Omicron-induced interferon signaling prevents influenza A H1N1 and H5N1 virus infection.

Recent findings in permanent cell lines suggested that SARS-CoV-2 Omicron BA.1 induces a stronger interferon response than Delta. Here, we show that BA.1 and BA.5 but not Delta induce an antiviral state in air-liquid interface cultures of primary human bronchial epithelial cells and primary human monocytes. Both Omicron subvariants caused the production of biologically active types I (α/ß) and III (λ) interferons and protected cells from super-infection with influenza A viruses. Notably, abortive Omicron infection of monocytes was sufficient to protect monocytes from influenza A virus infection. Interestingly, while influenza-like illnesses surged during the Delta wave in England, their spread rapidly declined upon the emergence of Omicron. Mechanistically, Omicron-induced interferon signaling was mediated via double-stranded RNA recognition by MDA5, as MDA5 knockout prevented it. The JAK/STAT inhibitor baricitinib inhibited the Omicron-mediated antiviral response, suggesting it is caused by MDA5-mediated interferon production, which activates interferon receptors that then trigger JAK/STAT signaling. In conclusion, our study (1) demonstrates that only Omicron but not Delta induces a substantial interferon response in physiologically relevant models, (2) shows that Omicron infection protects cells from influenza A virus super-infection, and (3) indicates that BA.1 and BA.5 induce comparable antiviral states.

Repurposing of the antibiotic nitroxoline for the treatment of mpox.

The antiviral drugs tecovirimat, brincidofovir, and cidofovir are considered for mpox (monkeypox) treatment despite a lack of clinical evidence. Moreover, their use is affected by toxic side-effects (brincidofovir, cidofovir), limited availability (tecovirimat), and potentially by resistance formation. Hence, additional, readily available drugs are needed. Here, therapeutic concentrations of nitroxoline, a hydroxyquinoline antibiotic with a favourable safety profile in humans, inhibited the replication of 12 mpox virus isolates from the current outbreak in primary cultures of human keratinocytes and fibroblasts and a skin explant model by interference with host cell signalling. Tecovirimat, but not nitroxoline, treatment resulted in rapid resistance development. Nitroxoline remained effective against the tecovirimat-resistant strain and increased the anti-mpox virus activity of tecovirimat and brincidofovir. Moreover, nitroxoline inhibited bacterial and viral pathogens that are often co-transmitted with mpox. In conclusion, nitroxoline is a repurposing candidate for the treatment of mpox due to both antiviral and antimicrobial activity.

Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform.

Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a readout for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in numerous cell culture models, independently of cytopathogenic effect formation. Compared to other models, the Caco-2 subline Caco-2-F03 displayed superior performance. It possesses a stable SARS-CoV-2 susceptibility phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1,796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of phosphoglycerate dehydrogenase (PHGDH), CDC like kinase 1 (CLK-1), and colony stimulating factor 1 receptor (CSF1R). The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the hexokinase II (HK2) inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2-F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false-positive hits.

Differences between intrinsic and acquired nucleoside analogue resistance in acute myeloid leukaemia cells.

BACKGROUND: SAMHD1 mediates resistance to anti-cancer nucleoside analogues, including cytarabine, decitabine, and nelarabine that are commonly used for the treatment of leukaemia, through cleavage of their triphosphorylated forms. Hence, SAMHD1 inhibitors are promising candidates for the sensitisation of leukaemia cells to nucleoside analogue-based therapy. Here, we investigated the effects of the cytosine analogue CNDAC, which has been proposed to be a SAMHD1 inhibitor, in the context of SAMHD1. METHODS: CNDAC was tested in 13 acute myeloid leukaemia (AML) cell lines, in 26 acute lymphoblastic leukaemia (ALL) cell lines, ten AML sublines adapted to various antileukaemic drugs, 24 single cell-derived clonal AML sublines, and primary leukaemic blasts from 24 AML patients. Moreover, 24 CNDAC-resistant sublines of the AML cell lines HL-60 and PL-21 were established. The SAMHD1 gene was disrupted using CRISPR/Cas9 and SAMHD1 depleted using RNAi, and the viral Vpx protein. Forced DCK expression was achieved by lentiviral transduction. SAMHD1 promoter methylation was determined by PCR after treatment of genomic DNA with the methylation-sensitive HpaII endonuclease. Nucleoside (analogue) triphosphate levels were determined by LC-MS/MS. CNDAC interaction with SAMHD1 was analysed by an enzymatic assay and by crystallisation. RESULTS: Although the cytosine analogue CNDAC was anticipated to inhibit SAMHD1, SAMHD1 mediated intrinsic CNDAC resistance in leukaemia cells. Accordingly, SAMHD1 depletion increased CNDAC triphosphate (CNDAC-TP) levels and CNDAC toxicity. Enzymatic assays and crystallisation studies confirmed CNDAC-TP to be a SAMHD1 substrate. In 24 CNDAC-adapted acute myeloid leukaemia (AML) sublines, resistance was driven by DCK (catalyses initial nucleoside phosphorylation) loss. CNDAC-adapted sublines displayed cross-resistance only to other DCK substrates (e.g. cytarabine, decitabine). Cell lines adapted to drugs not affected by DCK or SAMHD1 remained CNDAC sensitive. In cytarabine-adapted AML cells, increased SAMHD1 and reduced DCK levels contributed to cytarabine and CNDAC resistance. CONCLUSION: Intrinsic and acquired resistance to CNDAC and related nucleoside analogues are driven by different mechanisms. The lack of cross-resistance between SAMHD1/ DCK substrates and non-substrates provides scope for next-line therapies after treatment failure.

SAMHD1 is a key regulator of the lineage-specific response of acute lymphoblastic leukaemias to nelarabine.

The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.

Selective inactivation of hypomethylating agents by SAMHD1 provides a rationale for therapeutic stratification in AML.

Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.