Amy K. Keating

TAM Receptor Tyrosine Kinases: Biologic Functions, Signaling, and Potential Therapeutic Targeting in Human Cancer

Tyro-3, Axl, and Mer constitute the TAM family of receptor tyrosine kinases (RTKs) characterized by a conserved sequence within the kinase domain and adhesion molecule-like extracellular domains. This small family of RTKs regulates an intriguing mix of processes, including cell proliferation/survival, cell adhesion and migration, blood clot stabilization, and regulation of inflammatory cytokine release. Genetic or experimental alteration of TAM receptor function can contribute to a number of disease states, including coagulopathy, autoimmune disease, retinitis pigmentosa, and cancer. In this chapter, we first provide a comprehensive review of the structure, regulation, biologic functions, and downstream signaling pathways of these receptors. In addition, we discuss recent evidence which suggests a role for TAM receptors in oncogenic mechanisms as family members are overexpressed in a spectrum of human cancers and have prognostic significance in some. Possible strategies for targeted inhibition of the TAM family in the treatment of human cancer are described. Further research will be necessary to evaluate the full clinical implications of TAM family expression and activation in cancer.

Taking aim at Mer and Axl receptor tyrosine kinases as novel therapeutic targets in solid tumors

Importance of the field: Axl and/or Mer expression correlates with poor prognosis in several cancers. Until recently, the role of these receptor tyrosine kinases (RTKs) in development and progression of cancer remained unexplained. Studies demonstrating that Axl and Mer contribute to cell survival, migration, invasion, metastasis and chemosensitivity justify further investigation of Axl and Mer as novel therapeutic targets in cancer. Areas covered in this review: Axl and Mer signaling pathways in cancer cells are summarized and evidence validating these RTKs as therapeutic targets in glioblastoma multiforme, NSCLC, and breast cancer is examined. A discussion of Axl and/or Mer inhibitors in development is provided. What the reader will gain: Potential toxicities associated with Axl or Mer inhibition are addressed. We propose that the probable action of Mer and Axl inhibitors on cells within the tumor microenvironment will provide a therapeutic opportunity to target both tumor cells and the stromal components that facilitate disease progression. Take home message: Axl and Mer mediate multiple oncogenic phenotypes and activation of these RTKs constitutes a mechanism of chemoresistance in a variety of solid tumors. Targeted inhibition of these RTKs may be effective as anti-tumor and/or anti-metastatic therapy, particularly if combined with standard cytotoxic therapies.

Inhibition of Mer and Axl Receptor Tyrosine Kinases in Astrocytoma Cells Leads to Increased Apoptosis and Improved Chemosensitivity

Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal–regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development. Mol Cancer Ther; 9(5); 1298–307. ©2010 AACR.

Ectopic Expression of the Proto-oncogene Mer in Pediatric T-Cell Acute Lymphoblastic Leukemia

Purpose: The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples. Experimental Design: Reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to determine expression of Mer in sorted human thymocyte populations, lymphocytes, and lymphocytes activated by phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. Mer expression in 34 T-cell ALL (T-ALL) patient samples was evaluated by reverse transcription-PCR, and Mer protein expression in a separate cohort of 16 patient samples was assayed by flow cytometry and Western blot. Results: Mer expression was absent in normal thymocytes or lymphocytes, and in T cells activated with phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. In contrast, Jurkat cells and T-ALL patient samples expressed unique 180 to 185 kDa Mer protein glycoforms. Substantial Mer RNA levels were principally observed in a subset of T-ALL patient samples that expressed B220 (P = 0.004) but lacked surface expression of CD3 (P = 0.02) and CD4 (P = 0.006), a phenotypic profile consistent with immature lymphoblasts. In addition, 8 of 16 T-ALL patient samples had Mer protein detected by flow cytometry and Western blot. Conclusions: Transforming Mer signals may contribute to T-cell leukemogenesis, and abnormal Mer expression may be a novel therapeutic target in pediatric ALL therapy.

Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase

Mer (MerTK) is a receptor tyrosine kinase important in platelet aggregation, as well as macrophage cytokine secretion and clearance of apoptotic cells. Mer is not normally expressed in thymocytes or lymphocytes; however, ectopic Mer RNA transcript and protein expression is found in a subset of acute lymphoblastic leukemia cell lines and patient samples, suggesting a role in leukemogenesis. To investigate the oncogenic potential of Mer in vivo, we created a transgenic mouse line (MerTg) that expresses Mer in the hematopoietic lineage under control of the Vav promoter. Ectopic expression and activation of the transgenic Mer protein was demonstrated in lymphocytes and thymocytes of the MerTg mice. At 12–24 months of age, greater than 55% of the MerTg mice, compared to 12% of the wild type, developed adenopathy, hepatosplenomegaly, and circulating lymphoblasts. Histopathological analysis and flow cytometry were consistent with T-cell lymphoblastic leukemia/lymphoma. Mer may contribute to leukemogenesis by activation of Akt and ERK1/2 anti-apoptotic signals, which were upregulated in MerTg mice. Additionally, a significant survival advantage was noted in MerTg lymphocytes compared to wild-type lymphocytes after dexamethasone treatment. These data suggest that Mer plays a cooperative role in leukemogenesis and may be an effective target for biologically based leukemia/lymphoma therapy.

Aberrant Mer receptor tyrosine kinase expression contributes to leukemogenesis in acute myeloid leukemia.

Acute myeloid leukemia (AML) continues to be extremely difficult to treat successfully, and the unacceptably low overall survival rates mandate that we assess new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation in AML has been associated with poor prognosis and provides strategic targets for novel therapy development. We found that Mer receptor tyrosine kinase was over-expressed in a majority of pediatric (29/36, 80%) and adult (10/10, 100%) primary AML patient blasts at the time of diagnosis, and 100% of patient samples at the time of relapse. Mer was also found to be expressed in 12 of 14 AML cell lines (86%). In contrast, normal bone marrow myeloid precursors expressed little to no Mer. Following AML cell line stimulation with Gas6, a Mer ligand, we observed activation of prosurvival and proliferative signaling pathways, including phosphorylation of ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. To assess the phenotypic role of Mer in AML, two independent short-hairpin RNA (shRNA) constructs were used to decrease Mer expression in the AML cell lines Nomo-1 and Kasumi-1. Reduction of Mer protein levels significantly increased rates of myeloblast apoptosis two to threefold in response to serum starvation. Furthermore, myeloblasts with knocked-down Mer demonstrated decreased colony formation by 67–87%, relative to control cell lines (P<0.01). NOD-SCID-gamma mice transplanted with Nomo-1 myeloblasts with reduced levels of Mer had a significant prolongation in survival compared with mice transplanted with the parental or control cell lines (median survival 17 days in parental and control cell lines, versus 32–36 days in Mer knockdown cell lines, P<0.0001). These data suggest a role for Mer in acute myeloid leukemogenesis and indicate that targeted inhibition of Mer may be an effective therapeutic strategy in pediatric and adult AML.

Mer receptor tyrosine kinase inhibition impedes glioblastoma multiforme migration and alters cellular morphology

Glioblastoma multiforme (GBM) is an aggressive brain tumor, fatal within 1 year from diagnosis in most patients despite intensive multimodality therapy. The migratory and microscopically invasive nature of GBM as well as its resistance to chemotherapy renders conventional therapies inadequate in its treatment. Although Mer receptor tyrosine kinase (RTK) inhibition has been shown to decrease the long-term survival and improve the chemosensitivity of GBM in vitro, its role in malignant cellular migration has not been previously evaluated. In this study, we report for the first time a role for Mer RTK in brain tumor migration and show that Mer inhibition profoundly impedes GBM migration and alters cellular morphology. Our data demonstrate that Mer RTK inhibition results in altered signaling through focal adhesion kinase (FAK) and RhoA GTPase and a transformation of cytoskeletal organization, suggesting both molecular and structural mechanisms for the abrogation of migration. We also describe a novel and translational method of Mer RTK inhibition using a newly developed monoclonal antibody, providing proof of principle for future evaluation of Mer-targeted translational therapies in the treatment of GBM. Previous findings implicating Mer signaling in glioblastoma survival and chemotherapy resistance coupled with our discovery of the role of Mer RTK in GBM cellular migration support the development of novel Mer-targeted therapies for this devastating disease.

Mer receptor tyrosine kinase is a therapeutic target in pre-B cell acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is currently treated with an intense regimen of chemotherapy yielding cure rates near 85%. However, alterations to treatment strategies using available drugs are unlikely to provide significant improvement in survival or decrease therapy-associated toxicities. Here, we report ectopic expression of the Mer receptor tyrosine kinase in pre-B-cell ALL (B-ALL) cell lines and pediatric patient samples. Inhibition of Mer in B-ALL cell lines decreased activation of AKT and MAPKs and led to transcriptional changes, including decreased expression of antiapoptotic PRKCB gene and increase in proapoptotic BAX and BBC3 genes. Further, Mer inhibition promoted chemosensitization, decreased colony-forming potential in clonogenic assays, and delayed disease onset in a mouse xenograft model of leukemia. Our results identify Mer as a potential therapeutic target in B-ALL and suggest that inhibitors of Mer may potentiate lymphoblast killing when used in combination with chemotherapy. This strategy could reduce minimal residual disease and/or allow for chemotherapy dose reduction, thereby leading to improved event-free survival and reduced therapy-associated toxicity for patients with B-ALL. Additionally, Mer is aberrantly expressed in numerous other malignancies suggesting that this approach may have broad applications.

Targeting paediatric acute lymphoblastic leukaemia: novel therapies currently in development

Modifications to the treatment of acute lymphoblastic leukaemia (ALL) in children have led to a dramatic increase in survival in the past 40 years. Despite this success, a significant subset of paediatric leukaemia patients either relapse or fail to ever achieve a complete remission. Additionally, some patients necessitate treatment with intensified chemotherapy regimens due to clinical or laboratory findings which identify them as high risk. These patients are unlikely to respond to further minor adjustments to the dosing or timing of administration of the same chemotherapy medications. Many novel targeted therapies for the treatment of childhood ALL provide potential mechanisms to further improve cure rates, and provide the possibility of minimizing toxicity to non‐malignant cells, given their specificity to malignant cell phenotypes. This article explores many of the potential targeted therapies in varying stages of development, from those currently in clinical trials to those still being refined in the research laboratory.

TAM receptor tyrosine kinases: Expression, disease and oncogenesis in the central nervous system

Receptor tyrosine kinases (RTKs) are cell surface proteins that tightly regulate a variety of downstream intra-cellular processes; ligand-receptor interactions result in cascades of signaling events leading to growth, proliferation, differentiation and migration. There are 58 described RTKs, which are further categorized into 20 different RTK families. When dysregulated or overexpressed, these RTKs are implicated in disordered growth, development, and oncogenesis. The TAM family of RTKs, consisting of Tyro3, Axl, and MerTK, is prominently expressed during the development and function of the central nervous system (CNS). Aberrant expression and dysregulated activation of TAM family members has been demonstrated in a variety of CNS-related disorders and diseases, including the most common but least treatable brain cancer in children and adults: glioblastoma multiforme.