Cytidine Deaminase Resolves Replicative Stress and Protects Pancreatic Cancer from DNA-Targeting Drugs.

Cytidine deaminase (CDA) functions in the pyrimidine salvage pathway for DNA and RNA syntheses and has been shown to protect cancer cells from deoxycytidine-based chemotherapies. In this study, we observed that CDA was overexpressed in pancreatic adenocarcinoma from patients at baseline and was essential for experimental tumor growth. Mechanistic investigations revealed that CDA localized to replication forks where it increased replication speed, improved replication fork restart efficiency, reduced endogenous replication stress, minimized DNA breaks, and regulated genetic stability during DNA replication. In cellular pancreatic cancer models, high CDA expression correlated with resistance to DNA-damaging agents. Silencing CDA in patient-derived primary cultures in vitro and in orthotopic xenografts in vivo increased replication stress and sensitized pancreatic adenocarcinoma cells to oxaliplatin. This study sheds light on the role of CDA in pancreatic adenocarcinoma, offering insights into how this tumor type modulates replication stress. These findings suggest that CDA expression could potentially predict therapeutic efficacy and that targeting CDA induces intolerable levels of replication stress in cancer cells, particularly when combined with DNA-targeted therapies. SIGNIFICANCE: Cytidine deaminase reduces replication stress and regulates DNA replication to confer resistance to DNA-damaging drugs in pancreatic cancer, unveiling a molecular vulnerability that could enhance treatment response.

The Antitumor Effect of the DNA Polymerase Alpha Inhibitor ST1926 in Glioblastoma: A Proteomics Approach.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant brain tumor. The median survival rate does not exceed two years, indicating an imminent need to develop novel therapies. The atypical adamantyl retinoid ST1926 induces apoptosis and growth inhibition in different cancer types. We have shown that ST1926 is an inhibitor of the catalytic subunit of DNA polymerase alpha (POLA1), which is involved in initiating DNA synthesis in eukaryotic cells. POLA1 levels are elevated in GBM versus normal brain tissues. Therefore, we studied the antitumor effects of ST1926 in several human GBM cell lines. We further explored the global protein expression profiles in GBM cell lines using liquid chromatography coupled with tandem mass spectrometry to identify new targets of ST1926. Low sub-micromolar concentrations of ST1926 potently decreased cell viability, induced cell damage and apoptosis, and reduced POLA1 protein levels in GBM cells. The proteomics profiles revealed 197 proteins significantly differentially altered upon ST1926 treatment of GBM cells involved in various cellular processes. We explored the differential gene and protein expression of significantly altered proteins in GBM compared to normal brain tissues.

A Case Report of Successful Use of Twice-Daily Letermovir in the Treatment of Resistant Cytomegalovirus in a Small Bowel Transplant Recipient.

Cytomegalovirus (CMV) is considered one of the most notable pathogens that affect patients after solid organ transplantation (SOT), especially small bowel transplant patients with a risk of high mortality rate. Its management relies historically on the use of CMV DNA polymerase inhibitors (namely, ganciclovir and valganciclovir). Second-line options include foscarnet and cidofovir, which are highly nephrotoxic and thus less preferred and only used in ganciclovir intolerance or resistance cases. Letermovir is a novel antiviral agent approved for CMV prophylaxis in hematopoietic stem cell transplant, but not for SOT (neither for prophylaxis nor for treatment). We report the first case on the successful use of letermovir in treating CMV disease in a small bowel transplant patient who failed to achieve viral clearance due to ganciclovir resistance and severe intolerance to foscarnet.

Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors.

Ribosome biogenesis is an energetically expensive program that is dictated by nutrient availability. Here we report that nutrient deprivation severely impairs precursor ribosomal RNA (pre-rRNA) processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, pre-rRNAs stored under starvation are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient stress leads to perturbed ribosome assembly upon nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine is dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which is in turn dependent on Raf/MEK/ERK signaling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that tumor cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.

5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

A Novel Molecular Target in EGFR-mutant Lung Cancer Treated With the Combination of Osimertinib and Pemetrexed.

BACKGROUND/AIM: Synergistic effects of epidermal growth factor receptor tyrosine kinase inhibitors and chemotherapy have been reported. Here, we evaluated the therapeutic potential of combining osimertinib with pemetrexed and investigated the molecular mechanisms. MATERIALS AND METHODS: We analyzed the antitumor effects of osimertinib± pemetrexed in PC-9 and H1975 cells. Gene expression on exposure to osimertinib±pemetrexed was assessed in these cultured cells. Cell lines resistant to osimertinib±pemetrexed were established to explore mechanisms of resistance. RESULTS: Osimertinib+pemetrexed treatment delayed the emergence of resistance relative to monotherapy in vitro and in vivo. Expression of the anti-apoptotic gene PLK1 was down-regulated in PC-9 and H1975 exposed to osimertinib+ pemetrexed, whereas it was up-regulated in resistant cells. Furthermore, inhibition of PLK1 induced apoptosis and inhibited proliferation of resistant cells. CONCLUSION: Blocking PLK1 contributes to mediating the synergistic anti-proliferative effect of osimertinib+pemetrexed. PLK1 over-expression may be a critical mechanism for acquired resistance to osimertinib+pemetrexed.

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.

Durvalumab with platinum-pemetrexed for unresectable pleural mesothelioma: survival, genomic and immunologic analyses from the phase 2 PrE0505 trial.

Mesothelioma is a rare and fatal cancer with limited therapeutic options until the recent approval of combination immune checkpoint blockade. Here we report the results of the phase 2 PrE0505 trial ( NCT02899195 ) of the anti-PD-L1 antibody durvalumab plus platinum-pemetrexed chemotherapy for 55 patients with previously untreated, unresectable pleural mesothelioma. The primary endpoint was overall survival compared to historical control with cisplatin and pemetrexed chemotherapy; secondary and exploratory endpoints included safety, progression-free survival and biomarkers of response. The combination of durvalumab with chemotherapy met the pre-specified primary endpoint, reaching a median survival of 20.4 months versus 12.1 months with historical control. Treatment-emergent adverse events were consistent with known side effects of chemotherapy, and all adverse events due to immunotherapy were grade 2 or lower. Integrated genomic and immune cell repertoire analyses revealed that a higher immunogenic mutation burden coupled with a more diverse T cell repertoire was linked to favorable clinical outcome. Structural genome-wide analyses showed a higher degree of genomic instability in responding tumors of epithelioid histology. Patients with germline alterations in cancer predisposing genes, especially those involved in DNA repair, were more likely to achieve long-term survival. Our findings indicate that concurrent durvalumab with platinum-based chemotherapy has promising clinical activity and that responses are driven by the complex genomic background of malignant pleural mesothelioma.

Expression of claudin-8 is induced by aldosterone in renal collecting duct principal cells.

Fine tuning of Na+ reabsorption takes place along the aldosterone-sensitive distal nephron, which includes the collecting duct (CD), where it is mainly regulated by aldosterone. In the CD, Na+ reabsorption is mediated by the epithelial Na+ channel and Na+ pump (Na+-K+-ATPase). Paracellular ion permeability is mainly dependent on tight junction permeability. Claudin-8 is one of the main tight junction proteins expressed along the aldosterone-sensitive distal nephron. We have previously shown a coupling between transcellular Na+ reabsorption and paracellular Na+ barrier. We hypothesized that aldosterone controls the expression levels of both transcellular Na+ transporters and paracellular claudin-8 in a coordinated manner. Here, we show that aldosterone increased mRNA and protein levels as well as lateral membrane localization of claudin-8 in cultured CD principal cells. The increase in claudin-8 mRNA levels in response to aldosterone was prevented by preincubation with 17-hydroxyprogesterone, a mineralocorticoid receptor antagonist, and by inhibition of transcription with actinomycin D. We also showed that a low-salt diet, which stimulated aldosterone secretion, was associated with increased claudin-8 abundance in the mouse kidney. Reciprocally, mice subjected to a high-salt diet, which inhibits aldosterone secretion, or treated with spironolactone, a mineralocorticoid receptor antagonist, displayed decreased claudin-8 expression. Inhibition of glycogen synthase kinase-3, Lyn, and Abl signaling pathways prevented the effect of aldosterone on claudin-8 mRNA and protein abundance, suggesting that signaling of protein kinases plays a permissive role on the transcriptional activity of the mineralocorticoid receptor. This study shows that signaling via multiple protein kinases working in concert mediates aldosterone-induced claudin-8 expression in the CD.NEW & NOTEWORTHY In this study, we showed that aldosterone modulates claudin-8 expression in cultured collecting duct principal cells and in the mouse kidney. The upregulation of claudin-8 expression in response to aldosterone is dependent on at least glycogen synthase kinase-3, Lyn, and Abl signaling pathways, indicating the participation of multiple protein kinases to the effect of aldosterone.

Chronic Hepatitis B Treatment Strategies Using Polymerase Inhibitor-Based Combination Therapy.

Viral polymerase is an essential enzyme for the amplification of the viral genome and is one of the major targets of antiviral therapies. However, a serious concern to be solved in hepatitis B virus (HBV) infection is the difficulty of eliminating covalently closed circular (ccc) DNA. More recently, therapeutic strategies targeting various stages of the HBV lifecycle have been attempted. Although cccDNA-targeted therapies are attractive, there are still many problems to be overcome, and the development of novel polymerase inhibitors remains an important issue. Interferons and nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are the only therapeutic options currently available for HBV infection. Many studies have reported that the combination of interferons and NRTI causes the loss of hepatitis B surface antigen (HBsAg), which is suggestive of seroconversion. Although NRTIs do not directly target cccDNA, they can strongly reduce the serum viral DNA load and could suppress the recycling step of cccDNA formation, improve liver fibrosis/cirrhosis, and reduce the risk of hepatocellular carcinoma. Here, we review recent studies on combination therapies using polymerase inhibitors and discuss the future directions of therapeutic strategies for HBV infection.

Polymerase θ Coordinates Multiple Intrinsic Enzymatic Activities during DNA Repair.

The POLQ gene encodes DNA polymerase θ, a 2590 amino acid protein product harboring DNA-dependent ATPase, template-dependent DNA polymerase, dNTP-dependent endonuclease, and 5'-dRP lyase functions. Polymerase θ participates at an essential step of a DNA double-strand break repair pathway able to join 5'-resected substrates by locating and pairing microhomologies present in 3'-overhanging single-stranded tails, cleaving the extraneous 3'-DNA by dNTP-dependent end-processing, before extending the nascent 3' end from the microhomology annealing site. Metazoans require polymerase θ for full resistance to DNA double-strand break inducing agents but can survive knockout of the POLQ gene. Cancer cells with compromised homologous recombination, or other DNA repair defects, over-utilize end-joining by polymerase θ and often over-express the POLQ gene. This dependency points to polymerase θ as an ideal drug target candidate and multiple drug-development programs are now preparing to enter clinical trials with small-molecule inhibitors. Specific inhibitors of polymerase θ would not only be predicted to treat BRCA-mutant cancers, but could thwart accumulated resistance to current standard-of-care cancer therapies and overcome PARP-inhibitor resistance in patients. This article will discuss synthetic lethal strategies targeting polymerase θ in DNA damage-response-deficient cancers and summarize data, describing molecular structures and enzymatic functions.

REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.

BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.

Mobility of Nucleostemin in Live Cells Is Specifically Related to Transcription Inhibition by Actinomycin D and GTP-Binding Motif.

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Hypoxia-induced SETX links replication stress with the unfolded protein response.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.

Double-strand breaks induce short-scale DNA replication and damage amplification in the fully grown mouse oocytes.

Break-induced replication (BIR) is essential for the repair of DNA double-strand breaks (DSBs) with single ends. DSBs-induced microhomology-mediated BIR (mmBIR) and template-switching can increase the risk of complex genome rearrangement. In addition, DSBs can also induce the multi-invasion-mediated DSB amplification. The mmBIR-induced genomic rearrangement has been identified in cancer cells and patients with rare diseases. However, when and how mmBIR is initiated have not been fully and deeply studied. Furthermore, it is not well understood about the conditions for initiation of multi-invasion-mediated DSB amplification. In the G2 phase oocyte of mouse, we identified a type of short-scale BIR (ssBIR) using the DNA replication indicator 5-ethynyl-2'-deoxyuridine (EdU). These ssBIRs could only be induced in the fully grown oocytes but not the growing oocytes. If the DSB oocytes were treated with Rad51 or Chek1/2 inhibitors, both EdU signals and DSB marker γH2A.X foci would decrease. In addition, the DNA polymerase inhibitor Aphidicolin could inhibit the ssBIR and another inhibitor ddATP could reduce the number of γH2A.X foci in the DSB oocytes. In conclusion, our results showed that DNA DSBs in the fully grown oocytes can initiate ssBIR and be amplified by Rad51 or DNA replication.

Preparation and functional evaluation of monoclonal antibodies targeting Hepatitis B Virus Polymerase.

HBV pol plays a critical role in the replication of hepatitis B virus (HBV). Previous studies conducted on HBV pol have produced limited evidence on HBV pol expression due to the lack of effective detection methods. The present study used the HBV pol (159-406 aa) protein as a target to screen for specific monoclonal antibodies that recognize HBV pol and subsequently evaluate their diagnostic and therapeutic value. Four antibodies (P3, P5, P12, P20) against HBV pol were obtained. Among them, the P20 antibody indicated optimal binding with HBV pol as demonstrated by Western blotting (WB) in a cell model transfected with the HBV genome. We also expressed P5 and P12 antibodies in mouse liver cells by transfection and the results indicated significant antiviral effects caused by these two antibodies especially P12. In summary, the present study established an antibody which was denoted P20. This antibody can be used to detect HBV pol expression by four HBV genomes via WB analysis. In addition, the antibody denoted P12 could exert antiviral effects via intracellular expression, which may provide a promising approach for the treatment of chronic hepatitis B.

RNA-dependent RNA polymerase: Structure, mechanism, and drug discovery for COVID-19.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global pandemic. Although great efforts have been made to develop effective therapeutic interventions, only the nucleotide analog remdesivir was approved for emergency use against COVID-19. Remdesivir targets the RNA-dependent RNA polymerase (RdRp), an essential enzyme for viral RNA replication and a promising drug target for COVID-19. Recently, several structures of RdRp in complex with substrate RNA and remdesivir were reported, providing insights into the mechanisms of RNA recognition by RdRp. These structures also reveal the mechanism of RdRp inhibition by nucleotide inhibitors and offer a molecular template for the development of RdRp-targeting drugs. This review discusses the recognition mechanism of RNA and nucleotide inhibitor by RdRp, and its implication in drug discovery.

DNA polymerase eta: A potential pharmacological target for cancer therapy.

In the last two decades, intensive research has been carried out to improve the survival rates of cancer patients. However, the development of chemoresistance that ultimately leads to tumor relapse poses a critical challenge for the successful treatment of cancer patients. Many cancer patients experience tumor relapse and ultimately die because of treatment failure associated with acquired drug resistance. Cancer cells utilize multiple lines of self-defense mechanisms to bypass chemotherapy and radiotherapy. One such mechanism employed by cancer cells is translesion DNA synthesis (TLS), in which specialized TLS polymerases bypass the DNA lesion with the help of monoubiquitinated proliferating cell nuclear antigen. Among all TLS polymerases (Pol η, Pol ι, Pol κ, REV1, Pol ζ, Pol µ, Pol λ, Pol ν, and Pol θ), DNA polymerase eta (Pol η) is well studied and majorly responsible for the bypass of cisplatin and UV-induced DNA damage. TLS polymerases contribute to chemotherapeutic drug-induced mutations as well as therapy resistance. Therefore, targeting these polymerases presents a novel therapeutic strategy to combat chemoresistance. Mounting evidence suggests that inhibition of Pol η may have multiple impacts on cancer therapy such as sensitizing cancer cells to chemotherapeutics, suppressing drug-induced mutagenesis, and inhibiting the development of secondary tumors. Herein, we provide a general introduction of Pol η and its clinical implications in blocking acquired drug resistance. In addition; this review addresses the existing gaps and challenges of Pol η mediated TLS mechanisms in human cells. A better understanding of the Pol η mediated TLS mechanism will not merely establish it as a potential pharmacological target but also open possibilities to identify novel drug targets for future therapy.