High-fidelity Homologous Recombination Research Articles

RPS19 and RPL5 Contribute to DNA Double-Strand Break Repair

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome most often due to germline pathogenic variants (PVs) in ribosomal protein (RP) genes, with RPS19 and RPL5 being the most frequently mutated. In addition to hypoplastic anemia, individuals have a moderately increased risk of myelodysplastic syndrome, acute myeloid leukemia, and solid cancers, most commonly colorectal cancer and osteosarcoma. A causal link between RP deficiency and cancer predisposition has yet to be firmly established. Our work aims to examine the impact of deficiencies in RPS19 and RPL5 on DNA double-strand break (DSB) repair with the overarching hypothesis that disruptions in DSB repair could increase mutagenesis and ultimately contribute to cancer development in DBA. We found that DBA patient-derived lymphoblastoid cells were hypersensitive to ionizing radiation (IR) and had delayed resolution of the DNA damage marker, γ-H2AX, at IR-induced foci. Additionally, we found that both RPS19- and RPL5-knocked down (KD) bone marrow-derived CD34+ cells had delayed resolution of DSBs after IR in neutral comet assays. These data suggest that RPS19 and RPL5 deficiencies lead to impaired DSB repair. We, therefore, assessed the effect of RPS19- and RPL5-KD on the four main DSB repair pathways using well-established U2OS (osteosarcoma) DSB reporter cell lines. We found decreased homologous recombination (HR) and single-strand annealing (SSA) upon RPS19-KD, whereas the efficiencies of nonhomologous end-joining (NHEJ) and alternative end-joining (alt-EJ) were unchanged. Consistent with an HR defect, RPS19-KD cells were hypersensitive to PARP inhibition. In contrast, RPL5-KD cells had increased NHEJ and alt-EJ, whereas the efficiencies of HR and SSA were unchanged. Together, these results suggest that shifts in DSB repair pathway choice, with less high-fidelity HR, or more error-prone NHEJ or mutagenic alt-EJ, could contribute to mutagenesis in the context of RPS19 or RPL5 PVs. To investigate the mechanism(s) underlying these effects, we examined cell cycle profiles and found decreased G1 and increased G2/M cells following both RPS19- and RPL5-KD, which suggests altered cell cycle profiles are not responsible for DSB repair effects since NHEJ occurs principally in G1 and HR is restricted to G2. Given the essential roles of RPS19 and RPL5 in translation, we also examined levels of crucial proteins in each DSB repair pathway. Surprisingly, we found increased levels of NHEJ proteins DNA ligase IV (LIG4) and XRCC4 in both RPS19 and RPL5-KD cells. However, restoring LIG4/XRCC4 to normal levels via siRNA KD did not normalize the DSB repair efficiencies. Since HR and SSA require extensive resection of the 5' DNA strand at DSBs, we examined whether RPS19-KD impaired this key step by assessing the accumulation of nuclear RPA2, which associates with end-resected DNA. We found nuclear RPA2 levels were not reduced but rather increased following IR compared to the control, indicating the reduction in HR and SSA efficiency in these cells was not due to impaired end resection and may, instead, involve a downstream step in HR that we are currently investigating. To assess if RPS19 and RPL5 might directly contribute to repair, we monitored the recruitment of fluorescently tagged RPS19 and RPL5 to DSBs. We found that these proteins were rapidly recruited to sites of laser microirradiation and sites of chromatin associated Fok1-nuclease (Figure 1), both of which induce DSBs. The recruitment of both proteins was hindered by PARP inhibition and enhanced for RPS19 with PARG inhibition, suggesting RPS19, in particular, may be parylated or interact with parylated proteins. Similar studies were performed in Saos2 cells, which, in contrast to U2OS cells, are p53 null. Whereas RPS19 was recruited to sites of laser microirradiation in Saos2 cells, RPL5 was not. Interestingly, a transcriptionally hyperactive p53 (∆CTD), but not a wild-type or a dominant-negative form of p53 (p.R248Q), restored RPL5 recruitment in Saos2 cells, suggesting a p53 downstream target is required for its recruitment. We are currently investigating proteins that mediate RPS19 and RPL5 DSB recruitment. Together, our results support a model whereby altered DNA DSB repair in RPS19- and RPL5-deficient cells may contribute to cancer development in DBA.

Quantifying Cellular Repair, Misrepair and Apoptosis Induced by Boron Ions, Gamma Rays and PRIMA-1 Using the RHR Formulation

The recent interaction cross-section-based formulation for radiation-induced direct cellular inactivation, mild and severe sublethal damage, DNA-repair and cell survival have been developed to accurately describe cellular repair, misrepair and apoptosis in TP53 wild-type and mutant cells. The principal idea of this new non-homologous repairable-homologous repairable (RHR) damage formulation is to separately describe the mild damage that can be rapidly handled by the most basic repair processes including the non-homologous end joining (NHEJ), and more complex damage requiring longer repair times and high-fidelity homologous recombination (HR) repair. Taking the interaction between these two key mammalian DNA repair processes more accurately into account has significantly improved the method as indicated in the original publication. Based on the principal mechanisms of 7 repair and 8 misrepair processes presently derived, it has been possible to quite accurately describe the probability that some of these repair processes when unsuccessful can induce cellular apoptosis with increasing doses of γrays, boron ions and PRIMA-1. Interestingly, for all LETs studied (≈0.3-160 eV/nm) the increase in apoptosis saturates when the cell survival reaches about 10% and the fraction of un-hit cells is well below the 1% level. It is shown that most of the early cell kill for low-to-medium LETs are due to apoptosis since the cell survival as well as the non-apoptotic cells agree very well at low doses and other death processes dominate beyond D > 1 Gy. The low-dose apoptosis is due to the fact that the full activation of the checkpoint kinases ATM and Chk2 requires >8 and >18 DSBs per cell to phosphorylate p53 at serine 15 and 20. Therefore, DNA repair is not fully activated until well after 1/2 Gy, and the cellular response may be apoptotic by default before the low-dose hyper sensitivity (LDHS) is replaced by an increased radiation tolerance as the DNA repair processes get maximal efficiency. In effect, simultaneously explaining the LDHS and inverse dose rate phenomena. The partial contributions by the eight newly derived misrepair processes was determined so they together accurately described the experimental apoptosis induction data for γ rays and boron ions. Through these partial misrepair contributions it was possible to predict the apoptotic response based solely on carefully analyzed cell survival data, demonstrating the usefulness of an accurate DNA repair-based cell survival approach. The peak relative biological effectiveness (RBE) of the boron ions was 3.5 at 160 eV/nm whereas the analogous peak relative apoptotic effectiveness (RAE) was 3.4 but at 40 eV/nm indicating the clinical value of the lower LET light ions (15 ≤ LET ≤ 55 eV/nm, 2 ≤ Z ≤5) in therapeutic applications to maximize tumor apoptosis and senescence. The new survival expressions were also applied on mouse embryonic fibroblasts with key knocked-out repair genes, showing a good agreement between the principal non-homologous and homologous repair terms and also a reasonable prediction of the associated apoptotic induction. Finally, the formulation was used to estimate the increase in DNA repair and apoptotic response in combination with the mutant p53 reactivating compound PRIMA-1 and γ rays, indicating a 10-2 times increase in apoptosis with 5 µM of the compound reaching apoptosis levels not far from peak apoptosis boron ions in a TP53 mutant cell line. To utilize PRIMA-1 induced apoptosis and cellular sensitization for reactive oxygen species (ROS), concomitant biologically optimized radiation therapy is proposed to maximize the complication free tumor cure for the multitude of TP53 mutant tumors seen in the clinic. The experimental data also indicated the clinically very important high-absorbed dose ROS effect of PRIMA-1.

Fibroblast growth factor signalling influences homologous recombination-mediated DNA damage repair to promote drug resistance in ovarian cancer

BackgroundOvarian cancer patients frequently develop chemotherapy resistance, limiting treatment options. We have previously shown that individuality in fibroblast growth factor 1 (FGF1) expression influences survival and chemotherapy response.MethodsWe used MTT assays to assess chemosensitivity to cisplatin and carboplatin following shRNA-mediated knockdown or heterologous over-expression of FGF1 (quantified by qRT-PCR and immunoblot analysis), and in combination with the FGFR inhibitors AZD4547 and SU5402, the ATM inhibitor KU55933 and DNA-PK inhibitor NU7026. Immunofluorescence microscopy was used to quantify the FGF1-dependent timecourse of replication protein A (RPA) and γH2AX foci formation.ResultsPharmacological inhibition of FGF signalling reversed drug resistance in immortalised cell lines and in primary cell lines from drug-resistant ovarian cancer patients, while FGF1 over-expression induced resistance. Ataxia telangiectasia mutated (ATM) phosphorylation, but not DNA adduct formation was FGF1 dependent, following cisplatin or carboplatin challenge. Combining platinum drugs with the ATM inhibitor KU55933, but not with the DNA-PK inhibitor NU7026 re-sensitised resistant cells. FGF1 expression influenced the timecourse of damage-induced RPA and γH2AX nuclear foci formation.ConclusionDrug resistance arises from FGF1-mediated differential activation of high-fidelity homologous recombination DNA damage repair. FGFR and ATM inhibitors reverse platinum drug resistance, highlighting novel combination chemotherapy approaches for future clinical trial evaluation.

DNA Damage Clustering after Ionizing Radiation and Consequences in the Processing of Chromatin Breaks

Charged-particle radiotherapy (CPRT) utilizing low and high linear energy transfer (low-/high-LET) ionizing radiation (IR) is a promising cancer treatment modality having unique physical energy deposition properties. CPRT enables focused delivery of a desired dose to the tumor, thus achieving a better tumor control and reduced normal tissue toxicity. It increases the overall radiation tolerance and the chances of survival for the patient. Further improvements in CPRT are expected from a better understanding of the mechanisms governing the biological effects of IR and their dependence on LET. There is increasing evidence that high-LET IR induces more complex and even clustered DNA double-strand breaks (DSBs) that are extremely consequential to cellular homeostasis, and which represent a considerable threat to genomic integrity. However, from the perspective of cancer management, the same DSB characteristics underpin the expected therapeutic benefit and are central to the rationale guiding current efforts for increased implementation of heavy ions (HI) in radiotherapy. Here, we review the specific cellular DNA damage responses (DDR) elicited by high-LET IR and compare them to those of low-LET IR. We emphasize differences in the forms of DSBs induced and their impact on DDR. Moreover, we analyze how the distinct initial forms of DSBs modulate the interplay between DSB repair pathways through the activation of DNA end resection. We postulate that at complex DSBs and DSB clusters, increased DNA end resection orchestrates an increased engagement of resection-dependent repair pathways. Furthermore, we summarize evidence that after exposure to high-LET IR, error-prone processes outcompete high fidelity homologous recombination (HR) through mechanisms that remain to be elucidated. Finally, we review the high-LET dependence of specific DDR-related post-translational modifications and the induction of apoptosis in cancer cells. We believe that in-depth characterization of the biological effects that are specific to high-LET IR will help to establish predictive and prognostic signatures for use in future individualized therapeutic strategies, and will enhance the prospects for the development of effective countermeasures for improved radiation protection during space travel.

The Role of Recombinant AAV in Precise Genome Editing.

The replication-defective, non-pathogenic, nearly ubiquitous single-stranded adeno-associated viruses (AAVs) have gained importance since their discovery about 50 years ago. Their unique life cycle and virus-cell interactions have led to the development of recombinant AAVs as ideal genetic medicine tools that have evolved into effective commercialized gene therapies. A distinctive property of AAVs is their ability to edit the genome precisely. In contrast to all current genome editing platforms, AAV exclusively utilizes the high-fidelity homologous recombination (HR) pathway and does not require exogenous nucleases for prior cleavage of genomic DNA. Together, this leads to a highly precise editing outcome that preserves genomic integrity without incorporation of indel mutations or viral sequences at the target site while also obviating the possibility of off-target genotoxicity. The stem cell-derived AAV (AAVHSCs) were found to mediate precise and efficient HR with high on-target accuracy and at high efficiencies. AAVHSC editing occurs efficiently in post-mitotic cells and tissues in vivo. Additionally, AAV also has the advantage of an intrinsic delivery mechanism. Thus, this distinctive genome editing platform holds tremendous promise for the correction of disease-associated mutations without adding to the mutational burden. This review will focus on the unique properties of direct AAV-mediated genome editing and their potential mechanisms of action.

BRCA2 Promotes Spontaneous Homologous Recombination In Vivo.

Simple SummaryInherited mutation of either BRCA1 or BRCA2 is associated with familial breast and ovarian cancer. As a tumor suppressor, BRCA2 functions to maintain genome stability but we do not know the genomic impact of this deficiency on normal tissues in normal contexts, rather than cancer cells that carry other gene mutations. Numerous murine models of BRCA2 have been developed to investigate BRCA2 tumor suppressor function but have been met with difficulties due to the lethality of most of these models during embryogenesis. To circumvent embryonic lethality here, we employ the cre conditional system combined with an assay for measuring changes in stability of a large tandem DNA repeat by homology-directed recombination. The relevance of this assay is that it can measure DNA recombination events in a normal growing/developing tissue. Here, we show that BRCA2 is required for homologous recombination in a normal developing tissue long before cancer arises.Background: BRCA2 is known to be a tumor suppressor involved in homologous recombination repair and presumed to prevent genome instability in normal tissues prior to the development of tumors. Typical assessment of BRCA2 deficiency on the genome involves cell-based models using cancer cells with mixed genetic contexts, but the role in normal tissue in vivo has not been clearly demonstrated. Methods: Using conditional deletion of Brca2 exon 11, the region containing all eight BRC repeats, in the retinal pigment epithelium and the pink-eyed unstable mouse model, we evaluate the frequency of DNA deletion events. Results: In the current study, we show that conditional loss of Brca2 exon 11 results in a decreased frequency of spontaneous homologous recombination compared to wild-type mice. Of note, we observe no apparent concomitant increase in events that indicate single-strand annealing by the pink-eyed unstable mouse model. Conclusions: Therefore, our results demonstrate that BRCA2, as expected, is required for high-fidelity homologous recombination DNA repair in normal tissues, here in a tissue undergoing normal proliferation through normal development.

AVID200: A Novel TGF-β Inhibitor for the Treatment of Anemia Associated with Myelodysplastic Syndromes

AVID200 is an engineered TGF-β ligand trap comprised of TGF-β receptor ectodomains fused to a human Fc domain. AVID200 has antibody-like properties and is 1,000 times more selective for neutralizing TGF-β1 and TGF-β3 (pM potency), compared to TGF-b2. Since TGF-β1 and TGF-β3 are negative regulators of hematopoiesis, while TGF-b2 is a positive regulator of hematopoiesis, the unique isoform selectivity profile of AVID200 makes it an attractive agent for the treatment of Myelodysplastic syndrome (MDS) associated anemia. As TGF-β2 is implicated in normal cardiovascular function and tumor cell dormancy, the specificity profile of AVID200 also minimizes the potential for cardiotoxicity and promotion of metastasis, which have been observed with pan-neutralizing TGF- β agents.MDS consist of a group of disorders that lead to cytopenia through ineffective hematopoiesis. One critical factor that is commonly associated with the occurrence of these disorders is the pathological overproduction of TGF-β. Increased TGF-β results in the arrest of myeloprogenitor cells preventing hematopoiesis. This disruption of the differentiation program and resulting cytopenia can lead to development of acute myeloid leukemia (AML) in one-third of MDS patients.A critical feature of MDS is an increased level of non-homologous end-joining (NHEJ) activity, a low fidelity form of DNA damage repair, as compared to high-fidelity homologous recombination (HR) repair. The increased level of NHEJ repair in MDS cells results in a hypersensitivity to DNA damaging agents such as mitomycin C (MMC). Previously, in a model of MDS, treatment with TGF-β inhibitors was shown to restore the ratio of NHEJ:HR repair to normal, resulting in more accurate DNA repair. Therefore, the effect of increased TGF-β in patients with MDS is deleterious in two ways. First, elevated levels of TGF-β suppress erythropoiesis leading to anemia. Second, the shift from HR to NHEJ allows for less precise DNA repair and the development of mutations which could result in AML formation.Using a TGF-β responsive AML line, we have shown that AVID200 prevents the growth inhibition induced by TGF-β1. Additionally, AVID200 reversed the hypersensitivity to MMC, implying an improvement in DNA damage repair. Currently, we are investigating the effectiveness of treating two MDS cell lines (MDS-92 and MDS-L) with AVID200 to determine its ability to not only prevent the growth inhibition associated with TGF-β1, but also to enhance DNA damage repair.The safety of AVID200 has been evaluated in several preclinical models. In a chick embryo model of metastasis, AVID200 did not promote the growth of metastatic foci, whereas a pan-neutralizing TGF-β inhibitor did. This result is important since a significant percentage of patients with MDS go on to develop AML and other cancers. The cardiac safety of AVID200 was evaluated in a single-dose pilot non-human primate study. No adverse cardiovascular events were observed at doses of up to 30 mg/kg, the highest dose tested.These results indicate that AVID200 is a promising new agent for the safe and effective treatment for MDS associated anemia.(MJT is a Mitacs Accelerate Program Postdoctoral Fellow) DisclosuresThwaites:Formation Biologics: Employment, Research Funding. Koropatnick:Formation Biologics, Inc.: Research Funding. Tremblay:Formation Biologics: Employment. O'Connor-McCourt:Formation Biologics: Employment.

Targeting IDH1/2 mutant cancers with combinations of ATR and PARP inhibitors.

Mutations in the isocitrate dehydrogenase-1 and -2 (IDH1/2) genes were first identified in glioma and acute myeloid leukemia (AML), and subsequently found in multiple other tumor types. These neomorphic mutations convert the normal product of enzyme, α-ketoglutarate (αKG), to the oncometabolite 2-hydroxyglutarate (2HG). Our group recently demonstrated that 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state referred to as ‘BRCAness’, which confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we sought to elucidate sensitivity of IDH1/2-mutant cells to DNA damage response (DDR) inhibitors and, whether combination therapies could enhance described synthetic lethal interactions. Here, we report that ATR (ataxia telangiectasia and Rad3-related protein kinase) inhibitors are active against IDH1/2-mutant cells, and that this activity is further potentiated in combination with PARP inhibitors. We demonstrate this interaction across multiple cell line models with engineered and endogenous IDH1/2 mutations, with robust anti-tumor activity in vitro and in vivo. Mechanistically, we found ATR and PARP inhibitor treatment induces premature mitotic entry, which is significantly elevated in the setting of IDH1/2-mutations. These data highlight the potential efficacy of targeting HR defects in IDH1/2-mutant cancers and support the development of this combination in future clinical trials.

Abstract 1059: Targeting IDH1/2 mutant cancers with unique combinations of DNA repair inhibitors

Abstract Mutations in the Isocitrate Dehydrogenase-1 and -2 (IDH1/2) genes occur in the vast majority of solid tumors including low-grade and secondary high-grade gliomas. These neomorphic mutations occur early on in gliomagenesis leading to the production of 2-Hydroxyglutarate (2HG). 2HG has been implicated in tumorigenesis via inhibiting α-ketoglutarate (αKG)-dependent dioxygenases. Our group recently demonstrated that the production of 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state of “BRCAness”. We initially found that mutant IDH1/2-induced BRCAness confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, a finding which now has been replicated by multiple independent laboratories. Although IDH1/2 mutations were first identified in gliomas and acute myeloid leukemia (AML) cells, multiple other tumor types have subsequently been shown to harbor these mutations. Current clinical trials are testing the efficacy of PARP inhibitors as a monotherapy, as well as in combination with other DNA repair inhibitors. Here, we demonstrate that novel combinations of DNA repair inhibitors can be utilized to synergistically target IDH1/2-mutant glioma cells. In particular, we demonstrate potent synergy with ATRi and PARPis, a finding which was validated in multiple structurally unique drugs within these classes. We have shown that in an in vivo flank model bearing IDH1 R132H mutation, AZD6738 (ATRi) and Olaparib (PARPi) combination treatment reduces the tumor burden significantly relative to monotherapy and vehicle. As this combination is active in BRCA1/2-mutant cancers, in particular after the emergence of PARPi resistance, these data suggest are consistent with an underlying HR defect in IDH1/2-mutant gliomas. These preclinical investigations will provide a blueprint for future clinical trials combining PARP and ATR inhibitors in the treatment of IDH1/2 mutant cancers. Citation Format: Amrita D. Sule, Ranjini Sundaram, Ranjit Bindra. Targeting IDH1/2 mutant cancers with unique combinations of DNA repair inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1059.

A DNA Repair-Based Model of Cell Survival with Important Clinical Consequences.

This work provides a description of a new interaction, cross-section-based model for radiation-induced cellular inactivation, sublethal damage, DNA repair and cell survival, with the ability to more accurately elucidate different radiation-response phenomena. The principal goal of this work is to describe the damage-induction cross sections, as well as repair and survival, as Poisson processes with two main types of damage: mild damage that can be rapidly handled by the most basic repair processes; and more complex damage requiring longer repair times and the high-fidelity homologous recombination (HR) repair process to ensure accuracy and safety in the survival. This work is unique in its use of Poisson statistics to quantify the main repairable cell compartments that are exposed to simple and more complex sublethal hits, the cross section of which determines what is homologically and non-homologically repairable. The new method is applied to central radiation damage and survival data, such as in vitro cellular repair and survival with key DNA repair genes knocked out, low-dose hypersensitivity (LDHS), change in survival over the cell cycle, and variation with linear energy transfer (LET) for densely ionizing ions, all results supporting our basic assumptions. Among the results, it was shown that less than 1% of the simple DSBs are lethal at approximately 2 Gy and below for sparsely ionizing radiations, but their δ-electron track ends of between 1.5 and 0.5 keV can deliver 0.5 MGy to a few hundred nm3 volumes, mainly due to multiple scatter detours and multiple secondary electrons. They can cause dual double-strand breaks (DSBs) on the periphery of nucleosomes that are the most common multiply damaged sites, with an average of 1-2 δ-electron track ends per cell nucleus at 2 Gy. LDHS is most likely due to the normal lack of fast, efficient repair of sublethal damage below approximately 0.5 Gy, and requires largely intact key DNA repair genes to achieve significant repair recovery at higher doses. The new repair model describes this phenomenon quite accurately. Cells with key non-homologous end joining (NHEJ) genes knocked-out, lose LDHS but provoke HR repair, and cells with HR genes knocked out may lose some LDHS, but provoke NHEJ repair. The DNA duplication during the S phase results in a direct doubling as well of the total and sublethal hit cross sections. For the lowest LET carbon ions, NHEJ is reduced to where it is almost eliminated at maximum relative biological effectiveness (RBE), while HR is induced more than by X rays, due to complex damage and misrepair of DSBs produced by numerous δ electrons. The use of a lower LET such as electrons or photons during the final week of radiation treatment may potentially maximize complication-free cure. Optimally-designed weekly fractionation schedules are proposed to maximize the DNA repair potential in normal tissues. Additionally, the optimal therapeutic ion species, LET, apoptosis and permanent growth arrest/senescence window is identified with helium, lithium and boron ions and LETs at approximately 15-55 eV/nm, to maximize these quantities in the tumor and minimize them in the normal tissues, resulting in a very high probability of complication-free cure.

Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126.

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.

RDNA-06. TARGETING IDH1/2-MUTANT GLIOMAS WITH UNIQUE COMBINATIONS OF DNA REPAIR INHIBITORS

Abstract Mutations in the Isocitrate Dehydrogenase-1 and -2 (IDH1/2) genes occur in the vast majority of low-grade and secondary high-grade gliomas. These neomorphic mutations occur early on in gliomagenesis leading to the production of 2-Hydroxyglutarate (2HG). 2HG has been implicated in tumorigenesis via inhibiting α-ketoglutarate (αKG)-dependent dioxygenases. Our group recently demonstrated that the production of 2HG suppresses the high-fidelity homologous recombination (HR) DNA repair pathway, resulting in a state of “BRCAness”. We initially found that mutant IDH1/2-induced BRCAness confers exquisite sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors, a finding which now has been replicated by multiple independent laboratories. Although IDH1/2 mutations were first identified in gliomas and acute myeloid leukemia (AML) cells, multiple other tumor types have subsequently been shown to harbor these mutations. Current clinical trials are testing the efficacy of PARP inhibitors as a monotherapy, as well as in combination with other DNA repair inhibitors. Here, we demonstrate that novel combinations of DNA repair inhibitors can be utilized to synergistically target IDH1/2-mutant glioma cells. In particular, we demonstrate potent synergy with ATRi and PARPis, a finding which was validated in multiple structurally unique drugs within these classes. As this combination is active in BRCA1/2-mutant cancers, in particular after the emergence of PARPi resistance, these data suggest are consistent with an underlying HR defect in IDH1/2-mutant gliomas. These preclinical investigations will provide a blueprint for future clinical trials combining PARP and ATR inhibitors in the treatment of glioblastoma.

Abstract 338: A new investigational ATM Inhibitor, M3541, synergistically potentiates fractionated radiotherapy and chemotherapy in cancer cells and animal models

Abstract Physical or chemical DNA damaging agents are among the most widely used classes of cancer therapeutics today. Radiotherapy and topoisomerase inhibitors induce double strand breaks (DSB) considered most dangerous if left unrepaired because they can induce growth arrest, chromosomal abnormality and/or cell death. ATM is an essential kinase which orchestrates one of the two major pathways for repair of DSBs via the high fidelity homologous recombination (HR) mechanism. In addition, ATM acts as an upstream signaling kinase, regulating cell cycle checkpoint control in the response to DSB damage. Inhibitors of ATM kinase are expected to suppress DSB DNA repair, block checkpoint controls and enhance the therapeutic effect of radiation therapy and other DSB-inducing treatment modalities. We have developed a novel, highly potent and selective inhibitor of ATM, M3541, which modifies cellular response to DSB damage leading to potentiation of radiation therapy and DSB-inducing chemotherapy. Here, we present data from the preclinical evaluation of M3541 therapeutic potential in cellular and xenograft models of cancer. ATM inhibitor sensitized multiple tumor cell lines to ionizing radiation and a topoisomerase inhibitor in vitro. Oral administration of M3541 to nude mice bearing human tumor xenografts in a clinically relevant radiation regimen strongly enhanced the antitumor activity, leading to complete tumor regression. These activities resulted from inhibition of ATM kinase activity and modulation of its downstream effects in the xenograft tissues. M3541 is currently under clinical investigation in a Phase I trial. Citation Format: Astrid Zimmermann, Frank Zenke, Heike Dahmen, Christian Sirrenberg, Thomas Grombacher, Lyubomir T. Vassilev, Thomas Fuchss, Andree Blaukat. A new investigational ATM Inhibitor, M3541, synergistically potentiates fractionated radiotherapy and chemotherapy in cancer cells and animal models [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 338.

PO-147 Pinostilbene hydrate suppresses human oral cancer cell metastasis via downregulation matrix metalloproteinase-2 through the mitogen-activated protein kinase signalling pathway

IntroductionAcute myeloid leukaemia (AML) is the most common acute leukaemia in adults and has one of the poorest survival rates of all cancers. Despite advancements in treatments, the 5 year survival rate for AML remains at 26%, and is less than 8% for patients aged over 60. Approximately one-third of patients exhibit primary refractory disease, however, the mechanisms controlling this have not been fully elucidated. Overexpression of the brain and acute leukaemia, cytoplasmic (BAALC) gene is a negative prognostic factor for AML and is associated with primary refractory disease, but precisely how overexpression of BAALC is controlling sensitivity to chemotherapeutics is unknown. As AML cells with perturbations in DNA repair pathways exhibit alterations in sensitivity/resistance to a range of chemotherapeutics, we examined whether BAALC overexpression perturbs DNA repair pathways in AML cells.Material and methodsBAALC was overexpressed in a panel of AML cells and their ability to repair DNA damage was measured by western blot and comet assay. Alterations in expression of key DNA damage repair genes were measured by PCR in BAALC overexpressing and empty vector (EV) control cells. BAALC-mediated signalling complexes involved in DNA repair were identified by immunoprecipitation.Results and discussionsBAALC, but not EV, overexpression resulted in elevated DNA fragmentation (comet assay), and sustained gH2AX and ATM activation (pSer1981) post-daunorubicin and etoposide-induced DNA damage, indicative of an inability to repair the damage. Gene expression analyses in BAALC overexpressing cells identified that the expression of components (RAD51D, TP53BP1, BTG2) of the high-fidelity homologous recombination double strand break repair pathway were significantly decreased. Furthermore, BAALC interacts with several proteins (RAD51, CaMKII, dermcidin) implicated in DNA repair. Further supporting a role for BAALC overexpression in inducing defective homologous recombination repair, BAALC overexpressing AML cells were hypersensitive to cisplatin treatment.ConclusionAs perturbations in DNA repair pathways in AML have been associated with increased resistance to a range of chemotherapeutics, and BAALC overexpression is associated with primary refractory AML, our data suggests that BAALC overexpression induces decreased chemosensitivity by perturbing DNA repair pathways.

Developmental history and application of CRISPR in human disease.

Genome-editing tools are programmable artificial nucleases, mainly including zinc-finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short palindromic repeat (CRISPR). By recognizing and cleaving specific DNA sequences, genome-editing tools make it possible to generate site-specific DNA double-strand breaks (DSBs) in the genome. DSBs will then be repaired by either error-prone nonhomologous end joining or high-fidelity homologous recombination mechanisms. Through these two different mechanisms, endogenous genes can be knocked out or precisely repaired/modified. Rapid developments in genome-editing tools, especially CRISPR, have revolutionized human disease models generation, for example, various zebrafish, mouse, rat, pig, monkey and human cell lines have been constructed. Here, we review the developmental history of CRISPR and its application in studies of human diseases. In addition, we also briefly discussed the therapeutic application of CRISPR in the near future.

BRCA mutations in the manifestation and treatment of ovarian cancer.

BRCA genes are important for the integrity and stability of genetic material and play key roles in repairing DNA breaks via high fidelity homologous recombination. BRCA mutations are known to predispose carriers to gynecological malignancies, accounting for a majority of hereditary OC cases. Known to be lethal, OC is difficult to detect and control. Testing for BRCA mutations is a key step in the risk assessment, prognosis, treatment and prevention of OC and current clinical guidelines recommend BRCA mutation testing for all OCs of epithelial origin. Studies have established that ovarian tumors harboring BRCA mutations have distinct molecular and histo-pathological features that can be exploited for effective, targeted treatment. Deficiencies in DNA repair pathways that arise as a result of BRCA mutations make them hypersensitive to DNA-damaging treatments such as platinum chemotherapy and PARP inhibitors. Different combinations of treatment regimens which have the potential to greatly improve prognosis and disease outcomes are currently being evaluated. However, the issue of developing resistance to these treatments remains unresolved. This review emphasizes unique features of BRCA mutated OC and outlines the lay of the land in terms of diagnosis and treatment, while aiming to unravel the challenges that are part of its management.

Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.

Hematopoietic stem cells (HSCs) sustain lifelong blood cell regeneration by balancing their ability for self-renewal with their ability to differentiate into all blood cell types. To prevent organ exhaustion and malignant transformation, long-lived HSCs, in particular, must be protected from exogenous and endogenous stress, which cause severe DNA damage. When DNA is damaged, distinct DNA repair mechanisms and cell fate controls occur in adult HSCs compared with committed cells. Growth arrest and DNA damage-inducible 45 alpha (GADD45A) is known to coordinate a variety of cellular stress responses, indicating the molecule is an important stress mediator. So far, the function of GADD45A in hematopoietic stem and progenitor cells is controversial and appears highly dependent on the cell type and stress stimulus. Recent studies have analyzed its role in cell fate decision control of prospectively isolated HSCs and have revealed unexpected functions of GADD45A, as discussed here. The upregulation of GADD45A by DNA damage-causing conditions results in enhanced HSC differentiation, probably to efficiently eliminate aberrant HSCs from the system. These findings, in concert with a few studies on other stem cell systems, have led us to propose DNA damage-induced differentiation as a novel DNA damage response mechanism in stem cells that circumvents the fatal consequences of cumulative DNA damage in the stem cell compartment.

Nitric Oxide: Genomic Instability And Synthetic Lethality

Regardless of etiology, inflammatory conditions are characterized by overexpression of inducible nitric oxide synthase (iNOS) and overproduction of nitric oxide and reactive nitrogen species (NO/RNS) in epithelial and inflammatory cells at the site of carcinogenesis. NO/RNS produced in inflamed tissues can contribute to the process of carcinogenesis by different mechanisms. One of these mechanisms is NO-dependent stimulation of genomic instability by inhibiting of Breast Cancer type 1 Susceptibility protein (BRCA1) expression. Block of BRCA1 expression shifts DNA double-strand breaks (DSB) repair from error-free high-fidelity homologous recombination repair (HRR) to error-prone nonhomologous end joining (NHEJ). BRCA1 epigenetically block miRNA-155 expression via its association with HDAC2, which deacetylates histones H2A and H3 on the miRNA-155 promoter. The miRNA-155 is responsible for post-translational silencing of essential members of mismatch repair (MMR) core: MSH2, MSH6, and MLH1 proteins. They epigenetic inactivation induces DNA microsatellite instability (MSI). Recently, we demonstrated NO-dependent downregulation of MMR core proteins (MSH2, MSH6, and MLH1) through the ↓BRCA1/↑miRNA-155 signaling pathway. Hence, another NO-dependent mechanism of genomic instability is downregulation of MMR core proteins and stimulation of the DNA MSI. Loss or inhibition of Poly(ADP-ribose) polymerase 1 (PARP1) activity results in accumulation of DNA single-strand breaks, which are subsequently converted to DSB by the transcription machinery. In BRCA-positive cells, DSB are repaired by HRR, but they cannot be properly repaired in BRCA1-deficient cells, leading to genomic instability, chromosomal rearrangements, and cell death. Our data demonstrated that combination of NO-donors with PARP inhibitors significantly sensitized the BRCA1-positive cancer cells to DNA-damaging agents.

Genomic scars as biomarkers of homologous recombination deficiency and drug response in breast and ovarian cancers.

Poly (ADP-ribose) polymerase (PARP) inhibitors and platinum-based chemotherapies have been found to be particularly effective in tumors that harbor deleterious germline or somatic mutations in the BRCA1 or BRCA2 genes, the products of which contribute to the conservative homologous recombination repair of DNA double-strand breaks. Nonetheless, several setbacks in clinical trial settings have highlighted some of the issues surrounding the investigation of PARP inhibitors, especially the identification of patients who stand to benefit from such drugs. One potential approach to finding this patient subpopulation is to examine the tumor DNA for evidence of a homologous recombination defect. However, although the genomes of many breast and ovarian cancers are replete with aberrations, the presence of numerous factors able to shape the genomic landscape means that only some of the observed DNA abnormalities are the outcome of a cancer cell’s inability to faithfully repair DNA double-strand breaks. Consequently, recently developed methods for comprehensively capturing the diverse ways in which homologous recombination deficiencies may arise beyond BRCA1/2 mutation have used DNA microarray and sequencing data to account for potentially confounding features in the genome. Scores capturing telomeric allelic imbalance, loss of heterozygosity (LOH) and large scale transition score, as well as the total number of coding mutations are measures that summarize the total burden of certain forms of genomic abnormality. By contrast, other studies have comprehensively catalogued different types of mutational pattern and their relative contributions to a given tumor sample. Although at least one study to explore the use of the LOH scar in a prospective clinical trial of a PARP inhibitor in ovarian cancer is under way, limitations that result in a relatively low positive predictive value for these biomarkers remain. Tumors whose genome has undergone one or more events that restore high-fidelity homologous recombination are likely to be misclassified as double-strand break repair-deficient and thereby sensitive to PARP inhibitors and DNA damaging chemotherapies as a result of prior repair deficiency and its genomic scarring. Therefore, we propose that integration of a genomic scar-based biomarker with a marker of resistance in a high genomic scarring burden context may improve the performance of any companion diagnostic for PARP inhibitors.

Mismatch repair protein hMSH2-hMSH6 recognizes mismatches and forms sliding clamps within a D-loop recombination intermediate.

High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.