AXL Kinase Inhibitor Research Articles

Targeting AXL cellular networks in kidney fibrosis

IntroductionThe incidence of chronic kidney disease (CKD) is increasing, in parallel with risk factors including obesity and diabetes mellitus. AXL plays a central role in CKD, providing a rationale to evaluate clinical AXL targeting agents.MethodsTo determine the efficacy and underlying molecular mechanisms of AXL inhibition in CKD, we employed a murine unilateral ureteral obstruction (UUO) model preventively treated with a selective AXL kinase inhibitor (bemcentinib) during disease progression. We isolated kidneys at an early (3 days) or late (15 days) timepoint and profiled the cell populations using mass cytometry.ResultsPreventive treatment with bemcentinib significantly attenuated fibrosis in the UUO model. The anti-fibrotic effect correlated with a decrease in mesangial cells and inhibition of innate immune cell infiltration, while the proportion of epithelial cells increased. We mapped AXL expression to a unique network of cells in the kidney: mesangial cells, pericytes, macrophages and dendritic cells.DiscussionWe propose that AXL targeting affects an important cellular interaction network underlying fibrotic progression. These results support the clinical application of AXL targeting agents to treat CKD.

Machine Learning Approach to Predict AXL Kinase Inhibitor Activity for Cancer Drug Discovery Using Bayesian Optimization-XGBoost

Machine Learning Approach to Predict AXL Kinase Inhibitor Activity for Cancer Drug Discovery Using Bayesian Optimization-XGBoost

Phase I clinical study of FC084CSA, a high selective AXL kinase inhibitor that ameliorates tumor microenvironment and augments the effectivity of immunotherapy.

e15116 Background: NSCLC patients with STK11 gene mutations could not benefit from immunotherapy, according to the data of multiple clinical trials. This is associated with immunosuppressive tumor microenvironment (TME) due to elevated AXL expression, and deficiency in pro-inflammatory cytokines and effector T cells. AXL inhibitor bemcentinib (formerly BGB324) has been found to improve the effectiveness of immunotherapy of immune checkpoint inhibitors. FC084CSA is a high selective AXL inhibitor. Methods: This is an ongoing first-in-human, phase I study of FC084CSA in patients with advanced solid tumors. The primary objective is to evaluate the safety and tolerability of FC084CSA. The secondary objectives include preliminary efficacy and pharmacokinetics. Results: As of January 10th, 2024, 9 patients with a median age of 60 years (44-73) were enrolled in 4 different dose levels: 100mg QD, 200mg QD, 400mg QD and 600mg QD. All patients had ≥ 2 prior lines of prior therapy. No dosing-limiting toxicities were observed. 8 (88.9%) patients experienced treatment-related adverse events (TRAEs). Most TRAEs were grade 1 or 2 and only one grade 3 TRAE was observed. The most common TRAEs (≥20%) included increased blood lactate dehydrogenase (44.4%), elevated gamma-glutamyl transferase (33.3%), proteinuria (33.3%), anemia (33.3%), increased aspartate aminotransferase (22.2%), weight loss (22.2%), increased blood creatinine (22.2%) and vomiting (22.2%). A total of 8 patients had at least 1 post-baseline tumor assessment. Objective response rate (ORR) was 0% and disease control rate (DCR) was 62.5% (5/8). Conclusions: FC084CSA was well tolerated in patients with previously heavily treated solid tumors. Clinical trial information: NCT06231550 .

Abstract PO2-24-10: Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model

Abstract BACKGROUND The Gas6/AXL signaling pathway plays a substantial role in cancer cell survival, proliferation, immunosuppression, and metastasis. Elevated AXL expression is associated with advanced cancer stages and decreased overall survival rates, underscoring its potential as a therapeutic target for metastatic cancers. We investigated this possibility by utilizing AB-329, a selective small molecule AXL kinase inhibitor, to treat triple-negative breast cancer (TNBC). TNBC, typified by the lack of ER, PR, and HER2 expression, frequently displays mesenchymal characteristics and carries a significant risk of metastasis and an immunosuppressive tumor microenvironment. METHODS We assessed AXL protein expression in 28 human and 11 mouse breast cancer cell lines using Western blotting. The Microarray database was utilized to examine AXL gene expression in 40 human breast cancer cells. The Sulforhodamine B proliferation and soft agar assays measured the antiproliferative effect of AB-329 in TNBC cell lines. The in vitro anti-metastatic impact of AB-329 was evaluated through migration and invasion assays. We appraised the anti-tumor effects of AB-329 in combination with paclitaxel using 4T1 and E0771-LMB mouse TNBC xenograft models. We used FACS and CIBERSORTx to perform immune cell type profiling in E0771-LMB xenograft tumor tissues. To understand AB-329’s immunomodulatory effects in tumors, single-cell RNA sequencing was conducted in a humanized mouse model with SUM149 human TNBC xenografts. RESULTS Microarray data revealed higher AXL expression in TNBC cells compared to non-TNBC cells. Western blotting supported this, showing higher AXL protein expression, particularly in basal and mesenchymal subtypes of TNBC cells, compared to luminal breast cancer cells. AB-329 presented moderate antiproliferative effects as a monotherapy in the TNBC cell lines tested, with half-maximal inhibitory concentrations exceeding 5 µM. Nevertheless, combining AB-329 with paclitaxel resulted in significant growth inhibition in all tested TNBC cell lines (p < 0.05). Importantly, AB-329 alone could inhibit TNBC migration and invasion at a concentration of 1 µM (p < 0.01) and showed enhanced inhibition rates when paired with paclitaxel (p < 0.05). The combination of AB-329 and paclitaxel considerably reduced tumor growth in the 4T1 and E0771-LMB xenograft models compared to paclitaxel or AB-329. Total RNA sequencing and CIBERSORTx analysis indicated that the AB-329 and paclitaxel combination increased Natural Killer (NK) cells’ infiltration into E0771-LMB mouse TNBC xenograft tumors compared to single agent-treated tumors. A nearly 2.5-fold increase in NKT cell infiltration was also detected with AB-329 treatment compared to Control in SUM149 human TNBC xenografts. CONCLUSIONS Our preclinical data suggest that AB-329, an AXL inhibitor, could synergize effectively with paclitaxel in TNBC treatment, notably inhibiting tumor cell proliferation and migration while enhancing NK cell infiltration. Intriguingly, we observed an increased infiltration of NKT cells into tumors following AB-329 treatment, which may promote an anti-tumorigenic immuno-microenvironment. These results underscore the necessity for further investigation into the role of AB-329 in modulating the tumor immuno-microenvironment through AXL pathway inhibition and accentuate the potential for combining AB-329 with immunotherapy. Citation Format: Dileep Reddy Rampa, Jon Fuson, Huey Liu, Max Pan, Naoto Ueno, Jangsoon Lee. Anti-Tumorigenic Immuno-Microenvironment by AB-329, a Potent and Selective AXL inhibitor, combined with paclitaxel in Triple-Negative Breast Cancer Preclinical Model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-24-10.

Abstract 4182: Development and evaluation of orthotopically and subcutaneously implanted luciferase-labeled prostate cancer xenograft models for therapeutic assessment

Abstract Introduction: Prostate cancer is the most common male-specific cancer in many western countries including the US and the second most prevalent cause of cancer deaths in American males after lung cancer. The establishment of clinically relevant prostate cancer models is crucial for advancing our understanding of the disease and evaluating potential therapeutic interventions. In this in vivo project we characterize the tumor growth and response to therapy of two luciferase (luc) tagged human prostate cancer xenograft models of metastatic PC3M and non-metastatic DU145 cell lines. These models aim to replicate the variable clinical behavior of prostate cancer to provide valuable pre-clinical platforms to test existing and novel therapies. As a first step, these studies evaluate the effect of standard of care cytotoxic agents paclitaxel and carboplatin and the newer AXL kinase inhibitor bemcentinib alone and in combination on tumor growth kinetics and agent tolerability. Methods: PC3M-luc and DU145-luc cells were implanted subcutaneously on right flank or orthotopically into the prostate of NCG triple immunodeficient mice (Charles River). Subcutaneous tumor progression and treatment responses were monitored using calipers and bioluminescent imaging (BLI), with the capacity to detect developing metastases prior to necropsy. Orthotopic implants were monitored exclusively using BLI. Results: Orthotopic and subcutaneous implants of PC3M-luc demonstrated spontaneous metastasis to lungs and bones, replicating the established behavior of this cell line and effectively modeling metastases observed in prostate cancer patients. In contrast, orthotopic and subcutaneous implants of DU145-luc model did not metastasize. Both PC3M-luc and DU145-luc models independent of the tumor implant placement responded to paclitaxel treatment, with enhanced effects observed when combined with carboplatin. Notably, bemcentinib exhibited no activity as a single agent but showed promise when used in combination with cytotoxic agents. Conclusion: The development of orthotopic and subcutaneous PC3M-luc and DU145-luc prostate cancer models provides a valuable platform for assessing the effectiveness of established and novel therapies. These models mimic various clinical presentations of prostate cancer and promise to offer insights into disease progression and therapeutic strategies. Future studies will focus on durable responses and the assessment of other combination therapies for improved treatment outcomes. Citation Format: Kyle Draheim, Elizabeth Rainbolt, Bincy John, Cara Clouse, Andrew Wong, Brooke Wolff, Justin Avery, Chassidy Hall, Patrick Fadden, Anya Avrutskaya. Development and evaluation of orthotopically and subcutaneously implanted luciferase-labeled prostate cancer xenograft models for therapeutic assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4182.

Altered expression of AXL receptor tyrosine kinase in gastrointestinal cancers: a promising therapeutic target.

Gastrointestinal (GI) cancers that include all cancers of the digestive tract organs are generally associated with obesity, lack of exercising, smoking, poor diet, and heavy alcohol consumption. Treatment of GI cancers typically involves surgery followed by chemotherapy and/or radiation. Unfortunately, intrinsic or acquired resistance to these therapies underscore the need for more effective targeted therapies that have been proven in other malignancies. The aggressive features of GI cancers share distinct signaling pathways that are connected to each other by the overexpression and activation of AXL receptor tyrosine kinase. Several preclinical and clinical studies involving anti-AXL antibodies and small molecule AXL kinase inhibitors to test their efficacy in solid tumors, including GI cancers, have been recently carried out. Therefore, AXL may be a promising therapeutic target for overcoming the shortcomings of standard therapies in GI cancers.

Antitumor activity of AX-0085, a novel AXL kinase inhibitor: Analysis of apoptotic cell death in triple-negative breast cancer cells.

e15120 Background: Triple-negative breast cancer (TNBC) is insensitive to targeted therapies due to the negative expression of human epidermal growth factor receptor-2, estrogen receptor and progesterone receptor. TNBC cells express higher levels of AXL more often than other breast cancer subtypes. The receptor tyrosine kinase AXL plays a role in survival, invasion, migration, epithelial-mesenchymal transition (EMT), metastasis, resistance, and immune suppression in cancer cells including in TNBC. Thus, AXL is a promising therapeutic target for the treatment of TNBC, and we developed a new small-molecule AXL inhibitor, AX-0085. Methods: The interaction mechanism between AX-0085 and AXL was studied by molecular dynamic simulations, cell-based kinase assay, and western blotting in TNBC cells. We investigated the anti-tumor activity of AX-0085 on the AXL-dependent pro-tumorigenic properties such as cell proliferation, invasion, migration, EMT, and immune suppression (PD-L1 expression) in TNBC cell lines and investigated in vivo efficacy of AX-0085 with the 4T1 xenograft model. Results: In the molecular dynamic simulations, estimated binding energy of AX-0085 and cabozantinib to AXL was -10.1Kcal/mol and -8.1Kcal/mol, respectively. AX-0085 was made a hydrogen bond with catalytically important residue Lys567 and blocked the activation of AXL by inhibiting the salt bridge with Lys567-Glu585. AX-0085 (IC50: 4.4nM for AXL kinase in cell-based kinase assay) was one of the most potent AXL inhibitors. AX-0085 showed a high antitumor activity against all TNBC subtypes and was a more potent anti-proliferative activity than other kinase inhibitors in MDA-MB-231 cell line. AX-0085 effectively blocked the activation of AXL in TNBC and further inhibited AXL-dependent pro-tumorigenic events such as cell proliferation, invasion, migration, EMT, and PD-L1 expression in TNBC cell. AX-0085 induced cell cycle arrest at G1 phase and suppressed the expression of CDK2 and Cyclin E in TNBC. AX-0085 promoted apoptotic cell death by up-regulating cleaved caspase-9 and down-regulating Bcl-2 in TNBC. Pharmacological investigation showed that the AX-0085 treated tumors displayed a dose-dependent reduction in volume and weight of the tumors in the 4T1 mouse xenograft. Conclusions: Our results demonstrated that AX-0085 selectively blocks AXL activation which confers an effective therapeutic value in the treatment of TNBC. Currently, AX-0085 is undergoing non-clinical trials.

AX-0085, a novel AXL kinase inhibitor, and effects on osimertinib-resistance in non–small-cell lung cancer.

e15121 Background: Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) having high efficacy in treating patients with advanced non-small cell lung cancer (NSCLC) with EGFR activating mutations and EGFR T70M mutations. Although, osimertinib is a frontline anticancer agent for treating NSCLC, many patients inevitably develop osimertinib-resistance tumor recurrence. The activation of AXL was reported to be a major factor driving osimertinib-resistance in NSCLC. Thus, AXL is a promising therapeutic target for the treatment of osimertinib-resistant NSCLC, and we developed a new small-molecule AXL inhibitor, AX-0085. Methods: We established osimertinib-resistant cell lines from EGFR-mutated NSCLC cell lines, HCC827 and H1975, after high dose exposure and stepwise exposure to osimertinib in culture. We investigated the cell viability of AX-0085 and other kinase inhibitors, and the anti-tumor activity of AX-0085 on the proliferation, migration, apoptosis, and epithelial-mesenchymal transition (EMT) in osimertinib-resistant cells. Results: Compared to the parental cells, IC50 of resistant cell to osimertinib was significantly increased and osimertinib-resistant cells showed remarkably increased expression of AXL/pAXL and p-SRC as compared to the parental HCC827 cell. AX-0085 was completely blocked the AXL and SRC activation but EGFR inhibitors (gefitinib and osimertinib) did not inhibit the activation of AXL and SRC in the osimertinib-resistant cell line. AX-0085 (IC50: 4.4nM for AXL kinase in cell-based kinase assay) was one of the most potent AXL inhibitors and showed the strongest cytotoxicity against osimertinib-resistant cells compared to other kinase inhibitors. AX-0085 effectively down-regulated mRNA level of AXL and AXL regulating transcription factors in NSCLC cell lines such as HCC827 and H1975. AX-0085 treatment effectively inhibited the activation of AXL in acquired osimertinib-resistant cells and decreased the mRNA level of AXL and AXL regulating transcription factors. AX-0085 significantly inhibited the cell proliferation, apoptosis, migration, and EMT in acquired osimertinib-resistant cells. Conclusions: Our results showed that AX-0085 selectively inhibits the activation of AXL to overcome acquired resistance to osimertinib, suggesting an effective AXL-based therapy for patients with osimertinib-resistant tumors. Currently, AX-0085 is undergoing non-clinical trials.

Abstract 2255: Patient pharmacodynamic biomarker and pk evaluation results from an ongoing phase I dose-escalation study of q702, an axl, mer and csf1r kinase inhibitor in patients with advanced solid tumors

Abstract Background: Axl, Mer and CSF1 receptor tyrosine kinases play vital roles in promoting the immunosuppressive tumor microenvironment (TME) by affecting myeloid functions (e.g., tumor associated macrophage [TAM], myeloid derived suppression cell [MDSC]) and promoting epithelial-to-mesenchymal transition (EMT). Thus, simultaneous inhibition of Axl, Mer and CSF1R may be an effective strategy for TME modification. Q702 is a novel Axl/Mer/CSF1R kinase inhibitor that affects the immune components (modulating TAM and MDSC populations, inducing CD8+ T cell infiltration and increasing IFN-ɣ in CD8+ T cell) as well as changes in malignant cells such as increasing MHC I on the tumor cells of syngeneic mouse models. These nonclinical results suggest that Q702 monotherapy or Q702 combination with conventional therapies may have considerable potential as a novel treatment strategy for patients with advanced solid tumors. Methods: This is a Phase 1, Multicenter, Open-label, Dose-Escalation, Safety, Pharmacodynamic, Pharmacokinetic Study of Q702 with a Cohort Expansion at the recommended phase 2 dose (RP2D) in Patients with Advanced Solid Tumors (NCT04648254). Q702 was administered orally for seven days every other week. Peripheral blood samples were obtained on days 1,8,15, and 21. Axl, Mer and CSF1R target engagement is assessed by the quantifications of soluble Axl, Mer and M-CSF in plasma by Luminex xMAP® technology or ELISA. The pharmacodynamic biomarker changes are measured by flow cytometry for immune cell population shifts and IFN-ɣ levels in specific immune cells. Results: PK and PD biomarker samples from 22 patients with various tumor types (e.g. colon, pancreas, esophageal) from the dose escalation phase (4 mg to 240 mg) have been analyzed. Pharmacokinetic studies demonstrated dose proportional increase in Cmax and AUClast of Q702 and its two active metabolites which have activity against Axl and/or CSF1R. Axl and CSF1R target engagement by Q702 treatment is observed in a dose dependent manner. From the 60 mg cohort, target engagement for Axl and CSF1R reached a inhibitory level that was observed in nonclinical models. In the pharmacodynamic biomarker analysis, IFN-ɣ in CD8+ T cells and non-T cell populations is increased. Monocytes and M-MDSC population are decreased in peripheral blood. Conclusion: Up to 240 mg, Q702 has demonstrated the intended pharmacologic activity with acceptable safety profile. In biomarker analysis, immune modulation activity is exerted by Axl/Mer/CSF1R inhibition. Further assessment of pharmacokinetics, pharmacodynamics, safety and antitumor activity will be performed at the expansion phase at the RP2D in patients with selected advanced tumors. Citation Format: Bae Jung Choi, Devalingam Devalingam, Angela Alistar, Anthony El-Khoueiry, Alain Mita, Hwankyu Kang, Jinho Choi, Hyunji Ahn, Jeongjun Kim, Seung-Joo Lee, Yeong-In Yang, Jiye Ahn, Borami Jeon, Jaeseung Kim, Kiyean Nam. Patient pharmacodynamic biomarker and pk evaluation results from an ongoing phase I dose-escalation study of q702, an axl, mer and csf1r kinase inhibitor in patients with advanced solid tumors [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 2255.

Abstract 518: AB801 is a highly potent and selective AXL kinase inhibitor that demonstrates significant anti-tumor activity

Abstract Background: AXL receptor tyrosine kinase (AXL) is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer, stromal, and select immune cells, and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL can be mediated by its ligand, growth arrest specific protein 6 (GAS6), or via ligand-independent homo/hetero-dimerization, both of which facilitate AXL phosphorylation, initiation of signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the discovery and characterization of a novel, highly potent and selective AXL inhibitor, AB801. Materials and Methods: The potency and selectivity of AB801 against AXL and other kinases were determined using a panel of HTRF KinEASE-TK assays and via a competition binding assay utilizing DNA-tagged kinases. The effects of AB801 were further assessed by a cell-based phospho-AXL ELISA. The pharmacokinetic (PK) profile of the molecule was evaluated in preclinical species. AB801 was characterized in routine in vitro safety assays, including hERG inhibition. Downstream signaling of AXL was evaluated by phospho-array, Western blot, and qPCR. Pharmacodynamics (PD) and anti-tumor efficacy in combination with standard of care (SOC) therapies were assessed in murine cancer models. Results: The novel AXL inhibitor AB801 is potent, reversible, and selective. AB801 exhibits cellular activity at low nanomolar concentrations and retains significant activity in 100% human serum. Excellent selectivity was observed against MERTK (860x), TYRO3 (1,400x), and the overall kinome. Importantly, AB801 does not show significant CYP450 or hERG inhibition. Favorable preclinical PK is consistent with projected once-a-day oral administration in humans. AB801 increases sensitivity to SOC therapeutics such as chemotherapy, and results in increased DNA damage. Moreover, AB801 treatment sensitizes tumors to checkpoint blockade by increasing immune cell activation. Significant anti-tumor efficacy is observed in combination SOC therapies in multiple in vivo models. Conclusions: AXL inhibition is a promising therapeutic mechanism for impairing the growth of tumors resistant to SOC therapeutics. AB801 exhibits improved potency, selectivity, and safety profiles compared to other AXL inhibitors currently advancing into clinical development. Citation Format: Susan Lee Paprcka, Armon Goshayeshi, Suan Liu, Ruben Flores, Lauren Rocha, Jhansi L. Leslie, Dillon H. Miles, Corinne N. Foley, Shiwei Qu, Manjunath Lamani, Srinivas Paladugu, Hsin-Ting Huang, Nidhi Tribewal, Ada Chen, Joseph Kulusich, Stefan Garrido-Shaqfeh, Patricia Fabila, Salema Jafri, Anuja Devarajan, Ester Fernandez-Salas. AB801 is a highly potent and selective AXL kinase inhibitor that demonstrates significant anti-tumor activity [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 518.

Evaluation of combination treatment with DS-1205c, an AXL kinase inhibitor, and osimertinib in metastatic or unresectable EGFR-mutant non-small cell lung cancer: results from a multicenter, open-label phase 1 study

SummaryThe objective of this study was to evaluate the safety and tolerability of DS-1205c, an oral AXL-receptor inhibitor, in combination with osimertinib in metastatic or unresectable EFGR-mutant non-small cell lung cancer (NSCLC) patients who developed disease progression during EGFR tyrosine kinase inhibitor (TKI) treatment. An open-label, non-randomized phase 1 study was conducted in Taiwan, in which 13 patients received DS-1205c monotherapy at a dosage of 200, 400, 800, or 1200 mg twice daily for 7 days, followed by combination treatment with DS-1205c (same doses) plus osimertinib 80 mg once daily in 21-day cycles. Treatment continued until disease progression or other discontinuation criteria were met. At least one treatment-emergent adverse event (TEAE) was reported in all 13 patients treated with DS-1205c plus osimertinib; with ≥ 1 grade 3 TEAE in 6 patients (one of whom also had a grade 4 increased lipase level), and 6 patients having ≥ 1 serious TEAE. Eight patients experienced ≥ 1 treatment-related AE (TRAE). The most common (2 cases each) were anemia, diarrhea, fatigue, increased AST, increased ALT, increased blood creatinine phosphokinase, and increased lipase. All TRAEs were non-serious, with the exception of an overdose of osimertinib in 1 patient. No deaths were reported. Two-thirds of patients achieved stable disease (one-third for > 100 days), but none achieved a complete or partial response. No association between AXL positivity in tumor tissue and clinical efficacy was observed. DS-1205c was well-tolerated with no new safety signals in patients with advanced EGFR-mutant NSCLC when administered in combination with the EFGR TKI osimertinib. ClinicalTrials.gov; NCT03255083.

Repurposing Axl Kinase Inhibitors for the Treatment of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) infection persists as a common pathogen of pulmonary infection in infants and in the elderly with high morbidity and mortality. However, no specific therapeutics are available. Axl, a member of the TAM (Tyro3, Axl, and Mertk) family receptor kinases, is a pleiotropic inhibitor of the innate immune response and functions as a negative regulator of interferon pathway activation. In this report, we investigated Axl inhibitors for their effects against RSV infection. Axl inhibition with kinase inhibitors, including BMS-777607, R428, and TP-0903, or Axl ablation resulted in a significant reduction of RSV infection in cell-based assays. In an animal model of pulmonary RSV infection, treatment with BMS-777607, R428, or TP-0903 ameliorated pulmonary pathology with a significant reduction of RSV titers in the lung tissues and, consequently, decreased the expression of proinflammatory genes. The host promotes ISG expression for the antiviral response and for viral clearance. We found that Axl inhibition led to more robust IFN-β expression and antiviral gene induction. Thus, the results of this study imply that Axl kinase inhibitors may possess a broad spectrum of antiviral effects by promoting ISG expression.

Discovery of 3-Aminopyrazole Derivatives as New Potent and Orally Bioavailable AXL Inhibitors.

The receptor tyrosine kinase AXL is a promising target for anticancer drug discovery. Herein, we describe the discovery of 3-aminopyrazole derivatives as new potent and selective AXL kinase inhibitors. One of the representative compounds, 6li, potently inhibited AXL enzymatic activity with an IC50 value of 1.6 nM, and tightly bound with AXL protein with a Kd value of 0.26 nM, while was obviously less potent against most of the 403 wild-type kinases evaluated. Cell-based assays demonstrated that compound 6li potently inhibited AXL signaling, suppressed Ba/F3-TEL-AXL cell proliferation, reversed TGF-β1-induced epithelial-mesenchymal transition, and dose-dependently impeded cancer cell migration and invasion. Compound 6li also showed reasonable pharmacokinetic properties in rats and exhibited significant in vivo antitumor efficacy in a xenograft model of highly metastatic murine breast cancer 4T1 cells. Taken together, this study provides a new potent and selective AXL inhibitor for further anticancer drug discovery.

Identification of Novel AXL Kinase Inhibitors Using Ligand-Based Pharmacophore Screening and Molecular Dynamics Simulations

AXL kinase is a promising target in novel drug discovery for cancer. A ligand-based pharmacophore model was generated with the Pharmit web server. Its inbuilt PubChem molecule database was screened and led to 408 candidate molecules. Docking of the AXL kinase active sites with the identified list of candidate molecules was carried out with Autodock Vina docking software. This resulted in four compounds selected for further investigation. Molecular dynamics simulation of two ligands (PubChem-122421875 and PubChem-78160848) showed considerable binding with AXL kinase. From the MM-PBSA binding free energies investigation, the PubChem-122421875 (G = −179.3 kJ/mol) and PubChem-78160848 (G = −208.3 kJ/mol) ligands had favorable protein-ligand complex stability and binding free energy. Hence, PubChem-122421875 and PubChem-78160848 molecules identified in this work could be a potent starting point for developing novel AXL kinase inhibitor molecules.

MO434: Bemcentinib Targets Macrophage and Mesangial Cells in Renal Fibrosis

Abstract 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.

Integrated Proteomics-Based Physical and Functional Mapping of AXL Kinase Signaling Pathways and Inhibitors Define Its Role in Cell Migration.

Our study defines mechanisms of action of AXL in lung cancers which can be used to establish assays to measure drug targetable active AXL complexes in patient tissues and inform the strategy for targeting it's signaling as an anticancer therapy.

SKI-G-801, an AXL kinase inhibitor, blocks metastasis through inducing anti-tumor immune responses and potentiates anti-PD-1 therapy in mouse cancer models.

ObjectivesAXL‐mediated activation of aberrant tyrosine kinase drives various oncogenic processes and facilitates an immunosuppressive microenvironment. We evaluated the anti‐tumor and anti‐metastatic activities of SKI‐G‐801, a small‐molecule inhibitor of AXL, alone and in combination with anti‐PD‐1 therapy.Methods In vitro pAXL inhibition by SKI‐G‐801 was performed in both human and mouse cancer cell lines. Immunocompetent mouse models of tumor were established to measure anti‐metastatic potential of SKI‐G‐801. Furthermore, SKI‐G‐801, anti‐PD‐1 or their combination was administered as an adjuvant or neoadjuvant in the 4T1 tumor model to assess their potential for clinical application.ResultsSKI‐G‐801 robustly inhibited pAXL expression in various cell lines. SKI‐G‐801 alone or in combination with anti‐PD‐1 potently inhibited metastasis in B16F10 melanoma, CT26 colon and 4T1 breast models. SKI‐G‐801 inhibited the growth of B16F10 and 4T1 tumor‐bearing mice but not immune‐deficient mice. An antibody depletion assay revealed that CD8+ T cells significantly contributed to SKI‐G‐801‐mediated survival. Anti‐PD‐1 and combination group were observed the increased CD8+Ki67+ and effector T cells and M1 macrophage and decreased M2 macrophage, and granulocytic myeloid‐derived suppressor cell (G‐MDSC) compared to the control group. The neoadjuvant combination of SKI‐G‐801 and anti‐PD‐1 therapy achieved superior survival benefits by inducing more profound T‐cell responses in the 4T1 syngeneic mouse model.ConclusionSKI‐G‐801 significantly suppressed tumor metastasis and growth by enhancing anti‐tumor immune responses. Our results suggest that SKI‐G‐801 has the potential to overcome anti‐PD‐1 therapy resistance and allow more patients to benefit from anti‐PD‐1 therapy.

Abstract P253: Potent and selective AXL tyrosine kinase inhibition demonstrates significant anti-tumor efficacy in combination with standard of care therapeutics in preclinical models

Abstract Background. AXL receptor tyrosine kinase (AXL) is a transmembrane protein that is overexpressed in a variety of tumors and correlates with poor prognosis in cancer patients. AXL is expressed in cancer and stromal cells and has been implicated in the development of resistance to chemotherapy, targeted therapies & immunotherapies. Activation of AXL by its ligand, growth arrest specific protein 6 (Gas6), or ligand-independent dimerization facilitates AXL phosphorylation, initiates signaling cascades that promote cancer cell proliferation, survival, and an immunosuppressive microenvironment. Here we present the discovery and characterization of a novel, highly potent and selective novel AXL inhibitor. Materials and Methods. The potency and specificity of the novel Arcus inhibitor against AXL and other kinases was determined using a panel of HTRF KinEASE-TK assays. Intracellular target engagement was evaluated by monitoring displacement of a competitive fluorescent tracer using an AXL NanoBRETTM intracellular kinase assay. To further assess the inhibitory effect of the novel Arcus inhibitor on AXL kinase activity, AXL autophosphorylation induced SH2 domain translocation was measured in cells using a PathHunterÒ U2OS AXL Functional assay system. Inhibition of AXL phosphorylation in cancer cells was evaluated by Western blot and levels of soluble and surface AXL were assessed by ELISA and flow cytometry, respectively. Pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were evaluated in murine models. Results. A novel, potent, reversible, and highly selective AXL kinase inhibitor has been generated by Arcus. The novel inhibitor exhibits single-digit nanomolar potency in both biochemical and cell-based assays, retains significant activity in 100% human serum and does not show significant inhibition of the major CYP450 isoforms nor the hERG potassium channel. The novel Arcus molecule inhibits AXL phosphorylation mediated by both ligand-dependent Gas6 stimulation as well as ligand-independent autophosphorylation. AXL phosphorylation and subsequent signaling leads to receptor internalization, thereby decreasing both surface AXL expression and soluble AXL levels. AXL activity is inhibited in a concentration dependent manner significantly increasing both surface AXL expression and soluble AXL levels. More importantly, significant anti-tumor efficacy is observed in combination with targeted therapies in several in vivo models. Furthermore, AXL inhibition significantly reduces tumor growth after relapse to single-agent targeted therapy. Conclusions. A novel selective inhibitor of AXL tyrosine kinase activity has been developed that demonstrates single-digit nanomolar potency and inhibition of both ligand-dependent and ligand-independent AXL phosphorylation. Significant anti-tumor activity is observed in combination with targeted therapy and upon acquired resistance in xenograft models. Selective AXL inhibition is a promising therapeutic strategy to overcome resistance to chemotherapy, targeted therapy, and/or immunotherapy. Citation Format: Susan L. Paprcka, Subhasree Sridhar, Irene M. Luu, Salema Jafri, Dillon H. Miles, Suan Liu, Ruben Flores, Shiwei Qu, Manjunath Lamani, Sriivas Paladugu, Cesar Meleza, James Wu, Hema Singh, Yu Chen, Sean Cho, Akshata Udyavar, Angelo Kaplan, Enzo Stargnaro, Xiaoning Zhao, Lixia Jin, Manmohan R. Leleti, Stephen W. Young, Jay P. Powers, Matthew J. Walters, Ester Fernandez-Salas. Potent and selective AXL tyrosine kinase inhibition demonstrates significant anti-tumor efficacy in combination with standard of care therapeutics in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P253.

In-silico study of seaweed secondary metabolites as AXL kinase inhibitors

AXL kinase is an attractive cancer target for drug design and it is involved in different cancers. A set of molecule databases with 1072 secondary metabolites from seaweeds were screened against the AXL kinase active site and eight molecules were shortlisted for further studies. From the docking analysis of the complexes, four molecules GA011, BE005, BC010, and BC005 are showing prominent binging. From the 100 ns of molecular dynamics simulations and ligand-bound complex MM-PBSA free energy analysis, two molecules BC010 (ΔG = −135.38 kJ/mol) and BE005 (ΔG = −141.72 kJ/mol) are showing molecule stability in the active site also showing very strong binding free energies. It suggests these molecules could be the potent molecules for AXL kinase.

CTNI-07. ABTC-1701: PILOT SURGICAL PK STUDY OF BGB324 (BEMCENTINIB) IN RECURRENT GLIOBLASTOMA PATIENTS – RESULTS FROM INTERIM FUTILITY ANALYSIS

Abstract BACKGROUND Glioblastoma often can relapse as mesenchymal (MES) tumors, indicating a phenotypic shift during clinical progression. Glioma spheres, when they gain MES phenotypes, develop dependence on AXL for their growth both in vitro and in vivo. The first-in-class orally bioavailable AXL kinase inhibitor bemcentinib has IC50 of 14 nM and is > 100-fold selective for AXL over other kinases. Bemcentinib is currently under evaluation as a monotherapy and in combination with other treatments across various PhII trials in several oncology indications. METHODS This is an open-label, multicenter, intratumoral pharmacokinetic study of bemcentinib in patients with recurrent glioblastoma for whom a surgical resection is medically indicated. All subjects must have had histological confirmation of glioblastoma by either biopsy or resection that is progressive or recurrent following radiation therapy ± chemotherapy. Intratumoral drug levels were analyzed by LC/MS. RESULTS A planned analysis after the first 5 patients were enrolled with glioblastoma (4 IDH WT and 1 IDH-mutant), (3m, 2f, median age 57, KPS 80). Bemcentinib concentration in contrast enhancing brain tissue ranged from 3.1 to 43.0 uM with a geometric mean concentration of 11.1 uM (% CV, 132.1). The drug concentration in contrast enhancing tumor tissue exceeded the 1.0 uM threshold in all 5 patients, satisfying the criteria of the protocol to enroll an additional 15 patients into the surgical study. Total drug levels were approximately 2-fold greater in contrast enhancing tissue as compared to tissue from a non-enhancing region of the tumor (geometric mean, 5.8 uM; % CV, 187.7). The mean ratio of the drug concentration in brain tissue to plasma was 25.9 (% CV, 92.7) for contrast enhancing tissue and 13.4 (% CV, 126.8) for non-enhancing tissue. CONCLUSIONS Bemcentinib readily distributes in brain tumor tissue following oral administration. The study continues to complete accrual for the remaining 15 patients.