Lethal DNA Double-strand Breaks Research Articles

Inactivation of DNA Polymerase Theta (PolΘ) Is Synthetic Lethal in DNMT3A Mutated Myeloid Malignancies - Potential Clinical Applications

Somatic mutations in DNMT3A are associated with unfavorable outcome in patients with AML, MPN and CML. DNMT3A mutations promote resistance to anthracyclines (including daunorubicin, the component of standard “7+3” induction therapy), interferon alpha, and ABL1 kinase inhibitor imatinib. Thus, malignant clones carrying DNMT3A mutations may be difficult to eliminate using standard treatments. AML, MPN and CML cells harbor oncogenic tyrosine kinase (OTK) such as FLT3(ITD), JAK2(V617F) and BCR-ABL1, respectively. We reported before that elevated levels of formaldehyde generated by altered serine/one-carbon cycle metabolism contributed to accumulation of highly lethal DNA double-strand breaks (DSBs) in OTK-positive cells. To protect malignant cells from DSB-induced apoptosis, OTKs regulate DNA damage response (DDR) mechanisms involving DSBs sensing (ATM and ATR kinases) and repairing (RAD51-mediated homologous recombination = HR, RAD52-mediated transcription associated homologous recombination = TA-HR and single strand annealing = SSA, DNA-PK -mediated non-homologous end-joining = NHEJ, Polθ-dependent microhomology-mediated end-joining = TMEJ) as well as these activating cell cycle checkpoints (CHK1 and CHK2 kinases). Unfortunately, DNMT3A mutations caused resistance of OTK-positive cells to numerous DDR inhibitors (DDRis). DDRis sensitivity screen and synthetic lethal CRISPR/Cas9 screen revealed that OTK-positive cells with DNMT3A mutations are uniquely sensitive to the inhibition of DNA polymerase theta (Polθ encoded by POLQ gene). This effect was dependent on generation of formaldehyde by serine/one-carbon cycle metabolism. Abrogation of DNMT3A function by CRISPR/Cas9 targeting, Cre-loxP gene deletion, and heterozygous R882H mutation resulted in hypersensitivity of OTK-positive murine AML-like cells and human primary AML cells to Polθ inhibitors (Polθis) due to accumulation of toxic DSBs and activation of cGAS/STING pro-apoptotic pathway. Moreover, simultaneous loss of functional DNMT3A combined with inactivation of Polθ (by CRISPR/Cas9 targeting, insertion of neo-resistance gene, and D2230A+Y2231A polymerase inactive mutant) caused accumulation of DSBs, and reduced OTK-driven clonogenic potential and leukemogenic activity in mice. Polθ is abundantly overexpressed in OTK-positive DNMT3A-deficient cells due to enhanced POLQ mRNA stability and elevated translation of Polθ protein but not due to altered POLQ methylation and Polθ protein stability. Polθ is a key element not only in TMEJ of DSBs with limited end-resection, but also in replication fork restart and in single-strand DNA (ssDNA) gap filling. OTK-positive DNMT3A-deficient cells displayed hyperactivity of Polθ-mediated TMEJ and replication fork restart, but not ssDNA gap filling. These effects were accompanied by increased loading of Polθ on DNA damage detected by Polθ foci formation and chromatin extraction. Moreover, DNMT3A deficiency modulates chromatin architecture at DSBs to limit DNA end-resection thus favoring TMEJ over HR. Furthermore, we tested the effectiveness of Polθis combined with FDA approved drugs (quizartinib, etoposide, cytarabine, azacytidine) against FLT3(ITD)-positive DNMT3A-deficient cells (primary patient cells and cell lines) in vitro and in vivo. The combination of Polθis + quizartinib and Polθis + etoposide completely eradicated clonogenic activity of these cells while Polθis + cytarabine and Polθis + azacytidine exerted modest and weak effects, respectively, when compared to individual compound treatments. These drug combinations were only modestly toxic to normal bone marrow cells. Treatment with Polθi or etoposide reduced the percentage of GFP+ FLT3(ITD)-positive DNMT3A-deficient leukemia cells in peripheral blood of the mice by ~2-fold and prolonged survival time by ~1.5-fold. Remarkably, the combination of Polθi and etoposide eradicated leukemia cells below detectable levels in 6/12 mice with no visible toxicity. Median survival time of the mice will be recorded. Altogether, we discovered that Polθ protects OTK-positive DNMT3A-deficient myeloid malignant cells from the toxic effects of DSBs and identified Polθ as a novel therapeutic target.

Poly(ADP-Ribose) Glycohydrolase (PARG) Removes Repressive Poly-ADP Ribose Marks from DNA Polymerase Theta (PolΘ) to Stimulate DNA Double-Strand Breaks Repair in Myeloid Malignancies

Myeloid malignant cells (AML, MPN, CML) expressing oncogenic tyrosine kinases [OTKs: FLT3(ITD), JAK2(V617F), BCR/ABL1, respectively] accumulate DNA damage but altered DNA repair mechanisms protect them from apoptosis. We reported before that formaldehyde generated by altered serine/one-carbon cycle metabolism in malignant cells harboring OTKs contributed to accumulation of toxic DNA-protein crosslinks (DPCs) which were converted to highly lethal DNA double-strand breaks (DSBs). To counteract the toxicity of DSBs, OTKs enhanced the expression of DNA polymerase theta (PolΘ, encoded by POLQ gene), a unique DNA helicase-DNA polymerase fusion protein that promotes error-prone repair of DSBs by a mechanism referred to as PolΘ-mediated DNA end-joining (TMEJ). PolΘ plays an essential role in the initiation and maintenance of hematological malignancies harboring OTKs. Although the cellular activities of PolΘ have been widely studied, little is known about how PolΘ is regulated at the molecular level. For example, whether post-translational modifications of PolΘ are important for its TMEJ activity and regulation is unknown. Poly(ADP-ribose) polymerase 1 (PARP1) dependent poly-(ADP)-ribosylation (PARylation) of proteins is one of the major post-translational modification events involved in the DNA damage response (DDR). PARP1 - the founding member of the poly(ADP-ribose) polymerase family -transfers adenosine diphosphate (ADP)-ribose from nicotinamide adenine dinucleotide (NAD +) to substrate proteins enabling their PARylation. PARylation is tightly controlled by the glycohydrolase activity of poly(ADP-ribose) glycohydrolase (PARG). Thus, interplay between PARP1 and PARG is thought to regulate the PARylation status of relevant DDR proteins and multiple studies demonstrated that both PARP1 and PARG contribute to DDR. Here, we investigated whether the interplay of PARP1 and PARG is important for the regulation of TMEJ and the specific activities of PolΘ in myeloid malignancies harboring OTKs. We found that upon DNA damage OTKs exerted temporal effect on PolΘ, PARP1 and PARG detection in chromatin fractions. While both PolΘ and PARG displayed continuous time-dependent accumulation during 120 min. after irradiation, PARP1 levels sharply increased at 20 min. and dissipated at 120 min. We find that PARP1 binds to and directly PARylates PolΘ in vitro and in cells. However, PARylated PolΘ is unable to perform TMEJ in vitro due to its inability to bind DNA despite its PARylation dependent recruitment to DNA. Hence, PARG is needed to reactivate PolΘ DNA binding and its TMEJ activity by removing repressive PAR marks on PolΘ. In support of this, cellular studies show that PARG is essential for TMEJ and support a two-step mechanism of a PARP1-PARG regulatory axis of PolΘ and TMEJ. In the first step, the rate of PARP1-PolΘ spatiotemporal recruitment to DNA damage foci supports a rapid step whereby PARP1 PARylates PolΘ and facilitates its recruitment to the vicinity of DNA damage in an inactive state. The rate of PARG spatiotemporal recruitment supports a second step whereby subsequent recruitment of PARG corresponds to dissipation of PARP1 and PAR at DNA damage sites, dePARylation of PolΘ, and activation of TMEJ. These studies elucidate an unprecedented mechanism of PARP1-PARG activation of TMEJ and reveal the molecular basis by which PARP1 and PARG inhibition suppresses TMEJ. In conclusion, using intracellular and biochemical approaches we show here that both PARP1 and PARG are required for TMEJ despite their counteracting enzymatic activities. We also pinpointed a unique dynamic spatiotemporal interplay between PolΘ, PARP1 and PARG to regulate TMEJ activity. During the initial stage, PARP1 activated by DNA damage PARylates PolΘ and facilitates its recruitment to the vicinity of DNA damage. PARG removal of PAR from PolΘ reactivated its interaction with DNA resulting in robust TMEJ activity in vitro and in cells. Thus, DNA/chromatin binding and TMEJ activity of PolΘ were restored upon PARG-mediated removal of PAR repressive marks on PolΘ. This process seems to play a critical role in protecting OTK-positive hematopoietic malignant cells from genotoxic effect of formaldehyde generated by altered serine/one-carbon cycle metabolism.

Residual Foci of DNA Damage Response Proteins in Relation to Cellular Senescence and Autophagy in X-Ray Irradiated Fibroblasts.

DNA repair (DNA damage) foci observed 24 h and later after irradiation are called "residual" in the literature. They are believed to be the repair sites for complex, potentially lethal DNA double strand breaks. However, the features of their post-radiation dose-dependent quantitative changes and their role in the processes of cell death and senescence are still insufficiently studied. For the first time in one work, a simultaneous study of the association of changes in the number of residual foci of key DNA damage response (DDR) proteins (γH2AX, pATM, 53BP1, p-p53), the proportion of caspase-3 positive, LC-3 II autophagic and SA-β-gal senescent cells was carried out 24-72 h after fibroblast irradiation with X-rays at doses of 1-10 Gy. It was shown that with an increase in time after irradiation from 24 h to 72 h, the number of residual foci and the proportion of caspase-3 positive cells decrease, while the proportion of senescent cells, on the contrary, increases. The highest number of autophagic cells was noted 48 h after irradiation. In general, the results obtained provide important information for understanding the dynamics of the development of a dose-dependent cellular response in populations of irradiated fibroblasts.

Drug Discovery Targeting Post-Translational Modifications in Response to DNA Damages Induced by Space Radiation.

DNA damage in astronauts induced by cosmic radiation poses a major barrier to human space exploration. Cellular responses and repair of the most lethal DNA double-strand breaks (DSBs) are crucial for genomic integrity and cell survival. Post-translational modifications (PTMs), including phosphorylation, ubiquitylation, and SUMOylation, are among the regulatory factors modulating a delicate balance and choice between predominant DSB repair pathways, such as non-homologous end joining (NHEJ) and homologous recombination (HR). In this review, we focused on the engagement of proteins in the DNA damage response (DDR) modulated by phosphorylation and ubiquitylation, including ATM, DNA-PKcs, CtIP, MDM2, and ubiquitin ligases. The involvement and function of acetylation, methylation, PARylation, and their essential proteins were also investigated, providing a repository of candidate targets for DDR regulators. However, there is a lack of radioprotectors in spite of their consideration in the discovery of radiosensitizers. We proposed new perspectives for the research and development of future agents against space radiation by the systematic integration and utilization of evolutionary strategies, including multi-omics analyses, rational computing methods, drug repositioning, and combinations of drugs and targets, which may facilitate the use of radioprotectors in practical applications in human space exploration to combat fatal radiation hazards.

XRCC8 mutation causes hypersensitivity to PARP inhibition without Homologous recombination repair deficiency

PARP inhibitors inflict severe toxicity to homologous recombination (HR) repair deficient cells because DNA damages induced by PARP inhibition result in lethal DNA double strand breaks in the absence of HR repair during DNA replication. PARP inhibitors are the first clinically approved drugs designed for synthetic lethality. The synthetic lethal interaction of PARP inhibitors is not limited to HR repair deficient cells. We investigated radiosensitive mutants isolated from Chinese hamster lung origin V79 cells to identify novel synthetic lethal targets in the context of PARP inhibition. HR repair deficient BRCA2 mutant cells were used for positive control. Among tested cells, XRCC8 mutants presented hypersensitivity to PARP inhibitor, Olaparib. XRCC8 mutants showed elevated sensitivity to bleomycin and camptothecin similar to BRCA2 mutants. XRCC8 mutants presented an elevation of γ-H2AX foci formation frequency and S-phase dependent chromosome aberrations with Olaparib treatment. Enumerated damage foci following Olaparib treatment were observed to be elevated in XRCC8 as in BRCA2 mutants. Although this may suggest that XRCC8 plays a role in a similar DNA repair pathway as BRCA2 in HR repair, XRCC8 mutants presented functional HR repair including proper Rad51 foci formation and even elevated sister chromatid exchange frequencies with PARP inhibitor treatment. For comparison, RAD51 foci formation was suppressed in HR repair deficient BRCA2 mutants. Additionally, XRCC8 mutants did not display delayed mitotic entry with PARP inhibitors whereas BRCA2 mutants did. XRCC8 mutant cell line has previously been reported as possessing a mutation in the ATM gene. XRCC8 mutants displayed maximum cytotoxicity to ATM inhibitor among tested mutants and wild type cells. Furthermore, the ATM inhibitor sensitized XRCC8 mutant to ionzing radiation, however, XRCC8 mutant V-G8 expressed reduced levels of ATM protein. The gene responsible for XRCC8 phenotype may not be ATM but highly associated with ATM functions. These results suggest that XRCC8 mutation is a target for PARP inhibitor-induced synthetic lethality in HR repair independent manner via the disruption of cell cycle regulation. Our findings expand the potential application of PARP inhibitors in tumors lacking DNA damage responding genes other than HR repair, and further investigation of XRCC8 may contribute to this research.

Polθ Inhibition: An Anticancer Therapy for HR-Deficient Tumours.

DNA polymerase theta (Polθ)-mediated end joining (TMEJ) is, along with homologous recombination (HR) and non-homologous end-joining (NHEJ), one of the most important mechanisms repairing potentially lethal DNA double-strand breaks (DSBs). Polθ is becoming a new target in cancer research because it demonstrates numerous synthetically lethal interactions with other DNA repair mechanisms, e.g., those involving PARP1, BRCA1/2, DNA-PK, ATR. Inhibition of Polθ could be achieved with different methods, such as RNA interference (RNAi), CRISPR/Cas9 technology, or using small molecule inhibitors. In the context of this topic, RNAi and CRISPR/Cas9 are still more often applied in the research itself rather than clinical usage, different than small molecule inhibitors. Several Polθ inhibitors have been already generated, and two of them, novobiocin (NVB) and ART812 derivative, are being tested in clinical trials against HR-deficient tumors. In this review, we describe the significance of Polθ and the Polθ-mediated TMEJ pathway. In addition, we summarize the current state of knowledge about Polθ inhibitors and emphasize the promising role of Polθ as a therapeutic target.

Abstract 1843: Dianhydrogalactitol (VAL-083) for the treatment of glioblastoma multiforme (GBM): Impact of glucose transporters for crossing the blood brain barrier (BBB)

Abstract Glucose transporters at the blood brain barrier (BBB) maintain the continuous high glucose and energy demands of the brain. These transporters have been targets for the creation of therapeutic molecules as a means to cross the BBB. The BBB has been a major limitation for the successful development of treatments for brain tumors. To date, only a limited number of lipophilic small chemical entities, which can cross the BBB, have been approved for the treatment of GBM. These include the nitrosoureas such as carmustine (BCNU) and lomustine (CCNU) and temozolomide (TMZ). VAL-083 is a di-epoxide substituted hexitol sugar (galactitol). It has a molecular weight of 146 and is highly water soluble. VAL-083 is active against several GBM cell lines and animal models and acts as a DNA targeting agent rapidly forming cross-links at the N7 position of guanine, resulting in lethal DNA double strand breaks and cancer cell death. Its is active in GBM patients, both newly diagnosed and recurrent. Furthermore, its activity is independent of the DNA repair enzyme MGMT which limits the effectiveness of O6 guanine alkylating agents such as the nitrosoureas and TMZ.In clinical pharmacology studies, VAL-083 rapidly accumulates in the cerebrospinal fluid (CSF) and brain tumor tissue. Studies with radiolabeled VAL-083 have shown a half-life of ~ 20 h in CSF vs 2h for plasma post-IV infusion. Recent clinical studies in newly diagnosed GBM patients demonstrated levels of VAL-083 in CSF at least as high as in plasma two hours post infusion (ratio 1.24 ± 0.35). In comparison, peak TMZ concentrations in the CSF are ~30% of that of the plasma.Due to its structural similarity with glucose, we have initiated in vitro glucose transporter studies to identify if SGLTs (sodium dependent glucose cotransporters) or GLUTs (glucose transporters) are contributing to brain penetration. Our initial studies evaluated VAL-083 as a substrate of the human SGLT1 and SGLT2 SLC (solute carrier) transporters. HEK293 cells expressing either the SGLT1 or SGLT2 transporter were exposed to VAL-083 at 3 and 15 µM for 2 and 20 min. The probe substrate methyl-α-D- glucopyranoside (AMG) was used as a positive control, as well as comparison against the reference inhibitors phloridzin dihydrate (100 µM, 20 min) for SGLT1 and dapagliflozin (0.3 µM, 10 min) for SGLT2. Surprisingly, no transporter-specific accumulation of VAL-083 by SGLT1 or SGLT2 was observed. Ongoing studies in CaCo-2 cells are investigating whether VAL-083 is transported via other glucose transporters including GLUT1. GLUT1 is expressed on erythrocytes and the endothelial cells of the BBB and may be highly relevant to the uptake of VAL-083 into brain tissue. The results of these studies will be reported at the meeting. Citation Format: John M. Langlands, Sarath Kanekal, Anne Steino, Armando Mendez, Zoltan Karman, Beata Martonne Toth, Krisztina Heredi-Szabo, Dennis Brown. Dianhydrogalactitol (VAL-083) for the treatment of glioblastoma multiforme (GBM): Impact of glucose transporters for crossing the blood brain barrier (BBB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1843.

BAY-8400: A Novel Potent and Selective DNA-PK Inhibitor which Shows Synergistic Efficacy in Combination with Targeted Alpha Therapies

Eukaryotes have evolved two major pathways to repair potentially lethal DNA double-strand breaks. Homologous recombination represents a precise, DNA-template-based mechanism available during the S and G2 cell cycle phase, whereas non-homologous end joining, which requires DNA-dependent protein kinase (DNA-PK), allows for fast, cell cycle-independent but less accurate DNA repair. Here, we report the discovery of BAY-8400, a novel selective inhibitor of DNA-PK. Starting from a triazoloquinoxaline, which had been identified as a hit from a screen for ataxia telangiectasia and Rad3-related protein (ATR) inhibitors with inhibitory activity against ATR, ATM, and DNA-PK, lead optimization efforts focusing on potency and selectivity led to the discovery of BAY-8400. In in vitro studies, BAY-8400 showed synergistic activity of DNA-PK inhibition with DNA damage-inducing targeted alpha therapy. Combination of PSMA-targeted thorium-227 conjugate BAY 2315497 treatment of human prostate tumor-bearing mice with BAY-8400 oral treatment increased antitumor efficacy, as compared to PSMA-targeted thorium-227 conjugate monotherapy.

Ruxolitinib-Induced Defects in DNA Repair Cause Sensitivity to PARP Inhibitors in Myeloproliferative Neoplasms

Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), which are associated with mutations in JAK2, CALR and MPL genes. Current treatment options for Ph- MPNs include cytoreductive therapy with hydroxyurea, and the JAK2 kinase inhibitors (e.g., ruxolitinib), which produce durable reductions in splenomegaly and improvement of symptoms and probably of survival, but do not eliminate the disease-initiating cell population. All three disease “driver” mutations, [JAK2(V617F), CALR(del52), and MPL(W515L)] have been detected not only in mature MPN cells, but also in MPN stem cells, and therefore these cells must be eliminated to eradicate the disease. Since JAK2 kinase inhibitors did not eliminate the disease-initiating population novel therapeutic approaches are needed. We show here that cell lines expressing JAK2(V617F), MPL(W515L) or CALR(del52) accumulated reactive oxygen species-induced lethal DNA double-strand breaks (DSBs), which were efficiently repaired thus preventing MPN cell death. Inhibition of JAK2 kinase by ruxolitinib caused downregulation of BRCA1, RAD51 and LIG4, the key proteins in two major DSB repair mechanisms, BRCA-mediated homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (NHEJ). We recently reported (Nieborowska-Skorska et al., J.Clin.Invest., 2017) that NHEJ-deficient acute myeloid leukemia (AML) quiescent stem cells and HR/NHEJ-deficient AML proliefrating stem and progenitor cells were extremely sensitive to synthetic lethality triggered by poly-ADP-ribose polymerase (PARP) inhibitors olaparib and BMN673 (talazoparib). Therefore, we hypothesized that PARP inhibitors should be very effective against ruxolitinib-treated HR/NHEJ-deficient MPN quiescent and proliferating cells. Olaparib, when combined with ruxolitinib caused abundant accumulation of toxic DSBs resulting in enhanced elimination of MPN primary cells, including the disease-initiating cells from the majority of samples. Importantly, combination of BMN673, ruxolitinib and hydroxyurea was highly effective in vivo against JAK2(V617F)-positive murine MPN-like disease and also against pre-selected JAK2(V617F), CALR(del52), and MPL(W515L)-positive primary MPN xenografts. However, MPN samples from individual patients displayed high degree of variability in the sensitivity to JAK2 inhibitor combined with PARP inhibitor and/or hydroxyurea. The differences between susceptibility of individual MPNs to these drugs may depend on additional mutations accompanying the “driver” mutations (e.g., TET2 mutation) and on homo/heterozygosity of the “driver” mutation allele. In conclusion, we postulate that ruxolitinib-induced deficiencies in DSB repair pathways sensitized pre-selected MPN cells to synthetic lethality triggered by PARP inhibitors. DisclosuresKoschmieder:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche: Other: Clinical Trial participation. Skoda:Novartis: Consultancy, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Consultancy, Speakers Bureau.

Homologous recombination deficiency in epithelial ovarian cancer

SummarySince the introduction of poly-ADP-ribose polymerase (PARP) inhibitor therapy for epithelial ovarian cancer (EOC) patients, testing for aberrations of homologous recombination (HR) repair as a predictive biomarker of therapy response has become an area of particular clinical interest. As HR represents a crucial repair pathway of otherwise possibly lethal DNA double strand breaks, its deficiency triggers a phenotypic behavior of tumor cells resulting in the accumulation of genetic damage. PARP inhibitors target this emerging genomic instability by fostering DNA strand breaks. Whereas testing for mutations of the tumor-suppressor genes BRCA 1 and BRCA 2 as a pivotal part of the HR apparatus has entered clinical routine, approximately 30% more high-grade EOC patients harbor aberrations of the HR pathway other than BRCA mutations and may therefore respond to PARP inhibition therapy. In recent years, several double-blind, placebo-controlled trials investigating sizeable patient cohorts have reported positive results of PARP inhibitor therapy response in HR-positive patient subgroups. Therefore, introducing HR testing in both the primary and recurrent setting as a biomarker for PARP inhibitor response may expand the range of patients who may profit from this therapeutic option beyond BRCA-mutated tumors.

Eradication of LIG4-deficient glioblastoma cells by the combination of PARP inhibitor and alkylating agent.

Cancer cells often accumulate spontaneous and treatment-induced DNA damage i.e. potentially lethal DNA double strand breaks (DSBs). Targeting DSB repair mechanisms with specific inhibitors could potentially sensitize cancer cells to the toxic effect of DSBs. Current treatment for glioblastoma includes tumor resection followed by radiotherapy and/or temozolomide (TMZ) – an alkylating agent inducing DNA damage. We hypothesize that combination of PARP inhibitor (PARPi) with TMZ in glioblastoma cells displaying downregulation of DSB repair genes could trigger synthetic lethality. In our study, we observed that PARP inhibitor (BMN673) was able to specifically sensitize DNA ligase 4 (LIG4)-deprived glioblastoma cells to TMZ while normal astrocytes were not affected. LIG4 downregulation resulting in low effectiveness of DNA-PK–mediated non-homologous end-joining (D-NHEJ), which in combination with BMN673 and TMZ resulted in accumulation of lethal DSBs and specific eradication of glioblastoma cells. Restoration of the LIG4 expression caused loss of sensitivity to BMN673+TMZ. In conclusion, PARP inhibitor combined with DNA damage inducing agents can be utilized in patients with glioblastoma displaying defects in D-NHEJ.

Escherichia coli transcription factor NusG binds to 70S ribosomes.

Transcription and translation are coupled processes in bacteria. A role of transcription elongation cofactor NusG in coupling has been suggested by in vitro structural studies. NMR revealed association of the NusG carboxy-terminal domain with S10 (NusE), implying a direct role for NusG as a bridge linking RNAP and the lead ribosome. Here we present the first in vitro and in vivo evidence of full-length NusG association with mature 70S ribosomes. Binding did not require accessory factors in vitro. Mutating the NusG:S10 binding interface at NusG F165 or NusE M88 and D97 residues weakened NusG:S10 association in vivo and completely abolished it in vitro, supporting the specificity of this interaction. Mutations in the binding interface increased sensitivity to chloramphenicol. This phenotype was suppressed by rpoB*35, an RNAP mutation that reduces replisome-RNAP clashes. We propose that weakened NusG:S10 interaction leads to uncoupling when translation is inhibited, with resulting RNAP backtracking, replication blocks and formation of lethal DNA double-strand breaks.

ATM inhibition prevents interleukin-6 from contributing to the proliferation of glioblastoma cells after ionizing radiation.

Glioblastoma (GBM) is a highly fatal disease with a 5year survival rate of less than 22%. One of the most effective treatment regimens to date is the use of radiotherapy which induces lethal DNA double-strand breaks to prevent tumour growth. However, recurrence occurs in the majority of patients and is in-part a result of robust radioresistance mechanisms. In this study, we demonstrate that the multifunctional cytokine, interleukin-6 (IL-6), confers a growth advantage in GBM cells but does not have the same effect on normal neural progenitor cells. Further analysis showed IL-6 can promote radioresistance in GBM cells when exposed to ionising radiation. Ablation of the Ataxia-telangiectasia mutated serine/threonine kinase that is recruited and activated by DNA double-strand breaksreverses the effect of radioresistance andre-sensitised GBM to DNA damagethus leading to increase cell death.Our finding suggests targeting the signaling cascade of DNA damage response is a potential therapeutic approach to circumvent IL-6 from promoting radioresistance in GBM.

PARP-1 inhibition with or without ionizing radiation confers reactive oxygen species-mediated cytotoxicity preferentially to cancer cells with mutant TP53.

Biomarkers and mechanisms of poly (ADP-ribose) polymerase (PARP) inhibitor-mediated cytotoxicity in tumor cells lacking a BRCA-mutant or BRCA-like phenotype are poorly defined. We sought to explore the utility of PARP-1 inhibitor (PARPi) treatment with/without ionizing radiation in muscle-invasive bladder cancer (MIBC), which has poor therapeutic outcomes. We assessed the DNA damaging and cytotoxic effects of the PARPi olaparib in nine bladder cancer cell lines. Olaparib radiosensitized all cell lines with dose enhancement factors from 1.22 to 2.27. Radiosensitization was correlated with the induction of potentially lethal DNA double-strand breaks (DSB) but not with RAD51 foci formation. The ability of olaparib to radiosensitize MIBC cells was linked to the extent of cell kill achieved with drug alone. Unexpectedly, increased levels of reactive oxygen species (ROS) resulting from PARPi treatment were the cause of DSB throughout the cell cycle in-vitro and in-vivo. ROS originated from mitochondria and were required for the radiosensitizing effects of olaparib. Consistent with the role of TP53 in ROS regulation, loss of p53 function enhanced radiosensitization by olaparib in non-isogenic and isogenic cell line models and was associated with increased PARP-1 expression in bladder cancer cell lines and tumors. Impairment of ATM in addition to p53 loss resulted in an even more pronounced radiosensitization. In conclusion, ROS suppression by PARP-1 in MIBC is a potential therapeutic target either for PARPi combined with radiation or drug alone treatment. The TP53 and ATM genes, commonly mutated in MIBC and other cancers, are candidate biomarkers of PARPi-mediated radiosensitization.

Differential modes of photosensitisation in cancer cells by berberine and coralyne

In this study, we demonstrated that the cytotoxicity of the protoberberine alkaloids such as coralyne, berberine and jatrorrhizine to several human cancer cell lines can be improved significantly in combination with UVA exposure. However, the phototoxic property of coralyne was much higher than that of the other two alkaloids. The combination of coralyne and UVA (designated as CUVA) induced oxygen-independent cytotoxicity in the human lung cancer A549 cells by producing more lethal DNA double-strand breaks, and the effect was mediated via the replication machinery. In comparison, the berberine-induced phototoxicity to the A549 cells was mediated by reactive oxygen species generation, mitochondrial membrane permeabilisation and caspase-9/caspase-3 activation.

Highlights of This Issue

Patient-derived xenograft (PDX) and CTC-derived explant (CDX) models are powerful tools for studying tumors, particularly when used in combination with next-generation sequencing (NGS) approaches. This depends on data analysis pipelines that accurately distinguish between sequencing reads arising from the host, and those that come from the xenograft itself. Here, Khandelwal and colleagues reveal that failure to do this effectively leads to incorrect mutation calls and inaccurate measures of differential expression. Importantly, an open source software tool is described for removing contaminating host sequences and its utility as a highly sensitive and selective tool for correcting PDX/CDX-derived NGS data is demonstrated.Cancer-specific defects in DNA repair pathways create the opportunity to employ synthetic lethality, already applied against breast and ovarian cancer cells harboring deleterious mutations in BRCA1 and BRCA2, by using PARP1 inhibitors. The current Rapid Impact, demonstrates that Burkitt lymphoma/leukemia (BL) cells carrying the IGH/MYC translocation display low levels of BRCA2 tumor suppressor protein accompanied by inhibition of homologous recombination (HR) repair of potentially lethal DNA double-strand breaks. BRCA2-deficient BL cells are hypersensitive to PARP1 inhibitors used either alone or in combination with cytarabine. Therefore, patients with BL may benefit therapeutically from PARP1 inhibitors, such as the recently FDA-approved olaparib.Much of our understanding of metabolism in cancer involves the study of glucose. However, cancer cells often take up and metabolize increased levels of glutamine, relative to benign cells, to help satisfy the demands of a rapidly growing tumor. By mining clinical cohorts and functional testing in cell models, White and colleagues demonstrate that three well-established oncogenic drivers of prostate cancer (AR, MYC and mTOR) converge to increase the expression of the glutamine transporters SLC1A4 and SLC1A5, thereby promoting glutamine uptake and subsequent prostate cancer cell growth. Thus, SLC1A4 and SLC1A5 may offer promise as new therapeutic targets and/or biomarkers.The tumor suppressor p53 is mutated in about half of all cancers and its mutation drives tumorigenesis. In this study, Pappas and colleagues found that p53 maintains baseline expression of thirteen tumor suppressor genes through consensus DNA binding sites in enhancer and promoter elements. These tumor suppressor targets of basally expressed p53, together with stress-responsive targets of p53, mediate the ability of p53 to act as a tumor suppressor. Importantly, these results indicate that mutation of p53 results in reduced expression of multiple tumor suppressor genes in parallel, and helps to explain the high frequency of p53 mutations in cancer.

IGH/MYC Translocation Associates with BRCA2 Deficiency and Synthetic Lethality to PARP1 Inhibitors.

Burkitt lymphoma/leukemia cells carry t(8;14)(q24;q32) chromosomal translocation encoding IGH/MYC, which results in the constitutive expression of the MYC oncogene. Here, it is demonstrated that untreated and cytarabine (AraC)-treated IGH/MYC-positive Burkitt lymphoma cells accumulate a high number of potentially lethal DNA double-strand breaks (DSB) and display low levels of the BRCA2 tumor suppressor protein, which is a key element of homologous recombination (HR)-mediated DSB repair. BRCA2 deficiency in IGH/MYC-positive cells was associated with diminished HR activity and hypersensitivity to PARP1 inhibitors (olaparib, talazoparib) used alone or in combination with cytarabine in vitro Moreover, talazoparib exerted a therapeutic effect in NGS mice bearing primary Burkitt lymphoma xenografts. In conclusion, IGH/MYC-positive Burkitt lymphoma/leukemia cells have decreased BRCA2 and are sensitive to PARP1 inhibition alone or in combination with other chemotherapies.Implications: This study postulates that IGH/MYC-induced BRCA2 deficiency may predispose Burkitt lymphoma cells to synthetic lethality triggered by PARP1 inhibitors.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/15/8/967/F1.large.jpgMol Cancer Res; 15(8); 967-72. ©2017 AACR.

Abstract LB-170: A novel, nontoxic RXR agonist, UAB30, targets Fanconi anemia DNA repair pathway to act as potent cancer chemopreventive agent

Abstract UAB30, a synthetic analog of 9-cis-RA is a potent retinoid X receptors (RXR) agonist. It was found to have significantly low retinoic acid receptor (RAR)-binding activity. This unique property of UAB30 is responsible for its low lipid toxicity. Thus, it does not cause elevation in serum triglycerides both in humans and in experimental animals. These properties with favorable pharmacokinetics make it a suitable candidate for chronic use as chemopreventive agent. Here, we show that UAB30 is highly effective in inhibiting UVB-induced pathogenesis of skin cancers in Ptch1+/-/SKH-1 hairless mice. Similar to humans, this unique genetically engineered murine model develops both basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin following chronic UVB irradiation. Compared to controls, the UAB30-treated group showed a 48% reduction in tumor number and a 78% reduction in tumor volume. The number of papillomas, BCCs, and SCCs were reduced by 33%, 74%, and 76%, respectively. UAB30 also diminished UVB-induced skin inflammation, hyperplasia, and enhanced differentiation of suprabasal epidermal cells. The expression of cyclins A, D1, D2, and D3, and cdc25A were decreased in UAB30 treated SCCs, while pro-apoptotic proteins, Bax and cleaved caspase 3 were increased. UAB30 when topically applied 30 min after each UVB irradiation for 30 weeks, it showed significant reduction in cancer development. A 57% reduction in tumor number and 72% reduction in tumor volume were observed. BCCs and SCCs were reduced by 55% and 60%, respectively. We also demonstrate that UAB30 acts by a unique molecular mechanism. Induction of lethal DNA double strand breaks (DSBs) characterized by the presence of p-γH2AX was observed following UAB30 treatment. These effects were more prominent in cancer cells than normal keratinocytes. Induction of DSBs was associated with the activation of DNA damage response (DDR) signaling leading to increased phosphorylation of ATM, Rad50, and Chk2. These effects paralleled with the reduction in cell cycle proteins and cell cycle arrest in G2/M phase followed by apoptosis of arrested cells. UAB30-induced DSBs and DDR were not dependent on ROS as we did not observe significant induction of ROS in UAB30-treated cells and did not notice any protective effect of N-acetyl cysteine. These effects were similar to DDR and changes in proliferation/apoptosis response observed following treatment with cisplatin which is known inducer of DSBs. Moreover, other RXR agonists were significantly less potent in inducing DSBs and DDR responses. The underlying mechanism for the accumulation of DSBs was the diminished ability of cancer cells to repair these DNA lesions. Reduction in FANCD2 and FANCC, the Fanconi anemia core complex proteins which are known to be involved in DSBs repair appear to be the cause of extensive DNA damage associated tumor cell death. Citation Format: Sandeep C. Chaudhary, Craig A. Elmets, Mohammad Athar. A novel, nontoxic RXR agonist, UAB30, targets Fanconi anemia DNA repair pathway to act as potent cancer chemopreventive agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-170. doi:10.1158/1538-7445.AM2017-LB-170

Abstract 3072: A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death

Abstract The effectiveness of certain classes of cytotoxic cancer therapeutics likely depends on whether drug-induced DNA damage is successfully repaired or not, with the latter situation leading to mutations and strand breaks. However, double strand breaks (DSBs) also occur independently of genotoxic insults during apoptotic cell death caused by many drug classes, as well as natural biological processes. We have developed an immunofluorescent confocal microscopy assay that uses a biomarker profile suitable for individual cell analysis designed to distinguish between DSBs caused by apoptosis and those caused by direct DNA damage from cytotoxic drug action. γ-H2AX is an established biomarker for DSBs and activated cleaved caspase 3 is an executioner caspase important for apoptosis, which leads to nuclear condensation, DNA fragmentation, plasma membrane blebbing, and subsequent cell death. Our assay defines the DSBs of apoptosis by co-localized γ-H2AX and cleaved caspase 3 in individual cells, while defining the DSBs from early drug effects of DNA damaging chemotherapeutics by γ-H2AX induction in the absence of cleaved caspase 3. Building on our published findings that topotecan strongly induces γ-H2AX and DSBs within 1-4 hours in vitro and in vivo, we observed exposure-dependent increases in γ-H2AX /cleaved caspase 3 double positive cells at later time points, both in an HT29 in vitro spheroid model and an MDA-MB-231 xenograft model. Fit-for-purpose studies in the MDA-MB-231 xenograft model treated with birinipant, a SMAC mimetic and IAP deregulator that does not directly produce lethal DSBs, demonstrated a dose-dependent increase in cellular co-localization of γ-H2AX/cleaved caspase 3 consistent with birinipant induced apoptosis and the established mechanism of action of this compound. Clinical feasibility was established in a canine clinical trial using formalin-fixed paraffin-embedded (FFPE) 18-gauge needle biopsies: two novel indenoisoquinolines, indotecan (LMP400) and indimitecan (LMP776), increased tumor cell co-localization of γ-H2AX/cleaved caspase 3 in tumor samples obtained on day 5 of qdx5 treatment. This PD biomarker assay of early and late DSB response to drug exposure could have important applications for elucidation of mechanisms of action of anticancer drugs and the development of investigational agents. Funded by NCI Contract No. HHSN261200800001E. Citation Format: Angie B. Dull, Deborah Wilsker, Robert J. Kinders, Ralph E. Parchment, David Evans, Beverly A. Teicher, James H. Doroshow. A clinical pharmacodynamic biomarker assay that distinguishes potentially repairable, cytotoxic drug-induced DNA double strand breaks (DSBs) from DSBs associated with apoptotic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3072. doi:10.1158/1538-7445.AM2017-3072

Targeting DNA-Dependent Protein Kinase for Cancer Therapy.

The catalytic activity of DNA-dependent protein kinase (DNA-PK) is critical to its ability to repair lethal DNA double-strand breaks (DSBs). This includes repair of DSB lesions resulting from oxidative stress, oncogene-induced transcription, or following therapeutic treatment of cancer cells. Armed with this knowledge, many attempts have been made to identify small-molecule inhibitors of DNA-PK activity as an approach to induce tumour chemo- and radiosensitisation. This review examines the structures of known reversible and irreversible inhibitors, including those based on chromen-4-one, arylmorpholine, and benzaldehyde scaffolds. DNA-PK catalytic inhibitors, such as VX-984 (8-[(1S)-2-[[6-(4,6-dideuterio-2-methylpyrimidin-5-yl)pyrimidin-4-yl]amino]-1-methylethyl]quinoline-4-carboxamide) and M3814 ((S)-[2-chloro-4-fluoro-5-(7-morpholinoquinazolin-4-yl)phenyl]-(6-methoxypyridazin-3-yl)methanol), have now progressed into clinical development which should help to further advance our understanding of whether this approach is a promising therapeutic strategy for the treatment of cancer.