Mediated DNA Damage Research Articles

RFWD3 Participates in the Occurrence and Development of Colorectal Cancer via E2F1 Transcriptional Regulation of BIRC5.

Objectives: Colorectal cancer (CRC) is one of the most common human malignancies. It was reported that the alterations in the DNA damage response (DDR) pathways are emerging as novel targets for treatment across different cancer types including CRC. RFWD3 plays a critical role in replication protein A (RPA)-mediated DNA damage in cancer cells. More importantly, RFWD3 can response to DNA damage by positively regulating p53 stability when the G1 cell cycle checkpoint is activated. However, the functional significance of RFWD3 in CRC has not been reported in the existing documents.Materials and Methods: Here, we revealed high expression of RFWD3 in CRC tissues by IHC analysis and The Cancer Genome Atlas (TCGA) database. Besides, overexpression of RFWD3 in CRC cell lines was also confirmed by qRT-PCR and western blot assay. The Celigo cell counting method and wound-healing/transwell migration assay were applied to evaluate CRC cell proliferation and migration. The tumor growth indicators were quantified in nude mice xenografted with shRFWD3 and shCtrl RKO cells.Results: The results indicated that RFWD3 knockdown restricted CRC development in vitro and in vivo. In exploring the downstream mechanism of RFWD3’s action, we found that RFWD3 could transcriptionally activate BIRC5 by interacting with E2F transcription factor 1 (E2F1). Accordingly, we identified BIRC5 as a downstream gene of RFWD3 regulating CRC. Subsequent loss- and gain- of function experiments demonstrated that upon overexpressing BIRC5 in RKO cells with down-regulated RFWD3, the inhibitory effects of cell proliferation, migration and colony formation could be reversed, while the capacity of cell apoptosis was ameliorated, suggesting that the effects of RFWD3 depletion was mainly due to BIRC5 suppression.Conclusion: Taken together, this study revealed that RFWD3 participates in the occurrence and development of colorectal cancer via E2F1 transcriptional regulation of BIRC5.

SIRT1 protects the heart against doxorubicin-induced cardiotoxicity by mediating the DNA damage response via deacetylation of histone H2AX

Abstract Background Doxorubicin induces DNA damage not only in tumor cells but also in the cardiomyocyte, and accumulation of damaged DNA has been implicated in doxorubicin-induced cardiotoxicity. We previously found that cardiomyocyte-specific deletion of SIRT1, a NAD+-dependent histone/protein deacetylase, worsens doxorubicin-induced cardiotoxicity in mice. However, its molecular mechanism remains unclear. Phosphorylation of histone H2AX at Ser139 catalyzed by ATM (mutated in ataxia-telangiectasia) at the sites of DNA damage is a critical mediator for DNA repair. Purpose Here, we tested the hypothesis that deacetylation of H2AX by SIRT1 mediates DNA damage response to counteract doxorubicin-induced cardiotoxicity. Methods and results Wild-type (WT) mice and tamoxifen-inducible cardiomyocyte-specific SIRT1 knockout (SIRT1-cKO) mice at 3 month of age received doxorubicin (4 IP injections of 5 mg/kg/week) or a vehicle. Immunoblotting of myocardial lysates from mice 1 week after final doxorubicin showed that doxorubicin increased phospho-Ser139-H2AX level by 1.6-fold in WT, but such a response was blunted in SIRT1-cKO. Ser1981-phosphorylations of ATM induced by doxorubicin were similar in WT and SIRT1-cKO. DNA fragmentation evaluated by TUNEL staining revealed that the increase in TUNEL-positive nuclei by doxorubicin was more in SIRT1-cKO (0.13% to 0.38%) than those in WT (0.07% to 0.19%), suggesting higher DNA damage in SIRT1-cKO. In H9c2 cardiomyocytes, knockdown of SIRT1 also abolished the doxorubicin-induced Ser139-phosphorylation of H2AX without changing phospho-ATM levels. Increases in DNA damage evaluated by comet assay and cleavage of caspase-3 by doxorubicin were also enhanced in SIRT1-knockdown cells. Immunostaining for acetyl-Lys5-H2AX in the heart sections revealed that acetyl-Lys5-H2AX levels were increased in SIRT1-cKO by 58% compared with those in WT. In H9c2 cells, acetyl-Lys5-H2AX level was also increased by SIRT1 knockdown and reduced by expression of wild-type SIRT1. To test the role of the increased acetyl-Lys5-H2AX level under SIRT1 inhibition, we generated a mutant in which Lys5 was substituted to glutamine (K5Q H2AX) as a mimic of acetylated Lys5. In COS7 cells expressing WT or K5Q H2AX, Ser139-phosphorylation induced by doxorubicin was suppressed in K5Q mutant. In addition, doxorubicin-induced cleavage of caspase-3 was enhanced in H9c2 cells expressing K5Q H2AX as well as S139A H2AX, that cannot be phosphorylated at Ser139, compared with cells expressing WT H2AX. Conclusions These findings suggest that the increased Lys5 acetylation of H2AX via SIRT1 inhibition interferes Ser139 phosphorylation, leading to accumulation of damaged DNA and promotion of the apoptotic response. Such regulation of the DNA damage response contributes to protection by SIRT1 against doxorubicin-induced cardiotoxicity. Funding Acknowledgement Type of funding sources: None.

Correlation analysis of RDM1 gene with immune infiltration and clinical prognosis of hepatocellular carcinoma.

Purpose: Liver hepatocellular carcinoma (LIHC) is one of the most common primary malignant liver tumors worldwide. The RAD52 motif-containing protein 1 (RDM1) has been shown to play a role in mediating DNA damage repair and homologous recombination. The present study was designed to determine the expression of RDM1 and its prognostic value as well as its relationship with immune infiltration in LIHC patients.Methods: Oncomine and Tumor Immunoassay Resource were used to assess the expression of RDM1. PrognoScan and Kaplan–Meier bioinformatics database were used to analyze the impact of clinical influencing factors on prognosis. Finally, the Tumor Immune Assessment Resource (TIMER) and Gene Expression Analysis Interactive Analysis (GEPIA) databases were used to detect the correlation between the expression of RDM1 and expression of marker genes related to immune infiltration. Immunohistochemistry (IHC) method was used to detect the expression level of RDM1 in 90 cases of hepatocellular carcinoma and adjacent normal liver tissues.Results: RDM1 expression was up-regulated in most cancers. The expression of RDM1 was remarkably higher than that of the corresponding normal control genes in LIHC tissues. The increase in RDM1 messenger RNA (mRNA) expression was closely related to the decreases in overall survival (OS) and progression-free survival (PFS). Additionally, the increase in RDM1 mRNA expression was closely related to the infiltration levels of macrophages, CD8+ T cells and B cells and was positively correlated with a variety of immune markers in LIHC.Conclusion: The findings of the present study demonstrate that RDM1 is a potentially valuable prognostic biomarker that can help determine the progression of cancer and is associated with immune cell infiltration in LIHC.

Prodrug of ICRF-193 provides promising protective effects against chronic anthracycline cardiotoxicity in a rabbit model in vivo.

The anthracycline (ANT) anticancer drugs such as doxorubicin or daunorubicin (DAU) can cause serious myocardial injury and chronic cardiac dysfunction in cancer survivors. A bisdioxopiperazine agent dexrazoxane (DEX) has been developed as a cardioprotective drug to prevent these adverse events, but it is uncertain whether it is the best representative of the class. The present study used a rabbit model of chronic ANT cardiotoxicity to examine another bisdioxopiperazine compound called GK-667 (meso-(butane-2,3-diylbis(2,6-dioxopiperazine-4,1-diyl))bis(methylene)-bis(2-aminoacetate) hydrochloride), a water-soluble prodrug of ICRF-193 (meso-4,4'-(butan-2,3-diyl)bis(piperazine-2,6-dione)), as a potential cardioprotectant. The cardiotoxicity was induced by DAU (3 mg/kg, intravenously, weekly, 10 weeks), and GK-667 (1 or 5 mg/kg, intravenously) was administered before each DAU dose. The treatment with GK-667 was well tolerated and provided full protection against DAU-induced mortality and left ventricular (LV) dysfunction (determined by echocardiography and LV catheterization). Markers of cardiac damage/dysfunction revealed minor cardiac damage in the group co-treated with GK-667 in the lower dose, whereas almost full protection was achieved with the higher dose. This was associated with similar prevention of DAU-induced dysregulation of redox and calcium homeostasis proteins. GK-667 dose-dependently prevented tumor suppressor p53 (p53)-mediated DNA damage response in the LV myocardium not only in the chronic experiment but also after single DAU administration. These effects appear essential for cardioprotection, presumably because of the topoisomerase IIβ (TOP2B) inhibition provided by its active metabolite ICRF-193. In addition, GK-667 administration did not alter the plasma pharmacokinetics of DAU and its main metabolite daunorubicinol (DAUol) in rabbits in vivo. Hence, GK-667 merits further investigation as a promising drug candidate for cardioprotection against chronic ANT cardiotoxicity.

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.

Wild-type IDH2 protects nuclear DNA from oxidative damage and is a potential therapeutic target in colorectal cancer.

Although the role of isocitrate dehydrogenase (IDH) mutation in promoting cancer development has been well-characterized, the impact of wild-type IDH on cancer cells remains unclear. Here we show that the wild-type isocitrate dehydrogenase 2 (IDH2) is highly expressed in colorectal cancer (CRC) cells, and plays an unexpected role in protecting the cancer cells from oxidative damage. Genetic abrogation of IDH2 in CRC cells leads to reactive oxygen species (ROS)-mediated DNA damage and an accumulation of 8-oxoguanine with DNA strand breaks, which activates DNA damage response (DDR) with elevated γH2AX and phosphorylation of ataxia telangiectasia-mutated (ATM) protein, leading to a partial cell cycle arrest and eventually cell senescence. Mechanistically, the suppression of IDH2 results in a reduction of the tricarboxylic acid (TCA) cycle activity due to a decrease in the conversion of isocitrate to α-ketoglutarate (α-KG) with a concurrent decrease in NADPH production, leading to ROS accumulation and oxidative DNA damage. Importantly, abrogation of IDH2 inhibits CRC cell growth in vitro and in vivo, and renders CRC cells more vulnerable to DNA-damaging drugs. Screening of an FDA-approved drug library has identified oxaliplatin as a compound highly effective against CRC cells when IDH2 was suppressed. Our study has uncovered an important role of the wild-type IDH2 in protecting DNA from oxidative damage, and provides a novel biochemical basis for developing metabolic intervention strategy for cancer treatment.

The Cell Cycle Checkpoint Gene, RAD17 rs1045051, Is Associated with Prostate Cancer Risk.

Human RAD17, as an agonist of checkpoint signaling, plays an essential role in mediating DNA damage. This hospital-based case-control study aimed to explore the association between RAD17 rs1045051, a missense sin-gle nucleotide polymorphism (SNP), and prostate cancer risk. Subjects were 358 prostate cancer patients and 314 cancer-free urology patients undergoing treatment at the Zhujiang Hospital of Southern Medical University in China. RAD17 gene polymorphism rs1045051 was evaluated by the SNaPshot method. Compared with the RAD17 gene polymorphism rs1045051 AA genotype, there was a higher risk of prostate cancer for the CC gen-otype (adjusted odds ratio [AOR] = 1.731, 95% confidence interval [95%CI] = 1.031-2.908, p = 0.038). Compared with the A allele, the C allele was significantly associated with the disease status (AOR = 1.302, 95%CI = 1.037-1.634, p = 0.023). All these findings indicate that in the SNP rs1045051, both the CC genotype and C allele may have a substantial influence on the prostate cancer risk.

Abstract 2583: Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo

Abstract Head and neck cancer (HNC) is the seventh most prevalent cancer worldwide. The five-year survival rate is less than fifty percent despite significant advances in therapeutic approaches. Early diagnosis and treatment can help in prevention and disease management. Studies have reported that dietary intake of Allium vegetables or processed garlic that contains diallyl trisulfide (DATS) lowered the risk of various cancers. However, it has not been investigated for its efficacy and molecular mechanisms against HNC. Herein, we investigated the effect of DATS on HNC UMSCC-22A, UMSCC-22B, and Cal33 cells in vitro. DATS treatment significantly reduced the cell viability of HNC cells. DATS induced significant G2/M phase cell cycle arrest, which resulted in the accumulation of cyclin B1 and induced levels of p21. DATS-induced M phase arrest was attenuated by N-acetyl cysteine (NAC) treatment, which suggested that DATS mediates growth-suppressive effects by reactive oxygen species (ROS) generation. DATS treatment induced mitochondrial dysfunction. DATS induced ROS production led to apoptosis, which was attenuated by NAC treatment. DATS increased proapoptotic protein cleaved caspase-3, cleaved PARP, and Bax/Bcl2 ratio and decreased the levels of antiapoptotic protein XIAP. DATS induced ROS mediated DNA damage which was attenuated by NAC treatment. Further, DATS mediated increase in level of cleaved PARP, accumulation of Cyclin B1 and decrease in XIAP was attenuated by NAC treatment. DATS (1-10 μM) treatment resulted in inhibition of self renewal of HNC cancer stem cells (HNC CSCs) sphere formation. DATS reduced aldehyde dehydrogenase 1 (ALDH1) activity and CD133high/CD44high HNC CSC fraction. DATS-treated SCID mice tumor xenograft also revealed it's in vivo efficacy on HNC CSCs. Further, DATS treatment reduced the tumor weight, volume and reduced the levels of Ki67 cell proliferation marker in UMSCC22B head and neck cancer CD1 nude mice tumor xenograft. DATS treatment decreased tumor incidence and inhibited tumor promotion and progression in twenty two week C57BL6 4-Nitroquinoline 1-oxide (4-NQO)-induced mouse oral carcinogenesis model. DATS treatment reduced the cell proliferation marker Ki67 and increased TUNEL positive apoptotic cells in treatment groups compared to control group. Collectively, DATS inhibited cell proliferation, induced cell cycle arrest, and cell death via apoptosis involving DNA damage, mitochondrial dysfunction, and ROS generation. DATS treatment also reduced the HNC CSC fraction, sphere formation and ALDH1 activity. More importantly, DATS inhibited cancer incidence and progression in 4-NQO oral carcinogenesis study. Further, DATS inhibited HNC tumor growth and HNC CSC fraction in vivo. Thus, DATS could be a potential chemopreventive and chemotherapeutic agent against head and neck cancer. Citation Format: Sivapar V. Mathan, Su-Hyeong Kim, Shivendra V. Singh, Rana P. Singh. Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo [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 2583.

Microtubule disruption upon CNS damage triggers mitotic entry via TNF signaling activation.

Repair after traumatic injury often starts with mitotic activation around the lesion edges. Early midline cells in the Drosophila embryonic CNS can enter into division following the traumatic disruption of microtubules. We demonstrate that microtubule disruption activates non-canonical TNF signaling by phosphorylation of TGF-β activated kinase 1 (Tak1) and its target IkappaB kinase (Ik2), culminating in Dorsal/NfkappaB nuclear translocation and Jra/Jun expression. Tak1 and Ik2 are necessary for the damaged-induced divisions. Microtubule disruption caused by Tau accumulation is also reported in Alzheimer's disease (AD). Human Tau expression in Drosophila midline cells is sufficient to induce Tak1 phosphorylation, Dorsal and Jra/Jun expression, and entry into mitosis. Interestingly, activation of Tak1 and Tank binding kinase 1 (Tbk1), the human Ik2 ortholog, and NfkappaB upregulation are observed in AD brains.

Inflammatory Signature, Oxidative Stress, and DNA Damage Response in DBA Pathogenesis

Diamond-Blackfan anemia (DBA) is an inherited pure red cell aplasia caused mostly by pathogenic variants in genes encoding for ribosomal proteins (RPs). It was previously published that haploinsufficiency of RP leads to increased apoptosis of erythroid precursors and p53 upregulation in CD34+cells from DBA patients and in animal models. However, the exact mechanisms of p53 activation are still not completely understood. In our study, we examined the link between activation of p53 pathway, inflammation-evoked oxidative stress and DNA damage in DBA patients and cellular model.To investigate the pathogenesis of DBA, we prepared the RPL5- and RPS19-deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. The particular clones were heterozygous for different nucleotide size deletions in RPL5 or RPS19 genes resulting in decreased levels of these proteins to half when compared to control cells thus mimicking the RP haploinsuficiency in DBA patients. We examined the pathological consequences of RPL5 and RPS19 deficiency in both undifferentiated and differentiated cells; the differentiation was induced by either dimethyl sulfoxide - DMSO or hexamethylene bisacetamide - HMBA for at least 48 hours. Significantly decreased proliferation capacity and significantly increased apoptosis rate was detected for all undifferentiated RPL5- and RPS19-deficient clones in comparison with controlcells. Upon induction of erythroid differentiation, RPL5- and RPS19-deficient clones showed abnormal GATA1 expression (2-times lower than in control cells) and higher percentage of TUNEL+ apoptotic cells (RPL5-deficient cells 2.04%; RPS19-deficient cells 1.57%;controlcells 0.50%).The data from cellular models were consistent with an increased rate of apoptosis detected in the bone marrow of RPL5 and RPS19 mutant DBA patients by TUNEL assay (15-times more TUNEL+ cells in RPL5 and RPS19 mutant patients than in normal controls). These patients showed also induction of p53 in their bone marrow (p53 positive cells: 8.4% in RPL5-mutant patient and 7.3% in RPS19-mutant patient vs. 0.4% in normal controls). The activation of p53 was also confirmed in RPL5- and RPS19-deficient clones by detection of increased level of p53 phosphorylation.Because oxidative stress is known to induce p53, we examined oxidative DNA damage marker 8-oxoguanine (8-oxoG) in MEL-DBA clones. Cytospin slides of differentiated RPL5- and RPS19-deficient clones revealed elevated positivity for 8-oxoG in contrast to control cells. It is known that oxidative damage is caused by exposure of cells to inflammatory cytokines, which activate DNA damage response (DDR) through reactive oxygen species (ROS)-mediated DNA damage. We found upregulation of pro-inflammatory cytokines TNF-α and IL6 in RPL5- and RPS19-deficient cells and IL1b in RPS19-deficient cells. The increased expression of Map3k8 kinase or increased phosphorylation of p38 was detected in RPL5-deficient or RPS19-deficient cells; both of these molecules are known to play essential roles in the production of pro-inflammatory cytokines. We then analyzed the levels of pro-inflammatory cytokines in our DBA patients and observed that besides TNF-α, IL1b, and IL6 also other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF-γ, and GM-CSF) were upregulated in their serum. We also detected higher levels of ROS in differentiated MEL-DBA clones in comparison with controlcells. Increased ROS levels were also present in the serum of all DBA patients with elevated inflammatory cytokines in contrast to healthy controls. As a result of ROS production, DNA double-strand breaks can occur responding by phosphorylation of Ser-139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RPL5- and RPS19-deficient cells in comparison with control cells (mean intensity of fluorescence for RPL5-deficient cells: 2.63 ±0.34; RPS19-deficient cells: 3.29 ±0.41; and control cells: 1.02 ±0.22).Our results suggest that the defective ribosomal biogenesis is associated with the induction of inflammatory cytokine signature, oxidative stress, increased DNA damage and activation of DDR signaling. Further research is needed to elucidate the hierarchy of these processes.This study was supported by project GACR 15-13732S, AZV 16-32105A, IGA_LF_2017_015, and in part by LTAUSA17142 (BK). DisclosuresNo relevant conflicts of interest to declare.

Apobec Family of Proteins Mediate Genomic Changes in Multiple Myeloma By Inducing DNA Damage and C>T Mutations

Multiple myeloma (MM) is a heterogeneous disease characterized by unstable genome. Spontaneous DNA damage coupled with dysregulated repair accentuates the genomic instability seen in MM. The resulting clonal heterogeneity plays a critical role in resistance to treatment and relapse. Understanding the molecular factors that contribute to genomic instability in myeloma is therefore of great importance in developing better treatment strategies. Recently, a role for the AID/APOBEC family of cytidine deaminases in generating somatic mutations has been described with in MM as well as number of other cancers. We and others have previously shown that significant number of mutations bear APOBEC signature and interestingly, the APOBEC mutation signature correlates with sub clonal diversity in MM. We have now further explored role of APOBEC in MM with special focus on its ability to induce clonal diversity.We observed that APOBECs 3A, 3B, 3C, and 3G are highly expressed in MM. Using a fluorescent oligonucleotide based APOBEC deaminase assay to measure deaminase activity, we also observed significantly increased APOBEC activity in MM cell lines as well as primary patient MM cells. Using shRNA-mediated knockdown, we report that depletion of APOBECs in MM cell lines results in decreased DNA double strand breaks. We next investigated the impact of overexpression of APOBEC genes on genome integrity in MM cells. Over expression of APOBECs 3B or 3G in U266 and LP1 MM cell lines led to 3-5 fold increase in deaminase activity in MM cell extracts measured by APOBEC deaminase assay. Furthermore, APOBEC overexpression resulted in elevated double strand breaks as evidenced by increased gH2AX nuclear foci and by western blot. In order to determine the functional impact of APOBEC-induced mutations, we co-transfected EGFP plasmid along with APOBEC expression vectors in myeloma cells. 72-hrs after transfection, we isolated the EGFP plasmid, transformed bacteria, and sequenced the plasmid DNA isolated from 5 colonies from each group. Overexpression of APOBECs resulted in increased number of C>T mutations (3-5 fold) in the EGFP plasmid co-transfected into these cells. Interestingly, APOBEC over expression also resulted in the loss of GFP fluorescence in 20-30% of the cells compared to control cells, suggesting that overexpression of APOBECs can cause deleterious mutations leading to loss of function.APOBEC-mediated deamination of cytosine to uracil leads to generation of abasic sites that are cleaved by abasic endonuclease (APE1), a regulator of homologous recombination pathway dysregulated in MM contributing to genomic instability. Therefore, we investigated how modulating APOBEC activity affects downstream DNA damage repair pathways in the gain-of and loss-of-function cells. Using in vitro activity assays, we measured APE1 and HR activity in extracts collected from APOBEC overexpression and knockdown cells. Extracts from APOBEC overexpressing cells showed elevated APE1 and HR activity (>50%), whereas depletion of APOBEC proteins resulted in up to 80% reduction in HR and APE1 activity in the extracts. Interestingly, using a small molecule HR inhibitor we found that inhibiting HR pathway can reduce the DNA breaks induced by APOBEC overexpression. Furthermore, we investigated the acquisition of genomic changes such as amplifications, deletions, and SNVs over time, in control and APOBEC knockdown cells, using SNP arrays and whole genome sequencing (WGS). Our results show that knockdown of APOBECs reduces the accumulation of copy number changes, structural variants and mutations over time. This suggests that elevated expression of APOBECs may be contributing to genomic instability in MM cells.In summary, our results suggest that APOBEC mediated DNA damage could contribute to increased dysregulation of these important repair pathways and genome stability in myeloma. Inhibiting APOBEC activity could reduce the rate of accumulation of the ongoing genomic changes. DisclosuresNo relevant conflicts of interest to declare.

C-Terminal BRE Overexpression in 11q23-Rearranged and t(8;16) Acute Myeloid Leukemia Is Caused By Intragenic Transcription Initiation

Abstract TextOverexpression of the BRE (Brain and Reproductive organ-Expressed ) gene defines a distinct pediatric and adult acute myeloid leukemia (AML) subgroup (Balgobind et al, Leukemia 2010, and Noordermeer et al, Blood 2011 and 2012). BRE overexpression specifically associates with 11q23/MLL/KMT2A-rearranged AML, occurring in 70% and 50% of 11q23 childhood and adult AML cases, respectively. In adult 11q23-rearranged AML, BRE overexpression significantly associates with younger age. BRE functions in BRCA1-A (Breast Cancer 1) complex mediated DNA damage repair, BRISC (BRCC3 isopeptidase complex) complex mediated mitotic spindle assembly and in protection from apoptosis and senescence. Another constituent of the BRCA1-A/BRISC complexes is BRCC3 which is recurrently mutated in AML, especially in cases with a TET2 mutation (Huang et al., Haematologica, 2015). BRE is relatively highly expressed in blood cells in general but its exact function in hematopoiesis is unknown.By performing genome-wide ChIP-sequencing analyses on primary KMT2A-MLLT3 patient material (n=9) we identified a promoter enriched for active chromatin marks (H3K4me3/H3K27Ac) in BRE intron 4, exclusively in AML cases with BRE overexpression. Both 5' RACE end RNA-sequencing identified strong biallelic expression of a previously unknown C-terminal BRE transcript. This transcript starts near the intragenic promoter, with BRE intron 4 sequences spliced to exon 5 and downstream sequences. RNA-seq and qRT-PCR showed that the downstream transcript is expressed 5-8-fold higher compared to the full-length transcript, explaining BRE overexpression. Of note, two start codons in exon 5 of the downstream BRE transcript may cause the formation of N-terminally truncated BRE isoforms.By performing a qRT-PCR that specifically recognizes the new BRE transcript on samples from a large cohort of pediatric and adult AML (n=195) we observed that it was highly expressed in 60% of 11q23/ KMT2A -rearranged (n=58) and 70% of t(8;16)/ KAT6A-CREBBP cases (n=20), while it was virtually absent from other AML subsets (n=101) and normal hematopoietic tissues (n=14). In gene reporter assays, the AML-specific fusion protein KMT2A-MLLT3 transactivated the intragenic BRE promoter. Further epigenome analyses revealed 97 additional intragenic promoter marks frequently bound by KMT2A in AML with C-terminal BRE overexpression. The bound genes may be part of a context-dependent KMT2A-MLLT3-driven oncogenic program, because they were higher expressed in this AML subtype compared to other cytogenetically-defined AML groups (p<0.01). C-terminal BRE might be a contributor to this oncogenic program because in a case with relapsed AML, we observed an ins(11;2) fusing CHORD1 in frame to BRE exon 5. The CHORDC1 fusion to BRE exon 5 is remarkable because the alternate BRE transcript starts with exon 1b also fused to exon 5. This means that if the CHORDC1-BRE and alternate BRE transcripts would be translated, they would contain a similar C-terminal part of the BRE protein. The CHORDC1 disruption may also contribute to AML pathogenesis because it was recently shown that heterozygous Chord1 (a.k.a. Morgana) inactivation in mice results in the spontaneous development of atypical chronic myeloid leukemia (Di Savino, Blood, 2015). We conclude that abnormal expression of C-terminal alternate BRE sequences specific for AML is caused by distinct, non-random events; by a chromosomal abnormality and by intragenic transcription initiation in 11q23/ KMT2A -rearranged and t(8;16)/ KAT6A-CREBBP AML. These findings warrant further studies to determine whether leukemia-specific alternate BRE contributes to disease pathogenesis and how abnormal intragenic transcription activation in AML is brought about. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

AURKA Regulates the Mechanism of ATR Mediated DNA Damage Response about Drug Resistance in Multiple Myeloma

Objective: Multiple myeloma (MM) is a cancer formed by malignant plasma cells. So far, drug resistance and relapse are major clinical problems in MM. Our previous studies showed that some chromosomal instability genes are involved in drug resistance, which AURKA expression is closely related survival. But the correlation between AURKA expression and clinical and how to participate in the mechanism of proliferation and drug resistance are unknown.Methods: Analyzing the Gene expression profiling (GEP) data and the relationship between AURKA expression and prognosis in MM. Bone marrow plasma cells from MM patients and health donor were collected by CD138. Detecting oncogene AURKA in presence of deletion and amplification by FISH. Detecting expression of mRNA by real-time qPCR. Immunofluorescence (IF) was used to detect the expression of protein. Comet assay was used to evaluate DNA repair. Then using the AURKA inhibitors and targeting AURKA, CCK-8 is used to detect the proliferation, WB detection of apoptosis, DNA Damage protein, IF detection of spindle formation and analyzing whether the AURKA is correlated with chromosome instability and drug resistance. In vivo and in vitro, combined with clinical medication bortezomib, Melphalan, lenalidomide. We detect apoptosis and DNA damage related protein and tumor volume in NOD/SCID mice. Changes in the culture supernatant of DNA damage response factor is used by real-time qPCR technique and ELASA detection of cell mass.Results: 1: Analyzing 350 patients with GEP data, the prognosis of patients with the AURKA high expression are better than the lowers (P=0). Patients with high expression of AURKA are mainly distributed in the high proliferation group. 2: Detecting the expression of mRNA by Real time qPCR technique, AURKA expression is significantly higher in newly diagnosed patients with 27.5% (n =11/n=40). The expression of AURKA protein is detected by IF assay. The results show that AURKA expression was higher in newly diagnosed (36.84%, n=14/n=38) and relapsed (50%, n=20/n=40) than that in normal donors. Patients with high expression of AURKA are closely related to the clinical relevance of anemia, bone destruction, high CR and high LDH. To further detect the relationship between AURKA expression and chromosome DNA copy number, the results show that AURKA copy number is normal in newly diagnosed patients (n=20). But there are 10% patients (n=2/n=20) with AURKA amplified in relapsed patients. 3: In vivo and in vitro, AURKA inhibitor combined with Bomtzomib, lenalidomide and Melphalan could enhance the sensitivity of BTZ and xenograft tumor biopsy showed reduced group AURKA, Ki67 expression. After over expression of AURKA, the proportion of cells with a single nucleus was decreased and the formation of multiple nuclei increased. Knocking down the AURKA, spindle formation is disorder. The expression of gamma-H2AX molecules and 53BP1 related DNA damage is abnormal. WB suggests that DNA damage is inhibited, phosphorylation of ATR, ATM, CHK2 reduction, phosphorylation of gamma-H2AX and BRCA increased. Further, real time qPCR technique and ELASA detection of DNA damage response factor in cell culture supernatant is mainly changed, such as IL-1 and IL6.Conclusion: High expression of AURKA is associated with poor prognosis in MM; Inhibition of AURKA can promote apoptosis, DNA damage and reduce the secretion of IL-6 that could enhance the sensitivity to bortezomib, lenalidomide and Melphalan. The abnormal expression of AURKA related spindle formation disorder and cell nuclear phenomena that cause chromosomal instability. AURKA is mainly involved in the drug resistance through the DNA damage response ATR-CHK1-gammaH2AX pathway. DisclosuresNo relevant conflicts of interest to declare.

The Landscape of Circular RNAs in Drug Resistant TCF3-Rearranged Childhood Leukemia

Treatment resistance is associated with poor prognosis in acute lymphoblastic leukemia (ALL). The underlying causes are largely unknown and specific risk markers and treatment options are consequently lacking. Efforts need to be made to identify new therapeutic targets in drug resistant patients who are left without other treatment options. This applies in particular to ALL patients bearing the t(17;19)(q22;p13) translocation that leads to the expression of the oncogenic chimeric transcription factor TCF3-HLF . Due to treatment resistance these patients typically suffer from relapse and die within two years from diagnosis.Circular RNAs may present such novel targets. Similar to linear RNAs, circRNAs are derived from parental genes. They are circularized by backsplicing and are remarkably stable. CircRNAs may regulate the expression of their parental gene, e.g. by sequestration of miRNAs or RNA binding proteins. CircRNAs have been shown to play a role in cancer by regulating microRNAs with tumor suppressive or oncogenic function (including let-7 and miR-7) and dysregulated circRNA expression has been observed in a variety of human tumors.Therefore we analyzed the landscape of circular RNAs in the drug resistant TCF3-HLF + cell line HAL01 and compared our findings to RNA expression data of a cohort of TCF3-HLF + patients (n=5) at diagnosis and remission. As controls, we used a cell line (697) and data derived from matched diagnosis/remission samples of patients (n=5) with the drug sensitive ALL subtype TCF3-PBX1 .Cell lines underwent whole-exome, -transcriptome and -circRNome sequencing. Single nucleotide variation, indel and copy number analysis revealed only few and not previously in TCF3-HLF+ ALL reported somatic mutations (MSH6, EP300) in HAL01. Transcriptome sequencing confirmed the expression of a TCF3-HLF fusion transcript joining exon 16 of TCF3 to exon 4 of HLF. 54 inserted nontemplate nucleotides indicated terminal deoxynucleotide transferase activity characteristic of an early B cell stage. Similar to leukemic TCF3-HLF+ patient samples, expression of the B cell transcription factor PAX5 was halved, which may account for the block in B cell development. Gene expression analysis restricted to genes specifically expressed in HAL01 and not expressed in 697 revealed a functional cluster comprised of GO terms implicated in drug resistance as most highly enriched.We used a bioinformatic approach to identify circRNAs in RNAseq data of TCF3-HLF+ and TCF3-PBX1+ patient samples. We detected 556 putative circRNAs. 249 genes expressed specific circRNAs only in leukemic and not in remission samples. Of those, 109 circRNAs were exclusively expressed in drug resistant TCF3-HLF -positive leukemia and 9 derived from parental genes such as the lysine demethylase KDM1A involved in p53 mediated DNA damage signaling were recurrently detected.Enrichment of circRNAs by RNase R digest and subsequent sequencing led to detection of 14,311 circular RNAs in HAL01 and 697. 7,918 of those were described in the circBase database, 6,393 were novel. The circRNAs were derived from 4,652 parental genes. 1 to 39 circRNAs were transcribed per gene. The most highly, but ubiquitiously expressed circRNA was derived from the gene NRIP1, a corepressor that modulates a variety of transcription factors. Several circRNAs derived from genes known to be involved in ALL pathogenesis (PAX5, IKZF1 and RUNX1) were detected. CircRNAs derived from the drug transporter gene Multidrug resistance-associated protein 1 (MRP1) were expressed in drug resistant but not in sensitive cells. Two of those were also detected in a leukemic TCF3-HLF+ patient sample. MRP1 detoxifies chemotherapeutic drugs by export of these substances out of the cells. Clinically relevant substrates of MRP1 are anticancer drugs, including vincristine, etoposide, topotecan and methotrexate. Drug screening of TCF3-HLF+ patient derived xenografts previously demonstrated explicit resistance against these drugs. Overexpression of MRP1 and similar transporters is considered a major cause of multidrug resistant tumor phenotypes and may be due to promoter hypomethylation or gene amplification. Upregulation of these transporters up to 10 fold has been observed in TCF3-HLF+ leukemia. Our results may add another layer of regulation and may indicate circular RNAs as novel biomarkers or therapeutic targets for patients with therapy resistant, fatal disease. DisclosuresNo relevant conflicts of interest to declare.

An Escherichia coli Effector Protein EspF May Induce Host DNA Damage via Interaction With SMC1.

Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is an important foodborne pathogen that causes human diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. EspF is one of the most important effector proteins injected by the Type III Secretion System. It can target mitochondria and nucleoli, stimulate host cells to produce ROS, and promote host cell apoptosis. However, the mechanism of the host-pathogen interaction leading to host oxidative stress and cell cytotoxic effects such as DNA damage remains to be elucidated. Here, we used Cell Counting Kit-8 (CCK-8) assays and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) ELISA to study cell viability and DNA oxidative damage level after exposure to EspF. Western blot and immunofluorescence were also used to determine the level of the DNA damage target protein p-H2AX and cell morphology changes after EspF infection. Moreover, we verified the toxicity in intestinal epithelial cells mediated by EspF infection in vivo. In addition, we screened the host proteins that interact with EspF using CoIP-MS. We found that EspF may more depend on its C-terminus to interact with SMC1, and EspF could activate SMC1 phosphorylation and migrate it to the cytoplasm. In summary, this study revealed that EspF might mediate host cell DNA damage and found a new interaction between EspF and the DNA damage repair protein SMC1. Thus, EspF may mediate DNA damage by regulating the subcellular localization and phosphorylation of SMC1.

TRESK Regulates Gm11874 to Induce Apoptosis of Spinal Cord Neurons via ATP5i Mediated Oxidative Stress and DNA Damage.

Reportedly, TWIK-related spinal cord K+ (TRESK) deficiency in spinal cord neurons positively correlates with the mechanism underlying neuropathic pain (NP). However, the precise effects of TRESK on neurons of the spinal cord remain elusive. In the present study, we investigated the impact of TRESK silencing on spinal cord neurons to further elucidate the downstream mechanisms of TRESK. Herein, neurons of the dorsal spinal cord were cultured as a cell model for investigations. Apoptosis, oxidative stress, and DNA damage-related proteins were evaluated. Additionally, flow cytometry, microarray profiling, real-time polymerase chain reaction (PCR), western blotting, fluorescence in situ hybridization (FISH), immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) were performed. In cultured neurons, the downregulation of TRESK mRNA expression induced apoptosis of dorsal spinal cord neurons. Using real-time PCR and western blotting, the upregulation of LncRNA Gm11874 (Gm11874) and ATP5i, screened from the gene chip, was confirmed. On silencing TRESK, expression levels of γ-H2AX, poly [ADP-ribose] polymerase 1 (PARP-1), FoxO1, FoxO3, MitoSOX, malondialdehyde (MDA), and 8-hydroxy-2' -deoxyguanosine (8-OHdG), which are known indices of oxidative stress and DNA damage, were significantly elevated. Moreover, ATP induced oxidative stress, DNA damage, and apoptosis were reduced by ATP5i siRNA. Finally, Gm11874 and ATP5i were co-expressed in spinal cord neurons in a FISH experiment, and the expression of ATP5i was positively regulated by Gm11874. These results implied that ATP5i induced oxidative stress and DNA damage, resulting in neuronal apoptosis, and Gm11874 was confirmed to act upstream of ATP5i. Our study revealed that TRESK silencing upregulated Gm11874 to induce apoptosis of spinal cord neurons, which resulted in ATP5i promoting oxidative stress and DNA damage. These findings could highlight the TRESK-mediated NP mechanism.

Investigating pY240‐PTEN:Ki‐67 Interaction and its Role in DNA Damage Response

Glioblastoma (GBM) is the most aggressive and common malignant primary brain tumor. Standard of care for GBM patients involves chemotherapy and ionizing radiation which induce DNA damage to kill tumor cells; however resistance to these treatment modalities commonly develop through various mechanisms. One novel mechanism of radioresistance identified in the Furnari lab is through enhanced homologous recombination DNA damage response mediated by nuclear-localized tyrosine-phosphorylated PTEN (pY240-PTEN) recruited to chromatin through interaction with the PP1 Binding Domain (PP1BD) of Ki-67. The objective of this study is to characterize the regulation and molecular interactions involved in pY240-PTEN and Ki-67 binding. Biotin-tagged synthetic peptides based on the Ki-67 PP1BD are being used in streptavidin pull down experiments to interrogate the critical amino acids required for pY240-PTEN:Ki-67 interaction. Orthogonally, a minigene expressing the Ki-67 PP1BD and Leucine-Rich Repeat domain (required for tethering to chromatin) fused to a C-terminal mNeonGreen is employed in glioma stem-like cell lines to interrogate the impact of specific amino acid mutations on PTEN:Ki-67 interaction and subsequent disruption of pY240-PTEN mediated DNA damage response. Since phosphorylation of the PP1BD has been shown to affect binding of Ki-67 to other phosphatases, namely PP1γ, the peptides and minigene construct will additionally be used to investigate how phosphorylation of the PP1BD can affect PTEN:Ki-67 interactions. Specifically, phosphorylation of the Ki-67 PP1BD by Aurora B Kinase and Cyclin-Dependent Kinase 1 will be investigated through phosphorylated synthetic peptides and minigene constructs harboring mutations of candidate residues to non-phosphorylatable alanine, or phosphomimetic glutamic acid or aspartic acid. The insight into this interaction will be leveraged in future studies to evaluate competitor peptides based on Ki-67 for their ability to disrupt this binding and sensitize cells to radiation, as well as to guide CRISPR/Cas9 Ki-67 knockin mutations to determine the effects of Ki-67 mutation in vivo.

ATM mediated DNA double‐strand breaks accumulate in LRRK2 G2019S Parkinson's disease

Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting over one million people in the US. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of inherited and idiopathic PD. We were the first to show that mitochondrial DNA (mtDNA) damage is caused by the most common mutation in LRRK2 (G2019S) and inhibition of LRRK2 kinase activity restores mtDNA integrity in PD models. However, whether aberrant LRRK2 kinase activity due to PD-linked mutations has broad impact on nuclear genome integrity is unknown. Using LRRK2G2019S/G2019S knock-in (KI) human embryonic kidney 293 (HEK293) cells obtained by CRISPR/Cas9 gene editing, our preliminary results indicate nuclear DNA damage is increased, including DNA double-strand breaks (DSBs) as assayed by a neutral comet assay. Consistent with DSB accumulation, we observed significantly increased γ-H2AX and 53BP1 foci. ATM is activated by DSBs and phosphorylates several key proteins that initiate the DNA damage response, cell cycle arrest, DNA repair or apoptosis. We found that basal levels of ATM pS1981 are increased in this in vitro LRRK2 G2019S model, which is an autophosphorylation site that correlates with DNA damage-mediated activation. Additionally, downstream substrates CHK2 (pT68), and P53 (pS15) are similarly increased in LRRK2G2019S/G2019S KI cells compared to isogenic wild-type control cells, substantiating that the ATM-mediated DNA damage response pathway has been up-regulated with the LRRK2 G2019S mutation. Blocking either LRRK2 or ATM kinase activity pharmacologically, significantly reversed LRRK2 G2019S-induced γ-H2AX foci. Overall these results suggest DSBs accumulate in LRRK2 PD, which in turn activate a sustained ATM mediated DNA damage response, which may lead to cell cycle arrest, aberrant DNA repair, and/or cell death. Further understanding of the functional relationship between LRRK2 and ATM offers new molecular insights into PD pathogenesis.

Acetaminophen Induces DNA Damage and ATM Mediated DNA Damage Repair in Mouse Hepatocytes

Acetaminophen (APAP) is a common analgesic/antipyretic that is widely used throughout the world. While safe at therapeutic doses, it presents a risk for acute liver failure at higher dosages. Accounting for 50% of the cases of liver failure, drug-induced liver toxicity is the most common cause of liver damage in western societies. With the only efficacious form of treatment being liver transplant, it is imperative to find alternative forms of treatment. Gaining a greater understanding of the molecular pathology contributing to the progression of liver failure, we allow insights on methods to enhance patient care and quality of life. Acute liver injury from APAP is associated with increased levels of inflammation, oxidative stress, and hepatic cell death. High levels of oxidative stress damages multiple systems within the cell, most notability the DNA. In response to DNA damage, DNA damage response (DDR) markers are activated leading to cell cycle arrest, DNA repair and the eventual death of the cell if damage is unable to be repaired. Our hypothesis is that the accumulation of DNA damage results in the activation of the DDR, which then contributes to the further progression of APAP-induced liver injury. Methods Mouse hepatocytes (FL86B cells) were treated with APAP (1mM-100mM) and viability was assessed. FL83B cells were then treated for 24 hours with 5mM APAP, a dose that gave 70% of viability after 24 hr. A DNA comet assay was performed on the treated cells to assay DNA damage. ATM, phospho-ATM, phospho-Chk2 and gH2AX were assessed via immunofluorescence. ATM, Chk2, and BP53 were assess via qPCR. To assess the effects that the removal of ATM has on APAP exposure, the FL86B cells were transfected with ATM siRNA for 48 hours prior to 24-hour exposure of 5mM APAP. ATM and Chk2 were assessed via qPCR and ATM, phospho-ATM, Chk2 and phospho-Chk2 were assessed via immunofluorescence. Results APAP exposure to mouse hepatocytes showed a marked increase in the accumulation of DNA damage when compared to untreated hepatocytes. An increase in the markers for DNA double strand breaks (DSB), gH2AX and 53BP, as well the comet assay confirmed the accumulation of DNA damage after exposure to APAP. An increase of ATM and phosphor-ATM and its downstream effector Chk2 and active form phospho-Chk2 were increased after exposure to APAP. The removal of ATM attenuated the phosphorylation of ATM and Chk2 and enhanced cell viability with exposure to APAP. Conclusion Exposure to APAP induces DNA damage within hepatocytes. This elevated amount of DNA damage results in the activation of ATM and the DDR through Chk2. ATM-mediated signaling may be a potential driver for hepatocellular death and ALF. Agents designed to inhibit ATM-mediated signaling or the DDR response need to be assessed as potential therapeutic targets for the treatment of APAP-induced liver damage.

Recruitment of KMT2C/MLL3 to DNA Damage Sites Mediates DNA Damage Responses and Regulates PARP Inhibitor Sensitivity in Cancer.

When recruited to promoters, histone 3 lysine 4 (H3K4) methyltransferases KMT2 (KMT2A-D) activate transcription by opening chromatin through H3K4 methylation. Here, we report that KMT2 mutations occur frequently in non-small cell lung cancer (NSCLC) and are associated with high mutation loads and poor survival. KMT2C regulated DNA damage responses (DDR) through direct recruitment to DNA damage sites by Ago2 and small noncoding DNA damage response RNA, where it mediates H3K4 methylation, chromatin relaxation, secondary recruitment of DDR factors, and amplification of DDR signals along chromatin. Furthermore, by disrupting homologous recombination (HR)-mediated DNA repair, KMT2C/D mutations sensitized NSCLC to Poly(ADP-ribose) polymerase inhibitors (PARPi), whose efficacy is unclear in NSCLC due to low BRCA1/2 mutation rates. These results demonstrate a novel, transcription-independent role of KMT2C in DDR and identify high-frequency KMT2C/D mutations as much-needed biomarkers for PARPi therapies in NSCLC and other cancers with infrequent BRCA1/2 mutations. SIGNIFICANCE: This study uncovers a critical role for KMT2C in DDR via direct recruitment to DNA damage sites, identifying high-frequency KMT2C/D mutations as biomarkers for response to PARP inhibition in cancer.