GAS6 signaling through AXL receptor contributes to the progression of nonalcoholic steatohepatitis (NASH). Soluble AXL significantly increases both in NASH patients and mouse models. Experimental AXL inhibition by bemcentinib diminishes inflammation and fibrosis, supporting its therapeutic use in NASH.BACKGROUND AND AIMS: GAS6 signaling, through the TAM receptor tyrosine kinases AXL and MERTK, participates in chronic liver pathologies. Here, we addressed GAS6/TAM involvement in Non-Alcoholic SteatoHepatitis (NASH) development.METHODS: GAS6/TAM signaling was analyzed in cultured primary hepatocytes, hepatic stellate cells (HSC) and Kupffer cells (KCs). Axl -/-, Mertk -/and wild-type C57BL/6 mice were fed with Chow, High Fat Choline-Deficient Methionine-Restricted (HFD) or methionine-choline-deficient (MCD) diet. HSC activation, liver inflammation and cytokine/chemokine production were measured by qPCR, mRNA Array analysis, western blotting and ELISA. GAS6, soluble AXL (sAXL) and MERTK (sMERTK) levels were analyzed in control individuals, steatotic and NASH patients. RESULTS:In primary mouse cultures, GAS6 or MERTK activation protected primary hepatocytes against lipid toxicity via AKT/STAT-3 signaling, while bemcentinib (small molecule AXL inhibitor BGB324) blocked AXL-induced fibrogenesis in primary HSCs and cytokine production in LPS-treated KCs. Accordingly; bemcentinib diminished liver inflammation and fibrosis in MCD-and HFD-fed mice. Upregulation of AXL and ADAM10/ADAM17 metalloproteinases increased sAXL in HFD-fed mice. Transcriptome profiling revealed major reduction in fibroticand inflammatory-related genes in HFDfed mice after bemcentinib administration. HFD-fed Mertk -/mice exhibited enhanced NASH, while Axl -/mice were partially protected. In human serum, sAXL levels augmented even at initial stages, whereas GAS6 and sMERTK increased only in cirrhotic NASH patients. In agreement, sAXL increased in HFD-fed mice before fibrosis establishment, while bemcentinib prevented liver fibrosis/inflammation in early NASH.CONCLUSION: AXL signaling, increased in NASH patients, promotes fibrosis in HSCs and inflammation in KCs, while GAS6 protects cultured hepatocytes against lipotoxicity via MERTK. Bemcentinib, by blocking AXL signaling and increasing GAS6 levels, reduces experimental NASH, revealing AXL as an effective therapeutic target for clinical practice.

Although TP53 mutations are rare in acute myeloid leukemia (AML), wild type p53 function is habitually annulled through overexpression of MDM2 or through various mechanisms including epigenetic silencing by histone deacetylases (HDACs). We hypothesized that co-inhibition of MDM2 and HDACs, with nutlin-3 and valproic acid (VPA) would additively inhibit growth in leukemic cells expressing wild type TP53 and induce p53-mediated apoptosis. In vitro studies with the combination demonstrated synergistic induction of apoptosis in AML cell lines and patient cells. Nutlin-3 and VPA co-treatment resulted in massive induction of p53, acetylated p53 and p53 target genes in comparison with either agent alone, followed by p53 dependent cell death with autophagic features. In primary AML cells, inhibition of proliferation by the combination therapy correlated with the CD34 expression level of AML blasts. To evaluate the combination in vivo, we developed an orthotopic, NOD/SCID IL2rg null xenograft model of MOLM-13 (AML FAB M5a; wild type TP53) expressing firefly luciferase. Survival analysis and bioluminescent imaging demonstrated the superior in vivo efficacy of the dual inhibition of MDM2 and HDAC in comparison with controls. Our results suggest the concomitant targeting of MDM2-p53 and HDAC inhibition, may be an effective therapeutic strategy for the treatment of AML.

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

Disclosure: Drs. Lorens and Micklem are founders, shareholders, and employees of BerGenBio ASA. Dr. Gausdal is employed by and stock option holder of BerGenBio ASA. Drs. Lorens, Micklem, Gausdal, Lotsberg, and Engelsen are co-inventors of patent(s) pending or issued to BerGenBio ASA. Drs. Wnuk-Lipinska and Hellesøy are former employees of BerGenBio ASA. Drs. Chouaib and Brekken signed Sponsored Research Agreements with BerGenBio ASA related to separate research projects. Dr. Bivona reports grants from National Institutes of Health during the conduct of the study; grants and other from Novartis, Revolution Medicines, personal fees from AstraZeneca, Takeda, Strategia, Springworks, Array, Pfizer, and Rain outside the submitted work. Dr. Minna reports grants from National Cancer Institute, Margot Johnson Foundation, and CPRIT during the conduct of the study, and personal fees from National Cancer Institute and University of Texas Southwestern Medical Center outside the submitted work. Dr. Thiery is the scientific founder of Biocheetah Pte. Ltd., Singapore and advisor of Biosyngen Pte. Ltd., Singapore. The remaining authors declare no conflict of interest.

Immune resistance may arise from both genetic instability and tumor heterogeneity. Microenvironmental stresses such as hypoxia and various resistance mechanisms promote carcinoma cell plasticity. AXL, a member of the TAM (Tyro3, Axl, and Mer) receptor tyrosine kinase family, is widely expressed in human cancers and increasingly recognized for its role in cell plasticity and drug resistance. To investigate mechanisms of immune resistance, we studied multiple human lung cancer clones derived from a model of hypoxia-induced tumor plasticity that exhibited mesenchymal or epithelial features. We demonstrate that AXL expression is increased in mesenchymal lung cancer clones. Expression of AXL in the cells correlated with increased cancer cell-intrinsic resistance to both natural killer (NK)-and cytotoxic T lymphocyte (CTL)-mediated killing. A small-molecule targeting AXL sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXLdependent immune resistance involved a molecular network comprising NF-kB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Higher ICAM1 and ULBP1 tumor expression correlated with improved patient survival in two non-small cell lung cancer (NSCLC) cohorts. These results reveal an AXL-mediated immune-escape regulatory pathway, suggest AXL as a candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC.