Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers.

DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors. SIGNIFICANCE: Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Elevated levels of mitochondrial CoQ10 induce ROS-mediated apoptosis in pancreatic cancer.

Reactive oxygen species (ROS) are implicated in triggering cell signalling events and pathways to promote and maintain tumorigenicity. Chemotherapy and radiation can induce ROS to elicit cell death allows for targeting ROS pathways for effective anti-cancer therapeutics. Coenzyme Q10 is a critical cofactor in the electron transport chain with complex biological functions that extend beyond mitochondrial respiration. This study demonstrates that delivery of oxidized Coenzyme Q10 (ubidecarenone) to increase mitochondrial Q-pool is associated with an increase in ROS generation, effectuating anti-cancer effects in a pancreatic cancer model. Consequent activation of cell death was observed in vitro in pancreatic cancer cells, and both human patient-derived organoids and tumour xenografts. The study is a first to demonstrate the effectiveness of oxidized ubidecarenone in targeting mitochondrial function resulting in an anti-cancer effect. Furthermore, these findings support the clinical development of proprietary formulation, BPM31510, for treatment of cancers with high ROS burden with potential sensitivity to ubidecarenone.

PDX-derived organoids model in vivo drug response and secrete biomarkers.

Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired patient-derived xenograft (PDX) and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between area under the curve value of organoid drug dose response and in vivo tumor growth, irrespective of the drug treatment. In addition, we analyzed the glycome of PDX and PXO models and demonstrate that PXOs recapitulate the in vivo glycan landscape. In addition, we identify a core set of 57 N-glycans detected in all 10 models that represent 50%-94% of the relative abundance of all N-glycans detected in each of the models. Last, we developed a secreted biomarker discovery pipeline using media supernatant of organoid cultures and identified potentially new extracellular vesicle (EV) protein markers. We validated our findings using plasma samples from patients with PDAC, benign gastrointestinal diseases, and chronic pancreatitis and discovered that 4 EV proteins are potential circulating biomarkers for PDAC. Thus, we demonstrate the utility of organoid cultures to not only model in vivo drug responses but also serve as a powerful platform for discovering clinically actionable serologic biomarkers.

Position-Dependent Effect of Guanine Base Damage and Mutations on Telomeric G-Quadruplex and Telomerase Extension.

Telomeres are hot spots for mutagenic oxidative and methylation base damage due to their high guanine content. We used single-molecule fluorescence resonance energy transfer detection and biochemical assays to determine how different positions and types of guanine damage and mutations alter telomeric G-quadruplex structure and telomerase activity. We compared 15 modifications, including 8-oxoguanine (8oxoG), O-6-methylguanine (O6mG), and all three possible point mutations (G to A, T, and C) at the 3' three terminal guanine positions of a telomeric G-quadruplex, which is the critical access point for telomerase. We found that G-quadruplex structural instability was induced in the order C < T < A ≤ 8oxoG < O6mG, with the perturbation caused by O6mG far exceeding the perturbation caused by other base alterations. For all base modifications, the central G position was the most destabilizing among the three terminal guanines. While the structural disruption by 8oxoG and O6mG led to concomitant increases in telomerase binding and extension activity, the structural perturbation by point mutations (A, T, and C) did not, due to disrupted annealing between the telomeric overhang and the telomerase RNA template. Repositioning the same mutations away from the terminal guanines caused both G-quadruplex structural instability and elevated telomerase activity. Our findings demonstrate how a single-base modification drives structural alterations and telomere lengthening in a position-dependent manner. Furthermore, our results suggest a long-term and inheritable effect of telomeric DNA damage that can lead to telomere lengthening, which potentially contributes to oncogenesis.

The Importance of Serial Time Point Quantitative Assessment of Cardiac Troponin I in the Diagnosis of Acute Myocardial Damage.

OBJECTIVE: The present study was aimed to establish a threshold value for cardiac troponin I (cTnI) for nonacute coronary syndrome (ACS) participants from the local population and also to determine the importance of serial time point estimation of cTnI in acute myocardial infarction (AMI), non-ST-elevated MI (NSTEMI), and unstable angina cases. METHODS: The present study included 194 cases, admitted in ICCU with the complaint of anginal pain; 31 were diagnosed with AMI with typical electrocardiography (ECG) changes; whereas, 48 cases were diagnosed with NSTEMI. The latter group of cases was selected for the time point study of cTnI release at 0-4 h, 6-12 h, 72 h, and 144 h of admission. cTnI levels were assessed using the Abbott ARCHITECT i1000SR system. RESULTS: ACS was clinically ruled out in 98 cases, and cTnI level for them was used to decide cTnI threshold for the non-ACS group. cTnI level was checked in 17 cases of unstable angina. The threshold value of cTnI for non-ACS participants was 0.1 ng/ml and can be considered as cut-off value for the regional population. The data suggested that the peak of cTnI levels in most of the AMI cases reached during 6-12 h. The cTnI levels were lower than 0.1 ng/ml, and no significant change in ECG was noticed in 17 cases of unstable angina. CONCLUSION: The present study suggested that the repeat of cTnI assay after 4-6 h of admission is required if the initial value is <3 ng/ml.

Molecular mechanisms by which oxidative DNA damage promotes telomerase activity.

Telomeres are highly susceptible to oxidative DNA damage, which if left unrepaired can lead to dysregulation of telomere length homeostasis. Here we employed single molecule FRET, single molecule pull-down and biochemical analysis to investigate how the most common oxidative DNA lesions, 8-oxoguanine (8oxoG) and thymine glycol (Tg), regulate the structural properties of telomeric DNA and telomerase extension activity. In contrast to 8oxoG which disrupts the telomeric DNA structure, Tg exhibits substantially reduced perturbation of G-quadruplex folding. As a result, 8oxoG induces high accessibility, whereas Tg retains limited accessibility, of telomeric G-quadruplex DNA to complementary single stranded DNA and to telomere binding protein POT1. Surprisingly, the Tg lesion stimulates telomerase loading and activity to a similar degree as an 8oxoG lesion. We demonstrate that this unexpected stimulation arises from Tg-induced conformational alterations and dynamics in telomeric DNA. Despite impacting structure by different mechanisms, both 8oxoG and Tg enhance telomerase binding and extension activity to the same degree, potentially contributing to oncogenesis.

Biosimilars: Key regulatory considerations and similarity assessment tools.

A biosimilar drug is defined in the US Food and Drug Administration (FDA) guidance document as a biopharmaceutical that is highly similar to an already licensed biologic product (referred to as the reference product) notwithstanding minor differences in clinically inactive components and for which there are no clinically meaningful differences in purity, potency, and safety between the two products. The development of biosimilars is a challenging, multistep process. Typically, the assessment of similarity involves comprehensive structural and functional characterization throughout the development of the biosimilar in an iterative manner and, if required by the local regulatory authority, an in vivo nonclinical evaluation, all conducted with direct comparison to the reference product. In addition, comparative clinical pharmacology studies are conducted with the reference product. The approval of biosimilars is highly regulated although varied across the globe in terms of nomenclature and the precise criteria for demonstrating similarity. Despite varied regulatory requirements, differences between the proposed biosimilar and the reference product must be supported by strong scientific evidence that these differences are not clinically meaningful. This review discusses the challenges faced by pharmaceutical companies in the development of biosimilars.

Translesion Synthesis of 2'-Deoxyguanosine Lesions by Eukaryotic DNA Polymerases.

With the discovery of translesion synthesis DNA polymerases, great strides have been made in the last two decades in understanding the mode of replication of various DNA lesions in prokaryotes and eukaryotes. A database search indicated that approximately 2000 articles on this topic have been published in this period. This includes research involving genetic and structural studies as well as in vitro experiments using purified DNA polymerases and accessory proteins. It is a daunting task to comprehend this exciting and rapidly emerging area of research. Even so, as the majority of DNA damage occurs at 2'-deoxyguanosine residues, this perspective attempts to summarize a subset of this field, focusing on the most relevant eukaryotic DNA polymerases responsible for their bypass.

Oxidative guanine base damage regulates human telomerase activity.

Changes in telomere length are associated with degenerative diseases and cancer. Oxidative stress and DNA damage have been linked to both positive and negative alterations in telomere length and integrity. Here we examined how the common oxidative lesion 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG) regulates telomere elongation by human telomerase. When 8-oxoG is present in the dNTP pool as 8-oxodGTP, telomerase utilization of the oxidized nucleotide during telomere extension is mutagenic and terminates further elongation. Depletion of MTH1, the enzyme that removes oxidized dNTPs, increases telomere dysfunction and cell death in telomerase-positive cancer cells with shortened telomeres. In contrast, a preexisting 8-oxoG within the telomeric DNA sequence promotes telomerase activity by destabilizing the G-quadruplex DNA structure. We show that the mechanism by which 8-oxoG arises in telomeres, either by insertion of oxidized nucleotides or by direct reaction with free radicals, dictates whether telomerase is inhibited or stimulated and thereby mediates the biological outcome.

Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase η in the Alternative Lengthening of Telomeres.

Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT) pathways for telomere maintenance and survival. ALT involves homologous recombination (HR)-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID), we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS) proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη). We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ)-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

Translesion Synthesis of the N(2)-2'-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1.

Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between the in vitro experiments using purified DNA polymerases, and the cellular results may arise from several factors including the crucial roles played by the accessory proteins in TLS.

Comparative Error-Free and Error-Prone Translesion Synthesis of N(2)-2'-Deoxyguanosine Adducts Formed by Mitomycin C and Its Metabolite, 2,7-Diaminomitosene, in Human Cells.

Mitomycin C (MC) is a cytotoxic and mutagenic antitumor agent that alkylates DNA upon reductive activation. 2,7-Diaminomitosene (2,7-DAM) is a major metabolite of MC in tumor cells, which also alkylates DNA. MC forms seven DNA adducts, including monoadducts and inter- and intrastrand cross-links, whereas 2,7-DAM forms two monoadducts. Herein, the biological effects of the dG-N(2) adducts formed by MC and 2,7-DAM have been compared by constructing single-stranded plasmids containing these adducts and replicating them in human embryonic kidney 293T cells. Translesion synthesis (TLS) efficiencies of dG-N(2)-MC and dG-N(2)-2,7-DAM were 38 ± 3 and 27 ± 3%, respectively, compared to that of a control plasmid. This indicates that both adducts block DNA synthesis and that dG-N(2)-2,7-DAM is a stronger replication block than dG-N(2)-MC. TLS of each adducted construct was reduced upon siRNA knockdown of pol η, pol κ, or pol ζ. For both adducts, the most significant reduction occurred with knockdown of pol κ, which suggests that pol κ plays a major role in TLS of these dG-N(2) adducts. Analysis of the progeny showed that both adducts were mutagenic, and the mutation frequencies (MF) of dG-N(2)-MC and dG-N(2)-2,7-DAM were 18 ± 3 and 10 ± 1%, respectively. For both adducts, the major type of mutation was G → T transversions. Knockdown of pol η and pol ζ reduced the MF of dG-N(2)-MC and dG-N(2)-2,7-DAM, whereas knockdown of pol κ increased the MF of these adducts. This suggests that pol κ predominantly carries out error-free TLS, whereas pol η and pol ζ are involved in error-prone TLS. The largest reduction in MF by 78 and 80%, respectively, for dG-N(2)-MC and dG-N(2)-2,7-DAM constructs occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down. This result strongly suggests that, unlike pol κ, these three TLS polymerases cooperatively perform the error-prone TLS of these adducts.

Cardiac troponin I: A potent biomarker for myocardial damage assessment following high voltage electric burn.

Myocardial infarction (MI) following high voltage electric burn is very rare, and its pathogenesis remains controversial. Electrical burns represent only 4% of all burns. Hence, clinical managements have taken a slow pace in developing. The recent guidelines laid down by the cardiology societies include cardiac troponin I (cTnI) as the gold standard marker for the assessment of myocardial damage assessment. Two patients were admitted to our hospital at the different time with the same kind of high voltage electric burn. Both patients had complained with chest discomfort during admission, and cardiac parameter assessment was done for both the patients. cTnI was also measured for both patients, and marked increase in the values was seen within 5 h of onset of myocardial damage and got into normal range within 72 h. Myocardial damage following electric burn needs to be suspected and assessed as early as possible. Hence, cTnI should be the valuable tool to detect the severity of myocardial damage incurred in the electric burn cases.

Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent human carcinogen. Metabolic activation of NNK generates a number of DNA adducts including O(2)-methylthymidine (O(2)-Me-dT) and O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT). To investigate the biological effects of these O(2)-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O(2)-Me-dT or O(2)-POB-dT in human embryonic kidney 293T (HEK293T) cells. The bulkier O(2)-POB-dT exhibited high genotoxicity and only 26% translesion synthesis (TLS) occurred, while O(2)-Me-dT was less genotoxic and allowed 55% TLS. However, O(2)-Me-dT was 20% more mutagenic (mutation frequency (MF) 64%) compared to O(2)-POB-dT (MF 53%) in HEK293T cells. The major type of mutations in each case was targeted T → A transversions (56% and 47%, respectively, for O(2)-Me-dT and O(2)-POB-dT). Both lesions induced a much lower frequency of T → G, the dominant mutation in bacteria. siRNA knockdown of the TLS polymerases (pols) indicated that pol η, pol ζ, and Rev1 are involved in the lesion bypass of O(2)-Me-dT and O(2)-POB-dT as the TLS efficiency decreased with knockdown of each pol. In contrast, MF of O(2)-Me-dT was decreased in pol ζ and Rev1 knockdown cells by 24% and 25%, respectively, while for O(2)-POB-dT, it was decreased by 44% in pol ζ knockdown cells, indicating that these TLS pols are critical for mutagenesis. Additional decrease in both TLS efficiency and MF was observed in cells deficient in pol ζ plus other Y-family pols. This study provided important mechanistic details on how these lesions are bypassed in human cells in both error-free and error-prone manner.

DNA polymerases κ and ζ cooperatively perform mutagenic translesion synthesis of the C8-2'-deoxyguanosine adduct of the dietary mutagen IQ in human cells.

The roles of translesion synthesis (TLS) DNA polymerases in bypassing the C8-2'-deoxyguanosine adduct (dG-C8-IQ) formed by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a highly mutagenic and carcinogenic heterocyclic amine found in cooked meats, were investigated. Three plasmid vectors containing the dG-C8-IQ adduct at the G1-, G2- or G3-positions of the NarI site (5'-G1G2CG3CC-3') were replicated in HEK293T cells. Fifty percent of the progeny from the G3 construct were mutants, largely G→T, compared to 18% and 24% from the G1 and G2 constructs, respectively. Mutation frequency (MF) of dG-C8-IQ was reduced by 38-67% upon siRNA knockdown of pol κ, whereas it was increased by 10-24% in pol η knockdown cells. When pol κ and pol ζ were simultaneously knocked down, MF of the G1 and G3 constructs was reduced from 18% and 50%, respectively, to <3%, whereas it was reduced from 24% to <1% in the G2 construct. In vitro TLS using yeast pol ζ showed that it can extend G3*:A pair more efficiently than G3*:C pair, but it is inefficient at nucleotide incorporation opposite dG-C8-IQ. We conclude that pol κ and pol ζ cooperatively carry out the majority of the error-prone TLS of dG-C8-IQ, whereas pol η is involved primarily in its error-free bypass.

Mutational analysis of the C8-guanine adduct of the environmental carcinogen 3-nitrobenzanthrone in human cells: critical roles of DNA polymerases η and κ and Rev1 in error-prone translesion synthesis.

3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G→T>G→A>G→C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol η, pol κ, pol ι, pol ζ, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ∼55% reduction) in the level of TLS occurred in cells with knockdown of pol ζ. Pol η and pol κ were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ζ appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.