Phosphatidylserine receptors enhance SARS-CoV-2 infection.

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.

Axl-inhibitor bemcentinib alleviates mitochondrial dysfunction in the unilateral ureter obstruction murine model.

Renal fibrosis is a progressive histological manifestation leading to chronic kidney disease (CKD) and associated with mitochondrial dysfunction. In previous work, we showed that Bemcentinib, an Axl receptor tyrosine kinase inhibitor, reduced fibrosis development. In this study, to investigate its effects on mitochondrial dysfunction in renal fibrosis, we analysed genome-wide transcriptomics data from a unilateral ureter obstruction (UUO) murine model in the presence or absence of bemcentinib (n = 6 per group) and SHAM-operated (n = 4) mice. Kidney ligation resulted in dysregulation of mitochondria-related pathways, with a significant reduction in the expression of oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), citric acid cycle (TCA), response to reactive oxygen species and amino acid metabolism-related genes. Bemcentinib treatment increased the expression of these genes. In contrast, AKT/PI3K signalling pathway genes were up-regulated upon UUO, but bemcentinib largely inhibited their expression. At the functional level, ligation reduced mitochondrial biomass, which was increased upon bemcentinib treatment. Serum metabolomics analysis also showed a normalizing amino acid profile in UUO, compared with SHAM-operated mice following bemcentinib treatment. Our data suggest that mitochondria and mitochondria-related pathways are dramatically affected by UUO surgery and treatment with Axl-inhibitor bemcentinib partially reverses these effects.

Inhibiting the GAS6/AXL axis suppresses tumor progression by blocking the interaction between cancer-associated fibroblasts and cancer cells in gastric carcinoma.

BACKGROUND: The effects of cancer-associated fibroblasts (CAF) on the progression of gastric carcinoma (GC) has recently been demonstrated. However, agents targeting the interaction between CAF and GC cells have not been applied in a clinical setting. Here, we examined if inhibition for Axl receptor tyrosine kinase (AXL) can suppress CAF-induced aggressive phenotype in GC. METHODS: We investigated the function of CAF-derived growth arrest-specific 6 (GAS6), a major ligand of AXL, on the migration and proliferation of GC cells. The effect of the AXL inhibitor, BGB324, on the CAF-induced aggressive phenotype of GC cells was also investigated. In addition, we performed immunohistochemistry to examine the expression of phosphorylated AXL protein in 175 GC tissues and evaluated its correlation with the prognosis. RESULTS: The qPCR and western blot analysis showed that GAS6 expression was higher in CAF relative to other cells. We found that co-culture with CAF increased the phosphorylation of AXL (P-AXL), differentiation into a mesenchymal-like phenotype, and cell survival in GC cell lines. When the expression of AXL was genetically inhibited in GC cells, the effect of CAF was reduced. BGB324, a small molecule inhibitor of AXL, suppressed the effects of CAF on GC cell lines. In GC tissues, high levels of P-AXL were significantly associated with poor overall survival (P = 0.022). CONCLUSIONS: We concluded that CAF are a major source of GAS6 and that GAS6 promotes an aggressiveness through AXL activation in GC. We suggested that an AXL inhibitor may be a novel agent for GC treatment.

Axl, a prognostic and therapeutic target in acute myeloid leukemia mediates paracrine crosstalk of leukemia cells with bone marrow stroma.

Acute myeloid leukemia (AML) represents a clonal disease of hematopoietic progenitors characterized by acquired heterogenous genetic changes that alter normal mechanisms of proliferation, self-renewal, and differentiation.(1) Although 40% to 45% of patients younger than 65 years of age can be cured with current therapies, only 10% of older patients reach long-term survival.(1) Because only very few novel AML drugs were approved in the past 2 decades, there is an urgent need to identify novel targets and therapeutic strategies to treat underserved AML patients. We report here that Axl, a member of the Tyro3, Axl, Mer receptor tyrosine kinase family,(2-4) represents an independent prognostic marker and therapeutic target in AML. AML cells induce expression and secretion of the Axl ligand growth arrest-specific gene 6 (Gas6) by bone marrow-derived stromal cells (BMDSCs). Gas6 in turn mediates proliferation, survival, and chemoresistance of Axl-expressing AML cells. This Gas6-Axl paracrine axis between AML cells and BMDSCs establishes a chemoprotective tumor cell niche that can be abrogated by Axl-targeting approaches. Axl inhibition is active in FLT3-mutated and FLT3 wild-type AML, improves clinically relevant end points, and its efficacy depends on presence of Gas6 and Axl. Axl inhibition alone or in combination with chemotherapy might represent a novel therapeutic avenue for AML.

Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets.

BACKGROUND: The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels. RESULTS: In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells. CONCLUSION: The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.

AXL expression reflects tumor-immune cell dynamics impacting outcome in non-small cell lung cancer patients treated with immune checkpoint inhibitor monotherapy.

Introduction: AXL receptor expression is proposed to confer immune-checkpoint inhibitor (ICI)-resistance in non-small cell lung cancer (NSCLC) patients. We sought to interrogate AXL expression in conjunction with mutational and tumor-microenvironmental features to uncover predictive mechanisms of resistance in ICI-treated NSCLC patients. Methods: Tumor samples from 111 NSCLC patients treated with ICI-monotherapy were analyzed by immunohistochemistry for tumor- and immune-AXL expression. Subsets of patients were analyzed by whole-exome sequencing (n = 44) and imaging mass cytometry (n = 14). Results were related to ICI-outcome measurements. Results: Tumor-cell AXL expression correlated with aggressive phenotypic features including reduced OS in patients treated with ICIs (P = 0.04) after chemotherapy progression, but conversely associated with improved disease control (P = 0.045) in ICI-treated, PD-L1 high first-line patients. AXL+ immune-cell infiltration correlated with total immune-cell infiltration and improved overall outcomes (PFS: P = 0.044, OS: P = 0.054). Tumor-cell AXL-upregulation showed enrichment in mutations associated with PD-L1-upregulation and ICI-response such as MUC4 and ZNF469, as well as adverse mutations including CSMD1 and LRP1B which associated with an immune-suppressed tumor phenotype and poor ICI prognosis particularly within chemotherapy-treated patients. Tumor mutational burden had no effect on ICI-outcomes and was associated with a lack of tumor-infiltrating immune cells. Spatial-immunophenotyping provided evidence that tumor-cell AXL-upregulation and adverse mutations modulate the tumor microenvironment in favor of infiltrating, activated neutrophils over anti-tumor immune-subsets including CD4 and CD8 T-cells. Conclusion: Tumor-cell AXL-upregulation correlated with distinct oncotypes and microenvironmental immune-profiles that define chemotherapy-induced mechanisms of ICI-resistance, which suggests the combination of AXL inhibitors with current chemoimmunotherapy regimens can benefit NSCLC patients.

In vivo turnover and biodistribution of soluble AXL: implications for biomarker development.

Soluble biomarkers are paramount to personalized medicine. However, the in vivo turnover and biodistribution of soluble proteins is seldom characterized. The cleaved extracellular domain of the AXL receptor (sAXL) is a prognostic biomarker in several diseases and a predictive marker of AXL targeting agents. Plasma sAXL reflects a balance between production in tissues with lymphatic transport into the circulation and removal from blood by degradation or excretion. It is unclear how this transport cycle affects plasma sAXL levels that are the metric for biomarker development. Radiolabeled mouse sAxl was monitored after intravenous injection to measure degradation and urinary excretion of sAxl, and after intradermal injection to mimic tissue or tumor production. sAxl was rapidly taken-up and degraded by the liver and kidney cortex. Surprisingly, intact sAxl was detectable in urine, indicating passage through the glomerular filter and a unique sampling opportunity. The structure of sAxl showed an elongated, flexible molecule with a length of 18 nm and a thickness of only 3 nm, allowing passage through the glomerulus and excretion into the urine. Intradermally injected sAxl passed through local and distant lymph nodes, followed by uptake in liver and kidney cortex. Low levels of sAxl were seen in the plasma, consistent with an extended transit time from local tissue to circulation. The rapid plasma clearance of sAxl suggests that steady-state levels in blood will sensitively and dynamically reflect the rate of production of sAxl in the tissues but will be influenced by perturbations of liver and kidney function.

AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.

Evaluating extracellular matrix influence on adherent cell signaling by cold trypsin phosphorylation-specific flow cytometry.

BACKGROUND: Tissue microenvironments comprise different extracellular matrix (ECM) proteins that regulate cellular responsiveness to growth factors. In vitro culture of adherent cells on ECM-coated substrata is commonly used to study microenvironmental influence on specific cell signaling responses. Phosphorylation-specific flow cytometry can be utilized to quantify intracellular phosphorylation-dependent signaling events in single cells. However this approach necessitates trypsinization of adherent cells to accommodate flow cytometric analysis. Trypsin is a potent activator of cell signaling and can obscure signal transduction events induced by other factors. RESULTS: To address this we developed a cold trypsin-phosphorylation-specific flow cytometry protocol, where adherent cells are prepared for flow cytometric analysis on ice (~0°C), a temperature where trypsin retains activity but where intracellular kinases are inactive. We show that this straightforward approach can be used to quantify intracellular pERK levels in single adherent primary human vascular smooth muscle cells grown on different ECM. CONCLUSIONS: Exploiting the limited temperature dependence of trypsin facilitated development of a generally applicable phosphorylation-specific flow cytometry method for analysis of adherent cell types including primary patient derived cells. We demonstrate the utility of cold trypsin-phosphorylation-specific flow cytometry analysis of cell signaling to measure microenvironmental influence in single adherent cells.

Contextual compound screening for improved therapeutic discovery.

Cellular behaviors are governed by combinations of systemic and microenvironmental factors; together, these regulate cell signaling responses to growth factors. This contextual microenvironmental influence also determines drug sensitivity. Hence using in vitro systems that model contextual cellular behavior is highly beneficial for effective therapeutic development. Angiogenesis (formation of blood vessels) is driven by a series of dynamic endothelial cell signaling responses to growth factors under the influence of the vascular extracellular matrix and adjacent pericytes. In vitro primary human vascular cell co-cultures self-assemble into capillary-like structures through reciprocal heterotypic interactions that mimic angiogenic context dynamics. By using temporal live-cell imaging-based analysis, unique angiogenic microenvironments can be delineated to quantify the contextual activity of compound inhibitors. We used this in vitro organotypic contextual screening approach to conduct structure-activity relationship analysis on a combretastatin A-4 analogue series to identify novel compounds with potent vascular disrupting activity in vivo.

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival.

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

Soluble mediators released by acute myeloid leukemia cells increase capillary-like networks.

Increased bone marrow angiogenesis is seen in several hematological malignancies, including acute myeloid leukemia (AML). We used a co-culture assay of endothelial and vascular smooth muscle cells (vSMC) to investigate the effects of AML-conditioned medium on capillary networks. We investigated primary AML cells derived from 44 unselected patients and observed that for a large subset of patients, the constitutive cytokine release by the leukemic cells stimulated endothelial cell organization into capillary-like networks, while there were only minor or no effects for other patients. We analyzed the constitutive AML cell release of 31 cytokines for all the patients and performed a hierarchical cluster analysis of the cytokine profile which identified two major patient subsets that differed in their ability to enhance capillary-like networks; increased capillary-like networks was then associated with high constitutive release of several cytokines and especially high levels of several pro-angiogenic chemokines. Significantly increased network formation was not seen for any of the 11 acute lymphoblastic leukemia patients investigated. The cytokine response by activated normal T cells inhibited endothelial network formation in our in vitro model of angiogenesis and activated normal monocytes had only a minor influence on tube formation. Our study shows that AML-derived cytokines can induce the organization of endothelial cells into vessel-like structures.

A combined targeted/phenotypic approach for the identification of new antiangiogenics agents active on a zebrafish model: from in silico screening to cyclodextrin formulation.

A combined targeted/phenotypic approach for the rapid identification of novel antiangiogenics with in vivo efficacy is herein reported. Considering the important role played by the tyrosine kinase c-Src in the regulation of tumour angiogenesis, we submitted our in-house library of c-Src inhibitors to a sequential screening approach: in silico screening on VEGFR2, in vitro screening on HUVEC cells, ADME profiling, formulation and in vivo testing on a zebrafish model. A promising antiangiogenic candidate able to interfere with the vascular growth of a zebrafish model at low micromolar concentration was thus identified.

Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines.

Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines.

Dissecting the Role of AXL in Cancer Immune Escape and Resistance to Immune Checkpoint Inhibition.

The development and implementation of Immune Checkpoint Inhibitors (ICI) in clinical oncology have significantly improved the survival of a subset of cancer patients with metastatic disease previously considered uniformly lethal. However, the low response rates and the low number of patients with durable clinical responses remain major concerns and underscore the limited understanding of mechanisms regulating anti-tumor immunity and tumor immune resistance. There is an urgent unmet need for novel approaches to enhance the efficacy of ICI in the clinic, and for predictive tools that can accurately predict ICI responders based on the composition of their tumor microenvironment. The receptor tyrosine kinase (RTK) AXL has been associated with poor prognosis in numerous malignancies and the emergence of therapy resistance. AXL is a member of the TYRO3-AXL-MERTK (TAM) kinase family. Upon binding to its ligand GAS6, AXL regulates cell signaling cascades and cellular communication between various components of the tumor microenvironment, including cancer cells, endothelial cells, and immune cells. Converging evidence points to AXL as an attractive molecular target to overcome therapy resistance and immunosuppression, supported by the potential of AXL inhibitors to improve ICI efficacy. Here, we review the current literature on the prominent role of AXL in regulating cancer progression, with particular attention to its effects on anti-tumor immune response and resistance to ICI. We discuss future directions with the aim to understand better the complex role of AXL and TAM receptors in cancer and the potential value of this knowledge and targeted inhibition for the benefit of cancer patients.

Intrinsic Differences in Spatiotemporal Organization and Stromal Cell Interactions Between Isogenic Lung Cancer Cells of Epithelial and Mesenchymal Phenotypes Revealed by High-Dimensional Single-Cell Analysis of Heterotypic 3D Spheroid Models.

The lack of inadequate preclinical models remains a limitation for cancer drug development and is a primary contributor to anti-cancer drug failures in clinical trials. Heterotypic multicellular spheroids are three-dimensional (3D) spherical structures generated by self-assembly from aggregates of two or more cell types. Compared to traditional monolayer cell culture models, the organization of cells into a 3D tissue-like structure favors relevant physiological conditions with chemical and physical gradients as well as cell-cell and cell-extracellular matrix (ECM) interactions that recapitulate many of the hallmarks of cancer in situ. Epidermal growth factor receptor (EGFR) mutations are prevalent in non-small cell lung cancer (NSCLC), yet various mechanisms of acquired resistance, including epithelial-to-mesenchymal transition (EMT), limit the clinical benefit of EGFR tyrosine kinase inhibitors (EGFRi). Improved preclinical models that incorporate the complexity induced by epithelial-to-mesenchymal plasticity (EMP) are urgently needed to advance new therapeutics for clinical NSCLC management. This study was designed to provide a thorough characterization of multicellular spheroids of isogenic cancer cells of various phenotypes and demonstrate proof-of-principle for the applicability of the presented spheroid model to evaluate the impact of cancer cell phenotype in drug screening experiments through high-dimensional and spatially resolved imaging mass cytometry (IMC) analyses. First, we developed and characterized 3D homotypic and heterotypic spheroid models comprising EGFRi-sensitive or EGFRi-resistant NSCLC cells. We observed that the degree of EMT correlated with the spheroid generation efficiency in monocultures. In-depth characterization of the multicellular heterotypic spheroids using immunohistochemistry and high-dimensional single-cell analyses by IMC revealed intrinsic differences between epithelial and mesenchymal-like cancer cells with respect to self-sorting, spatiotemporal organization, and stromal cell interactions when co-cultured with fibroblasts. While the carcinoma cells harboring an epithelial phenotype self-organized into a barrier sheet surrounding the fibroblasts, mesenchymal-like carcinoma cells localized to the central hypoxic and collagen-rich areas of the compact heterotypic spheroids. Further, deep-learning-based single-cell segmentation of IMC images and application of dimensionality reduction algorithms allowed a detailed visualization and multiparametric analysis of marker expression across the different cell subsets. We observed a high level of heterogeneity in the expression of EMT markers in both the carcinoma cell populations and the fibroblasts. Our study supports further application of these models in pre-clinical drug testing combined with complementary high-dimensional single-cell analyses, which in turn can advance our understanding of the impact of cancer-stroma interactions and epithelial phenotypic plasticity on innate and acquired therapy resistance in NSCLC.

C/EBPB-dependent adaptation to palmitic acid promotes tumor formation in hormone receptor negative breast cancer.

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2. Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network.

Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

AXL targeting restores PD-1 blockade sensitivity of STK11/LKB1 mutant NSCLC through expansion of TCF1+ CD8 T cells.

Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.

Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome.

Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits beta5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138+ tumor cells. Although specific inhibition of either LMP7 or beta5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both beta5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2alpha suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors.