Mer Tyrosine Kinase Inhibitors Research Articles

P1.12A.09 A Phase 1b Study of the MER Tyrosine Kinase Inhibitor, MRX-2843, in Combination with Osimertinib in Advanced EGFR Mutant Non-Small Cell Lung Cancer

P1.12A.09 A Phase 1b Study of the MER Tyrosine Kinase Inhibitor, MRX-2843, in Combination with Osimertinib in Advanced EGFR Mutant Non-Small Cell Lung Cancer

Anti-inflammatory effects of MerTK by inducing M2 macrophage polarization via PI3K/Akt/GSK-3β pathway in gout

Mer tyrosine kinase (MerTK) has been found to regulate the secretion of inflammatory factors and exert immunosuppressive effects, but its role in gout remains unclear. In this study, we aimed to clarify the immnue effects of MerTK in gout. MerTK in synovium or serum of gout patients was determined by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and real-time quantitative polymerase chain reaction (RT-qPCR). In monosodium urate (MSU)-induced gout mice, the effect of MerTK inhibitor (UNC2250) on inflammation and polarization was also assessed. After inhibition, knockdown or overexpression of MerTK, inflammatory response and polarization level in THP1-derived macrophages were evaluated by RT-qPCR and flow cytometry. Regulation of MerTK inhibitors on mitochondrial function and downstream pathway in THP1-derived macrophages were detected. MerTK in synovium and serum of gout patients were increased. MerTK inhibitor stimulated the inflammation and M1 polarization in MSU-induced gout mice. MerTK inhibition, knock-down, or overexpression affected inflammatory response, polarization and mitochondrial function in vitro in gout model. The PI3K/Akt/GSK-3β pathway was identified to reduce after MerTK inhibition and the relevant results were as expected, validated by knock-down or overexpressing MerTK. In conclusion, MerTK was detected to increase in both gout patients and model. MerTK influenced inflammatory response and polarization markers through PI3K/Akt/GSK-3β pathway. Interfering MerTK/PI3K/Akt/GSK-3β axis may provide a new therapeutic target for gout.

HuoXueTongFu formula induces M2c macrophages via the MerTK/PI3K/AKT pathway to eliminate NETs in intraperitoneal adhesion in mice

Ethnopharmacological relevanceHuoXueTongFu Formula (HXTF) is a traditional Chinese herbal formula that has been used as a supplement and alternative therapy for intraperitoneal adhesion (IA). However, its specific mechanism of action has not been fully understood. Aim of the studyIn surgery, IA presents an inevitable challenge, significantly impacting patients' physical and mental well-being and increasing the financial burden. Our previous research has confirmed the preventive effects of HXTF on IA formation. However, the precise mechanism of its action still needs to be understood. MethodsIn this study, the IA model was successfully established by using the Ischemic buttons and treated with HXTF for one week with or without Mer Tyrosine Kinase (MerTK) inhibitor. We evaluated the pharmacodynamic effect of HXTF on IA mice. The MerTK/phosphoinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway-associated proteins were detected by Western blotting. Neutrophil extracellular traps (NETs) were detected by immunofluorescence. Macrophage phenotype was assessed by immunohistochemistry and flow cytometry. Inflammatory cytokines were detected by Real Time Quantitative PCR and Western blotting. ResultsHXTF reduced inflammatory response and alleviated IA. HXTF significantly enhanced MerTK expression, increased the number of M2c macrophages, and decreased the formation of NETs. In addition, the MerTK/PI3K/AKT pathway was significantly activated by HXTF. However, after using MerTK inhibitors, the role of HXTF in inducing M2c macrophage through activation of the PI3K/AKT pathway was suppressed and there was no inhibitory effect on NETs formation and inflammatory responses, resulting in diminished inhibition of adhesion. ConclusionHXTF may improve IA by activating the MerTK/PI3K/AKT pathway to induce M2c polarization, which removes excess NETs and attenuates the inflammatory response.

Effective Reaction-Based De Novo Strategy for Kinase Targets: A Case Study on MERTK Inhibitors.

Reaction-based de novo design is the computational generation of novel molecular structures by linking building blocks using reaction vectors derived from chemistry knowledge. In this work, we first adopted a recurrent neural network (RNN) model to generate three groups of building blocks with different functional groups and then constructed an in silico target-focused combinatorial library based on chemical reaction rules. Mer tyrosine kinase (MERTK) was used as a study case. Combined with a scaffold enrichment analysis, 15 novel MERTK inhibitors covering four scaffolds were achieved. Among them, compound 5a obtained an IC50 value of 53.4 nM against MERTK without any further optimization. The efficiency of hit identification could be significantly improved by shrinking the compound library with the fragment iterative optimization strategy and enriching the dominant scaffold in the hinge region. We hope that this strategy can provide new insights for accelerating the drug discovery process.

The Proto-Oncogene Mer Is a Novel Therapeutic Target in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is B-cell derived malignant tumor of the hematologic and lymphatic system, which is an incurable disease with most patients eventually relapsing and needing rescue therapy, so new therapeutic targets are emerging quickly nowadays. Mer tyrosine kinase (MerTK) is one of the TAM (Tyro-3, Axl, and MerTK) receptor tyrosine kinase family, and is often overexpressed or ectopic expressed in various malignant tumors, such as melanoma, gastric cancer, leukemia, lung cancer et.al, and is not expressed in normal B or T lymphocytes.To determine the role of MerTK in MCL, firstly, we analyzed MerTK expression in MCL cell lines by western-blot, and MerTK expression in MCL tumor tissues by Immunohistochemistry (IHC). Three (Z-138, Mino, JVM-2) of 6 (Z-138, Mino, JVM-2, Granta519, JVM-13, JeKo-1) MCL cell lines showed strong MerTK expression. Out of 132 MCL patients, 65 patients (48.9%) showed MerTK expression (positive percentage>10%). The correlation between clinical features and MerTK expression in MCL patients is currently under analysis.Lentivirus-mediated knockdown of MerTK in Z-138 and Mino cells decreased activation of AKT, ERK 1/2 and P38. Moreover, knockdown of MerTK resulted in inhibition of proliferation and invasion ability in Z-138, Mino and JVM-2 cells, as well as promoted chemosensitization in Z-138 and Mino cells. UNC2250, a MerTK-selective small molecular tyrosine kinase inhibitor, inhibited proliferation and invasion in Z-138, Mino and JVM-2 cells. In cell cycle analysis, UNC2250 induced apparently G2 phase arrest in Z-138, Mino and JVM-2 cells, and also induced polyploidy in JVM-2 cells. Furthermore, western-blot analysis revealed that the G2/M arrest was associated with decrease in pCdc2 and Cyclin B1 protein levels. Meanwhile, UNC2250 showed a dose- and time-dependent apoptotic death in Z-138, Mino and JVM-2 cells, which was associated with increase in Bax, cleaved forms of Caspase 3 and poly (ADP-ribose) polymerase (PARP) protein levels, and decrease in Bcl-2, Mcl-1 and Bcl-xL protein levels. UNC2250 also sensitized Z-138 and Mino cells to treatment with Vincristine and Doxorubicin, which may be a convincing evidence for UNC2250 or other new UNC2250-like MerTK inhibitor being applied in clinical therapy for MCL, allowing for dose reduction in combination with common cytotoxic chemotherapy. Further studies need to be completed in protein assay associated with MerTK inhibition in MCL cells, and the effect of UNC2250 in murine MCL xenograft models are ongoing.Our data proves that MerTK is highly expressed in MCL patients, and ectopic MerTK expression may be a novel therapeutic target in MCL. Our data also support further pre-clinical or even clinical research about UNC2250 or new MerTK inhibitor. DisclosuresNo relevant conflicts of interest to declare.

A critical role of the Gas6-Mer axis in endothelial dysfunction contributing to TA-TMA associated with GVHD

AbstractEndothelial dysfunction in the early phases of hematopoietic stem cell transplantation (HSCT) contributes to a common pathology between transplant-associated thrombotic microangiopathy (TA-TMA) and graft-versus-host disease (GVHD), which are serious complications of HSCT. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family: Tyro3, Axl, and Mer. Serum Gas6 levels were significantly increased in HSCT patients with grade II to IV acute GVHD (aGVHD), and Gas6 and Mer expression levels were upregulated in aGVHD lesions of the large intestine and skin. The increased serum Gas6 levels were also correlated with elevated lactate dehydrogenase, d-dimer, and plasmin inhibitor complex values in HSCT patients with aGVHD. In human umbilical vein endothelial cells (ECs), exogenous Gas6 or the exposure of sera isolated from patients with grade III aGVHD to ECs induced the downregulation of thrombomodulin and the upregulation of PAI-1, as well as the upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, which were inhibited by UNC2250, a selective Mer tyrosine kinase inhibitor. In mouse HSCT models, we observed hepatic GVHD with hepatocellular apoptosis, necrosis, and fibrosis, as well as TA-TMA, which is characterized pathologically by thrombosis formation in the microvasculature of the liver and kidney. Of note, intravenous administration of UNC2250 markedly suppressed GVHD and TA-TMA in these mouse HSCT models. Our findings suggest that the Gas6-Mer axis is a promising target for TA-TMA after GVHD.

A Critical Role of Growth Arrest-Specific Gene 6-Mer Axis in the Pathogenesis of Endothelial Damage Contributing to Thrombotic Microangiopathy Associated with Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Graft-versus-host disease (GVHD) and thrombotic microangiopathy (TMA) are severe complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Host endothelial cells are targets of alloreactive donor cytotoxic T lymphocytes or various proinflammatory cytokines following allo-SCT, and endothelial damage plays an important role in the pathogenic mechanism(s) of TMA associated with GVHD. However, the detailed mechanism(s) of TMA as well as GVHD have not yet been fully elucidated. Growth arrest-specific (Gas) 6 structurally belongs to the family of plasma vitamin K-dependent proteins working as a cofactor for activated protein C, and has growth factor-like properties through its interaction with receptor tyrosine kinases of the TAM family; Tyro3, Axl, and Mer. Gas6 and the TAM receptor tyrosine kinases have been reported to be associated with systemic inflammatory and autoimmune disorders, as well as hemostatic abnormalities. We hypothesized that Gas6-TAM pathway signaling contributed to endothelial dysfunction in the pathogenesis of TMA associated with GVHD. ELISA showed that the serum levels of Gas6 were markedly increased in the all-SCT patients with grades 2 or 3 GVHD at 21-35 days after stem-cell engraftment. The increased serum levels of Gas6 were also correlated with the elevated D-dimer and plasmin-α2 plasmin inhibitor complex values in the allo-SCT patients. Immunostaining showed that the expression of Gas6 and Mer was significantly upregulated in the intestinal acute GVHD lesion. The platelet aggregation test showed that UNC2250 (5 μM), a selective Mer tyrosine kinase inhibitor markedly suppressed increased platelet aggregation caused by exogenous Gas6 and collagen. Using Western blot analysis, exposure of human umbilical vein endothelial cells (ECs) to, Gas6 or 25-50% (vol/vol) serum isolated from patients with GVHD caused the downregulation of thrombomodulin (TM) and plasminogen activator inhibitor type-1 (PAI-1) as well as the upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as assessed by Western blot analyses, which were inhibited by UNC2250 or Mer siRNA. Furthermore, exogenous Gas6 or the addition of serum from patients with GVHD to ECs induced apoptosis of ECs with activation of caspase-3 and poly (ADP-ribose) polymerase, which were inhibited by UNC2250 or Mer siRNA. These results indicate that the Gas6-Mer axis may be crucial for platelet activation, endothelial damage and subsequent hypercoagulation in the pathogenesis of TMA associated with GVHD after allo-SCT. Furthermore, in the murine models of allo-SCT, we observed hepatic GVHD with hepatocellular apoptosis, necrosis and fibrosis and TMA characterized by thrombosis formation in the microvasculature of liver and kidney. The intravenous administration of 3 mg/kg UNC2250 markedly inhibited hepatic GVHD and TMA formations of liver and kidney in murine models of allo-SCT at 21 days after allo-SCT. The present study provides new evidence that the Gas6-Mer axis may be involved in the mechanism(s) underlying the progression of the pathogenesis of TMA associated with GVHD after all-SCT. We suggest that Gas6-Mer axis may be an attractive therapeutic target for treating TMA associated with GVHD caused by endothelial damage. DisclosuresNo relevant conflicts of interest to declare.

The proto-oncogene Mer tyrosine kinase is a novel therapeutic target in mantle cell lymphoma

BackgroundMantle cell lymphoma (MCL) is an incurable B cell-derived malignant tumor with a median overall survival of 4–5 years. Mer tyrosine kinase (MerTK) has been reported to be aberrantly expressed in leukemia, melanoma, and gastric cancer, and plays a pivotal role in the process of oncogenesis. This study assessed the role of MerTK in MCL for the first time.MethodsImmunohistochemistry and western blot were performed to figure out expression of MerTK in MCL. MerTK inhibition by either shRNA or treatment with UNC2250 (a MerTK-selective small molecular inhibitor) was conducted in MCL cell lines. MCL-cell-derived xenograft models were established to evaluate the anti-tumor effects of UNC2250 in vivo.ResultsMerTK was ectopically expressed in four of six MCL cell lines. Sixty-five of 132 (48.9%) MCL patients showed positive expression of MerTK. MerTK inhibition by either shRNA or treatment with UNC2250 decreased activation of downstream AKT and p38, inhibited proliferation and invasion in MCL cells, and sensitized MCL cells to treatment with vincristine in vitro and doxorubicin in vitro and in vivo. UNC2250 induced G2/M phase arrest and prompted apoptosis in MCL cells, accompanied by increased expression of Bax, cleaved caspase 3 and poly (ADP-ribose) polymerase, and decreased expression of Bcl-2, Mcl-1 and Bcl-xL. Moreover, UNC2250 delayed disease progression in MCL-cell-derived xenograft models.ConclusionsOur data prove that ectopic MerTK may be a novel therapeutic target in MCL, and further pre-clinical or even clinical studies of UNC2250 or new MerTK inhibitors are essential and of great significance.

MerTK inhibition by RXDX-106 in MerTK activated gastric cancer cell lines.

RXDX-106 is a potent and selective type II pseudo-irreversible (slow off-rate) inhibitor of TYRO3, AXL, MER and c-MET. MER tyrosine kinase (MerTK) is expressed in a variety of malignancies, including gastric cancer (GC). The oncogenic potential of MerTK is supported by various lines of evidence. First, we surveyed 10 GC cell lines for MerTK protein overexpression and MerTk phosphorylation. We next evaluated the change of downstream signaling molecules including (p)-ERK and (p)-AKT, following RXDX-106 treatment. We also investigated the effect of RXDX-106 in patient-derived cell lines to mimic the in vivo condition. The prevalence of MerTK protein overexpression was evaluated in 229 cancer tissue specimens. We have found that MerTK inhibitor treatment resulted in considerable inhibition of cell growth and downstream signaling. In addition, MerTK phosphorylation, not total MerTK expression, is likely more predictive of therapeutic success. p-MerTK protein overexpression by IHC was found in 18% (17/87) of GC patients. Lastly, RXDX-106 inhibited cell proliferation in MerTK activated gastric cancer cell line. These findings provide further evidence of oncogenic roles for MerTK in GC, and demonstrate the importance of kinase activity for MerTK tumorigeneicity and validate RXDX-106, a novel MerTK inhibitor, as a potential therapeutic agent for treatment of GC.

Design and Synthesis of Novel Macrocyclic Mer Tyrosine Kinase Inhibitors.

Mer tyrosine kinase (MerTK) is aberrantly elevated in various tumor cells and has a normal anti-inflammatory role in the innate immune system. Inhibition of MerTK may provide dual effects against these MerTK-expressing tumors through reducing cancer cell survival and redirecting the innate immune response. Recently, we have designed novel and potent macrocyclic pyrrolopyrimidines as MerTK inhibitors using a structure-based approach. The most active macrocycles had an EC50 below 40 nM in a cell-based MerTK phosphor-protein ELISA assay. The X-ray structure of macrocyclic analogue 3 complexed with MerTK was also resolved and demonstrated macrocycles binding in the ATP binding pocket of the MerTK protein as anticipated. In addition, the lead compound 16 (UNC3133) had a 1.6 h half-life and 16% oral bioavailability in a mouse PK study.

Synthesis, biological evaluation, and physicochemical property assessment of 4-substituted 2-phenylaminoquinazolines as Mer tyrosine kinase inhibitors

Current results identified 4-substituted 2-phenylaminoquinazoline compounds as novel Mer tyrosine kinase (Mer TK) inhibitors with a new scaffold. Twenty-one 2,4-disubstituted quinazolines (series 4–7) were designed, synthesized, and evaluated against Mer TK and a panel of human tumor cell lines aimed at exploring new Mer TK inhibitors as novel potential antitumor agents. A new lead, 4b, was discovered with a good balance between high potency (IC50 0.68μM) in the Mer TK assay and antiproliferative activity against MV4-11 (GI50 8.54μM), as well as other human tumor cell lines (GI50<20μM), and a desirable druglike property profile with low logP value (2.54) and high aqueous solubility (95.6μg/mL). Molecular modeling elucidated an expected binding mode of 4b with Mer TK and necessary interactions between them, thus supporting the hypothesis that Mer TK might be a biologic target of this kind of new active compound.

Synthesis, biological evaluation, and physicochemical property assessment of 4-substituted 2-phenylaminoquinazolines as Mer tyrosine kinase inhibitors

Synthesis, biological evaluation, and physicochemical property assessment of 4-substituted 2-phenylaminoquinazolines as Mer tyrosine kinase inhibitors

Design and Synthesis of Novel Macrocyclic Mer Tyrosine Kinase Inhibitors

Design and Synthesis of Novel Macrocyclic Mer Tyrosine Kinase Inhibitors

Abstract 1742: Inhibition of Mer tyrosine kinase with a novel small molecule inhibitor is efficacious in pre-clinical models of non-small cell lung cancer

Abstract Background: Mer tyrosine kinase is frequently overexpressed and activated in non-small cell lung cancer (NSCLC). In addition, genetic inhibition of Mer reduces NSCLC cell growth in vitro and tumor xenograft growth in vivo. In this study, we examined anti-tumor effects mediated by a first-in-class Mer-selective small molecule tyrosine kinase inhibitor (TKI) in pre-clinical models of NSCLC. Methods: The effects of Mer TKI treatment on activation of Mer and related members of the TAM-family of kinases, Axl and Tyro3, and effects on downstream proliferative and pro-survival signaling pathways were analyzed by immunoblot. In addition, Mer TKI-mediated anti-tumor activity was determined in a panel of NSCLC cell lines using soft-agar and clonogenic assays. Cells were stained with YoPro-1-iodide and propidium iodide dyes and induction of apoptosis was determined using flow cytometry. Finally, a subcutaneous murine xenograft model was employed to determine therapeutic effects in vivo. Results: The Mer TKI blocked Mer autophosphorylation in numerous cell lines at sub-micromolar concentrations and was highly selective for Mer over Axl and Tyro3. Treatment also inhibited downstream pro-survival signaling through the ERK1/2 and AKT pathways, which resulted in induction of apoptosis. Additionally, treatment reduced colony-forming potential in soft-agar and clonogenic assays by 85% to 99% in a large panel of cell lines. Sensitivity to the Mer TKI was independent of driver oncogene status, as cell lines positive for EGFR mutations, KRAS mutations, and gene fusions all responded to treatment. Interestingly, RNAi mediated knock-down of Axl enhanced sensitivity to Mer TKI treatment in biochemical and functional assays. Finally, in animals treatment decreased tumor progression resulting in a significant decrease in tumor volume. Conclusions: This Mer TKI is a novel and potent small molecule inhibitor that is selective for Mer over other TAM family kinases in cell-based assays. Treatment with this compound resulted in decreased downstream pro-oncogenic signaling, increased apoptosis, and decreased colony-forming potential in NSCLC cell lines. In addition, treatment was therapeutically effective in murine xenografts. Taken together, these data indicate that Mer inhibition may be an effective strategy for treatment of lung cancer. Sensitivity to the Mer TKI did not depend on driver oncogene status. Development of Mer TKIs for clinical application may therefore provide a molecularly-targeted treatment option for patients without known oncogenic mutations. In addition, Axl inhibition sensitized NSCLC cells to treatment with Mer TKI, suggesting a functional and/or physical interaction between Mer and Axl. In summary, the data presented here validate this Mer TKI as a potential treatment for NSCLC and provide critical data to support its continued development toward clinical application. Citation Format: Christopher T. Cummings, Kurtis D. Davies, Jacqueline Carrico, Deborah DeRyckere, Weihe Zhang, Xiaodong Wang, Stephen Frye, H. Shelton Earp, Douglas K. Graham. Inhibition of Mer tyrosine kinase with a novel small molecule inhibitor is efficacious in pre-clinical models of non-small cell lung cancer. [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 1742. doi:10.1158/1538-7445.AM2014-1742

Abstract 1736: A novel Mer tyrosine kinase inhibitor mediates increased cell killing in combination with FGFR inhibition

Abstract Purpose of Study: Although therapies targeting recently identified oncogenic drivers of non-small cell lung cancer (NSCLC) are in clinical use, a significant proportion of patients still lack a molecularly-targeted therapeutic option. Therefore, there is a continued need for development of new therapeutic strategies. We recently demonstrated oncogenic roles for Mer receptor tyrosine kinase in NSCLC. More specifically, we showed aberrant expression of Mer in approximately 70% of NSCLC patient samples compared to normal lung. Additionally, shRNA-mediated Mer inhibition resulted in increased cell death, decreased colony formation in clonogenic assays, increased chemosensitivity, and decreased tumorigenesis in murine xenografts. These data validate Mer as a potential therapeutic target in NSCLC. Fibroblast growth factor receptors (FGFR) are another class of tyrosine kinases that are aberrantly expressed and function to promote tumorigenesis in NSCLC. FGFR inhibition has been validated as a therapeutic strategy in preclinical NSCLC models and several FGFR inhibitors are currently in clinical development for treatment of NSCLC. Although Mer can signal through both the MEK/ERK and PI3K/AKT pathways, in NSCLC Mer signals mainly through PI3K/AKT. Because Mer and FGFR signal primarily through complementary pathways that mediate survival and proliferation (PI3K/AKT and MEK/ERK, respectively), we hypothesized that dual inhibition of FGFR and Mer may provide a therapeutic advantage relative to inhibition of either kinase alone. In this study we investigated the interaction between a novel Mer-selective small molecule tyrosine kinase inhibitor (TKI) and AZD-4547, an FGFR TKI, in NSCLC cell lines. Methods Used: Colo699 (Mer+, FGFR+) and H226 (Mer+, FGFR+) NSCLC cells were cultured for 14 days in soft agar in the presence of Mer TKI and/or AZD-4547, alone or in combination, and colonies were stained and counted. Changes in the activity of downstream signaling pathways, including PI3K/AKT, MEK/ERK, and STAT proteins were evaluated by immunoblotting. Results and Conclusions: In the soft agar assay, Colo699 and H226 colony formation was inhibited in the presence of Mer TKI and AZD-4547, both as single agents and in combination. Importantly, concurrent treatment with Mer TKI and AZD-4547 resulted in a greater decrease in colony-formation relative to either single agent. Immunoblotting revealed increased inhibition of pro-survival signaling in cells treated with both inhibitors relative to the single agents. Taken together, these data suggest that combination therapies targeting Mer kinase and FGFR may be effective for treatment of NSCLC and indicate biochemical mechanisms by which the combination therapy may mediate increased anti-tumor activity. Citation Format: Timothy P. Newton, Christopher T. Cummings, Gregory D. Kirkpatrick, Trista K. Hinz, Deborah DeRyckere, Weihe Zhang, Xiaodong Wang, Stephen Frye, H. Shelton Earp, Lynn Heasley, Douglas K. Graham. A novel Mer tyrosine kinase inhibitor mediates increased cell killing in combination with FGFR inhibition. [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 1736. doi:10.1158/1538-7445.AM2014-1736

Abstract 1740: 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 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 &amp;lt; 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 &amp;lt; 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

Abstract 947: A small molecule Mer tyrosine kinase inhibitor (UNC MerTKi) effectively inhibits growth of murine melanoma

Abstract The Mer Receptor Tyrosine Kinase (RTK) is overexpressed in hematologic and epithelial malignancies. Mer is not a proliferative driver but rather produces a cancer cell-intrinsic survival signal. In addition, tumor-associated macrophages (TAMs) express Mer, which upon binding ligand-associated with apoptotic material, triggers engulfment (effercytosis). The Mer signal also polarizes macrophages towards an M2-like, wound healing, anti-inflammatory state, calming the innate immune response when ingesting “self”. We have previously shown that syngeneic polyoma middle T (PyVmT) breast cancer cells demonstrate markedly attenuated growth when orthotopically implanted into Mer knock-out mice, with Mer-/- TAMs of PyVmT tumors producing M1-like inflammatory cytokines (JCI 123:3231, 2013). This result suggests that Mer inhibition could enhance innate anti-tumor responses, and toward that end, we examine the activity of first-in-class, orally bioavailable MerTK inhibitor on tumor growth in autochthonous murine tumor models. UNC MerTKi is 5-fold selective for Mer vs. Axl/Tyro3 and has favorable pharmacokinetics. Once daily, oral dosing inhibits the growth of Mer-expressing leukemia and NSCLC xenografts. UNC MerTKi was assessed in immune-competent, genetically engineered murine models (GEMMs) in the UNC Lineberger Mouse Phase 1 Unit (MP1U). After dose-finding studies in wild-type mice established an MTD, the inhibitor was given at 120 mpk/d in mouse chow, a dose which did not cause weight loss and produced a measurable effect (i.e. inhibition of second phase platelet aggregation, a known Mer pharmacodynamics marker). This dose did not exhibit single agent activity in a murine model of breast cancer (C3TAg), but exhibited pronounced single agent activity in RAS-driven, INK4a/Arf null melanoma GEMM (TRIA). The MP1U has previously reported the efficacy of 15 chemotherapeutic and/or targeted regimens in a large (&amp;gt;220) cohort of TRIA mice (CCR 18:5290, 2012). The overall response was 10% (partial responses and stable disease). There were no complete responses. A combination of MEK (AZD 6244) and PI3K/mTOR (BEZ235) inhibitors were the most active previous regimen (responses seen in 9/18 mice = 50%, with 0 CRs) with moderate toxicity. UNC MerTKi exhibited greater activity (6/8 mice = 75%, with 3 CRs) with mild, well-tolerated toxicity in the TRIA model. TRIA cell lines do not express Mer, suggesting that UNC MerTKi as a monotherapy may induce responses via Mer inhibition in TAMs and the tumor microenvironment, or via inhibition of Axl, Tyro or an unknown target. In summary, a potent and selective Mer inhibitor exhibited greater pre-clinical efficacy in a highly faithful model of RAS-mutant melanoma than any other drug tested to date, including several compounds that are FDA approved for use in metastatic melanoma. Citation Format: H Shelton Earp, David Darr, Albert Zimmermann, Kelly Clark, Norman E. Sharpless, Wolfgang Bergmeier, Weihe Zhang, Xiaodong Wang, Deborah DeRyckere, Stephen Frye, Douglas Graham. A small molecule Mer tyrosine kinase inhibitor (UNC MerTKi) effectively inhibits growth of murine melanoma. [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 947. doi:10.1158/1538-7445.AM2014-947

UNC2025, a potent and orally bioavailable MER/FLT3 dual inhibitor.

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.

UNC569, a novel small-molecule mer inhibitor with efficacy against acute lymphoblastic leukemia in vitro and in vivo.

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.

Evaluation of UNC569, a Novel Small Molecule Mer Inhibitor for the Treatment of ALL in Vitro and in Vivo.

Abstract 2607 Introduction:Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Current chemotherapeutic regimens are associated with short- and long-term toxicities. Therefore, novel, less toxic therapies are needed. Mer receptor tyrosine kinase (RTK) is ectopically expressed in ALL cell lines and patient samples. Inhibition of Mer expression reduces pro-survival signaling, increases chemosensitivity, and delays development of leukemia in vivo. Mer tyrosine kinase inhibitors (TKIs) are excellent candidates for targeted therapies. We report here the first small molecule inhibitor for Mer RTK (UNC569) and demonstrate efficacy as a novel strategy in the treatment of ALL. Methods:UNC569 is a substituted pyrazolopyrimidine. Inhibition of Mer kinase activity by UNC569 was determined by a microfluidic capillary electrophoresis assay. Western blot analysis was used to determine inhibition of phospho-Mer and downstream signaling by UNC569 in 697 (B-ALL) and Jurkat (T-ALL) cells. UNC569-mediated anti-leukemia activity was determined in short- (MTT) and long-term (colony-formation) assays. Diagnostic bone marrow or peripheral blood samples were obtained at the Children’s Hospital Colorado. We established a luciferase expressing ALL xenograft model in NOD scid gamma (NSG) mice to evaluate the effects of UNC569 on leukemia progression. NSG mice were transplanted with luciferase-tagged 697 cells and treated for three weeks with an orally bioavailable UNC569 formulation (15 mg/kg body weight, qd). Response was monitored twice weekly using the IVIS200 Imaging System. To examine effects of UNC569 on leukemia progression in a second model, we used transgenic zebrafish ectopically expressing human MYC. These fish develop T-cell malignancy at high penetrance. Cancers in these fish are labeled by T-lymphocyte-specific enhanced green fluorescent protein (EGFP), allowing measurement of treatment responses. Leukemic adult zebrafish were treated over three weeks with 3 μM or over 2 weeks with 4 μM UNC569. Tumor responses were monitored weekly using an Olympus MVX10 Imaging System. Results:UNC569 is a novel Mer TKI with potent activity against Mer RTK (IC50 = 2.9 nM). In cell-based assays, UNC569 inhibited accumulation of phospho-Mer in ALL cell lines (697 IC50 = 141±15 nM, Jurkat IC50 = 193±56 nM). Treatment with UNC569 resulted in inhibition of phosphorylation of Erk1/2 and Akt. Reduced proliferation/survival was observed in ALL cells treated with UNC569 (697 IC50 = 0.5±0.1 μM, Jurkat IC50 = 1.2±0.2 μM). Treatment with UNC569 also resulted in a statistically significant, dose-dependent decrease in colony-formation compared to control cultures in Jurkat (100.1±23.4 vs 25.6±6.4 colonies, p=0.04, n=3) and 697 cells (95.9±16.8 vs 14.8±12.8 colonies, p=0.02, n=3). Similarly, treatment with UNC569 reduced colony formation in methylcellulose compared to control cultures in one of three Mer RTK positive primary ALL patient samples (270.1±18.9 vs 134.0±6.4 colonies). No significant reduction of colony formation was observed in five Mer negative primary ALL patient samples. NSG mice transplanted with luciferase-expressing 697 B-ALL cells and treated with UNC569 (15 mg/kg/d) had significantly decreased leukemia burden compared to vehicle-treated control mice as measured by bioluminescence imaging (86.1×106±19.2×106 photons/second (n=15) vs 29.6×106±9.0×106 photons/second (n=10), p=0.04). Similarly, in the zebrafish T-ALL model, responses to UNC569 treatment (defined as >25% decrease in disease burden) were observed in 84% of animals treated with 4 μM UNC569 for 14 days (n = 16/19) and 77% of animals treated with 3 μM UNC569 for 21 days (n = 7/9). Conclusion:UNC569 is an effective Mer TKI that inhibits activation of Mer in ALL cells, mediates anti-leukemia activity against ALL cells in culture, and decreases colony-formation in methylcellulose. In addition, UNC569 has an anti-leukemia effect in primary ALL patient samples that express Mer protein, in a murine xenograft B-ALL model, and in a transgenic zebrafish model. Taken together, these data support further development of Mer TKI as a novel and effective ALL therapy. Disclosures:Wang:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Kireev:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Liu:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Yang:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties. Frye:WO: Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011, Pyrazolopyrimidine Compounds for the Treatment of Cancer. WO Patent 2011146313, 2011 Patents & Royalties.