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.

Biologically active carbazole derivatives: focus on oxazinocarbazoles and related compounds.

Four series of carbazole derivatives, including N-substituted-hydroxycarbazoles, oxazinocarbazoles, isoxazolocarbazolequinones, and pyridocarbazolequinones, were studied using diverse biological test methods such as a CE-based assay for CK2 activity measurement, a cytotoxicity assay with IPC-81 cell line, determination of MIC of carbazole derivatives as antibacterial agents, a Plasmodium falciparum susceptibility assay, and an ABCG2-mediated mitoxantrone assay. Two oxazinocarbazoles Ib and Ig showed CK2 inhibition with IC50 = 8.7 and 14.0 µM, respectively. Further chemical syntheses were realized and the 7-isopropyl oxazinocarbazole derivative 2 displayed a stronger activity against CK2 (IC50 = 1.40 µM). Oxazinocarbazoles Ib, Ig, and 2 were then tested against IPC-81 leukemia cells and showed the ability to induce leukemia cell death with IC50 values between 57 and 62 µM. Further investigations were also reported on antibacterial and antiplasmodial activities. No significant inhibitory activity on ABCG2 efflux pump was detected.

Performance of super-SILAC based quantitative proteomics for comparison of different acute myeloid leukemia (AML) cell lines.

As a direct consequence of the high diversity of the aggressive blood cancer acute myeloid leukemia (AML), proteomic samples from patients are strongly heterogeneous, rendering their accurate relative quantification challenging. In the present study, we investigated the benefits of using a super-SILAC mix of AML derived cell lines as internal standard (IS) for quantitative shotgun studies. The Molm-13, NB4, MV4-11, THP-1, and OCI-AML3 cell lines were selected for their complementarity with regard to clinical, cytogenetic, and molecular risk factors used for prognostication of AML patients. The resulting IS presents a high coverage of the AML proteome compared to single cell lines allied with high technical reproducibility, thus enabling its use for AML patient comparison. This was confirmed by comparing the protein regulation between the five cell lines and by applying the IS to patient material; hence, we were able to reproduce specific functional regulations known to be related to disease progression and molecular genetic abnormalities. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD000441.

Dynamic changes in immune cell populations by AXL kinase targeting diminish liver inflammation and fibrosis in experimental MASH.

Background and aims: Metabolic dysfunction-associated steatohepatitis (MASH) is a significant health concern with limited treatment options. AXL, a receptor tyrosine kinase activated by the GAS6 ligand, promotes MASH through activation of hepatic stellate cells and inflammatory macrophages. This study identified cell subsets affected by MASH progression and the effect of AXL inhibition. Methods: Mice were fed chow or different fat-enriched diets to induce MASH, and small molecule AXL kinase inhibition with bemcentinib was evaluated. Gene expression was measured by qPCR. Time-of-flight mass cytometry (CyTOF) used single cells from dissociated livers, acquired on the Fluidigm Helios, and cell populations were studied using machine learning. Results: In mice fed different fat-enriched diets, liver steatosis alone was insufficient to elevate plasma soluble AXL (sAXL) levels. However, in conjunction with inflammation, sAXL increases, serving as an early indicator of steatohepatitis progression. Bemcentinib, an AXL inhibitor, effectively reduced proinflammatory responses in MASH models, even before fibrosis appearance. Utilizing CyTOF analysis, we detected a decreased population of Kupffer cells during MASH while promoting infiltration of monocytes/macrophages and CD8+ T cells. Bemcentinib partially restored Kupffer cells, reduced pDCs and GzmB- NK cells, and increased GzmB+CD8+ T cells and LSECs. Additionally, AXL inhibition enhanced a subtype of GzmB+CD8+ tissue-resident memory T cells characterized by CX3CR1 expression. Furthermore, bemcentinib altered the transcriptomic landscape associated with MASH progression, particularly in TLR signaling and inflammatory response, exhibiting differential cytokine expression in the plasma, consistent with liver repair and decreased inflammation. Conclusion: Our findings highlight sAXL as a biomarker for monitoring MASH progression and demonstrate that AXL targeting shifted liver macrophages and CD8+ T-cell subsets away from an inflammatory phenotype toward fibrotic resolution and organ healing, presenting a promising strategy for MASH treatment.

Activation of both protein kinase A (PKA) type I and PKA type II isozymes is required for retinoid-induced maturation of acute promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) is characterized by granulopoietic differentiation arrest at the promyelocytic stage. In most cases, this defect can be overcome by treatment with all-trans-retinoic acid (ATRA), leading to complete clinical remission. Cyclic AMP signaling has a key role in retinoid treatment efficacy: it enhances ATRA-induced maturation in ATRA-sensitive APL cells (including NB4 cells) and restores it in some ATRA-resistant cells (including NB4-LR1 cells). We show that the two cell types express identical levels of the Cα catalytic subunit and comparable global cAMP-dependent protein kinase A (PKA) enzyme activity. However, the maturation-resistant NB4-LR1 cells have a PKA isozyme switch: compared with the NB4 cells, they have decreased content of the juxtanuclearly located PKA regulatory subunit IIα and PKA regulatory subunit IIß, and a compensatory increase of the generally cytoplasmically distributed PKA-RIα. Furthermore, the PKA regulatory subunit II exists mainly in the less cAMP-responsive nonautophosphorylated state in the NB4-LR1 cells. By the use of isozyme-specific cAMP analog pairs, we show that both PKA-I and PKA-II must be activated to achieve maturation in NB4-LR1 as well as NB4 cells. Therefore, special attention should be paid to activating not only PKA-I but also PKA-II in attempts to enhance ATRA-induced APL maturation in a clinical setting.

The lipopeptide toxins anabaenolysin A and B target biological membranes in a cholesterol-dependent manner.

The two novel cyanobacterial cyclic lipopeptides, anabaenolysin (Abl) A and B permeabilised mammalian cells, leading to necrotic death. Abl A was a more potent haemolysin than other known biodetergents, including digitonin, and induced discocyte-echinocyte transformation in erythrocytes. The mitochondria of the dead cells appeared intact with regard to both ultrastructure and membrane potential. Also isolated rat liver mitochondria were resistant to Abl, judged by their ultrastructure and lack of cytochrome c release. The sparing of the mitochondria could be related to the low cholesterol content of their outer membrane. In fact, a supplement of cholesterol in liposomes sensitised them to Abl. In contrast, the prokaryote-directed cyclic lipopeptide surfactin lysed preferentially non-cholesterol-containing membranes. In silico comparison of the positions of relevant functional chemical structures revealed that Abl A matched poorly with surfactin in spite of the common cyclic lipopeptide structure. Abl A and the plant-derived glycolipid digitonin had, however, predicted overlaps of functional groups, particularly in the cholesterol-binding tail of digitonin. This may suggest independent evolution of Abl and digitonin to target eukaryotic cholesterol-containing membranes. Sub-lytic concentrations of Abl A or B allowed influx of propidium iodide into cells without interfering with their long-term cell viability. The transient permeability increase allowed the influx of enough of the cyanobacterial cyclic peptide toxin nodularin to induce apoptosis. The anabaenolysins might therefore not only act solely as lysins, but also as cofactors for the internalisation of other toxins. They represent a potent alternative to digitonin to selectively disrupt cholesterol-containing biological membranes.

Functional p53 is required for rapid restoration of daunorubicin-induced lesions of the spleen.

BACKGROUND: The tumour suppressor and transcription factor p53 is a major determinant of the therapeutic response to anthracyclines. In healthy tissue, p53 is also considered pivotal for side effects of anthracycline treatment such as lesions in haematopoietic tissues like the spleen. We used a Trp53null mouse to explore the significance of p53 in anthracycline (daunorubicin) induced lesions in the spleen. METHODS: Mice with wild type or deleted Trp53 were treated with the daunorubicin (DNR) for three consecutive days. Spleens were collected at various time points after treatment, and examined for signs of chemotherapy-related lesions by microscopic analysis of haematoxylin-eosin or tunel-stained paraffin sections. Expression of death-inducing proteins was analysed by immunoblotting. Changes between Trp53 wild type and null mice were compared by t-tests. RESULTS: Signs of cell death (pyknotic nuclei and tunel-positive cells) in the white pulp of the spleen occurred earlier following DNR exposure in wt-mice compared to Trp53-null mice. While the spleen of wt-mice recovered to normal morphology, the spleen of the Trp53-null animals still had lesions with large necrotic areas and disorganised histologic appearance eight days after treatment. Immunoblotting showed that only Trp53-wt mice had significant increase in p21 after DNR treatment. However, both wt and null mice had elevated p63 levels following DNR exposure. CONCLUSIONS: p53 protects against severe and enduring cellular damage of the spleen parenchyma after DNR treatment, and initial DNR-induced apoptosis is not predictive of tissue lesions in the spleen. Our data indicate that p53 induction following DNR treatment serves to protect rather than to destroy normal tissue.

Iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from Streptosporangium sp. induces apoptosis selectively in myeloid leukemia cell lines and patient cells.

Despite recent improvement in therapy, acute myeloid leukemia (AML) is still associated with high lethality. In the presented study, we analyzed the bioactive compound iodinin (1,6-dihydroxyphenazine 5,10-dioxide) from a marine actinomycetes bacterium for the ability to induce cell death in a range of cell types. Iodinin showed selective toxicity to AML and acute promyelocytic (APL) leukemia cells, with EC50 values for cell death up to 40 times lower for leukemia cells when compared with normal cells. Iodinin also successfully induced cell death in patient-derived leukemia cells or cell lines with features associated with poor prognostic such as FLT3 internal tandem duplications or mutated/deficient p53. The cell death had typical apoptotic morphology, and activation of apoptotic signaling proteins like caspase-3. Molecular modeling suggested that iodinin could intercalate between bases in the DNA in a way similar to the anti-cancer drug daunorubicin (DNR), causing DNA-strand breaks. Iodinin induced apoptosis in several therapy-resistant AML-patient blasts, but to a low degree in peripheral blood leukocytes, and in contrast to DNR, not in rat cardiomyoblasts. The low activity towards normal cell types that are usually affected by anti-leukemia therapy suggests that iodinin and related compounds represent promising structures in the development of anti-cancer therapy.

Mutated HRAS Activates YAP1-AXL Signaling to Drive Metastasis of Head and Neck Cancer.

The survival rate for patients with head and neck cancer (HNC) diagnosed with cervical lymph node (cLN) or distant metastasis is low. Genomic alterations in the HRAS oncogene are associated with advanced tumor stage and metastasis in HNC. Elucidation of the molecular mechanisms by which mutated HRAS (HRASmut) facilitates HNC metastasis could lead to improved treatment options for patients. Here, we examined metastasis driven by mutant HRAS in vitro and in vivo using HRASmut human HNC cell lines, patient-derived xenografts, and a novel HRASmut syngeneic model. Genetic and pharmacological manipulations indicated that HRASmut was sufficient to drive invasion in vitro and metastasis in vivo. Targeted proteomic analysis showed that HRASmut promoted AXL expression via suppressing the Hippo pathway and stabilizing YAP1 activity. Pharmacological blockade of HRAS signaling with the farnesyltransferase inhibitor tipifarnib activated the Hippo pathway and reduced the nuclear export of YAP1, thus suppressing YAP1-mediated AXL expression and metastasis. AXL was required for HRASmut cells to migrate and invade in vitro and to form regional cLN and lung metastases in vivo. In addition, AXL-depleted HRASmut tumors displayed reduced lymphatic and vascular angiogenesis in the primary tumor. Tipifarnib treatment also regulated AXL expression and attenuated VEGFA and VEGFC expression, thus regulating tumor-induced vascular formation and metastasis. Our results indicate that YAP1 and AXL are crucial factors for HRASmut-induced metastasis and that tipifarnib treatment can limit the metastasis of HNC tumors with HRAS mutations by enhancing YAP1 cytoplasmic sequestration and downregulating AXL expression. SIGNIFICANCE: Mutant HRAS drives metastasis of head and neck cancer by switching off the Hippo pathway to activate the YAP1-AXL axis and to stimulate lymphovascular angiogenesis.

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.

AXL targeting by a specific small molecule or monoclonal antibody inhibits renal cell carcinoma progression in an orthotopic mice model.

AXL tyrosine kinase activation enhances cancer cell survival, migration, invasiveness, and promotes drug resistance. AXL overexpression is typically detected in a high percentage of renal cell carcinomas (RCCs) and is strongly associated with poor prognosis. Therefore, AXL inhibition represents an attractive treatment option in these cancers. In this preclinical study, we investigated the antitumor role of a highly selective small molecule AXL inhibitor bemcentinib (BGB324, BerGenBio), and a newly developed humanized anti-AXL monoclonal function blocking antibody tilvestamab, (BGB149, BerGenBio), in vitro and an orthotopic RCC mice model. The 786-0-Luc human RCC cells showed high AXL expression. Both bemcentinib and tilvestamab significantly inhibited AXL activation induced by Gas6 stimulation in vitro. Furthermore, tilvestamab inhibited the downstream AKT phosphorylation in these cells. The 786-0-Luc human RCC cells generated tumors with high Ki67 and vimentin expression upon orthotopic implantation in athymic BALB/c nude mice. Most importantly, both bemcentinib and tilvestamab inhibited the progression of tumors induced by the orthotopically implanted 786-0 RCC cells. Remarkably, their in vivo antitumor effectiveness was not significantly enhanced by concomitant administration of a multi-target tyrosine kinase inhibitor. Bemcentinib and tilvestamab qualify as compounds of potentially high clinical interest in AXL overexpressing RCC.

Phosphatidylserine Receptors Enhance SARS-CoV-2 Infection: AXL as a Therapeutic Target for COVID-19.

Phosphatidylserine (PS) receptors are PS binding proteins that mediate uptake of apoptotic bodies. Many enveloped viruses utilize this PS/PS receptor mechanism to adhere to and internalize into the endosomal compartment of cells and this is termed apoptotic mimicry. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. We evaluated if PS receptors serve as cell surface receptors for SARS-CoV-2 and found that the PS receptors, AXL, TIM-1 and TIM-4, facilitated virus infection when low concentrations of the SARS-CoV-2 cognate receptor, ACE2, was present. Consistent with the established mechanism of PS receptor utilization by other viruses, PS liposomes competed with SARS-CoV-2 for binding and entry. We demonstrated that this PS receptor enhances SARS-CoV-2 binding to and infection of an array of human lung cell lines and is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.

Association of AXL and PD-L1 Expression with Clinical Outcomes in Patients with Advanced Renal Cell Carcinoma Treated with PD-1 Blockade.

PURPOSE: A minority of patients currently respond to single-agent immune-checkpoint blockade (ICB), and strategies to increase response rates are urgently needed. AXL is a receptor tyrosine kinase commonly associated with drug resistance and poor prognosis in many cancer types, including in clear-cell renal cell carcinoma (ccRCC). Recent experimental cues in breast, pancreatic, and lung cancer models have linked AXL with immune suppression and resistance to antitumor immunity. However, its role in intrinsic and acquired resistance to ICB remains largely unexplored. EXPERIMENTAL DESIGN: In this study, tumoral expression of AXL was examined in ccRCC specimens from 316 patients who were metastatic receiving the PD-1 inhibitor nivolumab in the GETUG AFU 26 NIVOREN trial after failure of antiangiogenic therapy. We assessed associations between AXL and patient outcomes following PD-1 blockade, as well as the relationship with various markers, including PD-L1; VEGFA; the immune markers CD3, CD8, CD163, and CD20; and the mutational status of the tumor-suppressor gene von Hippel-Lindau (VHL). RESULTS: Our results show that high AXL-expression level in tumor cells is associated with lower response rates and a trend to shorter progression-free survival following anti-PD-1 treatment. AXL expression was strongly associated with tumor-PD-L1 expression, especially in tumors with VHL inactivation. Moreover, patients with tumors displaying concomitant PD-L1 expression and high AXL expression had the worst overall survival. CONCLUSIONS: Our findings propose AXL as candidate factor of resistance to PD-1 blockade, and provide compelling support for screening both AXL and PD-L1 expression in the management of advanced ccRCC.See related commentary by Hahn et al., p. 6619.

A Functional Role of GAS6/TAM in Nonalcoholic Steatohepatitis Progression Implicates AXL as Therapeutic Target.

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 fibrotic- and inflammatory-related genes in HFD-fed 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.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.

AXL Targeting Abrogates Autophagic Flux and Induces Immunogenic Cell Death in Drug-Resistant Cancer Cells.

INTRODUCTION: Acquired cancer therapy resistance evolves under selection pressure of immune surveillance and favors mechanisms that promote drug resistance through cell survival and immune evasion. AXL receptor tyrosine kinase is a mediator of cancer cell phenotypic plasticity and suppression of tumor immunity, and AXL expression is associated with drug resistance and diminished long-term survival in a wide range of malignancies, including NSCLC. METHODS: We aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC. RESULTS: We found that EGFRi resistance was mediated by up-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset of acquired resistance to long-term EGFRi treatment in vivo. AXL-expressing EGFRi-resistant cells revealed phenotypic and cell signaling heterogeneity incompatible with a simple bypass signaling mechanism, and were characterized by an increased autophagic flux. AXL kinase inhibition by the small molecule inhibitor bemcentinib or siRNA mediated AXL gene silencing was reported to inhibit the autophagic flux in vitro, bemcentinib treatment blocked clonogenicity and induced immunogenic cell death in drug-resistant NSCLC in vitro, and abrogated the transcription of autophagy-associated genes in vivo. Furthermore, we found a positive correlation between AXL expression and autophagy-associated gene signatures in a large cohort of human NSCLC (n = 1018). CONCLUSION: Our results indicate that AXL signaling supports a drug-resistant persister cell phenotype through a novel autophagy-dependent mechanism and reveals a unique immunogenic effect of AXL inhibition on drug-resistant NSCLC cells.

A Kinase Inhibitor with Anti-Pim Kinase Activity is a Potent and Selective Cytotoxic Agent Toward Acute Myeloid Leukemia.

More than 40 years ago, the present standard induction therapy for acute myeloid leukemia (AML) was developed. This consists of the metabolic inhibitor cytarabine (AraC) and the cytostatic topoisomerase 2 inhibitor daunorubucin (DNR). In light of the high chance for relapse, as well as the large heterogeneity, novel therapies are needed to improve patient outcome. We have tested the anti-AML activity of 15 novel compounds based on the scaffolds pyrrolo[2,3-a]carbazole-3-carbaldehyde, pyrazolo[3,4-c]carbazole, pyrazolo[4,3-a]phenanthridine, or pyrrolo[2,3-g]indazole. The compounds were inhibitors of Pim kinases, but could also have inhibitory activity against other protein kinases. Ser/Thr kinases like the Pim kinases have been identified as potential drug targets for AML therapy. The compound VS-II-173 induced AML cell death with EC50 below 5 µmol/L, and was 10 times less potent against nonmalignant cells. It perturbed Pim-kinase-mediated AML cell signaling, such as attenuation of Stat5 or MDM2 phosphorylation, and synergized with DNR to induce AML cell death. VS-II-173 induced cell death also in patients with AML blasts, including blast carrying high-risk FLT3-ITD mutations. Mutation of nucleophosmin-1 was associated with good response to VS-II-173. In conclusion new scaffolds for potential AML drugs have been explored. The selective activity toward patient AML blasts and AML cell lines of the pyrazolo-analogue VS-II-173 make it a promising drug candidate to be further tested in preclinical animal models for AML.

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction.

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (αSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfß), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.

AXL Targeting Overcomes Human Lung Cancer Cell Resistance to NK- and CTL-Mediated Cytotoxicity.

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 AXL-dependent immune resistance involved a molecular network comprising NF-κB 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.