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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.

Supplementary Table S2

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.

Supplementary Figures S1 to S6 with figure legends

Supplementary Materials and Methods

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.

This file contains supplementary Figures

Quantification of Pim levels in Molm13 cells

Table showing inhibition of kinases

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.

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

This file contains 6 supplementary figures and 5 supplementary Tables. Supplementary Figure S1 - Protein expression pattern of AXL in ccRCC specimens. Supplementary Figure S2 - Response rates, Survival and Biomarker analyses according to AXLneg, AXLlow and AXLhigh expression Supplementary Figure S3 - ORR and Progression-Free Survival according to PD-L1 Status alone or by grouping AXL expression plus PD-L1 status Supplementary Figure S4 - High AXL expression plus PD-L1 TC positivity is associated with worse OS in Nivolumab-treated patients in IMDC intermediate-risk/poor-risk group Supplementary Figure S5 - VHL status does not interfere with outcomes or AXL expression upon treatment with Nivolumab Supplementary Figure S6 - Biomarker distribution across AXL groups stratified based on VHL status

STK11 (LKB1) is a tumor suppressor, and loss-of-function mutations contribute to tumorigenesis. Mutations in the STK11 gene (STK11m) are present in ~ 20% of NSCLCs and are associated with poor response to chemotherapy and immune checkpoint inhibition (ICI) resulting in inferior survival outcomes. STK11m tumors are characterized by high oxidative stress/ROS, EMT, enhanced replication stress tolerance, resistance to DNA damage and a highly immunosuppressive tumor microenvironment with limited activation and expansion of anti-tumor CD8 T-cells. AXL, a member of the TAM family of receptor tyrosine kinases, is activated in response to cellular stress such as ROS and hypoxia. AXL expression and activation is associated with EMT and drug resistance, tumor tolerance towards oxidative stress and apoptosis, as well as an immunosuppressed tumor microenvironment. We have previously shown that selective AXL inhibition by bemcentinib potentiated ICI in STK11m NSCLC preclinical models and led to objective clinical response in individuals with STK11m NSCLC receiving bemcentinib and pembrolizumab (NCT03184571). Data from this clinical study, an independent real-world cohort of patients studied at the Haukeland Hospital in Bergen, Norway (REC 45562) and published results1, suggest AXL is expressed in ~ 80% of NSCLCs harboring a STK11m, indicating AXL expression is a characteristic of STK11m NSCLC and confirming that AXL is an attractive target in STK11m NSCLC. Inhibition of AXL in dendritic cells has been shown to increase ICI responses in preclinical models of STK11m NSCLC1. The relative contribution of targeting AXL in STK11m tumor cells vs the tumor microenvironment, and the impact on tumor cells after tumor cell targeting needs further exploration. Transcriptional analysis of STK11m and STK11wt sequences from public datasets, NCT03184571 patients and NSCLC cell lines identified transcriptional signatures consistent with the known roles of STK11 in DNA damage response and immunosuppression. Treatment of STK11m NSCLC cell lines with the AXL inhibitor bemcentinib led to a reduction in the STK11-associated DDR signature and increase in inflammatory signatures demonstrating the impact of AXL targeting on tumor cells. Due to the high unmet medical need in individuals harboring a STK11 mutation, the encouraging efficacy in the NCT03184571 clinical trial and the high incidence of AXL protein expression in STK11m tumors, a global, open-label Phase 1b/2a trial to determine the safety, tolerability and anti-tumor activity of bemcentinib with SOC (pembrolizumab, pemetrexed and carboplatin) in 1L advanced/metastatic non-squamous NSCLC patients with STK11 mutations and no actionable mutations is currently enrolling. The Phase 1b part of the study will evaluate the safety and tolerability of bemcentinib regardless of STK11 status, whereas the Phase 2a part will assess the efficacy in NSCLC patients with STK11 mutations. 1 Li et al., 2022; Cell Rep Med., PMID: 35492873 Citation Format: Magnus Blø, Austin Rayford, Noëlly Madeleine, Fabian Gärtner, Dana Bohan, Natalie Ruggio, Huiyu Li, Luc Girard, Rolf Brekken, John Minna, Marianne Ånerud, Wendy Maury, Claudia Gorcea-Carson, Gro Gausdal, David R. Micklem, Nigel McCracken. AXL as a therapeutic target in STK11 mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3245.

BACKGROUND AND AIMS Renal fibrosis, a progressive process of extracellular matrix accumulation leading to renal failure, lacks effective treatment. Expression of the AXL receptor tyrosine kinase has been implicated in kidney injury and mesangial proliferation [1]. Inhibition of AXL signalling with the selective AXL kinase inhibitor bemcentinib reduces fibrosis and inflammation in murine models of unilateral ureteral obstruction (UUO) and in glomerulonephritis [2, 3]. METHOD Male C57Bl/6 mice were subjected to UUO for 3 or 15 days and treated twice daily with vehicle or bemcentinib (50 mg/kg) by oral gavage. Kidneys were divided into pieces that were either dissociated into single cells for mass cytometry analysis or subjected to Sirius Red staining to evaluate collagen deposition. Samples for mass cytometry were stained with a 45-plex antibody panel, acquired on Helios, then cleaned and analysed using UMAP [4] for dimensionality reduction and PARC [5] for clustering. The effect of ligation was modelled with the least absolute shrinkage and selection operator (LASSO) using centered log-ratio transformed cluster compositions to predict the number of days of ligation. LASSO is a regression method that prevents overfitting by penalizing variables and is commonly used for variable selection. RESULTS Sirius Red staining confirmed, as previously published, reduced fibrosis development in kidneys from bemcentinib treated animals compared to the vehicle following 15 days of UUO. No significant effect of bemcentinib was observed after 3 days of obstruction. Mass cytometry analysis yielded 31 clusters representing immune, endothelial, pericyte, mesangial and epithelial cells from proximal tubules, loop of Henle, distal tubules and collecting duct. AXL was expressed by pericytes, endothelial cells, mesangial cells and macrophages. Modelling with LASSO suggested that the most important feature of ligation was the expansion of mesangial cells. Bemcentinib treatment did not result in any significant changes in non-ligated kidneys and kidneys ligated for 3 days. At 15 days, post-bemcentinib treatment, the number of mesangial cells and macrophages decreased, while the number of epithelial cells comprising the proximal tubules and loop of Henle increased. The LASSO model estimation corresponded to an apparent reduction in disease progression of 30% (corresponding to day 10 or 11 post-ligation). CONCLUSION AXL targeting macrophages and mesangial cells delays the progression of kidney fibrosis in the UUO model and represents a valid approach to treat kidney disease.

Epithelial-to-mesenchymal transition (EMT) contributes to tumor cell survival, immune evasion, migration, invasion, and therapy resistance. Across human cancer, tumors that are high grade, poorly differentiated, and have undergone EMT carry a worse prognosis with a higher likelihood of metastasis. AXL, a receptor tyrosine kinase, drives EMT and is implicated in tumor progression, metastasis, and therapy resistance in multiple cancer types including pancreatic cancer and breast cancer. TANK-binding kinase 1 (TBK1) is central to AXL-driven EMT yet, the mechanism of how TBK1 induces EMT remains unclear. Here, we report that AXL activation stimulates TBK1 binding and phosphorylation of AKT3. TBK1 activation of AKT3 drives binding and phosphorylation of slug/snail resulting in protection from proteasomal degradation and translocation of the complex into the nucleus. We show that nuclear translocation of AKT3 is required for AXL-driven EMT and metastasis. Congruently, nuclear AKT3 expression correlates with worse outcome in aggressive breast cancer. To advance AKT3 as a therapeutic target, an AKT3-isoform selective allosteric small molecule inhibitor, BGB214, was developed. BGB214 inhibits AKT3 nuclear translocation, EMT-TF stability, AKT3-mediated invasion of breast cancer cells and reduces tumor initiation in vivo. Our results suggest that AKT3 nuclear activity is an important feature of AXL-driven epithelial plasticity and that selective AKT3 inhibition represents a novel therapeutic avenue for treating aggressive cancer.SignificanceNuclear AKT3 activity is an important feature of AXL-TBK1 driven EMT and metastasis, thus selective AKT3 targeting represents a novel approach to treat aggressive cancer.

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. publishedVersion

AUTHOR SUMMARYPhosphatidylserine (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.

Mutations in

Background and Aims Interstitial fibrosis, characterised by the accumulation of extracellular matrix in the cortical interstitium, is directly correlated with progressive chronic kidney disease secondary to inflammatory, immunologic, obstructive or metabolic causes. An invariant histologic marker of this progression is the accumulation of fibroblasts, with the phenotypic appearance of activated myofibroblasts expressing alpha smooth muscle actin (αSMA) within intracellular contractile stress fibres. Once present, these myofibroblasts are prognostic indicators of expansion of fibrotic matrix and progressive tubular atrophy, leading towards end-stage disease. The Receptor Tyrosine Kinase AXL is involved in a range of kidney pathologies, with increased activity associated with Epithelial to Mesenchymal Transition (EMT) and tubular proliferation following podocyte loss. In mice treated with an angiotensin-converting enzyme (ACE) inhibitor, enhancement of AXL expression is localised to tubular segments within the medulla and there is evidence of parallel regulatory control of ACE and AXL. We have demonstrated enhanced expression of AXL and the mesenchymal marker, vimentin in diseased human kidney tissue secondary to diabetes or hypertension. Targeting AXL with a small-molecule inhibitor has previously been reported to attenuate fibrosis and reduce inflammation in the unilateral ureteric-outflow obstruction (UUO) model of kidney fibrosis in mice (Landolt et al., 2019). Tilvestamab is a novel function blocking humanized anti-AXL antibody. Tilvestamab blocks GAS6-mediated AXL receptor activation in fibroblasts and renal tubule epithelial cells and mediates AXL receptor internalization and degradation. In this study we aimed to further characterise AXL as a target in CKD and to investigate anti-fibrotic efficacy of tilvestamab. Method Eight weeks old male C57BL/6 mice underwent UUO operation. After 15 days, kidneys were dissociated and stained with a high dimensional single cell mass cytometry 33 markers antibody panel. Data were analysed using JMP Genomics (v.8.2). Precision Cut Kidney Slices (PCKSs) from explanted human kidney tissue were propagated in a bioreactor (Paish et al., 2019, FibroFind, UK). PCKS were incubated for 72hrs in the presence of investigational drugs. Secreted collagen1a1 were quantified by ELISA. RNA was reverse transcribed to cDNA and used in qPCRs to measure Col1a1 and αSMA. FFPE sections were stained for αSMA. High magnification images were taken of each slide and analysed for surface area covered by the stain. Results Expression pattern of AXL during development of kidney fibrosis in the UUO model was investigated using a mass cytometry antibody panel designed for identifying subpopulations of immune cells as well as cell populations of the fibrotic stroma. Two predominant cell populations were affected by ligation; the mesenchymal and the immune island. AXL was a marker characterising several of the key populations that expanded upon ligation supporting a role for AXL in kidney fibrosis pathogenesis. In an ex vivo model of human PCKS, tilvestamab dose-dependently reduced the levels of αSMA. When combined with the lower of two doses of the ACE inhibitor enalapril, the lowest dose of tilvestamab synergized to reduce αSMA levels further as well as reducing secreted Collagen 1a1. Conclusion AXL expression is induced in key cell populations during development of kidney fibrosis supporting AXL as a novel target in CKD. Tilvestamab represents a promising strategy for the pharmacologic intervention of kidney fibrosis, and the potential synergy with current reno-protective therapies warrants further exploration.

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

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., Author summary Phosphatidylserine (PS) receptors bind PS and 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. For viruses that have a mechanism(s) of endosomal escape, apoptotic mimicry is a productive route of virus entry. This clever use of this uptake mechanism by enveloped viruses is termed apoptotic mimicry. 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 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. PS is readily detectable on the surface of SARS-CoV-2 virions, and contrary to prior reports we were unable to identify any interaction between AXL and SARS-CoV-2 spike. Pharmacological inhibition of AXL activity and knockout of AXL expression suggest it is the preferred PS receptor during SARS-CoV-2 entry. We propose that AXL is an under-appreciated but potentially important host factor facilitating SARS-CoV-2 entry.

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. publishedVersion

Chemotherapy elicits anti-tumor immune responses by inducing immunogenic tumor cell death, targeting suppressive immune cells and activating Type 1 interferon (IFN) responses (1, 2, 3). Chemotherapeutic agents lead to cGAS - STING cytosolic DNA sensing pathway activation that results in Type 1 IFN release (4). IFN receptor (IFNAR1, IFNAR2) signaling mimics viral immune responses, is associated with clinical benefit (5, 6) and is exploited by chemo-immunotherapies (7, 8). The receptor tyrosine kinase AXL is associated with immune evasion and immunotherapy resistance (8). AXL serves as a critical regulatory checkpoint for TLR-induced IFN responses in dendritic cells, macrophages and natural killer cells (9, 10). IFN receptor signaling induces AXL expression (11) and AXL activation on dendritic cells is targeted by viruses (e.g. Zika) to abrogate IFN responses and inhibit anti-viral immunity. AXL serves as an IFN-response checkpoint by blocking IFNAR1 and IFNAR2 signaling. We hypothesized that tumor cells exploit AXL signaling to abrogate Type 1 IFN responses and inhibit antitumor immunity. We evaluated whether AXL inhibition promotes Type 1 IFN signalling and enhances immune checkpoint inhibitor efficacy. AXL kinase inhibition (bemcentinib) in combination with chemotherapy (doxorubicin) increased IFN response gene expression in mammary carcinoma and melanoma cell lines. In vivo, bemcentinib treatment potentiated the efficacy of immune checkpoint inhibitor treatment in combination with intratumoral doxorubicin injection (i.t) in the syngeneic myeloid derived suppressor cell (MDSC)- rich refractory mammary adenocarcinoma 4T1 model. In addition, bemcentinib treatment in conjunction with i.t. doxorubicin enhanced Type 1 IFN response, reduced cancer stemness and epithelial to mesenchymal (EMT) gene expression in this model. Furthermore, this combination treatment regimen sensitized the immune checkpoint inhibitor refractory Braf-mutant melanoma (YUMM1.7) by enhancing the type 1 IFN response resulting in significantly improved median overall survival. In conclusion, this study provides evidence that bemcentinib potentiates chemo-immunotherapy by enhancing tumor Type 1 IFN response and dampening EMT. References: 1) Galluzzi, Can Cell 2015. 2) Yan, Front Immunol 2018. 3) Fitzgerald, Nat Immun 2003. 4) Platanias, Nature Rev Immun 2005. 5) Sistigu, Nat Med. 2014. 6) Zitvogel L, Nature Rev Immun 2015. 7) Lazzari, Ther Adv Med Onc 2018. 8) Emens, CIR 2015. 9) Aguilera, Clin Can Res 2017. 10) Rothlin, Cell 2007. 11) Huang, Eur J Immunol 2015. 12) Cruz, JCI Insight. 2019. Citation Format: Sushil Dhakal, Sébastien Bougnaud, James B. Lorens, Gro Gausdal, Emmanuel E. Moutoussamy, Hani Gabra. AXL inhibition enhances Type 1 interferon (IFN) response and potentiates chemo-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2087.

IntroductionAcquired 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.MethodsWe aimed to investigate the mechanisms underlying AXL-mediated acquired resistance to first- and third-generation small molecule EGFR tyrosine kinase inhibitors (EGFRi) in NSCLC.ResultsWe found that EGFRi resistance was mediated byup-regulation of AXL, and targeting AXL reduced reactivation of the MAPK pathway and blocked onset ofacquired resistance to long-term EGFRi treatment invivo.AXL-expressing EGFRi-resistant cells revealed phenotypicand 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 toinhibit the autophagic flux invitro, 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).ConclusionOur 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.

Summary 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., Graphical Abstract, Highlights • AXL + mammary epithelial cells have multipotent activity conserved in women and mice • AXL allows accesses to epithelial-to-mesenchymal transition genes and prevents differentiation into luminal cells • Deletion of Axl reduced incidence of Wnt1-driven tumors in mice • Provides a rationale explaining the advantage to cancer cells that co-opt AXL signaling, Cell Biology; Stem Cells Research; Cancer

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., This study was funded by grants from Instituto de Salud Carlos III (PI16/00930 and PI19/01410 to Montserrat Marí) and CIBEREHD; Ministerio de Economía y Competitividad (SAF2015-66515-R and RTI2018-095672-B-I00 to Albert Morales and Pablo García de Frutos, and RTI2018-095572-B-100 to Anna Colell), and co-funded by FEDER (Fondo Europeo de Desarrollo Regional, Unión Europea), Fundació Marató de TV3 (to Pablo García de Frutos), AGAUR (2017_SGR_177 to Albert Morales), and CERCA Programme/ Generalitat de Catalunya

BackgroundMutations in tumor suppressor STK11/LKB1 are associated with negative predictive and prognostic impact in NSCLC patients receiving immune checkpoint inhibitors (CPI) in several published cohorts, although there have been some conflicting reports on the association of such mutations with patient outcomes in this setting [1–9]. STK11/LKB1 tumors are characterized by a suppressive tumor micro-environment devoid of cytotoxic T cells, and we hypothesized that targeting the receptor tyrosine kinase AXL, a known driver of an innate immune suppressive microenvironment, would restore sensitivity to PD-1 in syngeneic pre-clinical models as well as in patients harboring STK11/LKB1 mutated NSCLC.MethodsStk11/Lkb1 (L) mutation was introduced by CRISPR technology into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP). Sensitivity towards anti-PD-1 was evaluated in the absence and presence of the small molecule AXL inhibitor bemcentinib in the KPL model and in a human NSCLC xenograft model carrying a STK11/LKB1 mutation. The immune tumor landscape was mapped following introduction of the Stk11/Lkb1 mutation and therapeutic intervention with anti-PD-1/pembrolizumab and bemcentinib. FFPE fine-needle aspirate biopsies of target lesions were acquired from patients at screening immediately prior to enrollment in BGBC008, a PhII single-arm, 2-stage study with bemcentinib (200mg/d) and pembrolizumab (200 mg/q3wk) for previously-treated stage IV lung adenocarcinoma patients who were CPI naïve or CPI refractory. Patients were assessed for response according to RECIST1.1 criteria at scheduled scan intervals.ResultsIntroduction of a STK11/LKB1 mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss resulted in a PD-1 refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype that was recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of AXL with bemcentinib resulted in increased type I interferon secretion from dendritic cells resulting in expansion of tumor-associated TCF1+PD-1+CD8 T cells and restored therapeutic response to PD-1. This effect was observed in a syngeneic immunocompetent mouse model and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts.In an ongoing clinical trial (NCT03184571), 3 evaluable NSCLC patients with identified STK11/LKB1 mutations demonstrated objective clinical response/clinical benefit to the combination of AXL inhibitor bemcentinib and pembrolizumabConclusionsIn these models, AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC contributing to CPI resistance. Our results show that inhibition of AXL rescues this deficit and represents a new clinical strategy in combination with anti-PD-1 therapy in NSCLC patients carrying a STK11/LKB1 mutationAcknowledgementsThe authors would like to thank all patients and their caretakers for participating in this trial.Trial RegistrationPatients treated with bemcentinib and pembrolizumab combination therapy were enrolled in the BGBC008 clinical trial (BerGenBio ASA and Merck & Co., Inc., Kenilworth NJ, USA, NCT03184571)ReferencesGu M, Xu T, Chang P. KRAS/LKB1 and KRAS/TP53 co-mutations create divergent immune signatures in lung adenocarcinomas. Ther Adv Med Oncol. 2021;13:17588359211006950.Cho BC, Lopes G, Kowalski DM. Relationship between STK11 and KEAP1 mutational status and efficacy in KEYNOTE-042: pembrolizumab monotherapy as first-line therapy for PD-L1 positive advanced NSCLC. Cancer Res. 2020;80(16 Supplement):CT084.Aredo JV, Padda SK, Kunder CA. Impact of KRAS mutation subtype and concurrent pathogenic mutations on non-small cell lung cancer outcomes. Lung Cancer. 2019;133:144–150.Kwack WG, Shin SY, Lee SH. Primary Resistance to Immune Checkpoint Blockade in an STK11/TP53/KRAS-Mutant Lung Adenocarcinoma with High PD-L1 Expression. Oncol Targets Ther. 2020;13:8901–8905.Shire NJ, Klein AB, Golozar A. STK11 (LKB1) mutations in metastatic NSCLC: Prognostic value in the real world. PLoS One. 2020;15(9):e0238358. 6. Skoulidis F, Goldberg ME, Greenawalt DM. STK11/LKB1 Mutations and PD-1 Inhibitor Resistance in KRAS-Mutant Lung Adenocarcinoma. Cancer Discov. 2018;8(7):822–835. 7. Wang H, Guo J, Shang X. Less immune cell infiltration and worse prognosis after immunotherapy for patients with lung adenocarcinoma who harbored STK11 mutation. Int. Immunopharmacol. 2020;84:106574. 8. Kitajima S, Ivanova E, Gou S. Suppression of STING Associated with LKB1 Loss in KRAS-Driven Lung Cancer. Cancer Discov. 2019;9(1):34–45. 9. Mograbi B, Heeke S, Hofman P. The Importance of STK11/LKB1 Assessment in Non-Small Cell Lung Carcinomas. Diagnostics (Basel). 2021;11(2):196.Ethics ApprovalThis study was approved by the following ethical committees: Use of human cord blood: UT Southwestern (UTSW) Parkland Hospital, STU 112010-047Animal studies: UTSW Medical Center, Institutional Animal Care and Use Committee, APN 2015-100921Clinical study: London Bridge Research Ethics Committee (UK): 17/LO/0418; REC-South East (Norway): 2017/473; Drug Research Ethics Committee of the University Hospital Clinic of Barcelona (Spain): BGBC008/MK-3475_PN-531; Medical College of Wisconsin Institutional Review Board #4 (USA): PRO00029453

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.

The AXL receptor tyrosine kinase is associated with poor overall survival in breast cancer. Axl signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response. AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer cells, dendritic cells and tumor-associated macrophages. AXL is required for tumor immune evasion in mammary adenocarcinoma models and EMT-mediated resistance to cytotoxic T cell and natural killer (NK)-cell mediated cell killing. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms in breast cancer. We evaluated whether blocking AXL signaling with BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in the aggressive mammary adenocarcinoma (4T1) syngeneic (Balb/C) mouse modelthat display limited immunogenicity. Immune therapy with anti-CTLA-4/anti-PD-1 increased AXL and EMT-marker expression in 4T1 tumors, and correlated with lack of response to immune therapy. Combination treatment with BGB324 (50 mg/kg bid) significantly enhanced responsiveness to anti-CTLA-4/anti-PD-1 treatment (10 mg/kg of each, 4 doses) in Balb/C mice bearing established 4T1 tumors. The combination of BGB324 + anti-CTLA-4/anti-PD-1 resulted in durable primary tumor clearance in 23 % of treated mice versus 5.6% obtained with anti-CTLA-4/anti-PD-1 alone (p=0.0157). In a separate study, BGB324 + anti-CTLA-4 treated resulted in 22% long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of this model were concomitantly abrogated in the animals responding to the combination treatment. In addition, BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes (CTLs). Enhanced presence of CTLs was also detected in spleens from animals responding to treatment. BGB324 + anti-CTLA-4/anti-PD-1 treatment increased the number of NK cells, macrophages and polymorphonuclear neutrophils, but decreased the number of mMDSC. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. Together with recent results in other tumor types that support a prominent role for AXL in resistance to immune therapy and encouraging results from ongoing clinical trials with BGB324, support combining BGB324 with immune checkpoint inhibitors to improve treatment of breast cancer. Citation Format: Lorens JB, Lipinska KW, Davidsen K, Blø M, Hodneland L, Engelsen A, Kang J, Lie MK, Bougnaud S, Aguilera K, Ahmed L, Rybicka A, Nævdal EM, Deyna P, Boniecka A, Straume O, Chouaib S, Brekken RA, Gausdal G. BGB324, a selective small molecule inhibitor of the receptor tyrosine kinase AXL, enhances immune checkpoint inhibitor efficacy in mammary adenocarcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-08.

Clear cell renal cell carcinoma (ccRCC) represents the most common type of kidney cancer with high mortality in its advanced stages. Our study aim was to explore the correlation between tumor epithelial-to-mesenchymal transition (EMT) and patient survival. Renal biopsies of tumorous and adjacent nontumorous tissue were taken with a 16 g needle from our patients (n = 26) undergoing partial or radical nephrectomy due to ccRCC. RNA sequencing libraries were generated using Illumina TruSeq Access library preparation protocol and TruSeq Small RNA library preparation kit. Next generation sequencing (NGS) was performed on Illumina HiSeq2500. Comparative analysis of matched sample pairs was done using the Bioconductor Limma/voom R-package. Liquid chromatography-tandem mass spectrometry and immunohistochemistry were applied to measure and visualize protein abundance. We detected an increased generic EMT transcript score in ccRCC. Gene expression analysis showed augmented abundance of AXL and MMP14, as well as downregulated expression of KL (klotho). Moreover, microRNA analyses demonstrated a positive expression correlation of miR-34a and its targets MMP14 and AXL. Survival analysis based on a subset of genes from our list EMTrelated genes in a publicly available dataset showed that the EMT genes correlated with ccRCC patient survival. Several of these genes also play a known role in fibrosis. Accordingly, recently published classifiers of solid organ fibrosis correctly identified EMT-affected tumor samples and were correlated with patient survival. EMT in ccRCC linked to fibrosis is associated with worse survival and may represent a target for novel therapeutic interventions. Corrigendum: https://doi.org/10.14814/phy2.13671 publishedVersion

Background Upregulation of the receptor tyrosine kinase Axl has been linked with both a reduced response to immune checkpoint blockade as well as the development of therapy resistance to BRAF directed therapies in melanoma. Bemcentinib is a first-in-class orally bioavailable selective inhibitor of Axl which is currently being explored in several phase II clinical trials. BGBIL006 (NCT02872259) is an open label phase Ib/II trial designed to explore whether combinations with bemcentinib improves ORR and duration of response compared to standard of care therapies in patient (pts) with metastatic melanoma (MM). Trial design Patients are randomized 2:1 to receive D/T or pembro +/- bemcentinib, respectively, based on mutation status and tumour load. BRAF positive pts are allowed to switch D/T with pembrolizumab and vice versa upon progression. Tumour responses are assessed per investigator using RECIST v1.1. Plasma protein biomarker levels are measured using the DiscoveryMap v3.3 panel (Myriad RBM) in pts pre-dose and at C2D1. In June 2019, a formal interim analysis of clinical safety and efficacy data will be performed when at least 20 randomised patients have completed up to 12 cycles of treatment. Currently, 50 pts (92 planned) have been enrolled in the trial. Tolerability of the bemcentinib RP2D (200 mg daily) in combination with either D/T or pembro, and AE profiles for either therapeutic approach alone will be reported. Protein biomarkers candidates predictive of pt benefit following treatment and pre/post treatment changes of soluble proteins will be presented. Preliminary efficacy outcome data and safety data from the first formal preplanned interim analysis will be presented together with the recommendations from the Data Monitoring Committee. Clinical trial identification NCT02872259. Legal entity responsible for the study The authors. Funding Research Council Norway. Disclosure O. Straume: Travel / Accommodation / Expenses: BerGenBio. J.B. Lorens: Shareholder / Stockholder / Stock options: BerGenBio ASA. G. Gausdal: Full / Part-time employment: BerGenBio ASA. All other authors have declared no conflicts of interest.

As immunotherapies are now used to treat a large proportion of NSCLC patients, defining mechanisms of immune resistance is critical. Immune resistance may arise from both genetic instability and tumor heterogeneity driven by microenvironmental stresses such as hypoxia that promotes carcinoma cell plasticity as well as extrinsic or intrinsic mechanisms of immune resistance. AXL, a member of the TAM receptor tyrosine kinase family is widely expressed human cancers and increasingly recognized for its role in cell plasticity and drug resistance. In this study, we used a model of hypoxia-induced tumor plasticity to generate multiple lung cancer clones with mesenchymal and epithelial features to address mechanisms of immune resistance. We demonstrate that AXL expression is dramatically increased in mesenchymal lung cancer clones. Moreover, expression of AXL in the cells was correlated with an increased cancer cell intrinsic resistance to both NK and CTL-mediated killing, Notably, small molecule AXL targeting potently sensitized mesenchymal lung cancer cells to cytotoxic lymphocyte-mediated killing. Mechanistically, we showed that attenuation of AXL-dependent immune resistance to immune cells involved a novel molecular network comprising NF-κB activation, increased ICAM1 expression, and upregulation of ULBP1 expression coupled with MAPK inhibition. Congruently, higher ICAM1 and ULBP1 tumor expression, correlated with improved patient survival in two NSCLC cohorts. These results reveal a novel AXL-mediated immune escape regulatory pathway, suggest AXL as a novel candidate biomarker for tumor resistance to NK and CTL immunity, and support AXL targeting to optimize immune response in NSCLC. Citation Format: Sallem Chouaib, Stephane Terry, Stephanie Buart, Agnete Engelsen, Gro Gausdal, James Lorens, Jean Paul Thiery, Fathia Mami-Chouaib. AXL targeting enhances lymphocyte-mediated cytotoxicity of lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1200.

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 (alpha SMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgf beta), 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.

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 IL2rγ(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.

The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers. AXL signaling is important for tumor cell plasticity related to epithelial-to-mesenchymal transition (EMT), immune escape and intrinsic resistance to cytotoxic lymphocytes. AXL is expressed on several cells associated with the tumor immune microenvironment, including natural killer (NK) cells, dendritic cells and a subset of tumor-associated myeloid cells. AXL signaling enhances secretion of immune-suppressive cytokines from innate immune cells that limit antitumor immunity. Hence AXL resides uniquely at the nexus between tumor and microenvironmental antitumor immune suppression mechanisms. BGB324, a selective clinical-stage small-molecule Axl kinase inhibitor, is currently being evaluated in combination with pembrolizumab in three phase II clinical trials in patients with TNBC (NCT03184558), NSCLC (NCT03184571) and melanoma (NCT02872259). We show that BGB324 targets immune suppression mechanisms in the tumor microenvironment that improve immunotherapy in different murine tumor models. BGB324 treatment reduces myeloid-derived suppressor cells and tumor-associated macrophages, and lowers CCL11, IL-7, IL-1β, and IL-6 in murine pancreatic cancer models. This altered immune landscape is associated with increased tumor infiltration of NK and CD8+ T cells and enhanced therapy responses. Further, BGB324 targets tumor intrinsic immune resistance and enhances human CD8+ T cell and NK-cell mediated NSCLC tumor cell lysis. We are currently using high-dimensional mass cytometry analysis (CyTOF) to map adaptive Axl-dependent immune suppression during immune checkpoint blockade. Collectively these results highlight a prominent function for AXL in resistance to immune therapy and support continued clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment. Citation Format: Kjersti Davidsen, Katarzyna Wnuk-Lipinska, Wenting Du, Magnus Blø, Agnete Engelsen, Stephane Terry, Stacey D´mello, Maria Lie, Jing Kang, Linn Hodneland, Sebastien Bougnaud, Kristina Aguilera, Oddbjørn Straume, Salem Chouaib, Rolf A. Brekken, Gro Gausdal, James B. Lorens. BGB324, a selective small-molecule inhibitor of receptor tyrosine kinase AXL, targets tumor immune suppression and enhances immune checkpoint inhibitor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3774.

Background. The AXL receptor tyrosine kinase is associated with poor overall survival in breast cancer. AXL signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Upregulation of AXL has been associated with reduced response to anti-PD-1 therapy. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response, and AXL is expressed on several cells associated with the tumor immune microenvironment. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms. We show that AXL is required for tumor immune evasion in the 4T1/Balb/C mammary adenocarcinoma model and that blocking AXL signaling with BGB324, a selective clinical-stage small molecule AXL kinase inhibitor, enhanced the effect of immune checkpoint blockade. BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes and Natural Killer cells. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. These data provided a translational rationale for combining AXL targeted therapy with immune checkpoint inhibitors to enhance anti-cancer immune response. Study Design. BGBC007 (NCT03184558) is an open-label, single arm, multi-center phase II study designed to assess the anti-tumor activity of BGB324 in combination with pembrolizumab in patients with previously treated, locally advanced and unresectable, or metastatic TNBC or TN-IBC. Secondary objectives include safety and pharmacokinetic profile of BGB324 and pembrolizumab in combination. A single arm, extension of Simon's 2-stage design is employed with an interim and final analysis. Up to 56 evaluable patients will be enrolled. Recruitment will be halted once 28 evaluable patients have been entered to determine the Objective Response Rate (ORR, complete response and partial response). If 5 or fewer responses are observed in up to 28 patients, the trial will be terminated in favor of the null for futility. If 11 or more responses are observed, then the trial will be stopped in favor of the alternative for demonstration of activity. If 6 to 10 patients have an observed response then a further 28 patients may be evaluated. This design provides an overall power of 80.6% to test the stated null and alternative hypothesis. BGB324 will be administered orally, once daily, in a fasted state. Days 1, 2 and 3 of BGB324 administration consists of a 'loading' dose of 400 mg followed by a dose of 200 mg daily. A fixed dose of 200 mg pembrolizumab will be given by intravenous infusion over 30 minutes every 3 weeks. BGB324 and pembrolizumab will be given until disease progression, unacceptable dose toxicity, or until 106 weeks (35 cycles). Efficacy endpoints including ORR, Duration of Response, Progression Free Survival are based on tumor imaging evaluation by RECIST 1.1. Tumor specimens will be taken to assess AXL and PD-L1 expression. Citation Format: Yule M, Wnuk-Lipinska K, Davidsen K, Blø M, Hodneland L, Engelsen A, Kang J, Lie M, Bougnaud S, Aguilera K, Ahmed L, Rybicka A, Milde Nævdal E, Deyna P, Boniecka A, Straume O, Thiery J-P, Chouaib S, Brekken RA, Gausdal G, Lorens JB. A phase II multi-center study of BGB324 in combination with pembrolizumab in patients with previously treated, locally advanced and unresectable or metastatic triple negative breast cancer (TNBC) or triple negative inflammatory breast cancer (TN-IBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-01-03.

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.