ribose polymerase inhibitors, and bevacizumab in ovarian cancer. We present a detailed analysis of the most extensively investigated resistance mechanisms, including drug inactivation, drug target alterations, enhanced drug efflux pumps, increased DNA damage repair capacity, and reduced drug absorption/accumulation. The in-depth understanding of the molecular mechanisms associated with drug resistance is crucial to unveil new biomarkers capable of predicting and monitoring the kinetics during disease progression and discovering new therapeutic targets.

Abstract 711: HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells

Abstract Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 shows a dismal prognosis with 5-year survival of less than 30% compared to non-metastatic fusion-negative (FN)-RMS variant. In the last years, a lot of interest has focused on new targets identification to improve the radiotherapy (RT) efficacy. HDAC inhibitors (HDACi) radio-sensitize different cancer cell types including RMS. Recently, we reported that MS-275, a Class I and IV HDACi, in combination with RT affected cell survival, reduced colony formation ability, increased DNA damage repair inhibition and reactive oxygen species formation in FP-RMS cells. However, despite promising preclinical studies, HDAC inhibitors (HDACi) achieved only modest success in human clinical trials for solid tumors, frequently showing several toxicities, probably because of the limited specificity of many HDACi. Thus, a major effort is being directed toward identification of HDACi which are selective for HDAC isoforms often uniquely implicated in the radioresistance of specific cancers. In order to identify the class I HDAC responsible of radioresistance in RMS we knocked-down HDAC1, HDAC2, HDAC3, and HDAC8 expression in combination with RT. Interestingly, we observed an increased radiosensitivity only in HDAC3-depleted FP-RMS cells. Thus, we focused on HDAC3 to understand the mechanisms by which it promotes radioresistance in RMS. We observed that HDAC3 is overexpressed in RMS patients and cell lines compared to the normal counterpart. Furthermore, RMS cells are strongly dependent by HDAC3 expression while slight dependency has been observed with other class I HDACs in DepMap portal. We demonstrated that HDAC3 depletion by CRISPR in combination with RT in FP-RMS cells increases apoptosis, reduces colony formation ability, cancer stem cells population and anchorage independent growth and reduces cell growth in vivo and in vitro. Accordingly, HDAC3 depleted cells in combination with RT reduces levels and activation of key player of FP-RMS biology such as MYCN, ERK and AKT. Moreover, HDAC3 KD increases radiotherapy-induced DNA double strand break and impairs DNA repair mechanisms reducing levels and activation of both homologous recombination (HR) and non-homologous end joining (NHEJ) factors such as ATM, RAD51 and DNA-PKcs. Thus, we developed a new potent and highly specific HDAC3 inhibitor (HDAC3i). The new HDAC3i is highly specific in targeting FP-RMS cell growth in vitro while no effects have been observed in normal cells such as myoblasts and lung fibroblast. Moreover, the drug treatment in combination with radiotherapy phenotypically and molecularly recapitulated what observed in HDAC3 depleted cells.The study has been founded by Italian Association for Cancer Research (AIRC) to FM. Citation Format: Matteo Cassandri, Antonella Porrazzo, Silvia Pomella, Simona Camero, Francesca A. Aiello, Lucrezia D'Archivio, Clemens Zwergel, Beatrice Noce, Miriam Tomaciello, Francesca Vulcano, Luisa Milazzo, Francesca Pedini, Michele Signore, Alessandro Fanzani, Cinzia Marchese, Giuseppe Minniti, Sergio Valente, Antonello Mai, Francesca Megiorni, Rossella Rota, Francesco Marampon. HDAC3 sustains resistance to hypofractionated radiotherapy in fusion positve rhabdomyosarcoma cells [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 711.

Evidence for increased DNA damage repair in the postmortem brain of the high stress-response group of schizophrenia.

Schizophrenia (SZ) is a disorder diagnosed by specific symptoms and duration and is highly heterogeneous, clinically and pathologically. Although there are an increasing number of studies on the association between genetic and environmental factors in the development of SZ, the actual distribution of the population with different levels of influence of these factors has not yet been fully elucidated. In this study, we focused on stress as an environmental factor and stratified SZ based on the expression levels of stress-responsive molecules in the postmortem prefrontal cortex. We selected the following stress-responsive molecules: interleukin (IL) -1β, IL-6, IL-10, tumor necrosis factor-α, interferon-γ, glucocorticoid receptor, brain-derived neurotrophic factor, synaptophysin, S100 calcium-binding protein B, superoxide dismutase, postsynaptic density protein 95, synuclein, apolipoprotein A1 (ApoA1), ApoA2, and solute carrier family 6 member 4. We performed RNA sequencing in the prefrontal gray matter of 25 SZ cases and 21 healthy controls and conducted a hierarchical cluster analysis of SZ based on the gene expression levels of stress-responsive molecules, which yielded two clusters. After assessing the validity of the clusters, they were designated as the high stress-response SZ group and the low stress-response SZ group, respectively. Ingenuity Pathway Analysis of differentially expressed genes (DEGs) between clusters was performed, and Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was conducted on four cases each in the high and low stress-response SZ groups to validate DNA damage. We found higher prevalence of family history of SZ in the low stress-response SZ group (0/3 vs. 5/4, p = 0.04). Pathway analysis of DEGs between clusters showed the highest enrichment for DNA double-strand break repair. TUNEL staining showed a trend toward a lower percentage of TUNEL-positive cells in the high stress-response SZ group. Our results suggest that there are subgroups of SZ with different degrees of stress impact. Furthermore, the pathophysiology of these subgroups may be associated with DNA damage repair. These results provide new insights into the interactions and heterogeneity between genetic and environmental factors.

Loss of EPS8 sensitizes non-small-cell lung carcinoma to chemotherapy-induced DNA damage.

Epidermal growth factor receptor pathway substrate number 8 (EPS8) has been reported to be critical in mediating tumor progression. However, the molecular and biological consequences of EPS8 overexpression remain unclear. Here we evaluated whether EPS8 increased DNA damage repair in non-small-cell lung carcinoma (NSCLC) cells and the mechanism of EPS8-mediated DNA damage repair which influenced chemosensitivity. Serial studies of functional experiments revealed that EPS8 knockdown inhibited cell growth, induced cell-cycle arrest and increased cisplatin therapeutic effects on NSCLC. EPS8 was found to induce DNA damage repair via upregulation of phosphorylated-ATM and downregulation of the tumor suppressor p53 and G1 cell kinase inhibitor p21. Moreover, in conjunction with cisplatin, decreasing EPS8 protein levels further increased p53 protein level and inhibited ATM signaling. Transplanted tumor studies were also performed to demonstrate that EPS8 knockdown inhibited tumor growth and sensitized tumors to cisplatin treatment. In conclusion, we have described a novel molecular mechanism through which EPS8 is likely to be involved in cancer progression and chemoresistance via DNA damage repair, indicating that EPS8 expression may influence the response to chemotherapy. Therefore, targeting EPS8 may be a potential therapeutic approach for patients with NSCLC.

Glioblastoma recurrent cells switch between ATM and ATR pathway as an alternative strategy to survive radiation stress.

Primary treatment modality for glioblastoma (GBM) post-surgery is radiation therapy. Due to increased DNA damage repair capacity of resistant residual GBM cells, recurrence is inevitable in glioblastoma and unfortunately the recurrent tumours are resistant to the conventional therapy. Here we used our previously described in vitro radiation survival model generated from primary GBM patient samples and cell lines, which recapitulates the clinical scenario of therapy resistance and relapse. Using the parent and recurrent GBM cells from these models, we show that similar to parent GBM, the recurrent GBM cells also elicit a competent DNA damage response (DDR) post irradiation. However, the use of apical DNA damage repair sensory kinase (ATM and/or ATR) is different in the recurrent cells compared to parent cells. Consistently, we demonstrate that there is a differential clonogenic response of parent and recurrent GBM cells to the ATM and ATR kinase inhibitors with recurrent samples switching between these sensory kinases for survival emphasizing on the underlying heterogeneity within and across GBM samples. Taken together, here we report that recurrent tumours utilize an alternate DDR kinase to overcome radiation induced DNA damage. Since there is no effective treatment specifically for recurred GBM patients, these findings provide a rationale for developing newer treatment option to sensitize recurrent GBM samples by detecting in clinics the ability of cells to activate a DNA damage repair kinase different from their parent counterparts.

Molecular Effectors of Photodynamic Therapy-Mediated Resistance to Cancer Cells.

Photodynamic therapy (PDT) is currently enjoying considerable attention as the subject of experimental research to treat resistant cancers. The preferential accumulation of a non-toxic photosensitizer (PS) in different cellular organelles that causes oxidative damage by combining light and molecular oxygen leads to selective cell killing. However, one major setback, common among other treatment approaches, is tumor relapse and the development of resistance causing treatment failure. PDT-mediated resistance could result from increased drug efflux and decreased localization of PS, reduced light exposure, increased DNA damage repair, and altered expression of survival genes. This review highlights the essential insights of PDT reports in which PDT resistance was observed and which identified some of the molecular effectors that facilitate the development of PDT resistance. We also discuss different perceptions of PDT and how its current limitations can be overturned to design improved cancer resistant treatments.

MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Bcl-2-associated transcription factor 1 Ser290 phosphorylation mediates DNA damage response and regulates radiosensitivity in gastric cancer

BackgroundDNA damage response plays critical roles in tumor pathogenesis and radiotherapy resistance. Protein phosphorylation is a critical mechanism in regulation of DNA damage response; however, the key mediators for radiosensitivity in gastric cancer still needs further exploration.MethodsA quick label-free phosphoproteomics using high-resolution mass spectrometry and an open search approach was applied to paired tumor and adjacent tissues from five patients with gastric cancer. The dysregulated phosphoproteins were identified and their associated-pathways analyzed using Gene Set Enrichment Analysis (GSEA). The mostly regulated phosphoproteins and their potential functions were validated by the specific antibodies against the phosphorylation sites. Specific protein phosphorylation was further analyzed by functional and clinical approaches.Results832 gastric cancer-associated unique phosphorylated sites were identified, among which 25 were up- and 52 down-regulated. Markedly, the dysregulated phosphoproteins were primarily enriched in DNA-damage-response-associated pathways. Particularly, the phosphorylation of Bcl-2-associated transcription factor 1 (BCLAF1) at Ser290 was significantly upregulated in tumor. The upregulation of BCLAF1 Ser290 phosphorylation (pBCLAF1 (Ser290)) in tumor was confirmed by tissue microarray studies and further indicated in association with poor prognosis of gastric cancer patients. Eliminating the phosphorylation of BCLAF1 at Ser290 suppressed gastric cancer (GC) cell proliferation. Upregulation of pBCLAF1 (Ser290) was found in association with irradiation-induced γ-H2AX expression in the nucleus, leading to an increased DNA damage repair response, and a marked inhibition of irradiation-induced cancer cell apoptosis.ConclusionsThe phosphorylation of BCLAF1 at Ser290 is involved in the regulation of DNA damage response, indicating an important target for the resistance of radiotherapy.

Abstract 3098: Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML

Abstract Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML Relapse of acute myeloid leukemia (AML) is common and the main cause of mortality in this disease. Leukemia stem cells (LSCs) contribute to relapse however the mechanisms underpinning chemoresistance are not well understood. Here, we explored the DNA damage response in AML LSCs to investigate if enhanced DNA repair contributes to relapse and chemoresistance. We discovered that expression of DNA repair pathway genes were up-regulated in functionally defined LSC vs bulk AML cells (n= 227, accession number GSE76008), NES = 1.74, FDR = 0.057, and undifferentiated (n= 7) vs committed AML samples (n = 4, Princess Margaret Cancer Centre cohort), NES = 2.041, FDR = 0.000. Next, we investigated the expression of DNA damage repair genes and response to DNA damage in FACs sorted stem and bulk fractions of 8227 cells. 8227 cells are low passage primary AML cells that maintain a hierarchical organization with functionally defined stem cells in the CD34+CD38- fraction. Compared to bulk cells, the stem cell fraction of 8227 cells had increased expression of genes associated with DNA repair through homologous recombination (RAD51, XRCC2, XRCC3) and non-homologous end joining (XRCC4, XRCC5, PRKDC). Sorted 8227 cells were treated with daunorubicin, an intercalating anthracycline that causes double stranded breaks. DNA damage and repair were evaluated by measuring foci of 53BP1, RAD51 and γH2AX by fluorescent microscopy and quantified using image J. Compared to bulk cells, 8227 stem cells demonstrated increased DNA damage repair with increased foci of 53BP1 and RAD51 and decreased γH2AX foci. We recently discovered that the metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus to maintain stem cell number and function. Therefore, to understand how stemness impacts DNA damage repair, we selectively over-expressed HK2 in the nucleus of 8227 and NB4 cells by tagging HK2 with a nuclear localizing sequence. Over-expressing nuclear HK2 increased stem cell function as shown by clonogenic growth assays and engraftment into mouse marrow. We then treated these cells with daunorubicin and measured DNA damage repair. Increasing stem cell number and function by over-expressing nuclear HK2, increased 53BP1 and RAD51 foci and decreased γH2AX foci, similar to the phenotype observed in LSCs. As measured by the COMET assay, these cells had decreased levels of double strand DNA breaks under basal conditions and increased repair of DNA breaks after treatment with daunorubicin treatment. Over-expression of nuclear HK2 also conferred resistance to daunorubicin as measured by clonogenic growth assays. In summary, LSCs have increased levels of DNA repair genes and increased rates of DNA damage repair after exposure to chemotherapy. The accelerated DNA damage repair seen in LSCs may contribute to chemoresistance and subsequent disease relapse. Citation Format: Grace Egan, G.E. Thomas, Parasvi Patel, Veronique Voisin, Rose Hurren, Neil MacLean, Razq Hakem, Aaron D. Schimmer. Leukemia stem cells demonstrate enhanced DNA damage repair and chemoresistance in AML [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 3098.

Tyrosine Kinase c-MET as Therapeutic Target for Radiosensitization of Head and Neck Squamous Cell Carcinomas.

Simple SummaryThe overall five-year survival rate of patients with loco-regional advanced head and neck squamous cell carcinomas (HNSCC) is only around 40%. The curability of HNSCC with radiochemotherapy was previously found to be associated with clinical and biological parameters including tumor volume, hypoxia, epidermal growth factor receptor expression, and human papillomavirus infection status. Different signaling pathways, e.g., constitutively activated receptor tyrosine kinase signaling, increased DNA damage repair and intracellular defense against reactive oxygen species were identified as factors driving HNSCC progression and its resistance to therapy. c-MET was found to be hyperactivated in HNSCC and has been reported to drive tumor progression, therapy resistance, and metastatic spread. Here, we investigated the therapeutic potential of c-MET targeting strategies for HNSCC radiosensitization and discovered putative resistance mechanisms impeeding success of therapeutic intervention. This study highlights the importance of detailed knowledge about biological mechanisms and regulatory networks for future patient stratification and individualized treatment concepts.The receptor tyrosine kinase c-MET activates intracellular signaling and induces cell proliferation, epithelial-to-mesenchymal-transition and migration. Within the present study, we validated the prognostic value of c-MET in patients with head and neck squamous cell carcinoma (HNSCC) treated with radio(chemo)therapy using the Cancer Genome Atlas database and found an association of increased MET gene expression and protein phosphorylation with reduced disease-specific and progression-free survival. To investigate the role of c-MET-dependent radioresistance, c-MET-positive cells were purified from established HNSCC cell lines and a reduced radiosensitivity and enhanced sphere-forming potential, compared to the c-MET-depleted cell population, was found in two out of four analyzed cell lines pointing to regulatory heterogeneity. We showed that c-MET is dynamically regulated after irradiation in vitro and in vivo. Interestingly, no direct impact of c-MET on DNA damage repair was found. The therapeutic potential of eight c-MET targeting agents in combination with irradiation demonstrated variable response rates in six HNSCC cell lines. Amongst them, crizotinib, foretinib, and Pha665752 exhibited the strongest radiosensitizing effect. Kinase activity profiling showed an association of crizotinib resistance with compensatory PI3K/AKT and MAP kinase signaling. Overall, our results indicate that c-MET is conferring radioresistance in HNSCC through modulation of intracellular kinase signaling and stem-like features.

99529 The Role of ATF6-Mediated Signaling in PARP Inhibitor Resistant Ovarian Cancer

ABSTRACT IMPACT: This work has the potential to identify targetable pathways conveying resistance to PARP inhibitors that may improve ovarian cancer patient outcomes. OBJECTIVES/GOALS: High grade serous ovarian cancer is the deadliest gynecologic malignancy. PARP inhibitors are an FDA approved targeted therapy that is being used more and more frequently in the clinic. It is vital to understand mechanisms driving resistance to this therapy in order to develop treatments to improve patient responses. METHODS/STUDY POPULATION: RNA-sequencing and transcription factor analysis was used to identify pathways of interest. An AP-1 transcriptional reporter assays was used to confirm results of the transcription factor analysis. An unbiased lentiviral shRNA screen was used to identify AP-1 subunits promoting PARP inhibitor resistance. Lentiviral transduction allowed for the knockdown ATF6. Comet assays and two-plasmid systems were used to determine levels of DNA damage and levels of DNA damage repair respectively. RESULTS/ANTICIPATED RESULTS: PARP inhibitor resistant cell lines have increased WNT signaling which promotes to increased DNA damage repair. PARP inhibitor resistant cell lines also have increased AP-1 transcriptional activity, ATF6 expression, and active p38. ATF6 knockdown and p38 inhibition is sufficient to resensitize cells to PARP inhibition. Upon treatment with PARP inhibitors, ATF6 knockdown as well as p38 inhibition lead to increased DNA damage in PARP inhibitor resistant cell lines. RNA-sequencing reveals a signifcant overlap in downregulated genes in cells treated with a β-catenin inhibitor and cells with an ATF6 knockdown. DISCUSSION/SIGNIFICANCE OF FINDINGS: Due to the increasing prevalence of PARP inhibitors in the clinic, it is vital to uncover mechanisms contributing to resistance. This work has the potential to identify targetable pathways conveying resistance to PARP inhibitors that may improve ovarian cancer patient outcomes.

Leukemia Stem Cells Demonstrate Increased DNA Damage Repair and Chemoresistance in Acute Myeloid Leukemia

While most patients with AML achieve remission with standard induction chemotherapy, the majority ultimately relapse. Relapsed AML is due, at least in part, to the persistence of chemoresistant leukemia stem cells (LSCs). The mechanisms of chemoresistance in LSCs are not fully understood. Here, we explored DNA damage repair in LSCs. 8227 cells are low passage primary AML cells that maintain a hierarchical organization with functionally defined stem cells in the CD34+CD38- fraction. We FACS sorted 8227 cells into stem and bulk fractions and measured expression of DNA repair genes. LSCs were primed for DNA repair with increased expression of genes associated with homologous recombination (RAD51, XRCC2, XRCC3) and non-homologous end joining (XRCC4, XRCC5, PRKDC). Next, we treated the cell fractions with daunorubicin, an intercalating anthracycline that causes double stranded breaks. DNA damage and repair were evaluated by measuring foci of 53BP1, RAD51 and γH2AX by fluorescent microscopy and quantified using image J. Compared to bulk cells, 8227 stem cells demonstrated enhanced DNA damage repair with increased foci of 53BP1 and RAD51 and decreased γH2AX foci, compared to their basal levels. Similar findings were noted after exposing the stem and bulk cells to radiation. We recently discovered that the metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus to maintain stem cell number and function. Therefore, we selectively over-expressed HK2 in the nucleus of 8227 and NB4 cells by tagging HK2 with a nuclear localizing sequence (PAAKRVKLD). We confirmed selective over-expression of HK2 in the nucleus by immunoblotting and confocal microscopy. Over-expressing HK2 increased stem cell function as shown by clonogenic growth assays and engraftment into mouse marrow. We then treated these cells with daunorubicin and measured DNA damage repair. Over-expression of nuclear HK2 increased 53BP1 and RAD51 foci with decreased γH2AX foci, similar to the phenotype observed in LSCs. In addition, over-expression of nuclear HK2 conferred resistance to daunorubicin as measured by clonogenic growth assays. In summary, LSCs appear to be primed for DNA repair with increased levels of DNA damage repair genes. After exposure to chemotherapy and radiation, LSCs have increased repair of double strand DNA breaks compared to more differentiated blasts. This accelerated DNA damage repair may partly explain the increased chemoresistance seen in LSCs. Disclosures Schimmer: Takeda:Honoraria, Research Funding;Novartis:Honoraria;Jazz:Honoraria;AbbVie Pharmaceuticals:Other: owns stock ;Otsuka:Honoraria;Medivir AB:Research Funding.

CMTM7 Knockout Decreases The Radiosensitivity Of A549 Non-Small Cell Lung Cancer Cells By Increasing Akt Signaling

Radiotherapy is the main treatment for patients with early non-surgical and locally advanced non-small cell lung cancer (NSCLC). However, radiotherapy resistance is the main factor that leads to treatment failures such as local recurrence and distant metastasis in patients with NSCLC. Studies have demonstrated that radioresistance is associated with the constitutive activity of Akt-signaling. We previously demonstrated that CMTM7 as a tumor suppressor decreased the activation of Akt in the NSCLC cells. However, whether CMTM7 plays a role in radiosensitivity is still unknown. The purpose of this study is to elucidate the effect of CMTM7 on the radiosensitivity of A549 NSCLC cells and its mechanism. CRISPER/Cas9-mediated genome editing was used to knock out CMTM7 in A549 cells. CCK8 and clone formation assays were used to detect the sensitivity of A549 to ionizing radiation (IR). Flow cytometry was used to test the cell cycle and apoptosis. Western blotting and immunofluorescent assay were used to detect the number of γH2AX foci and the level of Akt-phosphorylation. A xenograft model in nude mice was used to test the in vivo radiosensitivity. We knocked out CMTM7 in A549 NSCLC cells using CRISPR/Cas9 genome editing strategy. Through a series of screens, two Cas9-CMTM7 clones were selected and CMTM7 was undetectable by RT-PCR and Western blotting. CCK8 and clone formation assays showed that knockout of CMTM7 decreased the sensitivity of A549 cells to radiotherapy compared with WT control group. Mechanically, CMTM7 knockout increased DNA damage repair, prolonged G2/M arrest, and enhanced the Akt phosphorylation upon IR, and the PI3K inhibitor LY294002 promoted the radiosensitivity in CMTM7knockout cells to the levels similar as WT control cells. In addition, CMTM7 knockout also markedly decreased the inhibition of tumor xenograft growth by IR in nude mice. Together, we found that knockout of CMTM7 decreased the radiosensitivity of A549 cells in vitro and in vivo. The mechanism of radioresistance might be that the constitutive activation of Akt decreased the repair of DNA damage induced by IR, consequently leading to radiotherapy resistance.

Persistent STAG2 mutation despite multimodal therapy in recurrent pediatric glioblastoma

Similar to their adult counterparts, the prognosis for pediatric patients with high-grade gliomas remains poor. At time of recurrence, treatment options are limited and remain without consensus. This report describes the genetic findings, obtained from whole-exome sequencing of a pediatric patient with glioblastoma who underwent multiple surgical resections and treatment with standard chemoradiation, as well as a novel recombinant poliovirus vaccine therapy. Strikingly, despite the variety of treatments, there was persistence of a tumor clone, characterized by a deleterious STAG2 mutation, whose deficiency in preclinical studies can cause aneuploidy and aberrant mitotic progression, but remains understudied in the clinical setting. There was near elimination of an EGFR mutated and amplified tumor clone after gross total resection, standard chemoradiation, and poliovirus therapy, followed by the emergence of a persistently STAG2 mutated clone, with rare mutations in PTPN11 and BRAF, the latter composed of a novel deleterious mutation previously not reported in pediatric glioblastoma (p.D594G). This was accompanied by a mutation signature shift towards one characterized by increased DNA damage repair defects, consistent with the known underlying STAG2 deficiency. As such, this case represents a novel report following the clinical and genetic progression of a STAG2 mutated glioblastoma, including treatment with a novel and emerging immunotherapy. Although STAG2 deficiency comprises only a small subset of gliomas, this case adds clinical evidence to existing preclinical data supporting a role for STAG2 mutations in gliomagenesis and resistance to standard therapies.

The role of deubiquitinating enzymes in cancer drug resistance.

Drug resistance is a well-known phenomenon leading to a reduction in the effectiveness of pharmaceutical treatments. Resistance to chemotherapeutic agents can involve various intrinsic cellular processes including drug efflux, increased resistance to apoptosis, increased DNA damage repair capabilities in response to platinum salts or other DNA-damaging drugs, drug inactivation, drug target alteration, epithelial-mesenchymal transition(EMT), inherent cell heterogeneity, epigenetic effects, or any combination of these mechanisms. Deubiquitinating enzymes (DUBs) reverse ubiquitination of target proteins, maintaining a balance between ubiquitination and deubiquitination of proteins to maintain cell homeostasis. Increasing evidence supports an association of altered DUB activity with development of several cancers. Thus, DUBs are promising candidates for targeted drug development. In this review, we outline the involvement of DUBs, particularly ubiquitin-specific proteases, and their roles in drug resistance in different types of cancer. We also review potential small molecule DUB inhibitors that can be used as drugs for cancer treatment.

ATM Expression Is Elevated in Established Radiation-Resistant Breast Cancer Cells and Improves DNA Repair Efficiency.

Repair of damaged DNA induced by radiation plays an important role in the development of radioresistance, which greatly restricts patients' benefit from radiotherapy. However, the relation between radioresistance development and DNA double-strand break repair pathways (mainly non-homologous end joining and homologous recombination) and how these pathways contribute to radioresistance are unclear. Here, we established a radioresistant breast cancer cell line by repeated ionizing radiation and studied the alteration in DNA repair capacity. Compared with parental sham-treated cells, radioresistant breast cancer cells present elevated radioresistance, enhanced malignancy, increased expression of Ataxia-telangiectasia mutated (ATM), and increased DNA damage repair efficiency, as reflected by accelerated γ-H2AX kinetic. These defects can be reversed by ATM inhibition or ATM knockdown, indicating a potential link between ATM, DNA repair pathway and radiosensitivity. We propose that cancer cells develop elevated radioresistance through enhanced DNA damage repair efficiency mediated by increased ATM expression. Our work might provide a new evidence supporting the potential of ATM as a potential target of cancer therapy.

Abstract 3478: APC loss mediates doxorubicin response in breast cancer via changes in intracellular drug concentration and DNA repair pathways

Abstract Chemoresistance is one of the leading causes of breast cancer related deaths. Understanding the molecular basis for chemoresistance is essential for novel therapeutic advancement to improve patient outcome. The Adenomatous Polyposis Coli (APC) tumor suppressor is either mutated or hypermethylated in up to 70% of sporadic breast cancer; however, little is known about how APC loss contributes to chemoresistance. Using mammary tumor cells isolated from the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we demonstrated that APC loss decreased doxorubicin (DOX) induced apoptosis. DOX, a commonly used chemotherapeutic in breast cancer, inhibits topoisomerase IIa, resulting in double stranded DNA breaks, resulting in cell cycle arrest to allow repair or apoptosis. We made the novel observation that APC loss in MMTV-PyMT;ApcMin/+ cells activated signal transducer and activator of transcription 3 (STAT3) thereby increasing the expression of the drug efflux pump, multidrug resistance protein 1 (MDR1). Therefore, we hypothesized that APC loss prevents doxorubicin-mediated cell death through: 1) reduced intracellular DOX and 2) alterations in DNA damage and repair. To investigate the intracellular accumulation of DOX, we first used calcein incorporation assay and demonstrated that APC loss increased MDR1 activity, which was restored by an MDR1 inhibitor. In addition, MDR1 inhibition sensitized the MMTV-PyMT;ApcMin/+ cells to DOX-mediated apoptosis. To investigate the effect of APC loss on DNA damage repair pathways, we initially monitored damage recognition pathways after 24-hour DOX treatment. The MMTV-PyMT;ApcMin/+ cells exhibited decreased γH2AX and ataxia-telangiectasia mutated (ATM) phosphorylation following DOX treatment compared to controls, suggesting decreased DNA damage either through decreased intracellular DOX or enhanced DNA repair. Decreased phosphorylation of Chk1 and Chk2 was also observed in DOX-treated MMTV-PyMT;ApcMin/+ cells. Preliminary data to investigate if this decreased DNA damage was due to increased DNA damage repair, via monitoring γH2AX expression throughout treatment and recovery from DOX, suggest enhanced DNA repair in MMTV-PyMT;ApcMin/+ cells. Using the ATM inhibitor, we observed an increase in DOX sensitivity in MMTV-PyMT;ApcMin/+ cells. Alternatively, ATR inhibition did not affect DOX-mediated apoptosis. We will further investigate the efficiency of repair pathways through reporter plasmids and radiation induced damage to separate the drug transport alterations. Finally, we will test whether combination therapy of doxorubicin with an MDR1 inhibitor will reduce tumor burden in vivo. Taken together, APC loss mediates DOX resistance via reducing intracellular DOX and increasing DNA damage repair demonstrating the potential use of combination therapy to overcome chemoresistance. Citation Format: Casey Stefanski, Kaitlyn Keffler, Stephanie McClintock, Lauren Milac, Jenifer Prosperi. APC loss mediates doxorubicin response in breast cancer via changes in intracellular drug concentration and DNA repair pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3478.

Abstract 554: APC loss alters DNA damage repair in breast cancer cells

Abstract Breast cancer is the second leading cause of cancer deaths in U.S. women. Many of these deaths have been attributed to the development of chemotherapeutic resistance. Thus, understanding the process of chemotherapeutic resistance will help develop new approaches to combat breast cancer and ultimately cause a decrease in breast cancer related deaths. The Adenomatous Polyposis Coli (APC) tumor suppressor is silenced or mutated in up to 70% of sporadic breast cancer; however, the possible effects of APC loss on chemoresistance has not been elucidated. Using the ApcMin/+ mouse crossed to the Polyoma middle T antigen (PyMT) transgenic model, we previously showed that APC loss decreased cisplatin and doxorubicin-induced apoptosis. We also have shown that APC loss increased mRNA expression of the multidrug resistance protein 1 (MDR1), which was augmented by doxorubicin treatment. Treatment with doxorubicin and A69, a STAT3 inhibitor, normalized MDR1 gene expression in MMTV-PyMT;ApcMin/+ cells. Here, we hypothesize that loss of APC causes an increase in doxorubicin efflux and a decrease in DNA damage. First, we show that APC loss increased MDR1 activity as measured by calcein flux. To determine whether MDR1 inhibition would restore doxorubicin induced DNA damage, a combination treatment of doxorubicin and the MDR1 inhibitor, Valspodar, was able to restore doxorubicin sensitivity in MMTV-PyMT;ApcMin/+ cells compared to the control MMTV-PyMT;Apc+/+ cells. In addition, we have shown a decrease of γH2AX, a marker of damaged DNA, in MMTV-PyMT;ApcMin/+ cells treated with doxorubicin suggesting that the MMTV-PyMT;ApcMin/+ cells either have less damage due to increased drug efflux via MDR1 or due to increased DNA damage repair. Due to APC binding to Topoisomerase IIa, we also examined the degree of Topoisomerase IIa inhibition (via doxorubicin and etoposide) in MMTV-PyMT;ApcMin/+ compared to MMTV-PyMT;Apc+/+ cells. Topoisomerase IIa activity will also be measured to determine the effect of APC loss on Topoisomerase IIa. To test the hypothesis that loss of APC affected DNA damage repair, the expression of phosphorylated H2AX was measured in MMTV-PyMT;ApcMin/+ and MMTV-PyMT;Apc+/+ cells treated with the following: doxorubicin, etoposide, Valspodar, or a combination treatment. The longevity of doxorubicin-induced double stranded breaks was observed. Future studies will measure DNA damage repair pathway efficiency via reporter plasmids. We also will measure downstream signaling of yH2AX. Overall with an understanding of the role of APC in DNA damage repair, the use of a combination therapy could be advantageous to overcome chemotherapeutic resistance. Citation Format: Stephanie M. McClintock Batista. APC loss alters DNA damage repair in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 554.

Potential role of cyclin F mRNA expression in the survival of skin melanoma patients: Comprehensive analysis of the pathways altered due to cyclin F upregulation.

Cyclin F is a part of the Skp, Cullin, F-box containing ligase complex. The activity of cyclin F includes cell cycle control, centrosome duplication and response to DNA damage. The cyclin F expression pattern is very similar to cyclin A, but cyclin F is an orphan cyclin without its cyclin-dependent kinase partner. There is little evidence concerning the role of cyclin F in cancer. In the present study, for the first time, we present analysis from The Cancer Genome Atlas (TCGA) data in the context of expression of cyclin F mRNA in melanoma patients. Our original in silico analysis, not published elsewhere before, revealed that high expression of cyclin F in melanoma patients is associated with worse overall survival. Cyclin F and ribonucleotide reductase family member 2 (RRM2) compose a functional axis responsible for nucleotide metabolism. Impairment in this pathway may contribute to increased DNA damage repair and drug resistance. Additionally, we analyzed the expression of RRM2 mRNA and discovered that high expression of RRM2 is associated with worse overall survival. To shed more light on cyclin F overexpression in melanoma, we analyzed all protein data available in the TCGA melanoma dataset. It was found that in patients with upregulated cyclin F mRNA, we noted increased activity of pathways related to cell cycle and DNA damage repair. These data will support further in vitro and in vivo studies on the involvement of cyclin F in skin cutaneous melanoma.

Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani

Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL) in the Indian subcontinent. Paromomycin (PMM), an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R) and the corresponding PMM sensitive (PMM-S) parasites revealed modulated expression of 500 genes (1.5 fold cut off) in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a) reduced oxidative phosphorylation; b) increased glycosomal succinate fermentation and substrate level phosphorylation; c) dependency on lipids and amino acids for energy generation; d) reduced DNA synthesis and increased DNA damage repair and e) decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca2+ channel that are also modulators of ABC transporters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO) levels while reactive oxygen species (ROS) level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania.