NOTCH localizes to mitochondria through the TBC1D15-FIS1 interaction and is stabilized via blockade of E3 ligase and CDK8 recruitment to reprogram tumor-initiating cells.

The P53-destabilizing TBC1D15-NOTCH protein interaction promotes self-renewal of tumor-initiating stem-like cells (TICs); however, the mechanisms governing the regulation of this pathway have not been fully elucidated. Here, we show that TBC1D15 stabilizes NOTCH and c-JUN through blockade of E3 ligase and CDK8 recruitment to phosphodegron sequences. Chromatin immunoprecipitation (ChIP-seq) analysis was performed to determine whether TBC1D15-dependent NOTCH1 binding occurs in TICs or non-TICs. The TIC population was isolated to evaluate TBC1D15-dependent NOTCH1 stabilization mechanisms. The tumor incidence in hepatocyte-specific triple knockout (Alb::CreERT2;Tbc1d15Flox/Flox;Notch1Flox/Flox;Notch2Flox/Flox;HCV-NS5A) Transgenic (Tg) mice and wild-type mice was compared after being fed an alcohol-containing Western diet (WD) for 12 months. The NOTCH1-TBC1D15-FIS1 interaction resulted in recruitment of mitochondria to the perinuclear region. TBC1D15 bound to full-length NUMB and to NUMB isoform 5, which lacks three Ser phosphorylation sites, and relocalized NUMB5 to mitochondria. TBC1D15 binding to NOTCH1 blocked CDK8- and CDK19-mediated phosphorylation of the NOTCH1 PEST phosphodegron to block FBW7 recruitment to Thr-2512 of NOTCH1. ChIP-seq analysis revealed that TBC1D15 and NOTCH1 regulated the expression of genes involved in mitochondrial metabolism-related pathways required for the maintenance of TICs. TBC1D15 inhibited CDK8-mediated phosphorylation to stabilize NOTCH1 and protect it from degradation The NUMB-binding oncoprotein TBC1D15 rescued NOTCH1 from NUMB-mediated ubiquitin-dependent degradation and recruited NOTCH1 to the mitochondrial outer membrane for the generation and expansion of liver TICs. A NOTCH-TBC1D15 inhibitor was found to inhibit NOTCH-dependent pathways and exhibited potent therapeutic effects in PDX mouse models. This unique targeting of the NOTCH-TBC1D15 interaction not only normalized the perinuclear localization of mitochondria but also promoted potent cytotoxic effects against TICs to eradicate patient-derived xenografts through NOTCH-dependent pathways.

Identification of COVID-19 prognostic markers and therapeutic targets through meta-analysis and validation of Omics data from nasopharyngeal samples.

BACKGROUND: While our battle with the COVID-19 pandemic continues, a multitude of Omics data have been generated from patient samples in various studies. Translation of these data into clinical interventions against COVID-19 remains to be accomplished. Exploring host response to COVID-19 in the upper respiratory tract can unveil prognostic markers and therapeutic targets. METHODS: We conducted a meta-analysis of published transcriptome and proteome profiles of respiratory samples of COVID-19 patients to shortlist high confidence upregulated host factors. Subsequently, mRNA overexpression of selected genes was validated in nasal swabs from a cohort of COVID-19 positive/negative, symptomatic/asymptomatic individuals. Guided by this analysis, we sought to check for potential drug targets. An FDA-approved drug, Auranofin, was tested against SARS-CoV-2 replication in cell culture and Syrian hamster challenge model. FINDINGS: The meta-analysis and validation in the COVID-19 cohort revealed S100 family genes (S100A6, S100A8, S100A9, and S100P) as prognostic markers of severe COVID-19. Furthermore, Thioredoxin (TXN) was found to be consistently upregulated. Auranofin, which targets Thioredoxin reductase, was found to mitigate SARS-CoV-2 replication in vitro. Furthermore, oral administration of Auranofin in Syrian hamsters in therapeutic as well as prophylactic regimen reduced viral replication, IL-6 production, and inflammation in the lungs. INTERPRETATION: Elevated mRNA level of S100s in the nasal swabs indicate severe COVID-19 disease, and FDA-approved drug Auranofin mitigated SARS-CoV-2 replication in preclinical hamster model. FUNDING: This study was supported by the DBT-IISc partnership program (DBT (IED/4/2020-MED/DBT)), the Infosys Young Investigator award (YI/2019/1106), DBT-BIRAC grant (BT/CS0007/CS/02/20) and the DBT-Wellcome Trust India Alliance Intermediate Fellowship (IA/I/18/1/503613) to ST lab.