TAM Kinases Research Articles

Abstract B148: Activity of the TAM kinase-targeting compound, SLC-391, is mediated by the engagement of the immune system in CT-26 syngeneic mouse model

Abstract Overexpression of TAM family receptor tyrosine kinases such as Axl has been reported in numerous types of human cancer, and found to correlate with tumor progression and prognosis, metastasis, and drug resistance. Aside from the role in cell growth and survival, TAM kinases have long been recognized for their immunosuppressive activity as well. Hence, downregulation of their activity is expected to unleash antitumor immunity in the tumor microenvironment. As one of the several TAM-targeting small-molecule inhibitors identified, SLC-391 displays a relatively strong activity against Axl, as evidenced by the reductions of phosphotransferase activity in radiometric biochemical assay and the level of Axl Y779 phosphorylation in the cell-based assay. Contradictorily, proliferation of CT-26 murine colon carcinoma cells seemed to be unaffected by the compound in the thymidine incorporation assay with an IC50 of ~10 μM. Interestingly, SLC-391 inhibited CT-26 tumor growth by 37% in a 15-day efficacy study in which the compound was administered at 50 mg/kg p.o.. In comparison, a PD-1 antibody delayed tumor growth by 27%. Tumor-infiltrating lymphocyte phenotyping revealed increases in the number of NK cells and the ratio of M1/M2-polarized macrophages in SLC-391 treatment group, followed by the rise of CD8+ T/Treg ratio and reduction in immunosuppressive myeloid cells. This is indicative of sequential engagement and stimulation of proinflammatory innate immune response and adaptive immune response. In addition, a synergistic antitumor effect was observed when the anti-PD-1 insensitive CT-26 syngeneic model was treated with a combination of SLC-391 and an anti-PD-1 antibody and the overall survival rate of the combination group was prolonged dramatically in comparison with the vehicle control group. To summarize, the antitumor activity of SLC-391 is at least in part mediated by reversing the immunosuppressive tumor microenvironment in CT-26 colon carcinoma model. Citation Format: Shenshen Lai, Rick Li, Paromita Raha, Yuxiang Hu, Jun Yan, Hong Zhang, Anthony Marotta, Zaihui Zhang. Activity of the TAM kinase-targeting compound, SLC-391, is mediated by the engagement of the immune system in CT-26 syngeneic mouse model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B148.

Abstract 4196: Anti-tumor activity of a TAM kinase-targeting compound in CT-26 syngeneic mouse model

Abstract Aberrant expression of TAM family receptor tyrosine kinases, comprising Tyro3, Axl and Mer, is found in virtually every type of human cancer. The expression levels correlate positively with disease staging and prognostic outcome, attributing to metastasis and drug resistance. The TAM kinases have recently emerged as a dual oncological therapeutic target, owing to their immunosuppressive activity. Apart from targeting tumor-survival/growth pathways their inhibition also unleash the anti-tumor immunity in the tumor microenvironment. In our efforts to develop small-molecule inhibitors targeting the TAM kinases, we identified SLC-391 as one of the most promising preclinical candidates with potent activity towards both Axl and Mer, which play roles in maintaining tumor survival and immunosuppressive tumor microenvironment, respectively. Pharmacodynamic studies of SLC-391 in CT26 syngeneic mouse model revealed increase in the number of NK cells and the ratio of M1/M2-polarized macrophages in the treatment group relative to the vehicle control, followed by the rise of CD8+ T/Treg ratio and reduction in immunosuppressive myeloid cells. This is indicative of sequential engagement and stimulation of pro-inflammatory innate immune response and adaptive immune response. In addition, a synergistic anti-tumor effect was observed when the anti-PD-1 insensitive CT-26 model was treated with a combination of SLC-391 and an anti-PD-1 antibody, suggesting that blocking the Axl/Mer-mediated immunosuppressive pathway may significantly enhance the therapeutic efficacy of immune checkpoint inhibitors. In summary, the anti-tumor activity of SLC-391 is mediated by directly inhibiting tumor cell growth as well as reversing the immunosuppressive tumor microenvironment. Citation Format: Shenshen Lai, Hong Zhang, Jun Yan, Zaihui Zhang. Anti-tumor activity of a TAM kinase-targeting compound in CT-26 syngeneic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4196. doi:10.1158/1538-7445.AM2017-4196

Enhancing targeted therapy of tumors with activating FGFR alterations.

e23134 Background: Emerging evidence points to the Fibroblast Growth Factor Receptor (FGFR) kinase family as a promising target in multiple cancer types including lung squamous cell carcinoma, cholangiocarcinoma, gastric, bladder and breast cancer. Although several FGFR kinase inhibitors have entered clinical trials, single agent clinical efficacy has been modest and resistance invariably occurs. A comprehensive understanding of the molecular basis of resistance to FGFR inhibitors is urgently needed to enable the successful application of anti-FGFR therapy in the clinic. Methods: We utilized a systematic genome-wide open reading frame (ORF) screen to identify genes whose upregulation confers resistance to the FGFR inhibitors BGJ398 and FIIN-3 in FGFR1-amplified H2077 lung cancer cells. In an orthogonal approach, we established resistant clones of FGFR1-amplified and FGFR3-translocated cell lines by chronic exposure to BGJ398, and characterized these by high-throughput gene expression analysis. Results: We identified 34 candidate genes, including expected findings such as a mutant KRAS allele and MET overexpression. Intriguingly, we also identified novel candidate resistance mechanisms involving the genes of the TAM family, which encode the transmembrane receptors Tyro3, Axl and Mertk. These tyrosine kinases may serve as a secondary target to augment FGFR therapy. We validated that the TAM family confers resistance to FGFR inhibitors in different FGFR dependencies including FGFR1-amplified lung cancer, FGFR2-amplified gastric and colon cancer and FGFR 3-translocated bladder cancer, with AXL having the greatest rescue. Notably, gene profiling of resistant clones also implicates the TAM family, with both receptors and their ligands upregulated in the majority cases. Moreover, concurrent TAM blockade augments the response to FGFR inhibition in vitro and provides a promising therapeutic strategy to overcome resistance. Conclusions: TAM kinases are important mediators of resistance to FGFR inhibitors and the dual blockade of FGFR and TAM provides a novel approach to enhance the efficacy of anti-FGFR therapy.

Axl/ Mer Inhibitor ONO-9330547 As a Novel Therapeutic Agent in a Stromal Co-Culture Model of Primary Acute Myeloid Leukaemia (AML)

The TAM kinase family are a group of receptor kinases involved in the regulation of many cellular processes including proliferation, cell survival and cytokine production. Consequently this group is frequently overexpressed or aberrantly activated in a variety of cancers including AML, where overexpression of Axl or its ligand Gas6 confers a poor prognosis particularly amongst FLT3-ITD mutated AMLs. ONO-9330547 is a highly potent Axl/Mer inhibitor which shows improved target selectivity compared to other orally available Axl inhibitors in leukaemic cells and spares normal CD34+ bone marrow cells predicting minimal toxicity.We examined a cohort of 70 primary AMLs Ex vivo for ONO-9330547 drug sensitivity and determined low micromolar EC50s in 60% of patient samples (Average EC50=3.16µM+/-3.49). Phospho-Axl was rapidly reduced in response to ONO-9330547 and downstream pro-survival targets AKT, S6 and ERK1/2 were concurrently suppressed. Dose dependant induction of apoptosis was observed in conjunction with suppression of the Axl ligand, Gas6.Synergistic interaction of ONO-9330547 with the chemotherapeutic drug Cytarabine was observed (Mean combination index =0.59), suggesting combination therapy to be a useful strategy with the development of Axl inhibitors.There was no association of ONO-9330547 sensitivity with any clinical characteristics within the patient cohort, however the samples from the commonly-occurring NPM1 mutant/ cytogenetically normal AML subgroup were significantly more sensitive to ONO-9330547 than WT or FLT3-ITD samples (p=0.004 lower EC50).mRNA levels of TAM family members Axl, Mer, Tyro3 and Gas6 were generally low in diagnostic patient material, although those with high levels of ≥1 of the TAM kinases were associated with drug resistance. This was confirmed through flow cytometric analysis of phospho-Axl, where similarly high basal activation of Axl correlated with increased EC50 values.Given the high rate of relapse and drug resistance in AML and the potential for microenvironment mediated protection of AML blasts in the bone marrow niche, we investigated the effects of ONO-9330547 in stromal co-culture models. Stimulation of basal p-Axl and Gas6 was observed through the addition of cytokines and adhesion of AML blasts to the stromal layer. In contrast to low levels of Gas6 at the transcript level, Gas6 was readily detectable in patient plasma samples and blasts co-cultured on primary AML-derived stromal layers compared to normal bone marrow stroma. Co-culture completely abrogated the efficacy of ONO-9330547 on AML blasts, significantly blocked apoptotic response and Axl/Gas 6 knockdown. In summary, constitutive or stroma- inducible levels of Gas6 ligand direct ONO-9330547 sensitivity in diagnostic AML patient samples. These data provide a pre-clinical assessment of ONO-9330547 in AML and provide rationale for further investigation of this compound in combination with both traditional and novel therapies that may disrupt microenvironment-mediated up-regulation of the Axl/Gas6 signalling pathway. DisclosuresGilmour:ONO pharmaceuticals: Research Funding. Ottmann:Fusion Pharma: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Hills:TEVA: Honoraria. Knapper:ONO pharmaceuticals: Research Funding; Novartis: Honoraria, Other: Travel and expenses for international conferences. Zabkiewicz:ONO pharmaceuticals: Research Funding.

MERTK Inhibition Induces Polyploidy and Promotes Cell Death and Cellular Senescence in Glioblastoma Multiforme.

BackgroundMER receptor tyrosine kinase (MERTK) is expressed in a variety of malignancies, including glioblastoma multiforme (GBM). Our previous work demonstrated that inhibition of MERTK using RNA interference induced cell death and chemosensitivity in GBM cells, implicating MERTK as a potential therapeutic target. Here we investigate whether a novel MERTK-selective small molecule tyrosine kinase inhibitor, UNC2025, has similar anti-tumor effects in GBM cell lines.MethodsCorrelations between expression of GAS6, a MERTK ligand, and prognosis were determined using data from the TCGA database. GBM cell lines (A172, SF188, U251) were treated in vitro with increasing doses of UNC2025 (50-400nM). Cell count and viability were determined by trypan blue exclusion. Cell cycle profiles and induction of apoptosis were assessed by flow cytometric analysis after BrdU or Po-Pro-1/propidium iodide staining, respectively. Polyploidy was detected by propidium iodide staining and metaphase spread. Cellular senescence was determined by β-galactosidase staining and senescence-associated secretory cytokine analysis.ResultsDecreased overall survival significantly correlated with high levels of GAS6 expression in GBM, highlighting the importance of TAM kinase signaling in GBM tumorigenesis and/or therapy resistance and providing strong rationale for targeting these pathways in the clinic. All three GBM cell lines exhibited dose dependent reductions in cell number and colony formation (>90% at 200nM) after treatment with UNC2025. Cell cycle analysis demonstrated accumulation of cells in the G2/M phase and development of polyploidy. After extended exposure, 60–80% of cells underwent apoptosis. The majority of surviving cells (65–95%) were senescent and did not recover after drug removal. Thus, UNC2025 mediates anti-tumor activity in GBM by multiple mechanisms.ConclusionsThe findings described here provide further evidence of oncogenic roles for MERTK in GBM, demonstrate the importance of kinase activity for MERTK tumorigenicity and validate UNC2025, a novel MERTK inhibitor, as a potential therapeutic agent for treatment of GBM.

ChemInform Abstract: State‐of‐the‐Art of Small Molecule Inhibitors of the TAM Family: The Point of View of the Chemist

AbstractReview: 128 refs.

State-of-the-art of small molecule inhibitors of the TAM family: The point of view of the chemist

The TAM family of tyrosine kinases receptors (Tyro3, Axl and Mer) is implicated in cancer development, autoimmune reactions and viral infection and is therefore emerging as an effective and attractive therapeutic target. To date, only a few small molecules have been intentionally designed to block the TAM kinases, while most of the inhibitors were developed for blocking different protein kinases and then identified through selectivity profile studies. This minireview will examine in terms of chemical structure the different compounds able to act on either one, two or three TAM kinases with details about structure–activity relationships, drug-metabolism and pharmacokinetics properties where they exist.

Abstract 5393: Evaluation of compounds developed against the TAM family kinases

Abstract The TAM family receptor tyrosine kinases, consisting of Tyro3, Axl and Mer, are implicated in a diverse range of cellular processes such as cell survival, proliferation, migration, angiogenesis, and inflammatory response. Despite the debates over their role as a driver in carcinogenesis owing to some recent findings, their link to human cancer is strongly supported by direct correlation of the level of expression/activity with tumor grade and prognosis. In particular, Axl was found to underlie metastasis as an essential regulator of the epithelial-mesenchymal transition (EMT) process and also to confer acquired resistance to the TKI-mediated targeted therapies by up-regulation of associated kinases. Currently, none of FDA-approved kinase drugs were developed for the TAM family kinases. In our effort to develop such small-molecule inhibitors targeting the TAM kinases for oncology indications, we uncovered a series of novel potent compounds with low nM potency against Axl and Tyro3. Effect of these compounds in human cancer cell lines of different tissue origins was mediated mainly through the Axl-dependent signaling pathways. Detailed analysis of signaling proteins downstream of Axl revealed different mechanisms of action for these compounds. In addition to suppression of cell proliferation, these compounds inhibited anchorage-independent colony formation, cell migration and invasion. Four compounds were selected to be evaluated in pharmacokinetic studies in mice and the results indicated that these compounds possess reasonable PK properties with high exposure, reasonably long half-life, and good bioavailability. The results of the efficacy studies in suppressing tumor growth in a xenograft and an orthotropic mouse models will be discussed. Citation Format: Hong Zhang, Zaihui Zhang, Xiaoqing Shi, Erica Lee, Rick Li, Yuxiang Hu, Jun Yan, Jasbinder Sanghera. Evaluation of compounds developed against the TAM family kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5393. doi:10.1158/1538-7445.AM2015-5393

The TAM family: phosphatidylserine sensing receptor tyrosine kinases gone awry in cancer.

The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential.

Structural elucidation of the DFG-Asp in and DFG-Asp out states of TAM kinases and insight into the selectivity of their inhibitors.

Structural elucidation of the active (DFG-Asp in) and inactive (DFG-Asp out) states of the TAM family of receptor tyrosine kinases is required for future development of TAM inhibitors as drugs. Herein we report a computational study on each of the three TAM members Tyro-3, Axl and Mer. DFG-Asp in and DFG-Asp out homology models of each one were built based on the X-ray structure of c-Met kinase, an enzyme with a closely related sequence. Structural validation and in silico screening enabled identification of critical amino acids for ligand binding within the active site of each DFG-Asp in and DFG-Asp out model. The position and nature of amino acids that differ among Tyro-3, Axl and Mer, and the potential role of these residues in the design of selective TAM ligands, are discussed.

Abstract 1748: Novel Ig1 inhibitors of the Axl tyrosine kinase that block Gas6-inducible receptor activation

Abstract Tyro-3, Axl, and Mer (abbreviated TAMs) are a family of three homologous receptor tyrosine kinases (RTKs) expressed predominantly on myeloid-derived hematopoietic cells and epithelial cells that function as inhibitory receptors that dampen inflammatory responses and maintain tissue tolerance. Both TAMs and their ligands (vitamin K activated proteins that consist of Gas6 and Protein S) are overexpressed in many human malignancies and expression is associated with more aggressive cancer staging, drug resistance, and poorer patient survival. Based on their clinical importance, there has been considerable effort to generate anti-TAM cancer therapeutics in the form of small molecule tyrosine kinase inhibitors, monoclonal antibodies, and soluble receptor traps that capture TAM ligands and prevent receptor activation. However, whereas several tyrosine kinase inhibitors have been reported, the high degree of sequence conservation in the TAM kinase domain is associated with lack of specificity and off target effects. To generate more selective inhibitors of TAMs, we have targeted the Ig1 domain of Axl, a ligand-binding region in the extracellular domain that gives TAMs their unique structural features. From the X-ray crystallographic structure of Gas6 in complex with the human Axl ectodomain (PDB 2c5d), two unique sites were targeted that include (i) the major binding pocket around Glu59 in Axl that interacts with Arg310 and Lys312 in Gas6, and (ii) the main hydrophobic interface between the Ig1 and the Lg1 that is required for Gas6-inducible dimerization. Virtual screening of the ZINC molecular data base was performed, and several drug-like compounds were selected based on ligand-Gas6 docking. We report on the characterization of two novel types of Axl inhibitors that include a triazolo pyridazine compound that targets the Ig1 binding pocket, and a 3-cyanocoumarin compound that targets the Ig1-Lg1 interface. Both compounds show activity on human Axl reporter lines, and therefore may represent a new class of Axl inhibitors with potential applications for anticancer therapy. Citation Format: William J. Welsh, Vladyslav Kholodovych, Raymond Birge, Tom Comollo, Stanley Kimani, Kamal Singh. Novel Ig1 inhibitors of the Axl tyrosine kinase that block Gas6-inducible receptor activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2014-1748

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.