TAM Family Of Receptor Tyrosine Kinases Research Articles

Development Of a Novel Small Molecule Inhibitor Of The Mer Tyrosine Kinase For Treatment Of Acute Lymphoblastic Leukemia

In children, acute lymphoblastic leukemia (ALL) is currently treated with an intense regimen of chemotherapy yielding cure rates near 85%, yet significant hurdles remain, including decreasing toxicity and improving patient outcome in relapsed or refractory disease. Alternative strategies using available drugs are unlikely to provide significant improvements while more targeted therapies have the potential to reduce the risk of severe therapy-associated toxicities including infertility, organ damage, and secondary malignancy. We have identified Mer, a member of the TAM-family of receptor tyrosine kinases, as a potential therapeutic target in pediatric ALL. Mer is ectopically expressed in pediatric B- and T- cell ALL. Using a genetic approach (shRNA-mediated Mer knockdown), we previously demonstrated anti-leukemia effects in B-ALL and T-ALL models. MerTK inhibition decreased pro-survival signaling and lymphoblast cell survival, inhibited growth in colony forming assays, increased resistance to chemotherapy, and increased leukemia-free survival in animal models. Here we report testing of a novel, first-in-class Mer-selective small molecule inhibitor (Mer TKI) that has effects similar to Mer knock-down in B-ALL pre-clinical models. Our current lead candidate for clinical development mediates potent inhibition of Mer with an IC50 of 0.74 nM. It has ≥10-fold selectivity for Mer over other TAM-family members, has limited off-target activity against other tyrosine kinases (with the exception of FLT3), and inhibits phosphorylation/activation of Mer in 697 B-ALL cells with an IC50 of 2.6 nM. Treatment with this Mer TKI also inhibits downstream signaling through the ERK1/2 MAP kinase and Akt pathways. In mouse models, our Mer TKI is 100% orally bioavailable and has a favorable pharmacokinetic profile with a maximum serum concentration of 1.75 µM after administration of a single 3 mg/kg dose and a half-life of 3.8 hours. Pharmacodynamic studies to assess effective target engagement in murine models demonstrate inhibition of Mer phosphorylation in leukemic blasts in the bone marrow up to 12 hours post-treatment with Mer TKI. The compound is well-tolerated up to a dose of 100 mg/kg administered once daily with the major observed side effects being anemia and reduced white blood cell count. In an orthotopic B-ALL murine xenograft model of minimal residual disease, treatment with Mer TKI significantly inhibited leukemogenesis and increased median survival from 25 days after inoculation with tumor cells to 65 days (p < 0.0001). In a similar model of existent disease in which leukemia was established for 11 days prior to initiation of treatment, median survival increased from 25 to 47 days in response to treatment with Mer TKI (p < 0.0001). In both models, tumor burden measured by bioluminescent imaging was significantly decreased in mice treated with Mer TKI relative to mice treated with vehicle, even after the development of advanced disease in the control animals (0.27 +/- 0.04 x107 photons/second in the minimal residual disease model and 0.98 +/- 0.23 x107 photons/second in the established disease model verses 26.75 +/- 4.54 x107 photons/sec in vehicle-treated mice, p < 0.0001). Its very high potency, relative selectivity, oral bioavailability, and favorable toxicity, pharmacokinetic, and pharmacodynamic profiles along with demonstrated therapeutic efficacy in murine ALL models make this novel Mer TKI an excellent candidate for clinical development. Disclosures:Wang:University of North Carolina at Chapel Hill: This author is an inventor on patent application relevant to this work, This author is an inventor on patent application relevant to this work Patents & Royalties. Zhang:University of North Carolina at Chapel Hill: This author is an inventor on a patent application relevant to this work, This author is an inventor on a patent application relevant to this work Patents & Royalties. Frye:University of North Carolina at Chapel Hill: This author is an inventor on a patent application relevant to this work, This author is an inventor on a patent application relevant to this work Patents & Royalties.

THU0178 Circulating free protein S levels may be linked to cardiovascular events and venous thrombosis in SLE

BackgroundSLE patients are at risk for diverse organ systems involvement, which increases the challenges for diagnosis and predictions for the development of specific clinical features. In recent studies we have...

Abstract 5543: Inhibition of the tyrosine kinase receptor Axl blocks cell invasion and promotes apoptosis in pancreatic cancer cells.

Abstract Pancreatic cancer is virtually a uniformly lethal disease and a better understanding of the molecular basis of this malignancy is needed to discover new ways to prevent or treat this deadly disease. A central feature of malignant cells is their ability to disseminate from the primary tumor and establish local and distant metastases. In most cases, cancer patients with localized disease have significantly better prognosis than those with metastatic tumors and the majority of cancer mortality is associated with metastatic disease rather than the primary tumor. The receptor tyrosine kinase Axl is overexpressed in over 50% of pancreatic cancers and expression of Axl in these cancers is highly associated with a poor prognostic outcome for patients. Axl is a TAM family receptor tyrosine kinase involved in multiple aspects of tumorigenesis. Increased expression of Axl is associated with increased oncogenic transformation, cell survival, proliferation, migration, angiogenesis, and cellular adhesion. The known ligand for Axl is the Growth Arrest Specific Gene-6 (Gas6) protein and it's binding to Axl leads to Axl autophosphorylation and activation of downstream signaling pathways including MAPK and PI3K/Akt pathways. We discovered and developed a small molecule Axl kinase inhibitor, HCI-2084, and explored it for the effectiveness of targeting the Axl kinase in cell-based models of pancreatic cancer. HCI-2084 is a 2-((2,5-substitutedpyrimidin-4-yl)amino)-N,N-dimethyl benzene sulfonamide that has low nanomolar (IC50 = 12 nM) activity against the Axl kinase in a biochemical assays. In further biochemical evaluation, HCI-2084 was shown to inhibit the entire TAM family of kinases (IC50 Axl = 12 nM; IC50 Mer = 60 nM; Tyro3 = 71% inhibition at 200 nM). HCI-2084 inhibits a small number of additional kinases when screened in a kinase panel of over 500 kinases and has demonstrated an ADMET profile suggesting it may be a potential clinical candidate. In cell proliferation assays, HCI-2084 significantly inhibited pancreatic cancer cell growth at concentrations as low as 30 nM. In pharmacodynamic assays, HCI-2084 dramatically inhibited Akt signaling (pAKT S473) downstream of GAS6 stimulation in pancreatic cancer cell lines. Consistent with the known function of Axl, HCI-2084 inhibited Gas6-induced migration and invasion of pancreatic cancer cells in vitro and potently induces apoptosis. Mechanistically, HCI-2084 decreases the expression of genes involved in Epithelial-Mesenchymal Transition (EMT) and induces cells to take on more epithelial phenotypes. HCI-2084 also significantly inhibited the growth of pancreatic cancer cell lines grown in xenograft tumor mouse model and taken together, these results suggest Axl is a potential therapeutic target in pancreatic cancer and that HCI-2084 is a potential agent treat this disease. Citation Format: Malia Anderson, Alex Ober, Alexis Mollard, Lee Call, Jared J. Bearss, Hariprasad Vankayalapati, Sunil Sharma, Steven Warner, David J. Bearss. Inhibition of the tyrosine kinase receptor Axl blocks cell invasion and promotes apoptosis in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5543. doi:10.1158/1538-7445.AM2013-5543

Axl/Gas6/NFκB signalling in schwannoma pathological proliferation, adhesion and survival

TAM family receptor tyrosine kinases comprising Tyro3 (Sky), Axl, and Mer are overexpressed in some cancers, correlate with multidrug resistance and contribute to tumourigenesis by regulating invasion, angiogenesis, cell survival and tumour growth. Mutations in the gene coding for a tumour suppressor merlin cause development of multiple tumours of the nervous system such as schwannomas, meningiomas and ependymomas occurring spontaneously or as part of a hereditary disease neurofibromatosis type 2. The benign character of merlin-deficient tumours makes them less responsive to chemotherapy. We previously showed that, amongst other growth factor receptors, TAM family receptors (Tyro3, Axl and Mer) are significantly overexpressed in schwannoma tissues. As Axl is negatively regulated by merlin and positively regulated by E3 ubiquitin ligase CRL4DCAF1, previously shown to be a key regulator in schwannoma growth we hypothesized that Axl is a good target to study in merlin-deficient tumours. Moreover, Axl positively regulates the oncogene Yes-associated protein, which is known to be under merlin regulation in schwannoma and is involved in increased proliferation of merlin-deficient meningioma and mesothelioma. Here, we demonstrated strong overexpression and activation of Axl receptor as well as its ligand Gas6 in human schwannoma primary cells compared to normal Schwann cells. We show that Gas6 is mitogenic and increases schwannoma cell-matrix adhesion and survival acting via Axl in schwannoma cells. Stimulation of the Gas6/Axl signalling pathway recruits Src, focal adhesion kinase (FAK) and NFκB. We showed that NFκB mediates Gas6/Axl-mediated overexpression of survivin, cyclin D1 and FAK, leading to enhanced survival, cell-matrix adhesion and proliferation of schwannoma. We conclude that Axl/FAK/Src/NFκB pathway is relevant in merlin-deficient tumours and is a potential therapeutic target for schwannoma and other merlin-deficient tumours.

Abstract LB-149: The OPCML tumor suppressor binds and negatively regulates the Axl receptor tyrosine kinase, sensitizing cells to foretinib in epithelial ovarian cancer (EOC)

Abstract OPCML (opioid binding protein/cell adhesion like molecule) is a tumour suppressor gene that is silenced almost by somatic methylation in more than 80% of EOC cases, with loss of heterozygosity (LOH) occurring in 49% of cases [1,2]. OPCML binds a specific repertoire of receptor tyrosine kinases (RTKs) consisting of HER2, HER4 EphA2, FGFR1 and FGFR3. OPCML switches RTKs from clathrin to non clathrin dependent endocytosis, resulting in polyubiquitination of the RTK and targeting to the proteasome for destruction with associated signalling and growth inhibition [3]. We show that Axl, a TAM family RTK that is frequently activated in ovarian cancer [4], also interacts with and is downregulated by OPCML. Axl is downregulated following transient and stable OPCML expression in ovarian cancer lines SKOV3 and PEO1, with reciprocally increased Axl expression following stable knockdown of physiological OPCML in normal ovarian surface epithelial line OSE-C2. We performed co-IP and mammalian 2-hybrid (M 2-H) analysis between Axl and OPCML and determined that the Ig and FNIII domains are crucial to this interaction. We previously reported a complete loss of function proline 95 to arginine mutation [2]. Transfection of OPCMLWT protein in OPCML deficient cells promotes intercellular adhesion and adhesion to Collagen I and Fibronectin matrix, in association with Axl downregulation, In contrast OPCMLP95R transfection did not repress Axl and did not promote intercellular or cell to matrix adhesion. M 2-H data demonstrated that OPCMLP95R expression abolishes the interaction seen between OPCMLWT and Axl. We explored the impact of OPCML expression on cell signalling response to Foretinib (GSK1363089), a potent inhibitor of Axl [5]; OPCML led to a 3-fold sensitization to the tyrosine kinase inhibitor. The finding that OPCML, a frequently inactivated ovarian cancer tumor suppressor negatively regulates Axl, a frequently activated RTK in ovarian cancer and sensitises Axl positive OPCML deficient cells to Foretinib defines a promising predictive and therapeutic role for OPCML in ovarian cancer.

Highlights of This Issue

Germline mutations in BRCA1 or BRCA2 tumor suppressor genes are found in approximately 10% of epithelial ovarian cancers (EOC). Patients with BRCA mutations (BRCA +) benefit from platinum-based treatment more than non-carriers due to the increased chemosensitivity that is related to the impaired ability to repair DNA by homologous recombination. Using a retrospective comparison of patients who were BRCA + or deemed nonhereditary, this trial investigated whether BRCA + predicts improved outcome following pegylated liposomal doxorubicin (PLD) for recurrence. In this retrospective analysis, recurrent EOC BRCA mutation carriers treated with PLD had an improved outcome, which appeared to be independent of platinum sensitivity.The mammalian TAM receptor tyrosine kinase family (RTK) includes three closely related members: Tyro-3, Axl, and Mer. Abnormal expression and ligand-induced activation of Axl or Mer can provide a survival advantage in a variety of cancers. Inhibition of Axl and Mer may enhance the sensitivity of cancer cells to cytotoxic agents and could potentially be a therapeutic strategy in targeting cancer cells. Verma and colleagues review the role of Axl and Mer in normal cellular function, their role in oncogenesis, and the potential to inhibit these RTKs as therapeutic strategies in treatment of cancer.MicroRNA (miRNA) roles in tumor biology remain largely unknown. Xu and colleagues introduce a systems biology approach based on the miRNA-target dysregulated network (MTDN) in specific tumor phenotypes to prioritize disease miRNAs. The MTDN is constructed by combining miRNA-target interactions with miRNA and mRNA expression profiles in tumor and nontumor tissues. Xu and colleagues' model achieves higher accuracy for classifying prostate cancer from NPC miRNAs in its application to prostate cancer. Predicted prostate cancer miRNAs are closely associated with oncogenesis, and miRNAs within families or from different families show combinatorial dysregulation of target genes. Finally, three miRNA target regulations are verified to hold in prostate cancer cells by transfection assays.Epithelial ovarian cancer is the most lethal gynecologic malignancy and often is not detected until late stages where current treatment modalities have limited effectiveness. Thrombospondin-1 (TSP-1) is an endogenous inhibitor of angiogenesis and reduced expression is often associated with aggressive tumor growth. In this paper, Campbell and colleagues show that the TSP-1 mimetic peptide ABT-898 was able to induce regression of advanced-stage ovarian tumors, cause destruction of abnormal tumor blood vessels, and significantly prolong survival. TSP-1 shows promise as an important tool in the treatment of advanced stage ovarian cancer.

Abstract 2577: Targeting the Axl tyrosine kinase receptor in pancreatic cancer

Abstract With the worst five-year survival rate of any cancer, pancreatic adenocarcinomas now rank as the fourth leading cause of cancer death in adults in the United States. This necessitates increased efforts to identify new leads that will serve as therapeutic targets in our fight against this aggressive cancer. Axl is a TAM family receptor tyrosine kinase involved in multiple aspects of tumorigenesis. Increased expression of Axl is associated with increased oncogenic transformation, cell survival, proliferation, migration, angiogenesis, and cellular adhesion. The oncogenic potential of Axl was first discovered in chronic myelogenous leukemia (CML), but it has been demonstrated to play a role in the progression and metastasis of other cancer types. The known ligand for Axl is the Growth Arrest Specific Gene-6 (Gas6) protein and its binding to Axl leads to Axl autophosphorylation and activation of downstream signaling pathways including MAPK and PI3K/Akt pathways. Furthermore, target validation studies of in vivo cancer models show that inhibition of Axl expression by RNAi blocked tumor growth in those models. Taken together, this information makes Axl kinase an exciting target for small molecule drug discovery. Using HCI-2084, a small molecule Axl kinase inhibitor, we explored the effectiveness of targeting the Axl kinase in pancreatic cancer. HCI-2084 demonstrates low nanomolar (IC50 = 12 nM) activity against the Axl kinase in a biochemical assay with good selectivity for Axl when screened in a kinase panel. In 2D and 3D cell proliferation assays, HCI-2084 significantly inhibited pancreatic cancer cell growth at concentrations as low as 30 nM. In pharmacodynamic endpoint assays, HCI-2084 dramatically inhibited Akt signaling (pAKT S473) downstream of GAS6 stimulation in pancreatic cancer cell lines. Inhibition of Axl autophosphorylation by HCI-2084 was also observed in an Axl-transfected cell line system. Consistent with the known function of Axl, HCI-2084 inhibited Gas6-induced migration and invasion of pancreatic cancer cells in vitro. The proteolytic processing of the extracellular domain of the Axl receptor is a known event downstream of Axl activation and results in the release of soluble Axl (sAxl) into the cell culture media (in vitro) or into the blood stream (in vivo). We hypothesized that sAxl levels could function as a biomarker for target inhibition. Indeed, conditioned media from pancreatic cancer cell lines treated with HCI-2084 showed significant dose-dependent reductions in sAxl levels compared to the vehicle treated controls. Taken together, these results suggest Axl is a potential therapeutic target in pancreatic cancer and that HCI-2084 is an exciting agent to potentially treat this disease. HCI-2084 is currently undergoing evaluation in animal efficacy and pharmacodynamic endpoint studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2577. doi:10.1158/1538-7445.AM2011-2577

TYRO3-mediated regulation of MITF: a novel target in melanoma?

<i>TYRO3</i>-mediated regulation of MITF: a novel target in melanoma?

Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer ( approximately 150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.