CRISPR-based kinome-screening revealed MINK1 as a druggable player to rewire 5FU-resistance in OSCC through AKT/MDM2/p53 axis.

Cisplatin, 5FU and docetaxel (TPF) are the most common chemotherapy regimen used for advanced OSCC. However, many cancer patients experience relapse, continued tumor growth, and spread due to drug resistance, which leads to treatment failure and metastatic disease. Here, using a CRISPR/Cas9 based kinome knockout screening, Misshapen-like kinase 1 (MINK1) is identified as an important mediator of 5FU resistance in OSCC. Analysis of clinical samples demonstrated significantly higher MINK1 expression in the tumor tissues of chemotherapy non-responders as compared to chemotherapy responders. The nude mice and zebrafish xenograft experiments indicate that knocking out MINK1 restores 5FU mediated cell death in chemoresistant OSCC. An antibody based phosphorylation array screen revealed MINK1 as a negative regulator of p53. Mechanistically, MINK1 modulates AKT phosphorylation at Ser473, which enables p-MDM2 (Ser 166) mediated degradation of p53. We also identified lestaurtinib as a potent inhibitor of MINK1 kinase activity. The patient derived TPF resistant cell based xenograft data suggest that lestaurtinib restores 5FU sensitivity and facilitates a significant reduction of tumor burden. Overall, our study suggests that MINK1 is a major driver of 5FU resistance in OSCC. The novel combination of MINK1 inhibitor lestaurtinib and 5FU needs further clinical investigation in advanced OSCC.

Selective autophagy of RIPosomes maintains innate immune homeostasis during bacterial infection.

The NOD1/2-RIPK2 is a key cytosolic signaling complex that activates NF-κB pro-inflammatory response against invading pathogens. However, uncontrolled NF-κB signaling can cause tissue damage leading to chronic diseases. The mechanisms by which the NODs-RIPK2-NF-κB innate immune axis is activated and resolved remain poorly understood. Here, we demonstrate that bacterial infection induces the formation of endogenous RIPK2 oligomers (RIPosomes) that are self-assembling entities that coat the bacteria to induce NF-κB response. Next, we show that autophagy proteins IRGM and p62/SQSTM1 physically interact with NOD1/2, RIPK2 and RIPosomes to promote their selective autophagy and limit NF-κB activation. IRGM suppresses RIPK2-dependent pro-inflammatory programs induced by Shigella and Salmonella. Consistently, the therapeutic inhibition of RIPK2 ameliorates Shigella infection- and DSS-induced gut inflammation in Irgm1 KO mice. This study identifies a unique mechanism where the innate immune proteins and autophagy machinery are recruited together to the bacteria for defense as well as for maintaining immune homeostasis.

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses.

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC-I antigen presentation and stress granule signaling are enhanced in IRGM-deficient cells, indicating a robust cell-intrinsic antiviral immune state. Consistently, IRGM-depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS-CoV-2, CHIKV, and Zika virus.

Diversity of virulence-associated genes in pathogenic Aeromonas hydrophila isolates and their in vivo modulation at varied water temperatures.

Most environmental parameters have no consistent effect on the expression of bacterial genes responsible for their virulence. However, as fish are poikilothermic, the possibility of temperature variation having a pronounced effect on the expression of virulence-associated gene(s) of bacteria infecting the host needs to be investigated. In this study, the diversity of virulence genes in seven Aeromonas hydrophila isolates collected from diseased fish from different parts of India was characterized, and the effect of temperature variation on the extent of expression of their virulence was investigated. All bacterial isolates were screened for a total of nine bacterial virulent genes {aerolysin, hemolysin, cytoen, outer membrane protein TS (Omp TS), elastase, flagellin, lipase, ß hemolysin and type 3 secretion system}, and the diversity in their presence or absence were marked at a particular in vitro condition. Three bacterial isolates (nos. 1, 7 and 2) were selected for further study, based on their ability to cause varied mortalities (20-100%) in Labeo rohita juveniles in intraperitoneal challenge study. Further, three isolates were injected intraperitoneally into L. rohita fingerlings at three different temperatures (i.e., 20, 28 and 37 °C) and at 6 h post-challenge, the kidney samples were collected to measure the levels of all nine bacterial virulence genes using semi-quantitative PCR. The maximum level of amplicons of virulence genes in all three A. hydrophila isolates was noticed at 28 °C as compared to 37 °C and 20 °C. It was also observed that haemolysin played a more prominent role in the expression of virulence, when compared to cytoen gene. Hence, it was concluded that water temperature does play a crucial role in governing virulence gene expression, and a temperature of 28 °C would be considered as suitable for looking into the pathogenicity of A. hydrophila for conducting any challenge study with this organism in tropical environment.