Molecular Pathways: MERTK Signaling in Cancer

Published OnlineFirst July 5, 2013; DOI: 10.1158/1078-0432.CCR-12-1451

Clinical Cancer Molecular Pathways Research

Christopher T. Cummings1, Deborah DeRyckere1, H. Shelton Earp2, and Douglas K. Graham1,3

Abstract MERTK is a receptor tyrosine kinase of the TAM (Tyro3, Axl, MERTK) family, with a deﬁned spectrum of normal expression. However, MERTK is overexpressed or ectopically expressed in a wide variety of cancers, including leukemia, non–small cell lung cancer, glioblastoma, melanoma, prostate cancer, breast cancer, colon cancer, gastric cancer, pituitary adenomas, and rhabdomyosarcomas, potentially resulting in the activation of several canonical oncogenic signaling pathways. These include the mitogen-activated protein kinase and phosphoinositide 3-kinase pathways, as well as regulation of signal transducer and activator of transcription family members, migration-associated proteins including the focal adhesion kinase and myosin light chain 2, and prosurvival proteins such as survivin and Bcl-2. Each has been implicated in MERTK physiologic and oncogenic functions. In neoplastic cells, these signaling events result in functional phenotypes such as decreased apoptosis, increased migration, chemoresistance, increased colony formation, and increased tumor formation in murine models. Conversely, MERTK inhibition by genetic or pharmacologic means can reverse these pro-oncogenic phenotypes. Multiple therapeutic approaches to MERTK inhibition are currently in development, including ligand "traps", a monoclonal antibody, and small-molecule tyrosine kinase inhibitors. Clin Cancer Res; 19(19); 1–6. 2013 AACR.

Background to the list of effectively targeted proteins in patients with In recent years, therapeutic agents targeting specific cancer. molecular aberrations in cancer cells have been effective at prolonging survival in multiple cancer types; however, Discovery of MERTK for the majority of patients with cancer, the oncogenic MERTK has been implicated in cancer pathogenesis since drivers are complex and identification of additional ther- it was first cloned from a human B-lymphoblastoid expres- apeutic targets has become a major research focus. One sion library and from a human glioblastoma library (7, 15). potential target is MERTK, a member of the TAM-family of Although ectopically expressed in multiple lymphoid leu- receptor tyrosine kinases, which also includes Axl and kemia cell lines and patient samples, MERTK is absent from Tyro3 (reviewed in ref. 1). The physiologic functions of normal B and T lymphocytes, suggesting that MERTK may MERTK have only recently been defined in platelets and be an attractive target for childhood acute lymphoblastic macrophages, but its overexpression and potential acti- leukemia (ALL) therapeutic development (7, 9, 27). MERTK vation in a wide variety of cancers indicate that MERTK sequencing from human and mouse revealed it to be the signaling confers an advantage on the tumor cell (2–26). human ortholog of the chicken c-eyk gene, the cellular Target validation studies, discussed below, suggest that proto-oncogene of v-eyk responsible for the oncogenic MERTK inhibition is a viable strategy for decreasing properties of the RPL30 avian retrovirus, which induces tumor burden in preclinical models. Clinically relevant lymphomas, sarcomas, and other tumor types in chickens agents are under development in an effort to add MERTK in vivo (28, 29). Unbiased gain-of-function retroviral insertion screens have also identified the oncogenic role of MERTK (30).

Authors' Afﬁliations: 1Department of Pediatrics, Section of Hematol- MERTK chimeric receptor signaling ogy, Oncology and Bone Marrow Transplantation, University of Color- Following cloning of the MERTK cDNA, multiple groups ado Anschutz Medical Campus, Aurora, Colorado; and 2Departments of Medicine and Pharmacology, UNC Lineberger Comprehensive Cancer investigated the signaling pathways downstream of MERTK Center, University of North Carolina at Chapel Hill, Chapel Hill, North activation (Fig. 1). As a ligand had not yet been identiﬁed, Carolina early studies used receptor chimeras with the intracellular Corresponding Author: Douglas K. Graham, Department of Pediatrics, domain of MERTK fused to the extracellular domain of Section of Hematology, Oncology and Bone Marrow Transplantation, University of Colorado Anschutz Medical Campus, Mail Stop 8302, proteins with a known ligand or dimerization capability. 12800 East 19th Avenue, P18-4400, Aurora, CO 80045. Phone: 303- An early strategy combined the transmembrane and cyto- 724-4006; Fax: 303-724-4015; E-mail: [email protected] plasmic domains of MERTK with the extracellular domain doi: 10.1158/1078-0432.CCR-12-1451 of human colony-stimulating factor 1 (CSF-1) receptor 2013 American Association for Cancer Research. (Fms; ref. 15). In this model, CSF-1 treatment induced

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Ligand trap Gas6, Protein S, Tubby, Tulp1, Galectin-3

Small–molecule inhibitors, miR126, miR335 monoclonal antibody MERTKMERTK EGFR

P P Grb2 Shc STAT5 P NFkB STAT6 P Raf P CREB P Pl3K P P ATF1 P FAK PLCy MEK Transcription P Akt P P P RhoA MLC2 Erk p38 P Ack1 p90RSK mTOR GSK3- Migration α/β Survivin Bcl-2 P P MSK1 p70S6 Bcl-xL Wwox c-Fos c-Jun Survival Proliferation/survival

Figure 1. The MERTK Signaling Network. Multiple pro-oncogenic signaling pathways have been implicated downstream of MERTK activation. These include pathways promoting survival, increasing migration, and inhibiting apoptosis. The ligands Gas6, protein S, Tubby, Tulp1, and Galectin-3 all induce MERTK autophosphorylation to initiate the signaling cascades depicted below. More recently, MERTK has been shown to be regulated by two miRNAs, miR126 and miR335, and may also have direct effects on gene transcription. Inhibitors of MERTK, including ligand traps, a monoclonal antibody, and small molecule inhibitors, are currently in preclinical development.

MERTK autophosphorylation, transformed NIH 3T3 cells, Finally, our group constructed a chimeric receptor com- and activated phospholipase Cg, phosphatidylinositol 3- posed of the extracellular and transmembrane domains of kinase (PI3K), and p70 S6 kinase. Recruitment of growth the EGF receptor (EGFR) fused to the intracellular domain factor receptor binding protein 2 (Grb2) and phosphory- of MERTK (34). Ligand activation prevented apoptosis in lation of Src homology and collagen (Shc) led to Raf-1, the myeloblast-like 32D cells promoting IL-3-indepen- mitogen-activated protein/extracellular signal–regulated dence and stimulated Erk, Akt, and p38 activity. In this kinase (MEK), and extracellular signal-regulated kinase model, MERTK signaling had a primary role in cell survival (Erk) activation. and cytoskeletal alterations without a substantial effect on Another MERTK chimeric protein was made by fusing the proliferation. extracellular domain of CD8 to the intracellular domain of MERTK creating a constitutively active MERTK chimera MERTK ligands (31). This MERTK chimera conferred an interleukin 3 Although studies using chimeric receptors provided (IL-3)–independent phenotype to Ba/F3 pro-B lympho- important information on MERTK signaling, identification cytes. The Grb2, MEK1, and Erk pathways as well as PI3K of authentic MERTK ligands permitted analysis in a more were also activated. NF-kB–mediated transcription was physiologic context. The first ligand described was Gas6, increased as assessed by a luciferase-reporter assay with identified by purification of Axl-activating conditioned potential activation of antiapoptotic signaling in tumor media. Gas6 binds MERTK, but with 3- to 10-fold lower cells (reviewed in ref. 32). The p38 pathway was also affinity (35, 36). Subsequently, protein S was identified as a activated and p38 inhibition decreased proliferation in MERTK ligand that did not activate Axl (37, 38). Both MERTK chimeric Ba/F3 cells. Similar to NF-kB, p38 has ligands are secreted by multiple cell types and are present complex effects on proliferation, migration, and survival in in human blood, although total protein S levels are 1,000- cancer cells (reviewed in ref. 33). fold higher; however, modifications of protein S may be

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necessary for it to activate Mer (39–41). More recently, a sion of MERTK increased the levels of phospho-Akt (21, 22). novel phagocytosis-based functional cloning screening Inhibition of MERTK by shRNA prevented the Gas6- strategy identified 3 new MERTK ligands: tubby, tubby-like induced increase of pAkt, pERK1/2, and pSTAT6, decreased protein 1 (Tulp1), and galectin-3 (42, 43). All known basal phosphorylation of Akt, mTOR, and p70S6 kinase, MERTK ligands induce MERTK autophosphorylation, increased PARP cleavage, and decreased CDC42 activity. In although to date, roles for tubby, Tulp1, and galectin-3 prostate cancer cells, MERTK associates with and facilitates have not been studied in MERTK-driven cancers. However, the activation of Ack1, a non–receptor tyrosine kinase. This overexpression of galectin-3 has been shown in many process results in an Ack activity-dependent degradation of cancers and galectin-3 is known to play roles in a wide the Wwox tumor suppressor, suggesting yet another onco- variety of oncogenic processes, consistent with the possi- genic mechanism, that is, control of a tumor suppressor (2). bility that these phenotypes may be mediated by MERTK An FMS-MERTK chimera expressed in a prostate cancer cell signaling (44). line induced Raf, MEK1/2, p90RSK, Erk1/2, and Akt phosphorylation and increased c-Fos and c-Jun transcription MERTK signaling in leukemia factor expression (25). The ectopic expression of MERTK in pediatric ALL led our group to investigate full-length MERTK receptor oncogenic MERTK migration and cellular traffic signaling in lymphocytes (9, 45). A transgenic model was MERTK signaling has been implicated in tumor cell made expressing MERTK from the Vav promoter; lympho- migration and invasion. In non–small cell lung cancer cells, cytes at all stages expressed MERTK. Lymphoblastic leuke- FAK is phosphorylated in response to Gas6 (23). Both total mia and lymphomas resulted from this ectopic MERTK and phosphorylated FAK and RhoA increase, whereas total expression and stimulation of these cells with Gas6 induced and phosphorylated myosin light chain 2 are decreased in MERTK autophosphorylation and downstream activation glioblastoma cells in response to shRNA-mediated MERTK of Erk1/2 and Akt. In human T-cell lymphoblastic leukemia inhibition, with impaired migration (17, 18). Melanoma cell lines, Gas6 also activated MERTK, Erk1/2, Akt, STAT5, cell migration and invasion are also decreased by shRNA and STAT6 (10). STAT activation within tumor cells con- MERTK expression (21, 22). MERTK’s physiologic role in tributes to prosurvival phenotypes (46–48). MERTK knock- ingesting apoptotic material in macrophages is an action down by short hairpin RNA (shRNA) resulted in decreased that is in part dependent on a MERTK:Vav activation process levels of phospho-STAT5 and phospho-Erk. In addition, in that stimulates Rac CDC42 and Rho GDP to GTP exchange acute myeloid leukemia cells that did not express Axl or (50). With respect to cellular trafficking, a recent report links Tyro3, Gas6 activated MERTK and resulted in the phos- MERTK action to EGFR surface levels (51). In the absence phorylation and activation of Erk1/2, p38, MSK1, cAMP- of MERTK, EGF treatment induced a higher rate of EGFR responsive element binding protein (CREB), activating internalization and degradation, resulting in decreased transcription factor 1 (ATF1), Akt, and STAT6 (49). shRNA levels of EGFR on the cell surface and reducing downstream knockdown of MERTK reduced the activation of p38, Erk, signaling. Related to the concept of receptor trafficking, a and CREB, further strengthening these signaling findings. recent study found MERTK to be located not only at the cell Taken together, these studies broaden and define endoge- surface, but also in the nucleus. Consensus nuclear local- nous MERTK signaling pathways. ization sequences have also been identified in the MERTK gene (52). This raises the possibility that MERTK may have MERTK signaling in solid tumors effects on gene transcription. MERTK signaling observed in leukemia cells is also seen in solid tumor cells; additional novel downstream effectors have also been identified. In non–small cell lung cancer, Clinical-Translational Advances p38, Erk1/2, GSK3a/b, MEK1/2, Akt, mTOR, CREB, and Tumor biology and target validation ATF1 phosphorylation were all induced following Gas6 Many studies have used shRNA to show critical onco- addition (23). shRNA-mediated MERTK inhibition resulted genic roles for MERTK in a variety of tumor types. In in decreased levels of CREB, Bcl-xL, survivin, and phospho- T-cell acute lymphoblastic leukemia, shRNA against Akt, and increased levels of Bcl-2, in response to serum MERTK resulted in increased apoptosis, decreased meth- starvation. These results suggest additional mechanisms by ylcellulose colony formation, increased sensitivity to which MERTK may impact tumor cell survival. In glioblas- cytotoxic chemotherapies, and delayed disease onset with toma cells, shRNA mediated reduction of MERTK protein increased survival in a murine leukemia model (9, 10). expression and also decreased levels of phosphorylated Erk Similarly, in acute myeloid leukemia, MERTK inhibition and Akt (16). In addition, MERTK expression correlated resulted in increased apoptosis, decreased colony forma- with Nestin and Sox2 expression in a glioblastoma spheroid tion, and increased survival in a mouse model (49). culture model, indicating possible roles for MERTK main- In glioblastoma, MERTK shRNA increased apoptosis and taining cells in an undifferentiated state (18). In melanoma, autophagy, decreased colony formation, increased che- Gas6-induced MERTK activation resulted in p38, ERK1/2, mosensitivity, altered morphology, and decreased migra- GSK3a/b, Akt, AMPK, STAT5, CHK-2, focal adhesion kinase tion (16–18). In non–small cell lung cancer, MERTK (FAK), and STAT6 phosphorylation, whereas overexpres- inhibition increased apoptosis, decreased colony

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formation, increased chemosensitivity, and decreased pyrazolopyrimidine sulfonamide derivative of UNC569, tumor formation in a mouse model (23). shRNA-medi- which has reduced human ether-a-go-go-related gene potas- ated inhibition of MERTK in melanoma cell lines resulted sium channel activity and therefore a more favorable tox- in increased cell death, decreased proliferation, decreased icity profile (60). This compound has shown promising colony formation in soft agar, decreased migration, and preclinical activity in a melanoma model, blocking MERTK decreased tumor formation in xenografts (21, 22). activation and downstream signaling via STAT6, Erk, and MERTK knockdown in breast cancer cells resulted in Akt, increasing apoptosis, decreasing colony formation in decreased formation of metastases and inhibited endo- soft agar, and decreasing collagen matrix invasion (22). thelial cell recruitment in mouse models (12). In breast cancer, miR-126 and miR-335 negatively regulate MERTK Summary expression; loss of these miRNAs contributes to breast The MERTK tyrosine kinase has been linked to the path- cancer progression and metastasis. Mechanistically, miR- ogenesis of cancer since it was first discovered by expression 126 loss increased MERTK expression. Increased MERTK cloning from a neoplastic cell nearly two decades ago. It has was followed by extracellular domain cleavage, and soluble since been shown to activate a wide variety of pro-oncogenic MERTK was postulated to act as a ligand sink, sequestering signaling pathways in an ever-expanding list of human Gas6 and preventing activation of TAM receptors on migrat- cancer types. Signaling pathways, including those involving ing endothelial cells. Decreased Gas6-induced signaling MAPK and p38, PI3K, Janus-activated kinase (JAK)/STAT, in endothelial cells increased their migration, resulting in FAK/RhoA/MLC2, and Bcl-2 family members, contribute to tumor-associated angiogenesis. increased proliferation and migration, and decreased apo- Inappropriate or overexpression of MERTK, one of its ptosis and chemosensitivity. Several types of MERTK inhi- ligands, or both, has been the most studied mechanism bitors are currently in development, including ligand traps, linking MERTK and cancer. However, recent studies in a monoclonal antibody, and small-molecule tyrosine multiple myeloma, melanoma, renal cancer, and head and kinase inhibitors. Such agents are designed to exploit the neck cancer have also identified mutation of MERTK as selective requirement for MERTK in tumor cells. Although another possible mechanism (21, 53, 54). Mutation has MERTK was infrequently studied in the first decade after its been recognized as a common method of activating mul- discovery due to a lack of understanding about ligand and tiple other receptor tyrosine kinases, and it will be interest- cellular biology, this is changing due to recent evidence ing to determine the frequency of and functional effects of linking MERTK to oncogenic progression. The hope is that these recently discovered MERTK mutations. these efforts will result in clinically available MERTK-targeted therapeutics in the near future, as well as a further Therapeutics in development understanding of how best to deploy them. One strategy to inhibit MERTK is to use a ligand trap, consisting of the extracellular domain of MERTK or an Disclosure of Potential Conflicts of Interest antibody against Gas6, to prevent ligand-dependent D. DeRyckere, H.S. Earp, and D.K. Graham hold equity in Meryx Incorporated. No potential conflicts of interest were disclosed by the MERTK activation (3). A monoclonal antibody against other author. MERTK has been used to decrease the levels of MERTK protein on the surface of glioblastoma cells, resulting in Authors' Contributions decreased tumor cell migration in vitro (17). In addition, Conception and design: C.T. Cummings, H.S. Earp, D.K. Graham several multikinase small-molecule inhibitors have been Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.T. Cummings shown to have inhibitory effects against MERTK, including Analysis and interpretation of data (e.g., statistical analysis, biosta- sunitinib, BMS-777607, R-428, and Compound-52 (55– tistics, computational analysis): C.T. Cummings, D.K. Graham Writing, review, and/or revision of the manuscript: C.T. Cummings, 58). Recently, several MERTK-selective small-molecule inhi- D. DeRyckere, H.S. Earp, D.K. Graham bitors have been developed using structure-aided design Study supervision: D.K. Graham algorithms. The first of these, UNC569, is a pyrazolopyrimidine derivative that functions as a competitive ATP Grant Support inhibitor (59). It is highly selective for MERTK, is orally This work was supported in part by a grant from the NIH (RO1CA137078; to D.K. Graham). bioavailable, has a favorable pharmacokinetic profile, and has shown promising preclinical activity against leukemia Received April 18, 2013; revised June 14, 2013; accepted June 14, 2013; in vitro. A second-generation compound, UNC1062, is a published OnlineFirst July 5, 2013.

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OF6 Clin Cancer Res; 19(19) October 1, 2013 Clinical Cancer Research

Molecular Pathways: MERTK Signaling in Cancer

Christopher T. Cummings, Deborah DeRyckere, H. Shelton Earp, et al.

Clin Cancer Res Published OnlineFirst July 5, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-12-1451

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