Douglas K. Graham

UNC2025, a MerTK small molecule inhibitor, is therapeutically effective alone and in combination with methotrexate in leukemia models

Purpose: MERTK tyrosine kinase is ectopically expressed in 30% to 50% of acute lymphoblastic leukemias (ALL) and more than 80% of acute myeloid leukemias (AML) and is a potential therapeutic target. Here, we evaluated the utility of UNC2025, a MERTK tyrosine kinase inhibitor, for treatment of acute leukemia. Experimental Design: Preclinical in vitro and in vivo assays using cell lines and primary leukemia patient samples were used to evaluate antileukemic effects of UNC2025. Results: UNC2025 potently inhibited prosurvival signaling, induced apoptosis, and reduced proliferation and colony formation in MERTK-expressing ALL and AML cell lines and patient samples. Approximately 30% of primary leukemia patient samples (78 of 261 total) were sensitive to UNC2025. Sensitive samples were most prevalent in the AML, T-ALL, and minimally differentiated (M0) AML subsets. UNC2025 inhibited MERTK in bone marrow leukemia cells and had significant therapeutic effects in xenograft models, with dose-dependent decreases in tumor burden and consistent two-fold increases in median survival, irrespective of starting disease burden. In a patient-derived AML xenograft model, treatment with UNC2025 induced disease regression. In addition, UNC2025 increased sensitivity to methotrexate in vivo, suggesting that addition of MERTK-targeted therapy to current cytotoxic regimens may be particularly effective and/or allow for chemotherapy dose reduction. Conclusions: The broad-spectrum activity mediated by UNC2025 in leukemia patient samples and xenograft models, alone or in combination with cytotoxic chemotherapy, supports continued development of MERTK inhibitors for treatment of leukemia. Clin Cancer Res; 23(6); 1481–92. ©2016 AACR.

MERTK inhibition induces polyploidy and promotes cell death and cellular senescence in glioblastoma multiforme

Background MER 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. Methods Correlations 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. Results Decreased 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. Conclusions The 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.

Targeting the TAM Receptors in Leukemia

Targeted inhibition of members of the TAM (TYRO-3, AXL, MERTK) family of receptor tyrosine kinases has recently been investigated as a novel strategy for treatment of hematologic malignancies. The physiologic functions of the TAM receptors in innate immune control, natural killer (NK) cell differentiation, efferocytosis, clearance of apoptotic debris, and hemostasis have previously been described and more recent data implicate TAM kinases as important regulators of erythropoiesis and megakaryopoiesis. The TAM receptors are aberrantly or ectopically expressed in many hematologic malignancies including acute myeloid leukemia, B- and T-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma. TAM receptors contribute to leukemic phenotypes through activation of pro-survival signaling pathways and interplay with other oncogenic proteins such as FLT3, LYN, and FGFR3. The TAM receptors also contribute to resistance to both cytotoxic chemotherapeutics and targeted agents, making them attractive therapeutic targets. A number of translational strategies for TAM inhibition are in development, including small molecule inhibitors, ligand traps, and monoclonal antibodies. Emerging areas of research include modulation of TAM receptors to enhance anti-tumor immunity, potential roles for TYRO-3 in leukemogenesis, and the function of the bone marrow microenvironment in mediating resistance to TAM inhibition.

Discovery of Macrocyclic Pyrimidines as MerTK-Specific Inhibitors

Macrocycles have attracted significant attention in drug discovery recently. In fact, a few de novo designed macrocyclic kinase inhibitors are currently in clinical trials with good potency and selectivity for their intended target. In this study, we successfully engaged a structure‐based drug design approach to discover macrocyclic pyrimidines as potent Mer tyrosine kinase (MerTK)‐specific inhibitors. An enzyme‐linked immunosorbent assay (ELISA) in 384‐well format was employed to evaluate the inhibitory activity of macrocycles in a cell‐based assay assessing tyrosine phosphorylation of MerTK. Through structure–activity relationship (SAR) studies, analogue 11 [UNC2541; (S)‐7‐amino‐N‐(4‐fluorobenzyl)‐8‐oxo‐2,9,16‐triaza‐1(2,4)‐pyrimidinacyclohexadecaphane‐1‐carboxamide] was identified as a potent and MerTK‐specific inhibitor that exhibits sub‐micromolar inhibitory activity in the cell‐based ELISA. In addition, an X‐ray structure of MerTK protein in complex with 11 was resolved to show that these macrocycles bind in the MerTK ATP pocket.

The small molecule MERTK inhibitor UNC2025 decreases platelet activation and prevents thrombosis

Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025s anti‐platelet effect is synergistic with inhibition of the ADP receptor, P2Y12.

GAS6/TAM Pathway Signaling in Hemostasis and Thrombosis

The GAS6/TYRO3-AXL-MERTK (TAM) signaling pathway is essential for full and sustained platelet activation, as well as thrombus stabilization. Inhibition of this pathway decreases platelet aggregation, shape change, clot retraction, aggregate formation under flow conditions, and surface expression of activation markers. Transgenic mice deficient in GAS6, or any of the TAM family of receptors that engage this ligand, exhibit in vivo protection against arterial and venous thrombosis but do not demonstrate either spontaneous or prolonged bleeding compared to their wild-type counterparts. Comparable results are observed in wild-type mice treated with pharmacological inhibitors of the GAS6-TAM pathway. Thus, GAS6/TAM inhibition offers an attractive novel therapeutic option that may allow for a moderate reduction in platelet activation and decreased thrombosis while still permitting the primary hemostatic function of platelet plug formation.

Roles for AXL and MERTK in Resistance to Cytotoxic and Targeted Therapies

Abstract AXL and MERTK are members of the TAM family (TYRO3, AXL, and MERTK) of receptor tyrosine kinases (RTKs). Ligand binding and subsequent activation of AXL and MERTK in human cancers lead to downstream signaling via pathways that mediate tumor cell survival and proliferation. TAM RTK signaling mediates resistance to conventional cytotoxic chemotherapies across a wide variety of both hematopoietic and nonhematopoietic malignancies. Similarly, TAM RTKs are upregulated and mediate bypass signaling in tumor cells with acquired resistance to a variety of different targeted kinase inhibitors. In addition, TAM RTKs can mediate therapeutic resistance through their physiological anti-inflammatory role in the immune system. These roles for AXL and MERTK in therapeutic resistance implicate them as attractive therapeutic targets, both in combination with other agents and in the context of acquired resistance. Accordingly, a number of TAM RTK-targeted agents are currently in development and several have progressed to clinical application, although challenges such as identification of appropriate biomarkers of TAM RTK inhibition remain.

MERTK promotes resistance to irreversible EGFR tyrosine kinase inhibitors in non-small cell lung cancers expressing wild-type EGFR-family members

Purpose: Lung cancer is the leading cause of cancer-related death. Non–small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and over 60% express wild-type EGFR (wtEGFR); however, EGFR tyrosine kinase inhibitors (TKIs) have limited effect in most patients with wtEGFR tumors. We previously identified MERTK tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843, a novel MERTK-selective inhibitor with favorable properties for clinical translation. The goal of this study was to determine whether MERTK and EGFR inhibitor combination therapy could provide antitumor efficacy against wtEGFR NSCLC. Experimental Design: An unbiased screen of 378 kinase inhibitors was conducted to identify synergistic interactions with MRX-2843 and biochemical and therapeutic effects were determined in vitro and in vivo. Results: Numerous irreversible EGFR TKIs, including CO-1686 and osimertinib, synergized with MRX-2843 to inhibit wtEGFR NSCLC cell expansion, irrespective of driver oncogene status. CO-1686 and MRX-2843 combination therapy inhibited MERTK, wtEGFR, and ERBB2/ERBB3 and decreased downstream PI3K-AKT, MAPK-ERK, and AURORA kinase (AURK) signaling more effectively than single agents. Inhibition of PI3K, AKT or AURK, but not MEK, synergized with CO-1686 to inhibit tumor cell expansion, suggesting their roles as key redundant resistance pathways. Treatment with MRX-2843 and CO-1686 or osimertinib prevented xenograft growth while single agents had limited effect. Tumor growth inhibition was durable even after treatment with combination therapy was stopped. Conclusions: Our data support the application of MRX-2843 in combination with an irreversible EGFR TKI as a novel strategy for treatment of patients with wtEGFR NSCLC.

Abstract A35: MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia

Background: Early T-precursor acute lymphoblastic leukemia (ETP-ALL) is a subclass of T-cell ALL (T-ALL) accounting for 15% of pediatric T-ALL cases and characterized by an immature phenotype, resistance to therapy, and high rates of induction failure and relapse (Wood B et al., Blood 2009). MERTK receptor tyrosine kinase is not expressed in normal T cells but is ectopically expressed in 40-50% of T-ALLs, particularly those with an immature T-cell phenotype (Graham DK et al., Clin Cancer Res 2006), suggesting a role in ETP-ALL. One potential role is regulation of the anti-apoptotic protein B-cell lymphoma-2 (BCL-2). BCL-2 is specifically expressed in double-negative T-cell precursors and is preferentially expressed in ETP-ALL compared to T-ALL (Chonghaile et al., Cancer Discovery 2014). Moreover, ETP-ALL cells are dependent on BCL-2 for survival. Our previous studies demonstrated regulation of BCL-2 and BCL-2 family members downstream of MERTK in B-ALL and acute myeloid leukemia cells (Linger RM et al., Blood 2013; Lee-Sherick AB et al., Oncotarget 2015). This interplay between MERTK and BCL-2 and their association with an immature T-ALL phenotype suggest that combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL. Methods: Publicly available mRNA expression data were used to assess MERTK and BCL-2 expression in T-ALL cell lines and patient samples. MERTK and BCL-2 protein expression were determined by immunoblot. ETP-ALL cell lines were cultured with vehicle or MRX-2843, a dual MERTK/FLT3 kinase inhibitor. MERTK protein was immunoprecipitated from cell lysates and phosphorylated and total proteins were assessed by immunoblot. Alternatively, cells were stained with PoPro-1-iodide and propidium iodide dyes and analyzed by flow cytometry to assess cell death. Orthotopic xenografts were established in NSGS mice using an ETP-ALL patient sample, and leukemia burden in peripheral blood (%hCD45+) was monitored by flow cytometry. After engraftment (1.86 +/- 0.43% peripheral blasts), mice were treated once daily with 75 mg/kg MRX-2843 or saline vehicle administered orally. Mice were euthanized when symptoms of advanced leukemia were evident and median survival was determined by Kaplan-Meier analysis. Results: MERTK mRNA was expressed at significantly higher levels in ETP-ALL cell lines and patient samples relative to other T-ALLs. Similarly, MERTK protein was ubiquitously expressed in ETP-ALL cell lines (n=2), ETP-ALL patient samples (n=2), and a near-ETP-ALL patient sample (n=1). In contrast, only 60% of other T-ALL cell lines (n=5) expressed MERTK. BCL-2 mRNA was expressed at significantly higher levels in ETP-ALL patient samples relative to other T-ALLs and BCL-2 protein was expressed in 2 of 2 ETP-ALL cell lines and 2 of 3 ETP-ALL and near-ETP-ALL patient samples. Treatment with MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK in ETP-ALL cells and induced dose-dependent cell death in the ETP-ALL cell lines PEER (43.2% vs 16% in vehicle-treated cultures, p Conclusions: MERTK and BCL-2 are preferentially expressed in ETP-ALL relative to T-ALL and MRX-2843, a dual MERTK/FLT3 kinase inhibitor, has robust therapeutic activity in cell culture and xenograft models of ETP-ALL. These data validate MRX-2843 as a novel agent with potential for clinical application in patients with ETP-ALL. In particular, combination therapy targeting MERTK and BCL-2 may be an effective therapeutic option for ETP-ALL. Citation Format: Ryan J. Summers, Katherine A. Minson, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A35.