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.

Protocol for generation of humanized HCC mouse model and cancer-driver mutations using CRISPR-Cas9.

We detail procedures for generating a humanized mouse model of hepatocellular carcinoma (HCC) recapitulating genetic mutations associated with metabolic liver diseases (MLD). We humanized liver parenchymal, non-parenchymal, and hematopoietic cells. We employed CRISPR-Cas9-based ARID1A knockout and constitutively active CTNNB1 knockin combined with an alcohol Western diet to generate cancer-driver mutations commonly found in MLD-HCC patients. This HCC model facilitates the study of tumor-promoting gene-environment interactions. For complete details on the use and execution of this protocol, please refer to Yeh et al.1.

ß-CATENIN stabilizes HIF2 through lncRNA and inhibits intravenous immunoglobulin immunotherapy.

Introduction: Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV neutralization in patients, their tumor-inhibitory effects have not yet been examined. Hepatitis B immunoglobulin (HBIG) therapy is employed to reduce hepatocellular carcinoma (HCC) recurrence in patients after living donor liver transplantations (LDLT). Hypothesis: We hypothesized that patient-derived intravenous immunoglobulin (IVIG) binding to HCC associated TICs will reduce self-renewal and cell viability driven by ß-CATENIN-downstream pathways. ß-CATENIN activity protected TICs from IVIG effects. Methods: The effects of HBIG and HCIG binding to TICs were evaluated for cell viability and self-renewal. Results: Inhibition of ß-CATENIN pathway(s) augmented TIC susceptibility to HBIG- and HCIG-immunotherapy. HBV X protein (HBx) upregulates both ß-CATENIN and NANOG expression. The co-expression of constitutively active ß-CATENIN with NANOG promotes self-renewal ability and tumor-initiating ability of hepatoblasts. HBIG bound to HBV+ cells led to growth inhibition in a TIC subset that expressed hepatitis B surface antigen. The HBx protein transformed cells through ß-CATENIN-inducible lncRNAs EGLN3-AS1 and lnc-ß-CatM. Co-expression of constitutively active ß-CATENIN with NANOG promoted self-renewal ability of TICs through EGLN3 induction. ß-CATENIN-induced lncRNAs stabilized HIF2 to maintain self-renewal of TICs. Targeting of EGLN3-AS1 resulted in destabilization of EZH2-dependent ß-CATENIN activity and synergized cell-killing of TICs by HBIG or HCIG immunotherapy. Discussion: Taken together, WNT and stemness pathways induced HIF2 of TICs via cooperating lncRNAs resulting in resistance to cancer immunotherapy. Therefore, therapeutic use of IVIG may suppress tumor recurrence through inhibition of TICs.

Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal.

The synergistic effect of alcohol and HCV mediated through TLR4 signaling transactivates NANOG, a pluripotency transcription factor important for the stemness of tumor-initiating stem-like cells (TICs). NANOG together with the PRC2 complex suppresses expression of oxidative phosphorylation (OXPHOS) genes to generate TICs. The phosphodegron sequence PEST domain of NANOG binds EED to stabilize NANOG protein by blocking E3 ligase recruitment and proteasome-dependent degradation, while the tryptophan-rich domain of NANOG binds EZH2 and SUZ12. Human ARID1A gene loss results in the resistance to combined FAO and PRC2 inhibition therapies due to reduction of mitochondrial ROS levels. CRISPR-Cas9-mediated ARID1A knockout and/or constitutively active CTNNB1 driver mutations promoted tumor development in humanized FRG HCC mouse models, in which use of an interface inhibitor antagonizing PRC2-NANOG binding and/or FAO inhibitor blocked tumor growth. Together, the PRC2-NANOG interaction becomes a new drug target for HCC via inducing differentiation-related genes, destabilizing NANOG protein, and suppressing NANOG activity.

GRP78 Inhibitor YUM70 Suppresses SARS-CoV-2 Viral Entry, Spike Protein Production and Ameliorates Lung Damage.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has given rise to many new variants with increased transmissibility and the ability to evade vaccine protection. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) chaperone that has been recently implicated as an essential host factor for SARS-CoV-2 entry and infection. In this study, we investigated the efficacy of YUM70, a small molecule inhibitor of GRP78, to block SARS-CoV-2 viral entry and infection in vitro and in vivo. Using human lung epithelial cells and pseudoviral particles carrying spike proteins from different SARS-CoV-2 variants, we found that YUM70 was equally effective at blocking viral entry mediated by original and variant spike proteins. Furthermore, YUM70 reduced SARS-CoV-2 infection without impacting cell viability in vitro and suppressed viral protein production following SARS-CoV-2 infection. Additionally, YUM70 rescued the cell viability of multi-cellular human lung and liver 3D organoids transfected with a SARS-CoV-2 replicon. Importantly, YUM70 treatment ameliorated lung damage in transgenic mice infected with SARS-CoV-2, which correlated with reduced weight loss and longer survival. Thus, GRP78 inhibition may be a promising approach to augment existing therapies to block SARS-CoV-2, its variants, and other viruses that utilize GRP78 for entry and infection.

Hepatic stellate cell stearoyl co-A desaturase activates leukotriene B4 receptor 2 - ß-catenin cascade to promote liver tumorigenesis.

Hepatocellular carcinoma (HCC) is the 3rd most deadly malignancy. Activated hepatic stellate cells (aHSC) give rise to cancer-associated fibroblasts in HCC and are considered a potential therapeutic target. Here we report that selective ablation of stearoyl CoA desaturase-2 (Scd2) in aHSC globally suppresses nuclear CTNNB1 and YAP1 in tumors and tumor microenvironment and prevents liver tumorigenesis in male mice. Tumor suppression is associated with reduced leukotriene B4 receptor 2 (LTB4R2) and its high affinity oxylipin ligand, 12-hydroxyheptadecatrienoic acid (12-HHTrE). Genetic or pharmacological inhibition of LTB4R2 recapitulates CTNNB1 and YAP1 inactivation and tumor suppression in culture and in vivo. Single cell RNA sequencing identifies a subset of tumor-associated aHSC expressing Cyp1b1 but no other 12-HHTrE biosynthetic genes. aHSC release 12-HHTrE in a manner dependent on SCD and CYP1B1 and their conditioned medium reproduces the LTB4R2-mediated tumor-promoting effects of 12-HHTrE in HCC cells. CYP1B1-expressing aHSC are detected in proximity of LTB4R2-positive HCC cells and the growth of patient HCC organoids is blunted by LTB4R2 antagonism or knockdown. Collectively, our findings suggest aHSC-initiated 12-HHTrE-LTB4R2-CTNNB1-YAP1 pathway as a potential HCC therapeutic target.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Profiling of Circulating Tumor Cells for Screening of Selective Inhibitors of Tumor-Initiating Stem-Like Cells.

A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.

LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells.

Chemoresistance and plasticity of tumor-initiating stem-like cells (TICs) promote tumor recurrence and metastasis. The gut-originating endotoxin-TLR4-NANOG oncogenic axis is responsible for the genesis of TICs. This study investigated mechanisms as to how TICs arise through transcriptional, epigenetic, and post-transcriptional activation of oncogenic TLR4 pathways. Here, we expressed constitutively active TLR4 (caTLR4) in mice carrying pLAP-tTA or pAlb-tTA, under a tetracycline withdrawal-inducible system. Liver progenitor cell induction accelerated liver tumor development in caTLR4-expressing mice. Lentiviral shRNA library screening identified histone H3K4 methylase SETD7 as central to activation of TLR4. SETD7 combined with hypoxia induced TLR4 through HIF2 and NOTCH. LIN28 post-transcriptionally stabilized TLR4 mRNA via de-repression of let-7 microRNA. These results supported a LIN28-TLR4 pathway for the development of HCCs in a hypoxic microenvironment. These findings not only advance our understanding of molecular mechanisms responsible for TIC generation in HCC, but also represent new therapeutic targets for the treatment of HCC.

Gut-derived Endotoxin-TLR4 Signaling Drives MYC-Ig Translocation to Promote Lymphoproliferation through c-JUN and STAT3 Activation.

Synergism between obesity and virus infection promotes the development of B-cell lymphoma. In this study, we tested whether obesity-associated endotoxin release induced activation-induced cytidine deaminase (AID). TLR4 activation in turn caused c-JUN-dependent and STAT3-dependent translocations of MYC loci to suppress transactivation of CD95/FAS. We used viral nucleocapside Core transgenic (Tg) mice fed alcohol Western diet to determine whether oncogenesis arising from obesity and chronic virus infection occurred through TLR4-c-JUN-STAT3 pathways. Our results showed B cell-specific, c-Jun and/or Stat3 disruption reduced the incidence of splenomegaly in these mice. AID-dependent t(8;14) translocation was observed between the Ig promoter and MYC loci. Comparison with human B cells showed MYC-immunoglobulin (Ig) translocations after virus infection with lipopolysaccharide stimulation. Accordingly, human patients with lymphoma with virus infections and obesity showed a 40% incidence of MYC-Ig translocations. Thus, obesity and virus infection promote AID-mediated translocation between the Ig promoter and MYC through the TLR4-c-JUN axis, resulting in lymphoproliferation. Taken together, preventative treatment targeting either c-JUN and/or STAT3 may be effective strategies to prevent tumor development. IMPLICATIONS: Obesity increases gut-derived endotoxin which induces Toll-like receptor-mediated MYC-Ig translocation via c-JUN-STAT3, leading to lymphoproliferation.

HCV and tumor-initiating stem-like cells.

Neoplasms contain tumor-initiating stem-like cells (TICs) that are characterized by increased drug resistance. The incidence of many cancer types have trended downward except for few cancer types, including hepatocellular carcinoma (HCC). Therefore mechanism of HCC development and therapy resistance needs to be understood. These multiple hits by hepatitis C virus (HCV) eventually promotes transformation and TIC genesis, leading to HCC development. This review article describes links between HCV-associated HCC and TICs. This review discusses 1) how HCV promotes genesis of TICs and HCC development; 2) how this process avails itself as a novel therapeutic target for HCC treatment; and 3) ten hall marks of TIC oncogenesis and HCC development as targets for novel therapeutic modalities.

DNMT and EZH2 inhibitors synergize to activate therapeutic targets in hepatocellular carcinoma.

The development of more effective targeted therapies for hepatocellular carcinoma (HCC) patients due to its aggressiveness is urgently needed. DNA methyltransferase inhibitors (DNMTis) represented the first clinical breakthrough to target aberrant cancer epigenomes. However, their clinical efficacies are still limited, in part due to an "epigenetic switch" in which a large group of genes that are demethylated by DNMTi treatment remain silenced by polycomb repressive complex 2 (PRC2) occupancy. EZH2 is the member of PRC2 that catalyzes the placement of H3K27me3 marks. EZH2 overexpression is correlated with poor HCC patient survival. We tested the combination of a DNMTi (5-aza-2'-deoxycytidine, DAC) and the EZH2 inhibitor (EZH2i) GSK126 in human HCC cell lines on drug sensitivity, DNA methylation, nucleosome accessibility, and gene expression profiles. Compared with single agent treatments, all HCC cell lines studied showed increased sensitivity after receiving both drugs concomitant with prolonged anti-proliferative changes and sustained reactivation of nascently-silenced genes. The increased number of up-regulated genes after combination treatment correlated with prolonged anti-proliferation effects and increased nucleosome accessibility. Combination treatments also activate demethylated promoters that are repressed by PRC2 occupancy. Furthermore, 13-31% of genes down-regulated by DNA methylation in primary HCC tumors were reactivated through this combination treatment scheme in vitro. Finally, the combination treatment also exacerbates anti-tumor immune responses, while most of these genes were downregulated in over 50% of primary HCC tumors. We have linked the anti-tumor effects of DAC and GSK126 combination treatments to detailed epigenetic alterations in HCC cells, identified potential therapeutic targets and provided a rationale for treatment efficacy for HCC patients.

Immunotherapy and Microbiota for Targeting of Liver Tumor-Initiating Stem-like Cells.

Cancer contains tumor-initiating stem-like cells (TICs) that are resistant to therapies. Hepatocellular carcinoma (HCC) incidence has increased twice over the past few decades, while the incidence of other cancer types has trended downward globally. Therefore, an understanding of HCC development and therapy resistance mechanisms is needed for this incurable malignancy. This review article describes links between immunotherapies and microbiota in tumor-initiating stem-like cells (TICs), which have stem cell characteristics with self-renewal ability and express pluripotency transcription factors such as NANOG, SOX2, and OCT4. This review discusses (1) how immunotherapies fail and (2) how gut dysbiosis inhibits immunotherapy efficacy. Gut dysbiosis promotes resistance to immunotherapies by breaking gut immune tolerance and activating suppressor immune cells. Unfortunately, this leads to incurable recurrence/metastasis development. Personalized medicine approaches targeting these mechanisms of TIC/metastasis-initiating cells are emerging targets for HCC immunotherapy and microbiota modulation therapy.

c-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity.

Metabolic syndrome is associated with obesity, insulin resistance, and the risk of cancer. We tested whether oncogenic transcription factor c-JUN metabolically reprogrammed cells to induce obesity and cancer by reduction of glucose uptake, with promotion of the stemness phenotype leading to malignant transformation. Liquid alcohol, high-cholesterol, fat diet (HCFD), and isocaloric dextrin were fed to wild-type or experimental mice for 12 months to promote hepatocellular carcinoma (HCC). We demonstrated 40% of mice developed liver tumors after chronic HCFD feeding. Disruption of liver-specific c-Jun reduced tumor incidence 4-fold and improved insulin sensitivity. Overexpression of c-JUN downregulated RICTOR transcription, leading to inhibition of the mTORC2/AKT and glycolysis pathways. c-JUN inhibited GLUT1, 2, and 3 transactivation to suppress glucose uptake. Silencing of RICTOR or c-JUN overexpression promoted self-renewal ability. Taken together, c-JUN inhibited mTORC2 via RICTOR downregulation and inhibited glucose uptake via downregulation of glucose intake, leading to self-renewal and obesity.

Activated and nonactivated MSCs increase survival in humanized mice after acute liver injury through alcohol binging.

The ability of the liver to regenerate after injury makes it an ideal organ to study for potential therapeutic interventions. Mesenchymal stem cells (MSCs) possess self-renewal and differentiation properties, as well as anti-inflammatory properties that make them an ideal candidate for therapy of acute liver injury. The primary aim of this study is to evaluate the potential for reversal of hepatic injury using human umbilical cord-derived MSCs. Secondary aims include comparison of various methods of administration as well as comparison of activated versus nonactivated human umbilical cord stem cells. To induce liver injury, humanized mice were fed high-cholesterol high-fat liquid diet with alcohol binge drinking. Mice were then treated with either umbilical cord MSCs, activated umbilical cord MSCs, or a placebo and followed for survival. Blood samples were obtained at the end of the binge drinking and at the time of death to measure alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Histology of all mouse livers was reported at time of death. Activated MSCs that were injected intravenously, intraperitoneally, or both routes had superior survival compared with nonactivated MSCs and with placebo-treated mice. AST and ALT levels were elevated in all mice before treatment and improved in the mice treated with stem cells. Conclusion: Activated stem cells resulted in marked improvement in survival and in recovery of hepatic chemistries. Activated umbilical cord MSCs should be considered an important area of investigation in acute liver injury.

A review of alcohol-pathogen interactions: New insights into combined disease pathomechanisms.

Progression of chronic infections to end-stage diseases and poor treatment results are frequently associated with alcohol abuse. Alcohol metabolism suppresses innate and adaptive immunity leading to increased viral load and its spread. In case of hepatotropic infections, viruses accelerate alcohol-induced hepatitis and liver fibrosis, thereby promoting end-stage outcomes, including cirrhosis and hepatocellular carcinoma (HCC). In this review, we concentrate on several unexplored aspects of these phenomena, which illustrate the combined effects of viral/bacterial infections and alcohol in disease development. We review alcohol-induced alterations implicated in immunometabolism as a central mechanism impacting metabolic homeostasis and viral pathogenesis in Simian immunodeficiency virus/human immunodeficiency virus infection. Furthermore, in hepatocytes, both HIV infection and alcohol activate oxidative stress to cause lysosomal dysfunction and leakage and apoptotic cell death, thereby increasing hepatotoxicity. In addition, we discuss the mechanisms of hepatocellular carcinoma and tumor signaling in hepatitis C virus infection. Finally, we analyze studies that review and describe the immune derangements in hepatotropic viral infections focusing on the development of novel targets and strategies to restore effective immunocompetency in alcohol-associated liver disease. In conclusion, alcohol exacerbates the pathogenesis of viral infections, contributing to a chronic course and poor outcomes, but the mechanisms behind these events are virus specific and depend on virus-alcohol interactions, which differ among the various infections.

State of the art treatment of hepatitis B virus hepatocellular carcinoma and the role of hepatitis B surface antigen post-liver transplantation and resection.

Chronic hepatitis B virus (HBV) infection is the major aetiology of hepatocellular carcinoma (HCC). The optimal goal of therapy, hepatitis B surface antigen (HBsAg) loss and anti-HBs production, is achieved rarely and HBsAg-associated HCC risk is well recognized. Here we review the role of HBsAg in HCC, the link between HBsAg and HCC recurrence post-liver transplantation or resection, and the implications for therapy. HBV-associated carcinogenesis is a multifactorial process. The observation that HBV-related HCC can occur in the absence of cirrhosis is compatible with a direct oncogenic effect of the virus, which may occur via multiple mechanisms, including those mediated by both mutated and unmutated HBsAg. HCC recurrence in HBsAg-positive patients post-liver transplantation has been reported in 10%-15% of patients and is likely to be because of expansion of residual HCC tumour cell populations containing integrated HBV DNA, which expand and independently replicate HBV, leading to the recurrence of both HCC and HBV. The direct role of HBsAg in HCC recurrence post-liver resection is less clear. Cirrhosis is the most important risk factor for HCC development, and precancerous cirrhotic liver remains after resection, with the potential to undergo malignant transformation regardless of the existence of HBV-derived oncogenic drivers. The role of HBsAg in the development of HCC and its recurrence post-surgical intervention has multiple implications for therapy and suggests a potential role for immunotherapy in the future management of HCC, in particular post-liver transplantation. Use of hepatitis B immunoglobulins that target HBsAg directly, alongside immune-oncology therapies, may be relevant in this setting.

The chaperone GRP78 is a host auxiliary factor for SARS-CoV-2 and GRP78 depleting antibody blocks viral entry and infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 global pandemic, utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for viral entry. However, other host factors might also play important roles in SARS-CoV-2 infection, providing new directions for antiviral treatments. GRP78 is a stress-inducible chaperone important for entry and infectivity for many viruses. Recent molecular docking analyses revealed putative interaction between GRP78 and the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (SARS-2-S). Here we report that GRP78 can form a complex with SARS-2-S and ACE2 on the surface and at the perinuclear region typical of the endoplasmic reticulum in VeroE6-ACE2 cells and that the substrate-binding domain of GRP78 is critical for this interaction. In vitro binding studies further confirmed that GRP78 can directly bind to the RBD of SARS-2-S and ACE2. To investigate the role of GRP78 in this complex, we knocked down GRP78 in VeroE6-ACE2 cells. Loss of GRP78 markedly reduced cell surface ACE2 expression and led to activation of markers of the unfolded protein response. Treatment of lung epithelial cells with a humanized monoclonal antibody (hMAb159) selected for its safe clinical profile in preclinical models depleted cell surface GRP78 and reduced cell surface ACE2 expression, as well as SARS-2-S-driven viral entry and SARS-CoV-2 infection in vitro. Our data suggest that GRP78 is an important host auxiliary factor for SARS-CoV-2 entry and infection and a potential target to combat this novel pathogen and other viruses that utilize GRP78 in combination therapy.

p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs.

Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15-NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15-NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC.