dsDNA Break Repair Research Articles

Does single-strand DNA break repair capacity influence oocyte maintenance and quality?

Human genome-wide association studies and evidence from animal models link ovarian ageing to double-strand (ds)DNA break repair capacity. Is there a connection between single-strand (ss)DNA repair mechanisms and ovarian function? We hypothesize that endogenous cellular processes subject oocytes to ssDNA lesions, and thus, ssDNA repair capacity is fundamental to their survival and maintenance.

Double-stranded DNA binding function of RAD51 in DNA protection and its regulation by BRCA2

RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.

Estrogen receptor inhibition mediates radiosensitization of ER-positive breast cancer models

Endocrine therapy (ET) is an effective first-line therapy for women with estrogen receptor-positive (ER + ) breast cancers. While both ionizing radiation (RT) and ET are used for the treatment of women with ER+ breast cancer, the most effective sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we sought to understand the effects of inhibiting estrogen receptor (ER) signaling in combination with RT in multiple preclinical ER+ breast cancer models. Clonogenic survival assays were performed using variable pre- and post-treatment conditions to assess radiosensitization with estradiol, estrogen deprivation, tamoxifen, fulvestrant, or AZD9496 in ER+ breast cancer cell lines. Estrogen stimulation was radioprotective (radiation enhancement ratios [rER]: 0.51–0.82). Conversely, when given one hour prior to RT, ER inhibition or estrogen depletion radiosensitized ER+ MCF-7 and T47D cells (tamoxifen rER: 1.50–1.60, fulvestrant rER: 1.76–2.81, AZD9496 rER: 1.33–1.48, estrogen depletion rER: 1.47–1.51). Combination treatment resulted in an increase in double-strand DNA (dsDNA) breaks as a result of inhibition of non-homologous end joining-mediated dsDNA break repair with no effect on homologous recombination. Treatment with tamoxifen or fulvestrant in combination with RT also increased the number of senescent cells but did not affect apoptosis or cell cycle distribution. Using an MCF-7 xenograft model, concurrent treatment with tamoxifen and RT was synergistic and resulted in a significant decrease in tumor volume and a delay in time to tumor doubling without significant toxicity. These findings provide preclinical evidence that concurrent treatment with ET and RT may be an effective radiosensitization strategy.

Dysregulation of X-ray repair cross-complementing 4 expression in the eutopic endometrium of women with endometriosis.

Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of dsDNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Toward this, we focused on the X-ray repair cross-complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (P < 0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the Gene Expression Omnibus database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AX and 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (hTERT immortalized human endometrial stromal cell line (ThESCs)) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.

Inhibition of Estrogen Receptor Signaling as a Strategy for Radiosensitization of ER+ Breast Cancers

Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of tumor initiation and progression and therefore a target of first-line therapies for ER-positive (ER+) breast cancer (BC) patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of women with ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in multiple preclinical models.Clonogenic survival assays were used to assess radiosensitization. ER inhibition was accomplished with tamoxifen and the selective ER degrader (SERD), fulvestrant, in ER+ MCF-7 and T47D cells or ER-negative (ER-) SUM-159 cells. DNA damage was assessed by yH2AX foci. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) was measured by RAD51 foci or using a pYFP reporter, respectively. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Western blotting was used to quantify protein expression. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo where mice were pretreated with tamoxifen for one or six days prior to starting RT. Synergy was determined using the fractional tumor volume method.One-hour pretreatment with tamoxifen prior to RT resulted in radiosensitization of ER+ MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER- SUM-159 cells (enhR: 0.99-1.02). MCF-7 and T47D cells treated with tamoxifen and RT did not alter the kinetics of dsDNA break repair as measured by yH2AX foci (P > 0.05) but demonstrated a decrease in NHEJ-mediated repair (P < 0.05). No changes were observed in HR-mediated repair by Rad51 foci (P > 0.05). While cell cycle arrest was induced at 24 hours after RT, no changes were observed with tamoxifen treatment in combination with RT. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with treatment of tamoxifen, RT, or the combination (P > 0.05). In vivo, an MCF-7 xenograft study demonstrated a significant delay in time to tumor doubling (17 days in control, 40 days with tamoxifen, 32 days with RT, and undefined with combination; P < 0.0001) and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT with synergy noted with combination treatment.Our data suggest that endocrine therapies may be effectively used to radiosensitize ER+ breast tumors. This work also supports further clinical investigation of the timing of RT for patients receiving ET as concurrent use may be more effective than sequential, especially as ET prior to RT is increasingly used as a bridging therapy during the COVID pandemic.

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine–aspartate domain–containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.

Structural and mechanistic insights into the Artemis endonuclease and strategies for its inhibition.

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-β-lactamase (MBL) and β-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its β-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.

Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting

E. coli RecBCD, a helicase/nuclease involved in double stranded (ds) DNA break repair, binds to a dsDNA end and melts out several DNA base pairs (bp) using only its binding free energy. We examined RecBCD-DNA initiation complexes using thermodynamic and structural approaches. Measurements of enthalpy changes for RecBCD binding to DNA ends possessing pre-melted ssDNA tails of increasing length suggest that RecBCD interacts with ssDNA as long as 17–18 nucleotides and can melt at least 10–11 bp upon binding a blunt DNA end. Cryo-EM structures of RecBCD alone and in complex with a blunt-ended dsDNA show significant conformational heterogeneities associated with the RecB nuclease domain (RecBNuc) and the RecD subunit. In the absence of DNA, 56% of RecBCD molecules show no density for the RecB nuclease domain, RecBNuc, and all RecBCD molecules show only partial density for RecD. DNA binding reduces these conformational heterogeneities, with 63% of the molecules showing density for both RecD and RecBNuc. This suggests that the RecBNuc domain is dynamic and influenced by DNA binding. The major RecBCD-DNA structural class in which RecBNuc is docked onto RecC shows melting of at least 11 bp from a blunt DNA end, much larger than previously observed. A second structural class in which RecBNuc is not docked shows only four bp melted suggesting that RecBCD complexes transition between states with different extents of DNA melting and that the extent of melting regulates initiation of helicase activity.

Abstract 737: Estrogen receptor inhibition with tamoxifen mediates radiosensitization of ER+ breast cancer models

Abstract Purpose: Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of tumor initiation and progression. Therefore, patients with ER-positive (ER+) breast cancers are given first-line therapies which target the ER and downstream ER signaling. Ionizing radiation (RT) has been shown to significantly improve locoregional control and increase overall survival in patients with ER+ breast cancer. Similarly, endocrine therapy (ET) has also been shown to improve metastasis-free and overall survival in women with ER+ breast cancer. While both radiation and ET are used in women with ER+ breast cancer, the effect of endocrine therapies on tumor radiosensitization remains unclear. Here we assessed the efficacy and mechanism of ER inhibition in ER+ breast cancers in combination with radiation therapy. Methods: Clonogenic survival assays were performed to assess radiosensitization and calculate radiation enhancement ratios (enhR) with the selective estrogen receptor modulator (SERM), tamoxifen, in ER+ MCF-7 and T47D cells or ER-negative (ER-) SUM-159 cells. DNA damage was assessed by yH2AX foci. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) was measured by RAD51 foci or using a pYFP reporter, respectively. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Western blotting was used to quantify expression of proteins and phospho-proteins involved in DNA repair, cell cycle, and apoptosis. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo. Results: ER inhibition with tamoxifen radiosensitized ER+ MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER- SUM-159 cells (enhR: 0.99-1.02). MCF-7 and T47D cells treated with tamoxifen did not have changes in the kinetics of dsDNA break repair as measured by yH2AX foci (p&amp;gt;0.05) but demonstrated a decrease in NHEJ-mediated repair (p&amp;lt;0.05). No changes were observed in HR-mediated repair by Rad51 foci (p&amp;gt;0.05). While cell cycle arrest was induced at 24 hours after RT, no changes were observed with tamoxifen treatment in combination with RT. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with treatment of tamoxifen, radiation, or the combination (p&amp;gt;0.05). In vivo xenograft studies demonstrate a significant delay in time to tumor doubling and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT. Conclusion: Our data suggest that tamoxifen may be effectively used to radiosensitize ER+ breast tumors. This work also supports further clinical investigation of the timing of radiation for patients receiving endocrine therapy as concurrent use may be more effective than sequential. Citation Format: Anna R. Michmerhuizen, Andrea M. Pesch, Rachel Schwartz, Kari Wilder-Romans, Meilan Liu, Ruth Azaria, Alexa Jelley, Lori J. Pierce, Corey W. Speers. Estrogen receptor inhibition with tamoxifen mediates radiosensitization of ER+ breast cancer models [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 737.

THE DEAD-Box PROTEIN Csha IN STAPHYLOCOCCUS AUREUS CONTAINS ATP-INDEPENDENT DNA STRAND ANNEALING AND EXCHANGE ACTIVITIES

DEAD-box proteins (DBPs) that are usually RNA helicases have important roles in eukaryotic and bacterial RNA metabolism. Recent studies have reported that certain prokaryotic DBPs exhibit ATP-independent nucleic acid displacement and annealing activities. We investigated one putative RNA helicase, CshA DEAD-box protein, from vancomycin-resistant Staphylococcus aureus strain Mu 50 for ATP-independent activities on nucleic acids. We herein report that CshA has two novel ATP-independent activities - annealing of complementary single-stranded DNA (ssDNA) and strand exchange on short double-stranded DNA (dsDNA). These DNA strand annealing and exchange activities are independent of Mg2+ ion or ATP binding and hydrolysis. ssDNA annealing activity as well as versatile DNA strand exchange activity of CshA suggests a possible role in dsDNA break repair processes.

Elevation of Intracellular dNTP Levels: A Mechanistic Role of SAMHD1 Cancer Mutations

Elevated intracellular dNTP level is a biochemical marker of cancer cells. Recently, a series of mutations in a highly multi-functional dNTPase, SAMHD1, have been reported in various cancers. Here we investigated the role of SAMHD1 in cancer by characterizing cancer-specific SAMHD1 mutations. R366C/H mutants, which were found in two cancer types, lost dNTPase activity and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely due to loss of interaction with γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1. However, these mutants retain dNTPase-independent functions including mediating dsDNA break repair, interacting with CtIP and Cyclin A2, and suppressing innate immune responses.Finally, SAMHD1 degradation in human primary activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study supports that the loss of SAMHD1 dNTPase activity mechanistically contributes to the elevated dNTP levels commonly found in cancer cells.

Probing the Biomechanics of RuvA-Holliday Junction Complex

DNA break is inevitable as it mainly occurs due to cells’ own reactive oxygen species (ROS). While DNA break can be single-stranded or double-stranded, the double-stranded DNA (dsDNA) breaks are more dangerous. If such damage is not repaired or not repaired correctly, it can lead to genetic instability and serious health issues including cancers. The repair of dsDNA breaks occurs via a process called homologous recombination (HR), which involves several DNA-binding proteins. Therefore, to have a better insight into the repair mechanism and origin of repair defects, we need a better understanding of how these proteins interact with DNA and DNA intermediates of the HR process such as Holliday junctions (HJs).

Regulation of DNA break repair by RNA

Genomic stability is critical for cell survival and its effective repair when damaged is a vital process for preserving genetic information. Failure to correctly repair the genome can lead to the accumulation of mutations that ultimately drives carcinogenesis. Life has evolved sophisticated surveillance, repair pathways, and mechanisms to recognize and mend genomic lesions to preserve its integrity. Many of these pathways involve a cascade of protein effectors that act to identify the type of damage, such as double-strand (ds) DNA breaks, propagate the damage signal, and recruit an array of other protein factors to resolve the damage without loss of genetic information. It is now becoming increasingly clear that there are a number of RNA processing factors, such as the transcriptional machinery, and microRNA biogenesis components, as well as RNA itself, that facilitate the repair of DNA damage. Here, some of the recent work unravelling the role of RNA in the DNA Damage Response (DDR), in particular the dsDNA break repair pathway, will be reviewed.

Seviteronel, a Novel CYP17 Lyase Inhibitor and Androgen Receptor Antagonist, Radiosensitizes AR-Positive Triple Negative Breast Cancer Cells.

Increased rates of locoregional recurrence (LR) have been observed in triple negative breast cancer (TNBC) despite multimodality therapy, including radiation (RT). Recent data suggest inhibiting the androgen receptor (AR) may be an effective radiosensitizing strategy, and AR is expressed in 15–35% of TNBC tumors. The aim of this study was to determine whether seviteronel (INO-464), a novel CYP17 lyase inhibitor and AR antagonist, is able to radiosensitize AR-positive (AR+) TNBC models. In cell viability assays, seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 μM). Using clonogenic survival assays, however, AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing cells with radiation enhancement ratios of 1.20–1.89 in models of TNBC with high AR expression. AR-negative (AR−) models, regardless of their estrogen receptor expression, were not radiosensitized with seviteronel treatment at concentrations up to 5 μM. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair with significant delays in repair at 6, 16, and 24 h as measured by immunofluorescent staining of γH2AX foci. Similar effects were observed in an in vivo AR+ TNBC xenograft model where there was a significant reduction in tumor volume and a delay to tumor doubling and tripling times in mice treated with seviteronel and radiation. Following combination treatment with seviteronel and radiation, increased binding of AR occurred at DNA damage response genes, including genes involved both in homologous recombination and non-homologous end joining. This trend was not observed with combination treatment of enzalutamide and RT, suggesting that seviteronel may have a different mechanism of radiosensitization compared to other AR inhibitors. Enzalutamide and seviteronel treatment also had different effects on AR and AR target genes as measured by immunoblot and qPCR. These results implicate AR as a mediator of radioresistance in AR+ TNBC models and support the use of seviteronel as a radiosensitizing agent in AR+ TNBC.

TBCRC 030: A randomized phase II study of preoperative cisplatin versus paclitaxel in TNBC—Evaluating the homologous recombination deficiency (HRD) biomarker.

507 Background: Cisplatin (C) and paclitaxel (T) have activity in TNBC, however predictive biomarkers are lacking. The HRD assay detects impaired dsDNA break repair and may identify BRCA1/2-proficient tumors for treatment with DNA targeting therapies. TBCRC 030 was designed to determine the association between HRD and response to preoperative chemotherapy (CT) in TNBC. Methods: This phase II study randomized patients (pts) with BRCA1/2-proficient/unknown stage I-III TNBC to 12 weeks (wks) of preoperative C or T, followed by surgery. HRD was performed on baseline tissue, with positive scores &gt; 33. Non-responders at 12 wks could crossover to alternative CT. The co-primary objectives were to detect a positive association of HRD with pathologic response (RCB 0-1) vs not (RCB 2-3) to C and a negative association to T. Target accrual of 160 pts was planned to yield 140 evaluable specimens for HRD, providing 90% power for the primary objectives. Analyses used logistic models and likelihood ratio tests with one-sided Type I errors of alpha = 0.05. Results: 140 pts initiated treatment, (72 Arm C, 68 Arm T; 81% T1-2, 62% node negative); 138 were evaluable for response at 12 wks. Post-enrollment testing showed 8 pts (5.8%) with germline DNA-repair pathway mutations. HRD results were available for 95 pts (68.8%, 23 inadequate tissue, 22 pending); 68 (71.6%) were HRD positive: 38 in Arm C, 30 in Arm T. In response-evaluable pts, 87 (63.0%) had surgery at 12 wks, and 51 (37.0%) crossed over. Response outcomes are shown in the Table. No association was seen between HRD score and RCB response to either neoadjuvant C (OR 2.78, [CI 0.61, 17.74]) or T (OR 0.98, CI [0.20, 5.06]). There was no evidence of an interaction between HRD and CT arms. Similarly, no association was observed between HRD score and pCR to either C (OR 1.47, CI [0.40, 5.59]) or T (OR 0.61, CI [0.14, 2.52]). There were no new safety signals. Conclusions: In this mostly BRCA1/2 proficient TNBC cohort, 12 wks of preoperative C or T led to a similar response rate of about 40%; baseline HRD was not predictive of response to preoperative CT, defined either by RCB 0-1 or pCR. Further data will be presented. Correlative analyses of research tissues for markers predictive of response to specific CT in TNBC is ongoing. Clinical trial information: NCT01982448. [Table: see text]

Azacitidine and Ascorbate Inhibit the Competitive Outgrowth of Human TET2 Mutant HSPCs in a Xenograft Model of Pre-Leukemia

The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine.To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in “Hit & Run,” we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis.First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p<0.02). This underscores the importance of TET2 in promoting erythroid differentiation and suggests TET2 mutations can exert a myeloid lineage skewing sensitive to ascorbate.Next, we investigated the effects of TET2 disruption on hematopoietic colony formation in methylcellulose. Both methods resulted in increased numbers of TET2-disrupted colonies compared to control (Hit & Run, n=4 independent experiments, p<0.0001; HDR, n=3 independent experiments, p<0.0001) and absence of erythroid BFU-E. Interestingly, analysis of indels in Hit & Run colonies showed that serial replating enriched for a 65 base pair deletion that results in a null allele, suggesting that TET2-disrupted cells outcompete normal HSPCs in vitro.Next, we transplanted control or TET2-disrupted Hit & Run CD34+ cells into NSG mice. Primary transplantation at 4 months showed no statistical differences in either engraftment rate (human CD45+) or differentiation (T/ B/ Myeloid cells), although the frequency of TET2 indels increased gradually in CD33+ cells. Intriguingly, 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells (lymphoid=22.1%, myeloid=73.0%, Mann-Whitney U, p=0.0485) and a particular increase in a CMML-like CD33highCD14+CD16- population. Furthermore, preliminary data from tertiary transplantation (8 weeks after transplantation) indicates persistent myeloid skewing in the bone marrow in some mice and expansion of TET2-mutant cells, suggesting a CMML-like disease.Finally, we used in vivo competition studies to determine if TET2-disrupted HSPCs are selectively targeted by azacitidine or ascorbate treatment compared to controls. NSG mice were intrafemorally transplanted with a one-to-one ratio of control and TET2-disrupted HSPCs, and 4 months later, these mice were treated with azacitidine (2.5mg/kg/dose, i.p. daily on days 1-5 of a 14-day cycle for 2 cycles) or ascorbate (4g/kg/dose, i.p. twice daily for a month). In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p=0.036), but did not eradicate established TET2 pre-leukemia in all evaluable mice. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p=0.14).Our data show that TET2 disruption in primary human HSPCs blocked erythroid differentiation, increased colony formation and replating, and caused myeloid skewing and a CMML-like disease in vivo after an extended period of time. In this model, azacitidine or ascorbate treatment slowed expansion of TET2-mutant human pre-leukemic clones raising the intriguing possibility of preventing CHIP progression to de novo AML. DisclosuresNo relevant conflicts of interest to declare.

Viral Proteins U41 and U70 of Human Herpesvirus 6A Are Dispensable for Telomere Integration.

Human herpesvirus-6A and -6B (HHV-6A and -6B) are two closely related betaherpesviruses that infect humans. Upon primary infection they establish a life-long infection termed latency, where the virus genome is integrated into the telomeres of latently infected cells. Intriguingly, HHV-6A/B can integrate into germ cells, leading to individuals with inherited chromosomally-integrated HHV-6 (iciHHV-6), who have the HHV-6 genome in every cell. It is known that telomeric repeats flanking the virus genome are essential for integration; however, the protein factors mediating integration remain enigmatic. We have previously shown that the putative viral integrase U94 is not essential for telomere integration; thus, we set out to assess the contribution of potential viral recombination proteins U41 and U70 towards integration. We could show that U70 enhances dsDNA break repair via a homology-directed mechanism using a reporter cell line. We then engineered cells to produce shRNAs targeting both U41 and U70 to inhibit their expression during infection. Using these cells in our HHV-6A in vitro integration assay, we could show that U41/U70 were dispensable for telomere integration. Furthermore, additional inhibition of the cellular recombinase Rad51 suggested that it was also not essential, indicating that other cellular and/or viral factors must mediate telomere integration.

Replication-transcription conflicts trigger extensive DNA degradation in Escherichia coli cells lacking RecBCD

Bacterial chromosome duplication is initiated at a single origin (oriC). Two forks are assembled and proceed in opposite directions with high speed and processivity until they fuse and terminate in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of replication-transcription conflicts. In addition, ΔrecB cells show substantial amounts of DNA degradation in the termination area. In this study we show that head-on encounters of replication and transcription at a highly-transcribed rrn operon expose fork structures to degradation by nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination area of ΔrecB cells. However, additional processes exacerbate degradation specifically in this location. Replication profiles from ΔrecB cells in which the chromosome is linearized at two different locations highlight that the location of replication termination can have some impact on the degradation observed. Our data improve our understanding of the role of RecBCD at sites of replication-transcription conflicts as well as the final stages of chromosome duplication. However, they also highlight that current models are insufficient and cannot explain all the molecular details in cells lacking RecBCD.

Abstract 3218: Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway

Abstract Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of radiation (RT); therefore, approaches that result in radiosensitizaton in TNBC may improve clinical outcomes. Despite some controversy as to the role of MELK kinase as an oncogene, we previously established its clear role in conferring radioresistance in TNBC. Here we extend those studies to understand the mechanism of conferred radioresistance. Methods: Clonogenic survival assays were used to quantify the degree of radiosensitivity after MELK inhibition. Mass spectrometry was utilized to identify proteins associated with MELK. NHEJ assays were performed using linearized pEYFP vector and quantitated by FACS. qRT-PCR was used to determine MELK RNA expression. Rad51 and γH2AX foci counting were performed to evaluate homologous recombination (HR). Neutral comet assay was used to quantitate dsDNA break repair. Laser microirradiation studies were performed to determine the location and kinetics of protein shuttle to and from sites of dsDNA breaks, including MELK, Ku70 and Ku80 proteins. Fluorescent microscopy was performed for localization of endogenous MELK protein post RT. To study the effect of MELK kinase activity on Ku70-Ku80 complex formation and recruitment at the DNA damage site, cells were lysed and immunoprecipitation was performed using MELK, Ku70 or Ku80-specific antibodies. Results: We previously demonstrated that genomic or pharmacologic inhibition of MELK confers significant radiosensitization. Mechanistic studies were undertaken to understand the pathways critical to MELK-mediated radioresistance. When MELK is genomically or pharmacologically inhibited, NHEJ and HR reporter assays demonstrate that this radiosensitization is driven through impaired NHEJ at 4, 16, and 24 hrs after RT and not mediated by HR in multiple TNBC cell lines. Tandem mass spectrometry studies of tagged MELK protein identify Ku70 and Ku80 as direct interactors with MELK protein, which is confirmed by Co-IP. Laser microirradiation studies confirm that MELK, Ku70 and Ku80 co-localize to sites of dsDNA breaks, and that MELK kinase function is required to stabilize the Ku70/Ku80 complex at these sites. Comet assay confirms that rapid dissolution of the Ku70/Ku80 complex when MELK is inhibited or functionally dead is not a result of accelerated dsDNA break repair. Conclusion: MELK kinase function is critical in conferring radioresistance in TNBC and inhibition of function confers radiosensitivity through an NHEJ-mediated pathway. MELK also stabilizes Ku70/Ku80 proteins at sites of dsDNA breaks and allows for more efficient repair of breaks induced by ionizing RT. These results further support the rationale for developing clinical strategies to inhibit MELK in combination with RT treatment as a novel radiosensitizing strategy in TNBC. Citation Format: Shyam Nyati, Ben Chandler, Eric Olsen, Leah Moubadder, Meilan Liu, Meleah Cameron, Kari Wilder-Romans, Theodore S. Lawrence, Powel H. Brown, Fellix Y. Fang, Lori J. Pierce, Corey Speers. Maternal embryonic leucine zipper kinase (MELK) confers radioresistance in triple-negative breast cancers (TNBC) through a nonhomologous end joining (NHEJ)-mediated pathway [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 3218.

Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.