Small Molecule Inhibitor Of AXL Research Articles

LBA52 A randomized phase Ib/II study of the selective small molecule AXL inhibitor bemcentinib in combination with either dabrafenib/trametinib or pembrolizumab in patients with metastatic melanoma

LBA52 A randomized phase Ib/II study of the selective small molecule AXL inhibitor bemcentinib in combination with either dabrafenib/trametinib or pembrolizumab in patients with metastatic melanoma

Phase 1 trial of bemcentinib (BGB324), a first-in-class, selective AXL inhibitor, with docetaxel in patients with previously treated advanced non-small cell lung cancer

ObjectivesAXL, a transmembrane receptor tyrosine kinase, is highly expressed and associated with poor prognosis in non-small cell lung cancer (NSCLC). Bemcentinib (BGB324), a selective orally bioavailable small molecule AXL inhibitor, synergizes with docetaxel in preclinical models. We performed a phase I trial of bemcentinib plus docetaxel in previously treated advanced NSCLC. Materials and MethodsEscalation of two dose levels of bemcentinib (200 mg load × 3 days then 100 mg daily, or 400 mg load × 3 days then 200 mg daily) in combination with docetaxel (60 or 75 mg/m2 every 3 weeks) followed a 3+3 study design. Due to hematologic toxicity, prophylactic G-CSF was added. Bemcentinib monotherapy was administered for one week prior to docetaxel initiation to assess pharmacodynamic and pharmacokinetic effects alone and in combination. Plasma protein biomarker levels were measured. Results21 patients were enrolled (median age 62 years, 67% male). Median treatment duration was 2.8 months (range 0.7–10.9 months). The main treatment-related adverse events were neutropenia (86%, 76% ≥G3), diarrhea (57%, 0% ≥G3), fatigue (57%, 5% ≥G3), and nausea (52%, 0% ≥G3). Neutropenic fever occurred in 8 (38%) patients. The maximum tolerated dose was docetaxel 60 mg/m2 with prophylactic G-CSF support plus bemcentinib 400 mg load × 3 days followed by 200 mg daily thereafter. Bemcentinib and docetaxel pharmacokinetics resembled prior monotherapy data. Among 17 patients evaluable for radiographic response, 6 (35%) patients had partial response and 8 (47%) patients had stable disease as best response. Bemcentinib administration was associated with modulation of proteins involved in protein kinase B signaling, reactive oxygen species metabolism, and other processes. ConclusionBemcentinib plus docetaxel with G-CSF support demonstrates anti-tumor activity in previously treated, advanced NSCLC. The role of AXL inhibition in the treatment of NSCLC remains under investigation.

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.

Dual Axl/MerTK inhibitor INCB081776 creates a proinflammatory tumor immune microenvironment and enhances anti-PDL1 efficacy in head and neck cancer.

The tyrosine kinase receptors Axl and MerTK are highly overexpressed in head and neck cancer (HNC) cells, where they are critical drivers of survival, proliferation, metastasis, and therapeutic resistance. We investigated the role of Axl and MerTK in creating an immunologically "cold" tumor immune microenvironment (TIME) by targeting both receptors simultaneously with a small molecule inhibitor of Axl and MerTK (INCB081776). Effects of INCB081776 and/or anti-PDL1 on mouse oral cancer (MOC) cell growth and on the TIME were evaluated. Targeting Axl and MerTK can reduce M2 and induce M1 macrophage polarization. In vivo, INCB081776 treatment alone or with anti-PDL1 appears to slow MOC tumor growth, increase proinflammatory immune infiltration, and decrease anti-inflammatory immune infiltration. This data indicates that simultaneous targeting of Axl and MerTK with INCB081776, either alone or in combination with anti-PDL1, slows tumor growth and creates a proinflammatory TIME in mouse models of HNC.

Trials in progress: TAM kinase inhibitor PF-07265807 and sasanlimab plus axitinib in patients with advanced or metastatic renal cell carcinoma—A phase 1, open-label, pharmacokinetic, safety and tolerability study.

TPS743 Background: Tumor-associated macrophage kinases (TAMK) are expressed on tumor cells during malignant transformation. Inhibition of AXL and MERTK, members of the TAMK family, may lower the immune activation threshold and promote antitumor immunity. PF-07265807 (ARRY-067) is a small-molecule inhibitor of MERTK and AXL showing antitumor activity in preclinical models as monotherapy or combined with anti-programmed cell death protein 1 (anti-PD-1) antibodies (ab). Sasanlimab is an anti-PD-1 ab with acceptable safety profile being tested (300 mg SC Q4W, Phase 3) in non-muscle invasive bladder cancer. Axitinib is a VEGF inhibitor approved (single agent and combined with anti-PD-1 inhibitors) for advanced renal cell carcinoma (RCC). This first-in-human Phase 1, open-label, multi-center study (NCT04458259) will evaluate the safety, tolerability, pharmacokinetics, and preliminary antitumor activity of PF-07265807 in patients (pts) with advanced or metastatic solid tumors as a single agent and in combination with sasanlimab with or without axitinib. We describe here the dose escalation (Part 3) and dose expansion (Part 4) for the triplet combination of PF-07265807 + sasanlimab + axitinib. Methods: Eligible participants for the triplet cohorts are adult pts with confirmed unresectable advanced or metastatic clear cell RCC, with estimated creatinine clearance ≥30 mL/min and urinary protein <2+ or ≥2+ but 24-h urine protein:creatinine ratio <2 g/24 h. Pts for Part 4 need to have intermediate and poor risk RCC and no prior systemic therapy for metastatic disease. Other key eligibility criteria: measurable disease by RECIST 1.1 or non-measurable disease, ECOG PS 0–2, adequate bone marrow and liver function, and resolved acute effects of prior therapy. Each cycle is 21 days. Part 3 will determine the maximum tolerated dose/recommended phase 2 dose (MTD/RP2D) of PF-07265807 in triplet therapy using a Bayesian logistic regression model and the escalation with overdose control principle. After the MTD/RP2D is identified, Part 4 will further evaluate the safety and preliminary efficacy of PF-07265807 combined with sasanlimab and axitinib. Treatment with study drug will continue until disease progression, consent withdrawal, or unacceptable toxicity, whichever occurs first. Primary endpoints of Part 3 are incidence of dose-limiting toxicities, adverse events (AE), and laboratory (lab) abnormalities. For Part 4, the primary endpoints are objective response rate and complete response rate; secondary endpoints include disease control rate; time-to-event endpoints; AEs; lab abnormalities; concentrations of PF-07265807 and its metabolite, sasanlimab, and axitinib; and incidence and titer of anti-sasanlimab anti-drug antibodies response. The study started in late 2020 and is recruiting. Clinical trial information: NCT04458259 .

Abstract 3535: Simultaneous inhibition of Axl and MerTK enhances anti-PDL1 efficacy and creates a pro-inflammatory tumor immune microenvironment in head and neck cancer

Abstract The tyrosine kinase receptors Axl and MerTK, known for their role on macrophages in regulating clearance of apoptotic cells, are highly overexpressed in head and neck cancer (HNC). Previous studies in our laboratory have shown that Axl is a critical driver of survival, proliferation, metastasis, and therapeutic resistance in HNC, and that MerTK is functionally redundant to Axl. In this study, we investigated the cooperative role of Axl and MerTK in creating an immunologically cold tumor immune microenvironment (TIME) by targeting both receptors simultaneously with a small molecule inhibitor of Axl and MerTK (INCB081776). Because Axl and MerTK are expressed on both macrophages and HNC cancer cells, we examined the effect of INCB081776 treatment on each cell type. In macrophages, Axl and MerTK signaling leads to M2-type polarization, an anti-inflammatory state that leads to the resolution of inflammation and, in cancer settings, promotes tumor growth. Our experiments suggest that treatment with INCB081776 can reduce M2 polarization and increase M1-type polarization, a pro-inflammatory state that promotes inflammation and tumor cell killing. Next, to determine the efficacy of INCB081776 on HNC cancer cells, mouse oral cancer (MOC) tumors were implanted in syngeneic mice and treated with INCB081776 alone or in combination with a monoclonal antibody against PDL1 (anti-PDL1), thereby mimicking current standard-of-care immune checkpoint inhibitor treatment. The results showed a marked effect of INCB081776 single-agent treatment on MOC tumor growth and an increase in several pro-inflammatory cell types (M1 macrophages, CD8+ T cells, total infiltrating leukocytes), suggesting that INCB081776 treatment can create an immunologically hot TIME. Further, in-depth analysis of tumor infiltrating leukocytes following INCB081776 treatment in both immunologically hot (MOC1) and cold (MOC2) HNC tumors suggested that INCB081776 has a greater effect in cold tumors. In cold tumors, levels of pro-inflammatory cells (CD8+ T cells, M1 macrophages, etc.) increased, and levels of anti-inflammatory cells (M2 macrophages, regulatory T cells, etc.) decreased, both to a greater extent than in hot tumors. Finally, the combination of INCB081776 and anti-PDL1 was superior to either treatment alone in slowing tumor growth. Together, these studies indicate that INCB081776 cooperates with anti-PDL1 in a syngeneic mouse model of HNC to slow tumor growth and create a pro-inflammatory environment, especially in immunologically cold tumors, thereby highlighting the potential clinical benefit of this therapeutic combination. Citation Format: Kourtney L. Kostecki, Mari Iida, Anne L. Wiley, Seungpyo Hong, Ravi Salgia, Paul M. Harari, Deric L. Wheeler. Simultaneous inhibition of Axl and MerTK enhances anti-PDL1 efficacy and creates a pro-inflammatory tumor immune microenvironment in head and neck cancer [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 3535.

Abstract 2602: Combined inhibition of AXL and ATR enhances cell death and immune response in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is an aggressive lung tumor of neuroendocrine origin with a dismal 5-year survival rate. Despite high response rates to initial therapy, rapid development of therapeutic resistance limits overall survival. There are also limited treatment options, particularly in the relapsed setting. Our group and others have shown that AXL, a TAM family receptor tyrosine kinase that is highly expressed in mesenchymal tumors, mediates resistance to chemotherapy, radiation and targeted therapies in SCLC, non-small cell lung cancer and other cancers, through its roles in driving epithelial to mesenchymal transition and DNA damage repair. AXL has also been implicated in immune escape. Based on these findings, we hypothesize that AXL targeting may be a potential therapeutic approach in SCLC. We screened BGB324 (bemcentinib), a selective small-molecule AXL inhibitor in clinical trials for various advanced solid tumors, in a panel of 60 human SCLC cell lines using cell viability assays. The SCLC cell lines showed a range of sensitivities, with IC50 values ranging from 41 nM to 10 µM (median IC50 3.1 µM) with NeuroD1-driven SCLC cell lines being highly sensitive to BGB324 (ANOVA p<0.05). BGB324 also showed potent inhibition of tumor growth in an in vivo SCLC model. BGB324 further combined synergistically with an ATR inhibitor (AZD3738) and induces significant DNA damage. In a syngeneic model of SCLC, the combination of BGB324, AZD6738 and an anti-PDL1 antibody combination also induced significant tumor regression. Together, these promising findings show that AXL inhibition may be effective in SCLC and support further investigation of AXL and ATR inhibitor combinations with immune checkpoint blockade. Citation Format: Kavya Ramkumar, Azusa Tanimoto, C. Allison Stewart, Qi Wang, Li Shen, Robert J. Cardnell, B. Leticia Rodriguez, Don L. Gibbons, Jing Wang, Carl M. Gay, Lauren A. Byers. Combined inhibition of AXL and ATR enhances cell death and immune response in small cell lung cancer [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 2602.

Phase 1 dose escalation and expansion study of bemcentinib (BGB324), a first-in-class, selective AXL inhibitor, with docetaxel in patients with previously treated advanced NSCLC.

9081 Background: AXL, a transmembrane receptor tyrosine kinase, is overexpressed and associated with poor prognosis and treatment resistance in non-small cell lung cancer (NSCLC). Bemcentinib (BGB324) is a selective orally bioavailable small molecule inhibitor of AXL, currently in phase 2 clinical development, that has demonstrated synergistic activity with docetaxel in in vivo models of NSCLC. This phase I dose escalation and expansion trial assessed the safety, tolerability, and preliminary efficacy of bemcentinib in combination with docetaxel in previously treated advanced NSCLC. Methods: Dose escalation of daily bemcentinib in combination with docetaxel (60 or 75 mg/m2 every 3 weeks) followed a standard 3+3 design. Bemcentinib monotherapy was administered for one week prior to docetaxel initiation to assess pharmacodynamic effects alone and in combination. Plasma protein biomarker levels were measured using the DiscoveryMap v3.3 panel (Myriad RBM) pre-dose (C1D-7), C1D1, and C2D1. Results: A total of 21 patients (pts) were enrolled with median age 62 (range 39-84) and 67% male. A median of 3 (range 1-13) cycles of therapy were administered. Principal treatment-related adverse events were neutropenia (86%, 76% ≥G3), diarrhea (57%, 0% ≥G3), fatigue (52%, 5% ≥G3), and nausea (52%, 0% ≥G3). Neutropenic fever ≥G3 occurred in 7 (33%) pts. The maximum tolerated dose was 60 mg/m2 docetaxel plus 200 mg bemcentinib daily with prophylactic G-CSF support. Pharmacokinetics of bemcentinib alone and in combination with docetaxel were highly similar. Among 15 evaluable pts, 4 (27%) pts had partial response and 9 (60%) pts had stable disease as the best radiographic response. Bemcentinib treatment resulted in changes in plasma levels of proteins associated with regulation of sterol synthesis and AKT signaling. Conclusions: Bemcentinib in combination with docetaxel and prophylactic G-CSF support has a manageable safety profile, leads to decreases in systemic factors associated with tumor growth and metastasis, and demonstrates evidence of anti-tumor activity in previously treated, advanced NSCLC. Further studies to identify the patient population most likely to benefit from this regimen are warranted. Clinical trial information: NCT02922777.

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.

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

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

Abstract 3154: Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis

Abstract Prostate and breast cancers are the most prevalent and the second leading causes of cancer-deaths for men and women in the United States. Cancer survival rates generally worsen significantly with advanced and disseminated disease with nearly 90% of all cancer deaths resulting from metastasis. For breast and prostate cancers, bone is the most common site of metastasis. A key signaling pathway involved in metastatic dissemination of tumor cells is the receptor tyrosine kinase Axl. Axl belongs to the Tyro3, Axl, and MerTK (TAM) subfamily, is expressed in a variety of tumor types, including breast and prostate, and is associated with poor prognosis, metastasis, and outcome. The goal of the present study was to characterize the role of Axl signaling in prostate and breast cancer cell dissemination to bone. This was accomplished using Axl knockdown neoplastic cells, a selective small molecule Axl inhibitor (BGB324), and osteoclast progenitor cells in vitro, as well as intracardiac injection of Axl knockdown tumor cells in vivo. The results showed that Axl inhibition significantly decreases tumor cell migration and invasion in vitro, and suppression of Axl signaling in osteoclast precursor cells reduces the formation of mature osteoclasts. In vivo, Axl knockdown in prostate and breast cancer cells significantly suppressed the formation of neoplastic bone lesions. Taken together our findings indicate that therapeutic targeting of Axl may impair tumor metastasis to the bones through neoplastic and host cell signaling axes. Citation Format: Mai Tanaka, Dietmar W. Siemann. Host and neoplastic cell Axl inhibition impair prostate and breast cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3154.

Macrophage AXL receptor tyrosine kinase inflames the heart after reperfused myocardial infarction.

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1β, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.

AXL Inhibition Induces DNA Damage and Replication Stress in Non-Small Cell Lung Cancer Cells and Promotes Sensitivity to ATR Inhibitors.

AXL, a TAM (TYRO3, AXL, and MERTK) family receptor tyrosine kinase, is increasingly being recognized as a key determinant of resistance to targeted therapies, as well as chemotherapy and radiation in non-small cell lung cancer (NSCLC) and other cancers. We further show here that high levels of AXL and epithelial-to-mesenchymal transition were frequently expressed in subsets of both treatment-naïve and treatment-relapsed NSCLC. Previously, we and others have demonstrated a role for AXL in mediating DNA damage response (DDR), as well as resistance to inhibition of WEE1, a replication stress response kinase. Here, we show that BGB324 (bemcentinib), a selective small-molecule AXL inhibitor, caused DNA damage and induced replication stress, indicated by ATR/CHK1 phosphorylation, more significantly in TP53-deficient NSCLC cell lines. Similar effects were also observed in large-cell neuroendocrine carcinoma (LCNEC) cell lines. High AXL protein levels were also associated with resistance to ATR inhibition. Combined inhibition of AXL and ATR significantly decreased cell proliferation of NSCLC and LCNEC cell lines. Mechanistically, combined inhibition of AXL and ATR significantly increased RPA32 hyperphosphorylation and DNA double-strand breaks and induced markers of mitotic catastrophe. Notably, NSCLC cell lines with low levels of SLFN11, a known predictive biomarker for platinum and PARP inhibitor sensitivity, were more sensitive to AXL/ATR cotargeting. These findings demonstrate a novel and unexpected role for AXL in replication stress tolerance, with potential therapeutic implications. IMPLICATIONS: These findings demonstrate that the combination of AXL and ATR inhibitors could be a promising therapeutic combination for NSCLC, LCNEC, and other cancers.

Efficacy and Safety of Bemcentinib in Patients with Myelodysplastic Syndromes or Acute Myeloid Leukemia Failing Hypomethylating Agents

Efficacy and Safety of Bemcentinib in Patients with Myelodysplastic Syndromes or Acute Myeloid Leukemia Failing Hypomethylating Agents

The Combination of AXL Inhibitor Bemcentinib and Low Dose Cytarabine Is Well Tolerated and Efficacious in Elderly Relapsed AML Patients: Update from the Ongoing BGBC003 Phase II Trial (NCT02488408)

The Combination of AXL Inhibitor Bemcentinib and Low Dose Cytarabine Is Well Tolerated and Efficacious in Elderly Relapsed AML Patients: Update from the Ongoing BGBC003 Phase II Trial (NCT02488408)

Role of AXL in metastatic melanoma and impact of TP-0903 as a novel therapeutic option for melanoma brain metastasis.

e22021 Background: Melanoma brain metastases (MBM) are common with a median overall survival of 4-5 months. Although immunotherapies have improved clinical outcomes and have doubled overall survival in MBM, there is a high incidence rate of relapse caused by drug resistance. AXL, a receptor tyrosine kinase (RTK), is associated with drug resistance and metastasis in many cancers. The activation of AXL via trans-phosphorylation regulates multiple signaling pathways that induce tumor survival, metastasis, drug resistance, and epithelial-to-mesenchymal transition (EMT). In MBM, AXL is upregulated and associated with disease progression, promoting cell invasion and migration. This suggests that targeting AXL can be a novel strategy to overcome treatment-related resistance in MBM. TP-0903, an investigational small molecule inhibitor of AXL, has shown efficacy in reversing the mesenchymal phenotype and re-sensitizing resistant cancer cells to targeted therapies in heme malignancies, pancreatic, and breast cancer. We aim to investigate the efficacy of TP-0903 in MBM. Methods: The Cancer Genome Atlas (TCGA) data was utilized to investigate the signaling pathways downstream of AXL that are upregulated in advanced melanoma. Nine signaling molecules including AKT1, mTOR, and PAK4 were analyzed to identify any correlation between gene expression levels and overall survival. Four metastatic melanoma cell lines were used to evaluate the effect of TP-0903 on cell viability and active AXL downregulation was assessed in vitro through MTS cell viability assays and Immunoblotting. Wound closure assays were executed to understand the functional consequences of AXL downregulation. Results: In all nine genes, high expression levels confer poor survival probability. Cell viability assays of four malignant melanoma cell lines showed that TP-0903 treatment resulted in IC50 values ranging from 32 – 692 nM. Western blot analysis indicated that TP-0903 reduced the levels of phosphorylated AXL in malignant melanoma cell lines. In addition, increasing TP-0903 concentrations reduced the rate of cell migration in these malignant melanoma cell lines. Conclusions: AXL plays a role in EMT, treatment resistance, and metastasis in MBM, resulting in poor survival. Our findings suggest TP-0903 is effective in reducing cell migration, inhibit metastasis, and can be a potential therapeutic option in MBM.

Metformin suppresses the growth of leukemia cells partly through downregulation of AXL receptor tyrosine kinase

Metformin is an anti-diabetic drug known to have anticancer activity by inhibiting mechanistic target of rapamycin (mTOR); however, other molecular mechanisms may also be involved. In this study, we examined the effects of metformin on the activity of receptor tyrosine kinases of the TAM (TYRO3, AXL, and MERTK) family, which have important roles in leukemia cell growth. The results indicated that metformin suppressed the in vitro growth of four leukemia cell lines, OCI/AML2, OCI/AML3, THP-1, and K562, in a dose-dependent manner, which corresponded to the downregulation of the expression and phosphorylation of AXL and inhibition of its downstream targets such as phosphorylation of STAT3. Furthermore, metformin augmented the suppressive effects of a small-molecule AXL inhibitor TP-0903 on the growth of OCI/AML3 and K562 cells and prevented doxorubicin-induced AXL activation in K562 cells, which induces chemoresistance in leukemia cells, thus potentiating doxorubicin anti-proliferative effects. Given that metformin also downregulated expression of TYRO3 and phosphorylation of MERTK, these findings indicate that anti-leukemic effects exerted by metformin could be partly due to the inhibition of TAM kinases. Thus, metformin has a clinical potential for patients with leukemia cells positive for AXL and the other TAM proteins as well as activated mTOR.

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

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

Kinetic and Mechanistic Characterization of a Potent and Selective Inhibitor for Human AXL Receptor Tyrosine Kinase

The AXL receptor tyrosine kinase (AXL) is a member of the TAM RTK family comprising TYRO3, AXL and MER. AXL signaling is activated by binding of the growth arrest‐specific protein 6 (Gas6) or vitamin K‐dependent protein S (PROS1), which subsequently induces homodimerization and autophosphorylation, thereby initiating a conventional RTK signaling pathway. Maximal stimulation requires an extracellular lipid, phosphatidylserine (PtdSer).Activation of AXL regulates an array of signal transduction pathways, including those mediating cell survival, proliferation and migration, as well as promoting an immunosuppressive phenotype within the tumor microenvironment 1, 2. AXL has been shown to be highly expressed in sarcoma, renal cell carcinoma, pancreatic adenocarcinoma and other types of tumors, correlative with poor prognosis in cancer patients. Furthermore, AXL has been shown to facilitate both intrinsic and acquired resistance to multiple anti‐cancer drugs 3, suggesting therapeutic targeting of AXL may be an effective anti‐cancer strategy.We have developed a potent, reversible and competitive small molecule AXL inhibitor. This inhibitor (Compound 1) inhibits the activity of recombinant human AXL kinase domain with nanomolar potency, as determined by detection of phosphorylated substrate using homogeneous time‐resolved fluorescence (HTRF). Compound 1 is also highly selective against TYRO3, MERTK and MET kinase domains. The compound binds to the intracellular AXL kinase domain with a nanomolar dissociation rate constant measured by monitoring displacement of a competitive fluorescent tracer using bioluminescence resonance energy transfer (BRET). In order to assess the inhibitory effect of the compound on downstream signal transduction, AXL autophosphorylation‐induced SH2 translocation was determined using an AXL PathHunter® assay and phosphorylation of AKT Ser473 induced by AXL activation was also assessed by AlphaLISA.Support or Funding Information