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.

Olaparib for adjuvant treatment of BRCA mutation-positive HER2-negative high-risk early breast cancer after chemotherapy

People with BRCA mutation-positive HER2­negative high-risk early breast cancer usually have chemotherapy followed by surgery (neoadjuvant chemotherapy), or surgery followed by chemotherapy (adjuvant chemotherapy). Clinical trial evidence shows that, compared with placebo, olaparib after neoadjuvant or adjuvant chemotherapy decreases the chance of the cancer returning or getting worse, and increases the length of time people live. The cost-effectiveness estimates for olaparib are within what NICE considers to be an acceptable use of NHS resources. So, olaparib is recommended.

Prospects of using the nucleoside analogue riamilovir in patients with SARS-CoV-2 infection.

AIM: To evaluate clinical efficacy of nucleoside analogues in therapy of moderate COVID-19 in in-patients. MATERIALS AND METHODS: Retrospective processing of 108 completed clinical cases with moderate novel coronavirus disease was carried out for the period 2020-2021. The duration of the disease at the time of admission did not exceed three days. Experimental group consisted of 53 patients who, in addition to standard therapy, were prescribed "off-label" riamilovir at a daily dosage of 1250 mg for 5 days by the decision of the medical commission. Comparison group included 55 patients who received a combination of umifenovir and ribavirin as antiviral therapy for 5 days. The duration of the main clinical manifestations of the disease, the indicators of clinical and biochemical blood tests, results of the SARS-CoV-2 virus RNA study using the nucleic acid amplification method (NAAT diagnostics). RESULTS: Significantly faster achievement of clinical improvement in the group of patients treated with riamilovir was shown, as well as faster sanitation from SARS-CoV-2 virus based on the results of etiological testing. CONCLUSION: The use of riamilovir for the treatment of patients with moderate novel coronavirus infection (COVID-19) resulted in a significant reduction of general infectious syndromes and respiratory symptoms. Patients from the experimental group significantly faster achieved clinical recovery and sanitation from the pathogen according to the results of NAAT diagnostics.

Brincidofovir y cidofovir para viruela símica / Brincidofovir and cidofovir for smallpox seismic

INTRODUÇÃO: Brincidofovir es un profármaco y un inhibidor de la polimerasa de ADN análogo de nucleótido de ortopoxvirus que se convierte intracelularmente en el análogo de nucleótido cidofovir, que se fosforila a difosfato de cidofovir, la fracción activa que tiene actividad antiviral contra el virus de la viruela. La incorporación de cidofovir en la cadena de ADN viral en crecimiento da como resultado reducciones en la tasa de síntesis de ADN viral. Se administra por vía oral en comprimidos o suspensión oral a 200 mg una vez a la semana en dos dosis para personas con 48 kg o más, mientras que para personas de 10 a 48 kg la suspensión oral se administra a 4 mg/kg una vez a la semana en dos dosis. La Administración de Alimentos y Medicamentos (FDA, su sigla del inglés Food and Drug Administration) de los Estados Unidos y la Agencia Europea de Medicamentos (EMA, su sigla del inglés European Medicines Agency) han autorizado para el año 2021 la comercialización brincidofovir (TEMBEXA®) para el tratamiento de la viruela, sin embargo, todavía no han ampliado su indicación a viruela símica. OBJETIVO: El objetivo del presente informe es evaluar parámetros de eficacia, seguridad, conveniencia y recomendaciones disponibles acerca del empleo de brincidofovir y cidofovir para el tratamiento de personas con viruela símica en Argentina. MÉTODOS: Teniendo en cuenta la velocidad con la que la información relacionada a la epidemia aparece y se modifica, se desarrollará un protocolo sustentado en proyectos que resume activamente la evidencia científica a medida que la misma se hace disponible. Desarrollaremos una síntesis de evidencia viva, dado el contexto actual de producción de la evidencia y necesidad actualizada de información de calidad para la toma de decisiones en salud.13,14 Con este fin se utilizará la plataforma L-ove de Epistemonikos (https://app.iloveevidence.com/topics) para identificar revisiones sistemáticas "vivas". Se seleccionarán aquellas con una calidad metodológica apropiada evaluada a través de la herramienta AMSTAR-2, y que a su vez llevaran un proceso de actualización frecuente. De cada una de las revisiones sistemáticas vivas identificadas se extractarán los efectos de la intervención sobre los desenlaces priorizados como importantes o críticos y la certeza en dichos efectos. Para la priorización de los desenlaces se adoptará una perspectiva desde el paciente considerando sus potenciales preferencias. La selección se realizará por consenso entre los autores y supervisores del informe considerando los protocolos de ensayos clínicos registrados y publicados, y las cohortes de pacientes con viruela símica. Se seleccionaron "mortalidad", "hospitalización", "mejoría clínica" (tiempo hasta la mejoría subjetiva, aparición de nuevas lesiones, todas las lesiones con costra y una capa de piel sana), "eventos adversos graves" como desenlaces críticos e importantes. Adicionalmente, se extractarán datos relacionados con efectos de subgrupo potencialmente relevantes para la toma de decisión, con especial énfasis en el tiempo de evolución, la severidad de la enfermedad y el estado inmunológico. Para confeccionar las conclusiones en el efecto de las intervenciones evaluadas sobre los desenlaces priorizados, utilizamos lineamientos publicados, específicamente desarrollados a tal fin. RECOMENDACIONES: Para la identificación de recomendaciones sustentadas en evidencia y actualizadas, se utilizarán motores de búsqueda de recomendaciones y directrices basadas en evidencia: ACCESSSS, ECRI, BIGG Database, MAGIC authoring and publication platform (MAGICapp), BIGG-REC, National Institute for Health and Clinical Excellence (NICE), National Guideline Clearinghouse (NGC), New Zealand Guidelines Group (NZGG), Scottish Intercollegiate Guidelines Network (SIGN), American College of Physicians (ACP), Association of the Scientific Medical Societies in Germany (AWMF) Guidelines Register, Institute for Clinical Systems Improvement (ICSI), Canadian Medical Association (CMA), GuiaSalud. Se seleccionarán aquellas guías con rigor metodológico apropiado (Dominios 3 y 4 según la herramienta AGREE II => 60%) y se incorporaron sus recomendaciones al informe. RESULTADOS: Se identificó una revisión sistemática que cumplió con los criterios de inclusión del presente informe y reportó resultados: Se identificó 1 ECA registrado en fase 1 que evalúa el uso de brincidofovir endovenoso. Las tecnologías no están autorizadas para su comercialización en Argentina y no se encuentran disponible al momento de la realización del presente informe. No se halló información relacionada con valores y preferencias de las personas con viruela símica. CONCLUSIONES: No se halló evidencia que permita evaluar los efectos en la salud relacionados con el empleo de brincidofovir y cidofovir en pacientes expuestos o con viruela símica. Las tecnologías no están autorizadas para su comercialización por la Administración Nacional de Medicamentos, Alimentos y Tecnología Médica de nuestro país al momento de la fecha de realización del presente informe. Se estima que brincidofovir y cidofovir cumplen criterios para ser utilizados en el marco del uso de emergencia monitoreado de intervenciones no registradas y experimentales (MEURI). No se identificaron guías de práctica clínica de adecuada calidad metodológica actualizadas que mencionen las tecnologías en la indicación evaluada.

Potency of the novel PolC DNA polymerase inhibitor CRS0540 in a disseminated Listeria monocytogenes intracellular hollow-fibre model.

BACKGROUND: Listeriosis is an orphan disease, which is nevertheless fatal in immunocompromised people. CRS0540 is a novel PolC DNA polymerase inhibitor that has demonstrated good in vitro and in vivo activity against Listeria monocytogenes. METHODS: Rodent-to-human allometry projection-based human population pharmacokinetics of CRS0540 were used for all studies. CRS0540 pharmacokinetics/pharmacodynamics studies in an intracellular hollow-fibre system model of disseminated listeriosis (HFS-Lister) examined the effect of eight treatment doses, administered daily over 7 days, in duplicate units. Total bacterial burden versus AUC/MIC exposures on each day were modelled using the inhibitory sigmoid Emax model, while CRS0540-resistant bacterial burden was modelled using a quadratic function. Ten thousand-subject Monte Carlo simulations were used to predict an optimal clinical dose for treatment. RESULTS: The mean CRS0540 intracellular/extracellular AUC0-24 ratio was 34.07 (standard error: 15.70) as measured in the HFS-Lister. CRS0540 demonstrated exposure-dependent bactericidal activity in the HFS-Lister, with the highest exposure killing approximately 5.0 log10 cfu/mL. The free drug AUC0-24/MIC associated with 80% of maximal kill (EC80) was 36.4. Resistance emergence versus AUC/MIC was described by a quadratic function, with resistance amplification at an AUC/MIC of 54.8 and resistance suppression at an AUC/MIC of 119. Monte Carlo simulations demonstrated that for the EC80 target, IV CRS0540 doses of 100 mg/kg achieved PTAs of >90% at MICs up to 1.0 mg/L. CONCLUSIONS: CRS0540 is a promising orphan drug candidate for listeriosis. Future PK/PD studies comparing it with penicillin, the standard of care, could lead to this drug as a new treatment in immunocompromised patients.

Molecular tension microscopy of the LINC complex in live cells.

We present a protocol to measure the effect of pharmacological treatments on the mechanical tension experienced by nesprins at the cytoplasmic surface of the nuclear envelope of mammalian cells in culture. We apply this protocol to MDCK epithelial cells exposed to the actin depolymerization agent cytochalasin D. To do so, we perform confocal spectral imaging of transiently expressed molecular tension sensors of mini-nesprin 2G and analyze the FRET signal from the sensors with a custom-made Fiji script. For complete details on the use and execution of this protocol, please refer to Déjardin et al. (2020).

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.

Dietary Isothiocyanates, Sulforaphane and 2-Phenethyl Isothiocyanate, Effectively Impair Vibrio cholerae Virulence.

Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.

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.

C60 fullerene against SARS-CoV-2 coronavirus: an in silico insight.

Based on WHO reports the new SARS-CoV-2 coronavirus is currently widespread all over the world. So far > 162 million cases have been confirmed, including > 3 million deaths. Because of the pandemic still spreading across the globe the accomplishment of computational methods to find new potential mechanisms of virus inhibitions is necessary. According to the fact that C60 fullerene (a sphere-shaped molecule consisting of carbon) has shown inhibitory activity against various protein targets, here the analysis of the potential binding mechanism between SARS-CoV-2 proteins 3CLpro and RdRp with C60 fullerene was done; it has resulted in one and two possible binding mechanisms, respectively. In the case of 3CLpro, C60 fullerene interacts in the catalytic binding pocket. And for RdRp in the first model C60 fullerene blocks RNA synthesis pore and in the second one it prevents binding with Nsp8 co-factor (without this complex formation, RdRp can't perform its initial functions). Then the molecular dynamics simulation confirmed the stability of created complexes. The obtained results might be a basis for other computational studies of 3CLPro and RdRp potential inhibition ways as well as the potential usage of C60 fullerene in the fight against COVID-19 disease.

Early dynamic changes of quasispecies in the reverse transcriptase region of hepatitis B virus in telbivudine treatment.

BACKGROUND: Telbivudine (LdT) - a synthetic thymidine ß-L-nucleoside analogue (NA) - is an effective inhibitor for hepatitis B virus (HBV) replication. The quasispecies spectra in the reverse transcriptase (RT) region of the HBV genome and their dynamic changes associated with LdT treatment remains largely unknown. METHODS: We prospectively recruited a total of 21 treatment-naive patients with chronic HBV infection and collected sequential serum samples at five time points (baseline, weeks 1, 3, 12, and 24 after LdT treatment). The HBV RT region was amplified and shotgun-sequenced by the Ion Torrent Personal Genome Machine (PGM)® system. We reconstructed full-length haplotypes of the RT region using an integrated bioinformatics framework, including de novo contig assembly and full-length haplotype reconstruction. In addition, we investigated the quasispecies' dynamic changes and evolution history and characterized potential NAs resistant mutations over the treatment course. RESULTS: Viral quasispecies differed obviously between patients with complete (n = 8) and incomplete/no response (n = 13) at 12 weeks after LdT treatment. A reduced dN/dS ratio in quasispecies demonstrated a selective constraint resulting from antiviral therapy. The temporal clustering of sequential quasispecies showed different patterns along with a 24-week observation, although its statistic did not differ significantly. Several patients harboring pre-existing resistant mutations showed different clinical responses, while NAs resistant mutations were rare within a short-term treatment. CONCLUSION: A complete profile of quasispecies reconstructed from in-depth shotgun sequencing may has important implications for enhancing clinical decision in adjusting antiviral therapy timely.

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.

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.

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.