Abstract: Unopposed platelet activation can be associated with pathologic thrombosis. An intact growth arrest–specific gene 6 (GAS6)/Mer receptor tyrosine kinase (MERTK) signaling pathway contributes importantly to potentiating platelet activation triggered by molecular agonists ex vivo and thrombus stabilization in vivo. We describe, herein, the inhibition of platelet function and stable thrombus formation conferred by iMer, a naturally occurring MERTK splice variant, that acts as a GAS6 decoy receptor and decreases phosphorylation of MERTK. Human and murine platelets incubated with this truncated protein demonstrate reduced activation in ex vivo assays including aggregometry (similar to treatment with anti-GAS6 antibody), expression of P-selectin, spreading on collagen, and accumulation on collagen at a venous shear rate. Wild-type C57BL/6 mice treated with iMer had improved survival in a collagen/epinephrine-induced pulmonary embolism model, without increase in tail bleeding time on preliminary analysis. Taken together, these findings confirm previous data suggesting the importance of GAS6-MERTK signaling in platelet activation and thrombus formation and highlighting the potential therapeutic implications of targeting this pathway as a means of treating or preventing thrombosis.

PDF - 358KB, Cells were plated in methylcellulose in the presence of the indicated concentrations of UNC1653 or vehicle only. Colonies were counted after eight days. Long-term colony growth of Jurkat cells in methylcellulose is shown. Mean values and standard errors were derived from 3 independent experiments.

PDF - 634KB, Eighteen fish (Myc1-Myc18) were treated with UNC569 for 2 weeks. Animals were imaged pre- and post-treatment, with GFP intensities quantified as described in the text. Ten of 18 fish (55.6%; Myc1-Myc8, Myc10, Myc16) showed >50% regressions. Six fish (33.3%; Myc9, Myc11-15) had 25-50% responses, and 2/18 (11.1%; Myc17, Myc18) progressed on treatment. Average diminution in tumor burden across the entire cohort was 47.8%. Four images are shown for each animal: Day 0 columns shows the raw image and GFP intensity plot for each fish prior to UNC treatment. Day 14 columns show post-treatment images and intensity plots. GFP Score day 14 columns show final responses, depicted as % of original cancer remaining.

PDF - 172KB, Jurkat cell cultures were treated with the indicated concentrations of UNC569 for 1 hour. Pervanadate was added to cell cultures for 3 minutes to stabilize the phosphorylated form of Tyro3. Tyro3 was immunoprecipitated from cell lysates and total Tyro3 protein and Tyro3 phosphoprotein (p-Tyro3) were detected by western blot. Numbers on the right indicate the location of molecular weight (kD) markers. Immunoblots are representative of three independent experiments.

PDF - 164KB, Jurkat and 697 cell cultures were treated with UNC569 (500 nM) for 1 hour. Pervanadate was added to cell cultures for 3 minutes to stabilize the phosphorylated form of Mer. Cells were harvested after 24 and after 48 hours. Mer was immunoprecipitated from cell lysates and total Mer protein and Mer phosphoprotein (p-Mer) were detected by western blot.

PDF - 294KB, TAM receptor expression in Jurkat cell line was detected by western blot analysis. Whole cell lysates were prepared and phosphorylated (denoted by p-) and total proteins were detected. Western blots representative of three independent experiments are shown. Blots were stripped and reprobed with anti-tubulin antibody to confirm similar protein loading.

We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.

Abstract In children, acute lymphoblastic leukemia (ALL) is 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 may reduce the risk of severe toxicities including infertility, organ damage, and secondary malignancy. We previously demonstrated ectopic expression of MER, a member of the TAM-family of receptor tyrosine kinases, in pediatric B- and T-cell ALL. Using shRNA-mediated MER knockdown, we also demonstrated anti-leukemia effects of MER inhibition in B- and T-ALL models, implicating MER as a novel therapeutic target. Here we report preclinical testing of a novel, first-in-class MER-selective small molecule tyrosine kinase inhibitor (UNC TKI) as a potential therapy for MER-expressing ALL. UNC TKI mediates potent inhibition of MER in enzymatic assays (IC50 = 0.74 nM), has ≥10-fold selectivity for MER over other TAM-family members, and has limited off-target activity against other tyrosine kinases, with the exception of FLT3. In 697 B-ALL cells, UNC TKI inhibited phosphorylation/activation of MER with an IC50 of 2.6 nM and decreased downstream signaling through the ERK and AKT pathways, leading to induction of apoptosis and reduced colony-formation in methylcellulose in MER-expressing ALL cell lines. In mouse models, UNC TKI is orally bioavailable and inhibits MER phosphorylation/activation in leukemic blasts in the bone marrow. In an orthotopic B-ALL xenograft model of minimal residual disease, treatment with UNC TKI resulted in a dose-dependent reduction in tumor burden and increased median survival from 27 days after inoculation with tumor cells to 70 days (p < 0.0001). In a similar model of existent disease in which leukemia was established for 14 days prior to initiation of treatment, median survival increased from 27.5 to 45 days in response to treatment with UNC TKI (p < 0.0001). In both models, tumor burden measured by bioluminescent imaging was significantly decreased in mice treated with UNC TKI relative to mice treated with vehicle, even after the development of advanced disease in the control animals. In addition, treatment with UNC TKI in combination with methotrexate, a chemotherapy that is currently in clinical use for treatment of pediatric ALL, resulted in reduced tumor burden and increased tumor-free survival relative to mice treated with either agent alone. The very high potency, relative selectivity, oral bioavailability, and demonstrated target inhibition and therapeutic efficacy in murine ALL models, both alone and in combination with chemotherapy, identify UNC TKI as an excellent candidate for clinical development in patients with MER-expressing ALL. Citation Format: Deborah A. DeRyckere, Amanda A. Hill, Xiaodong Wang, Weihe Zhang, Michael A. Stashko, Susan Sather, Christopher Cummings, Dmitri Kireev, William P. Janzen, Stephen V. Frye, H. Shelton Earp, Douglas K. Graham. Development of a novel small molecule MER tyrosine kinase inhibitor with therapeutic activity in cell culture and mouse models of acute lymphoblastic leukemia. [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 1740. doi:10.1158/1538-7445.AM2014-1740

The successes of targeted therapeutics against EGFR and ALK in non-small cell lung cancer (NSCLC) have demonstrated the substantial survival gains made possible by precision therapy. However, the majority of patients do not have tumors with genetic alterations responsive to these therapies, and therefore identification of new targets is needed. Our laboratory previously identified MER receptor tyrosine kinase as one such potential target. We now report our findings targeting MER with a clinically translatable agent - Mer590, a monoclonal antibody specific for MER. Mer590 rapidly and robustly reduced surface and total MER levels in multiple cell lines. Treatment reduced surface MER levels by 87%, and this effect was maximal within four hours. Total MER levels were also dramatically reduced, and this persisted for at least seven days. Mechanistically, MER down-regulation was mediated by receptor internalization and degradation, leading to inhibition of downstream signaling through STAT6, AKT, and ERK1/2. Functionally, this resulted in increased apoptosis, increased chemosensitivity to carboplatin, and decreased colony formation. In addition to carboplatin, Mer590 interacted cooperatively with shRNA-mediated MER inhibition to augment apoptosis. These data demonstrate that MER inhibition can be achieved with a monoclonal antibody in NSCLC. Optimization toward a clinically available anti-MER antibody is warranted.

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.

Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC50 = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development. (C) 2013 Elsevier Masson SAS. All rights reserved.

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.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although survival rates have improved, patients with certain biologic subtypes still have suboptimal outcomes. Current chemotherapeutic regimens are associated with short- and long-term toxicities and novel, less toxic therapeutic strategies are needed. Mer receptor tyrosine kinase is ectopically expressed in ALL patient samples and cell lines. Inhibition of Mer expression reduces prosurvival signaling, increases chemosensitivity, and delays development of leukemia in vivo, suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Brain and spinal tumors are the second most common malignancies in childhood. Multiple chemotherapy approaches and radiotherapies have been attempted, yet overall survival remains dismal. Mer is also abnormally expressed in atypical teratoid/rhabdoid tumors (AT/RT), providing a rationale for targeting Mer as a therapeutic strategy. We have previously described UNC569, the first small-molecule Mer inhibitor. This article describes the biochemical and biologic effects of UNC569 in ALL and AT/RT. UNC569 inhibited Mer activation and downstream signaling through ERK1/2 and AKT, determined by Western blot analysis. Treatment with UNC569 reduced proliferation/survival in liquid culture, decreased colony formation in methylcellulose/soft agar, and increased sensitivity to cytotoxic chemotherapies. MYC transgenic zebrafish with T-ALL were treated with UNC569 (4 μmol/L for two weeks). Fluorescence was quantified as indicator of the distribution of lymphoblasts, which express Mer and enhanced GFP. UNC569 induced more than 50% reduction in tumor burden compared with vehicle- and mock-treated fish. These data support further development of Mer inhibitors as effective therapies in ALL and AT/RT. Mol Cancer Ther; 12(11); 2367–77. ©2013 AACR.

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.

Abstract Background Growth Arrest Specific gene 6 (Gas6) is a ligand for the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases found on the surface of platelets. Previous studies have shown that stimulation of these receptors results in amplification of platelet activation and thrombus stabilization via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, leading to phosphorylation of the β3 integrin. Previous work (from our lab and others) demonstrated that inhibition of the Gas6/TAM pathway results in impaired platelet aggregation, reduced aggregate stability, and decreased platelet spreading. Additionally, knockout mice deficient in the receptor or ligand are protected from venous and arterial thrombosis, but retain normal tail bleeding times. Here, we describe development and characterization of novel Mer-selective small molecule inhibitors (SMIs) for thrombosis applications. Objectives To determine if Mer-selective SMIs can inhibit platelet aggregation and protect mice from thrombosis using in vitro and in vivo models Methods We used aggregometry and in vivo murine models of arterial and venous thrombosis to compare two Mer-selective SMIs (UNC Mer TKI1 and UNC Mer TKI2) and determine the most effective inhibitor of platelet aggregation and thrombus formation. The inhibitory effect of two doses (1µM and 5 µM) of the compounds were determined using standard light-transmission aggregometry after a 30 minute incubation with washed human platelets at 37 ¢ªC and compared to platelets treated with vehicle control or with a TKI control (UNC TKI Null), a SMI with similar structure but minimal anti-TAM activity. Both collagen/epinephrine-induced systemic venous thrombosis and FeCl3-induced carotid artery injury models were used to determine effects on thrombosis mediated by UNC TKIs. Wild type C57Bl/6 mice were treated with one of the two inhibitors and compared to mice treated with vehicle control. Mean values +/- SEM are shown and statistical significance (p<0.05) was determined using the student’s paired t-test. Results UNC Mer TKI1 exhibited more potent inhibition of platelet aggregation in vitro relative to UNC Mer TKI2, although both compounds mediated dose-dependent effects. At a concentration of 1uM, the maximum percent aggregation in UNC Mer TKI1-treated samples (n=7) was significantly greater than samples treated with UNC TKI Null (n=7), 20% DMSO vehicle (n=7), or UNC TKI2 (n=7), with mean values of 69 +/- 2.2%, 76.7 +/-1.8% (p<0.01), 76.9 +/- 2.1% (p=0.001), and 77 +/- 1.8% (p<0.001), respectively. At a concentration of 5 µM, UNC Mer TKI1-treated samples (n=7) exhibited a mean maximum percent aggregation of 23.7 +/- 2.4% compared to 50.4 +/- 4.8% for samples treated with UNC Mer TKI2 (n=7, p<0.001). UNC Mer TKIs also mediated protection from thrombus formation in mice. Following FeCl3 injury to the carotid artery, vehicle-treated mice (n=11) developed stable vessel occlusions with a mean time of 6.77 +/- 0.25 min. In contrast, stable occlusion occurred at a mean time of 46.6 +/- 7.72 min (n=9, p=0.001) for UNC Mer TKI1-treated mice. Survival times following venous injection of collagen and epinephrine were also significantly increased in mice treated with either UNC Mer TKI relative to the UNC TKI Null or vehicle controls. Mice pre-treated with UNC Mer TKI1 (n=9, p=0.04 compared to vehicle alone) or UNC Mer TKI2 (n=9, p=0.03 compared to vehicle alone) survived for 19.84 +/- 4.4 and 21.25 +/- 4.65 minutes, respectively. In contrast, mice given UNC TKI Null (n=3) or vehicle (n=21), only survived for 3.21 +/- 2.4 min and 3.09 +/- 0.22 minutes, respectively. Conclusion UNC Mer TKIs mediate dose-dependent inhibition of platelet aggregation and protect mice from arterial and venous thrombosis. Their pronounced activity compared to an inactive scaffold protein with minimal anti-TAM activity suggest that Gas6/TAM pathway inhibition is the mechanism of action for these novel compounds. UNC Mer TKI1 has more potent anti-thrombotic properties than UNC Mer TKI2. Disclosures: Branchford: University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Sather:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. DeRyckere:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Zhang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Liu:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Earp:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Wang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Frye:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Graham:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Di Paola:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties.