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High-grade gliomas (HGG) afflict both children and adults and respond poorly to current therapies. Epigenetic regulators have a role in gliomagenesis, but a broad, functional investigation of the impact and role of specific epigenetic targets has not been undertaken. Using a two-step, in vitro/in vivo epigenomic shRNA inhibition screen, we determine the chromatin remodeler BPTF to be a key regulator of adult HGG growth. We then demonstrate that BPTF knockdown decreases HGG growth in multiple pediatric HGG models as well. BPTF appears to regulate tumor growth through cell self-renewal maintenance, and BPTF knockdown leads these glial tumors toward more neuronal characteristics. BPTF’s impact on growth is mediated through positive effects on expression of MYC and MYC pathway targets. HDAC inhibitors synergize with BPTF knockdown against HGG growth. BPTF inhibition is a promising strategy to combat HGG through epigenetic regulation of the MYC oncogenic pathway.

Simple Summary Expression of MERTK and/or AXL (members of the TAM family of receptor tyrosine kinases) provides a survival advantage for non-small cell lung cancer (NSCLC) cells and correlates with lymph node metastasis, drug resistance, and disease progression. TAM receptors on host tumor infiltrating cells also play important roles in the immunosuppressive tumor microenvironment. Thus, MERTK and AXL are attractive biologic targets for NSCLC treatment, and clinical trials have recently been launched exploring the efficacy of MERTK/AXL inhibitors in NSCLC. This timely review will address the potential clinical impact of these agents as well as potential side effects to be monitored with the use of these novel drugs. Abstract MERTK and AXL are members of the TAM family of receptor tyrosine kinases and are abnormally expressed in 69% and 93% of non-small cell lung cancers (NSCLCs), respectively. Expression of MERTK and/or AXL provides a survival advantage for NSCLC cells and correlates with lymph node metastasis, drug resistance, and disease progression in patients with NSCLC. The TAM receptors on host tumor infiltrating cells also play important roles in the immunosuppressive tumor microenvironment. Thus, MERTK and AXL are attractive biologic targets for NSCLC treatment. Here, we will review physiologic and oncologic roles for MERTK and AXL with an emphasis on the potential to target these kinases in NSCLCs with activating EGFR mutations.

Molecularly-targeted agents have improved outcomes for a subset of patients with BRAF-mutated melanoma, but treatment of resistant and BRAF wild-type tumors remains a challenge. The MERTK receptor tyrosine kinase is aberrantly expressed in melanoma and can contribute to oncogenic phenotypes. Here we report the effect of treatment with a MERTK-selective small molecule inhibitor, UNC2025, in preclinical models of melanoma. In melanoma cell lines, treatment with UNC2025 potently inhibited phosphorylation of MERTK and downstream signaling, induced cell death, and decreased colony formation. In patient-derived melanoma xenograft models, treatment with UNC2025 blocked or significantly reduced tumor growth. Importantly, UNC2025 had similar biochemical and functional effects in both BRAF-mutated and BRAF wild-type models and irrespective of NRAS mutational status, implicating MERTK inhibition as a potential therapeutic strategy in tumors that are not amenable to BRAF-targeting and for which there are limited treatment options. In BRAF-mutated cell lines, combined treatment with UNC2025 and the BRAF inhibitor vemurafenib provided effective inhibition of oncogenic signaling through ERK, AKT, and STAT6, increased induction of cell death, and decreased colony-forming potential. Similarly, in NRAS-mutated cell lines, addition of UNC2025 to cobimetinib therapy increased cell death and decreased colony-forming potential. In a BRAF-mutated patient-derived xenograft, treatment with combined UNC2025 and vemurafenib was well-tolerated and significantly decreased tumor growth compared with vemurafenib alone. These data support the use of UNC2025 for treatment of melanoma, irrespective of BRAF or NRAS mutational status, and suggest a role for MERTK and targeted combination therapy in BRAF and NRAS-mutated melanoma.

MERTK is ectopically expressed and promotes survival in acute lymphoblastic leukemia (ALL) cells and is thus a potential therapeutic target. Here we demonstrate both direct therapeutic effects of MERTK inhibition on leukemia cells and induction of anti-leukemia immunity via suppression of the coinhibitory PD-1 axis. A MERTK-selective tyrosine kinase inhibitor, MRX-2843, mediated therapeutic anti-leukemia effects in immunocompromised mice bearing a MERTK-expressing human leukemia xenograft. In addition, inhibition of host MERTK by genetic deletion (Mertk-/- mice) or treatment with MRX-2843 significantly decreased tumor burden and prolonged survival in immune-competent mice inoculated with a MERTK-negative ALL, suggesting immune-mediated therapeutic activity. In this context, MERTK inhibition led to significant decreases in expression of the coinhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the leukemia microenvironment. Furthermore, although T cells do not express MERTK, inhibition of MERTK indirectly decreased PD-1 expression on CD4+ and CD8+ T cells and decreased the incidence of splenic FOXP3+ Tregs at sites of leukemic infiltration, leading to increased T cell activation. These data demonstrate direct and immune-mediated therapeutic activities in response to MERTK inhibition in ALL models and provide validation of a translational agent targeting MERTK for modulation of tumor immunity.

The receptor tyrosine kinase MERTK is aberrantly expressed in numerous human malignancies, and is a novel target in cancer therapeutics. Physiologic roles of MERTK include regulation of tissue homeostasis and repair, innate immune control, and platelet aggregation. However, aberrant expression in a wide range of liquid and solid malignancies promotes neoplasia via growth factor independence, cell cycle progression, proliferation and tumor growth, resistance to apoptosis, and promotion of tumor metastases. Additionally, MERTK signaling contributes to an immunosuppressive tumor microenvironment via induction of an anti-inflammatory cytokine profile and regulation of the PD-1 axis, as well as regulation of macrophage, myeloid-derived suppressor cell, natural killer cell and T cell functions. Various MERTK-directed therapies are in preclinical development, and clinical trials are underway. In this review we discuss MERTK inhibition as an emerging strategy for cancer therapy, focusing on MERTK expression and function in neoplasia and its role in mediating resistance to cytotoxic and targeted therapies as well as in suppressing anti-tumor immunity. Additionally, we review preclinical and clinical pharmacological strategies to target MERTK.

Introduction Despite recent improvement in outcomes for de novo disease, pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains challenging to treat at relapse. Investigation into genomic markers of treatment response and therapy resistance offers an opportunity to further enhance outcomes for these patients. We previously identified a T-ALL blast-associated gene signature at diagnosis (Dx) and characterized the immune microenvironment in Dx T-ALL marrow samples using single cell transcriptome analysis (Bhasin et al. Blood 2020(ASH)). This approach allowed us to generate a granular expression map of both the T-ALL landscape and the Dx bone marrow (BM) immune microenvironment. Here we expand this work by evaluating samples collected from the same patients Dx and End of Induction (EOI) BM samples from pediatric T-ALL patients. The use of paired samples provides insight into treatment-induced changes in the microenvironment. Further, the inclusion of both minimal residual disease (MRD) positive and MRD negative samples allowed us to compare differences between these groups. Methods Using the 10X genomics platform, we profiled the single cell transcriptome of ~18,000 BM and immune microenvironment cells from viably frozen samples collected from T-ALL patients at Dx or EOI. Five paired Dx and EOI samples and one EOI sample from a patient with relapsed T-ALL were evaluated, for a total of 11 samples. Three paired samples were MRD positive at EOI and two were MRD negative; the relapsed sample was MRD negative. Cell clustering was performed using the Seurat package and differential expression analysis was performed using R/Bioconductor packages (Hao et al. Cell 2021). Cell communication analysis was conducted using the CellChat R tool (v 1.0.0) to infer cell-cell communication within the EOI MRD positive and MRD negative subsets and compare their communication networks (Jin et al. Nature Comm 2021). Results Using our previously described blast-associated gene signature (Bhasin et al. ASH 2020) we were able to identify residual blast populations at EOI in MRD-positive samples. Comparative analysis of gene profiles at Dx and EOI showed significant changes in the microenvironment cell populations with highest increase in erythroid cell populations after induction therapy. The gene expression profiles were significantly different for immune cells at Dx and EOI and the relapsed sample had greater similarity to the Dx samples indicating a persistent immunosuppressive environment. Clustering analysis of the EOI samples (3 MRD positive and 2 MRD negative) demonstrated the presence of patient specific blast cells in MRD positive samples that retained patient-specific transcriptomeheterogeneity at EOI (Fig.1A). Analysis of communication networks between different cell types based on receptor and ligand expression levels between different cell types identified a CD34 + cluster of stem cells that had different interactions with other immune populations in the MRD positive and negative subsets. Differential expression analysis between the MRD positive and MRD negative cells in this CD34 + stem cell cluster identified higher expression of myeloid associated genes such as CEBPB, CEBPD, AZU1 in the MRD negative group relative to the MRD positive cells, which showed higher expression of B-cell related genes such as IGHM, VPREB1, CD79A/ B along with upregulation of P13K signaling in B-lymphocytes, B-cell receptor signaling and autophagy pathways. Analysis of upstream regulators based on the differential gene signature between the MRD positive and MRD negative group demonstrated upregulation of MYC and TCF3 activity and inhibition of TGFB1, CSF3 and CEBPA in MRD positive compared to MRD negative samples (Fig.1B). Conclusions: Leukemic blasts exhibit patient-specific gene expression signatures that are present at EOI in MRD positive samples. Exploration of the impact of minimal residual disease at EOI revealed differential gene expression patterns in stem cells from MRD positive samples, characterized by activation of B cell related signaling pathways and regulators such as MYC and TCF3. In contrast, a more myeloid-like expression signature was observed in stem cells from MRD negative samples. These findings open the avenues for exploration of therapeutic targets of T-ALL progression. Figure 1 Figure 1. Disclosures DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Introduction: Pediatric mixed phenotype acute leukemia (MPAL), a rare subgroup of leukemia, contains features of both myeloid and lymphoid lineage blasts, which makes the disease more difficult to diagnose/treat. More information is needed to understand the origins of the major pediatric MPAL subtypes, B/Myeloid (B-MPAL) and T/Myeloid (T-MPAL), and how they relate to other leukemias. Single-cell RNA sequencing (scRNA-seq) analysis of bone marrow (BM) can provide in-depth information about the leukemia microenvironment and reveal differences/similarities between MPAL subtypes and other types of leukemia that could be exploited to develop novel diagnostics/therapies. Methods: We analyzed ~16,000 cells from five pediatric MPAL BM samples to generate a transcriptomic landscape of B-MPAL and T-MPAL blasts and associated microenvironment cells. Samples collected at the time of diagnosis (Dx) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy et al. JCI 2019, Tellechea et al. JID, 2020). After data normalization, cell clusters were identified using principal component analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al. Nat. Biotech 2018). Meta-analysis was performed using single cell samples from ongoing studies in the Bhasin lab (Bhasin, et al. Blood 2020 (ASH), Thomas et al. Blood 2020 (ASH)) and publicly available single cell data from GEO biorepository. Unsupervised analysis using UMAP and PCA was performed to determine the overall relationship among B-MPAL, T-MPAL and other leukemias (acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell ALL (T-ALL)). Supervised differentially expressed gene (DEG) analysis was performed to identify B- and T- MPAL blast cell signatures (P value < 0.001 and log2 FC > 0.5). Transcriptomic profiles in MPAL samples and normal BM stem and immune cells were compared using data from the Human Cell Atlas Data Portal (humancellatlas.org). Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes (P value < 0.001). Results: PCA analysis showed transcriptome similarity between B-MPAL and both B-ALL and AML, while T-MPAL transcriptome correlated with T-ALL and AML (Fig. 1A). B- and T-MPAL subtype blasts clustered separately from each other in UMAP analyses, with T-MPAL blasts clustering with T-ALL blasts, and B-MPAL somewhat overlapping with B-ALL blasts. Subtype DEG analysis of leukemia blasts and healthy BM revealed distinct significantly upregulated gene signatures in B-MPAL (YBX3, SOCS2, BCL11A, and HIST1H1C) and T-MPAL (ITM2A, HPGD, PDLIM1, and TRDC) blasts (Fig. 1B). Pathway analysis showed upregulated gene activity related to TNFA signaling via NFKB, B-cell survival, and the AP1, FRA, and NGF transcription factors in B-MPAL blasts. In contrast, IL-17 and IL-12, T-cell apoptosis, and Stathmin pathways were upregulated in T-MPAL blasts (Fig. 1C). T-MPAL T-cells also expressed higher levels of T-cell exhaustion markers compared to T-cells in B-MPAL samples and healthy bone marrow. After filtering out genes that are significantly expressed in immune cells, we identified genes that are differentially expressed at diagnosis in MPAL blasts from patients that relapsed after treatment (Dx1) versus patients that achieved remission (Dx2). These genes are potential prognostic markers for B-MPAL and T-MPAL relapse/remission. These include MDM2 and NEIL1 from Dx1 and FOSL2 and CDKN1A in Dx2 B-MPAL blasts. In T-MPAL, expression of HES4 and SPINK2 is associated with Dx1 blasts and GNAQ and ITGA4 with Dx2 blasts. Pathway enrichment analysis on B-MPAL blasts revealed upregulation of interferon gamma and PD-1 signaling in Dx1 samples and increased HSP27 and Cell Cycle pathways in the Dx2 subset. T-MPAL Dx1 associated pathways included prostaglandin synthesis and IL-17, while cell-cell junction and extracellular matrix interactions were increased in T-MPAL Dx2 samples (Fig. 1D). Conclusion: Single-cell profiling was used to characterize the molecular landscapes of MPAL blasts and the bone marrow microenvironment and identified gene signatures and pathways that are specifically enriched in B- and T-MPAL subtypes. Figure 1 Figure 1. Disclosures DeRyckere: Meryx: Other: Equity ownership. Graham: Meryx: Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership.

Inhibition of MER receptor tyrosine kinase (MERTK) causes direct tumor cell killing and stimulation of the innate immune response. Therefore, MERTK has been identified as a therapeutic target in a wide variety of human tumors. Clinical trials targeting MERTK have recently been initiated, however, none of these drugs are MERTK-specific. Herein, we present the discovery of a highly MERTK-selective inhibitor UNC5293 (24). UNC5293 has subnanomolar activity against MERTK with an excellent Ambit selectivity score (S50 (100 nM) = 0.041). It mediated potent and selective inhibition of MERTK in cell-based assays. Furthermore, it has excellent mouse PK properties (7.8 h half-life and 58% oral bioavailability) and was active in bone marrow leukemia cells in a murine model.

Introduction: While advances in front-line conventional chemotherapy have increased the likelihood of attaining remission in pediatric AML, relapse rates remain high (25-35%), and novel therapies are needed (Zhang, Savage et al. 2019). The clinical and molecular heterogeneity of AML makes it complex to study and creates challenges for the development of novel therapies (Bolouri, Farrar et al. 2018). It is important to identify cells and pathways underlying relapse to facilitate development of novel therapies. Single-cell RNA Sequencing (scRNA-Seq) allows in-depth analysis of the heterogeneous AML landscape to provide a detailed view of the tumor microenvironment, revealing populations of blasts and immune cells which may be relevant to relapse or complete remission. Methods: We analyzed ~36,500 cells from 14 pediatric AML bone marrow samples in our institutional biorepository, spanning different AML subtypes and 3 healthy children to generate a comprehensive scRNA-Seq landscape of immature AML-associated blasts and microenvironment cells. Samples collected at the time of diagnosis (Dx), end of induction (EOI), and relapse (Rel) were used to generate scRNA-Seq data using a droplet-based barcoding technique (Panigrahy, Gartung et al. 2019). After normalization of scRNA-Seq data, the cell clusters were identified using principal component analysis and Uniform Manifold Approximation and Projection (UMAP) approach (Becht et al, 2018). Differential expression, pathways and systems biology analysis between relapsed and remission patients reveal differences for specific cell clusters (Panigrahy, Gartung et al. 2019). To determine the clinical outcome association of our AML blast specific markers, survival analysis was performed on AML TARGET data (https://ocg.cancer.gov/programs/target) using cox proportional hazard survival approach. To characterize AML blast cells with high accuracy, we used support vector machine (SVM), an Artificial Intelligence based feature extraction and model development approach (Bhasin, Ndebele et al. 2016). Results:ScRNA-Seq analysis of paired Dx and EOI samples using UMAP identified three blast cell clusters with significant gene expression differences among different patients, indicating heterogeneity of AML blast cells (Fig 1a, b). Comparative analysis of the three Dx enriched blast cell clusters with other cells identified a "core blast cell signature" with overexpression of genes like AZU1, CLEC11A, FLT3, and NREP (Fig 1c). These core AML-blast genes were linked to significant activation of the Wnt/Ca2+, Phospholipase C, and integrin signaling pathways (Z score >2 and P-value The scRNA-Seq of AML specific blast cells from relapsed and remission samples exhibited a different clustering pattern indicating different transcriptome landscapes. Relapse-associated AML cell clusters expressed high levels of AZU1, S100A4, LGALS1, and GRK2 genes (Fig 2a). Analysis of the non-AML tumor microenvironment demonstrated enrichment of T/NK in relapsed samples, with differential expression of T cell regulatory/activation genes (Fig 2b, c). ScRNA-Seq showed enrichment of monocyte/macrophage cell clusters in remission samples with distinct relapse- and remission-specific clusters. Remission associated macrophage/monocyte clusters showed overexpression of S100A10, FTH1, CST3 and IFITM2 genes (Fig 2d). Similarly, enrichment of T cell and monocyte/macrophage clusters was observed in relapse and remission samples respectively during EOI. Conclusions: Using single cell transcriptomics we developed a novel potential gene signature to characterize heterogenous AML blast populations with high sensitivity. These genes and the pathways they regulate implicate potential therapeutic targets in pediatric AML. Single cell transcriptome analysis also enabled identification of cell clusters with modulated gene expression at both Dx and EOI that may be useful in predicting relapse/remission. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is characterized by proliferation of immature T-cells and accounts for ~15% of pediatric ALL. T-ALL blasts are phenotypically diverse and are sub-classified into pro-, pre-, cortical and mature T-ALL based on the stage of differentiation of the leukemic clone. Early T precursor ALL (ETP-ALL) is a T-ALL subtype associated with higher risk of relapse (Raetz and Teachey, 2016). Bulk sequencing approaches have revealed valuable information about modulated genes in T-ALL; however, little is understood about the interplay between tumor cells and the immune microenvironment. We present a comprehensive single cell RNA sequencing (scRNASeq) analysis of T-ALL samples with the purpose of characterizing the heterogenous tumor and microenvironment cells in order to identify dysregulated genes in leukemia cells and investigate oncogenic signaling pathways. Methods: We profiled 16,280 cells from 5 diagnostic pediatric T-ALL bone marrow samples using the Chromium single cell transcriptomics platform (10x Genomics, Pleasanton, CA). To compare T-ALL versus healthy bone marrow profiles and identify T-ALL-specific malignant blast cell populations, we included data from 3 healthy pediatric bone marrow samples from a recent study (Caron et al, 2020). Dimension reduction using the Uniform Manifold Approximation and Projection (UMAP) approach was used to identify unique cell type clusters (Becht et al, 2018). Using the TARGET dataset (https://ocg.cancer.gov/programs/target), we further evaluated the prognostic significance of identified malignant blast-specific genes by performing Kaplan-Meier survival analysis and compared gene expression patterns and tumor microenvironment makeup between ETP-ALL and non-ETP T-ALL patient samples. Results: We successfully characterized leukemic blasts (CD7+, CD99+ and CD3D+) and other major immune cell types (T cells, B cells, monocytes, erythroid precursors) using the expression of established marker genes (Fig 1A, C). Clustering analysis revealed patient-specific leukemia blast cell clusters (Fig 1B). Differential expression analysis between CD3D+ patient-specific leukemia clusters and CD3D+ clusters comprised of normal T-cells identified a set of 385 promiscuous genes that are significantly differentially expressed between malignant and normal T-cells (p-value 1.3, p-value Conclusions: We identified a gene signature characterizing heterogenous T-ALL blast populations. External validation using the TARGET dataset identified genes within the signature that are associated with poor outcomes in T-ALL. Differences in the composition of the immune microenvironment in ETP-ALL versus non-ETP T-ALL samples provide a promising area for future study. Further studies will be carried out with relapsed and non-relapsed T-ALL samples to validate this leukemia signature. Disclosures Bhasin: Canomiiks Inc: Current equity holder in private company, Other: Co-Founder.

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.

The TAM family (TYRO3, AXL, MERTK) tyrosine kinases play roles in diverse biological processes including immune regulation, clearance of apoptotic cells, platelet aggregation, and cell proliferation, survival, and migration. While AXL and MERTK have been extensively studied, less is known about TYRO3. Recent studies revealed roles for TYRO3 in cancer and suggest TYRO3 as a therapeutic target in this context. TYRO3 is overexpressed in many types of cancer and functions to promote tumor cell survival and/or proliferation, metastasis, and resistance to chemotherapy. In addition, higher levels of TYRO3 expression have been associated with decreased overall survival in patients with colorectal, hepatocellular, and breast cancers. Here we review the physiological roles for TYRO3 and its expression and functions in cancer cells and the tumor microenvironment, with emphasis on the signaling pathways that are regulated downstream of TYRO3 and emerging roles for TYRO3 in the immune system. Translational agents that target TYRO3 are also described.

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.

Osimertinib (OSI) was recently FDA-approved as a front-line agent for newly diagnosed EGFRMT non-small cell lung cancer (NSCLC). However, unmet clinical needs have arisen in conjunction with OSI use, including understanding mechanisms of OSI resistance and developing novel approaches to prevent or reverse resistance and/or enhance OSI efficacy in responsive patients. To address these issues, osimertinib-resistant (osiR) derivatives of five EGFRMT NSCLC cell lines were generated and roles for MERTK, a receptor tyrosine kinase that has been implicated as a potential therapeutic target in NSCLC, were characterized. PI3K-AKT and MAPK-ERK signaling pathways were activated in osiR cells, even when EGFR was not active. Treatment with the MERTK ligands GAS6 or PROS1 stimulated AKT, ERK, and ribosomal S6 phosphorylation in parental cells treated with OSI and in osiR cells, implicating MERTK as a mediator of resistance to OSI. Downstream signaling was responsive to both EGF and GAS6 stimulation in parental cells but was only activated by GAS6 in osiR cells. OSI blocked EGF-dependent signaling through AKT, ERK and S6 in parental cells in the absence of GAS6, but combined treatment with OSI and MRX-2843, a novel MERTK inhibitor currently in Phase I clinical trials, was required to block signaling in the presence of GAS6. However, treatment with MRX-2843 alone had little impact on downstream signaling in the presence of activated EGFR. Thus, MERTK is not the dominant driver of downstream signaling in parental cells. In contrast, treatment with MRX-2843 alone was sufficient to inhibit downstream signaling in osiR cells and osiR cells were also more sensitive to treatment with MRX-2843 in clonogenic assays. Thus, osiR cells have increased dependence on MERTK kinase activity relative to parental cells. Interestingly, EGFR and MERTK co-precipitated from parental cell lysates and GAS6 stimulation enhanced this interaction. In contrast, MERTK and EGFR interaction was not detected in osiR cells, suggesting a more complex interplay between these two receptors. MERTK and the ligand PROS1 were dramatically upregulated in EGFRMT tumors treated with OSI in vivo, consistent with a role for autocrine MERTK activation in osiR tumor growth. Indeed, treatment with OSI alone or in combination with MRX-2843 was sufficient to block tumor growth in vivo, but when treatment was stopped, tumors treated with OSI alone started to grow, while treatment with the combination resulted in durable suppression of tumor growth. Together these data implicate MERTK as a mediator of resistance to OSI and suggest that combining MRX-2843 and OSI therapy will control tumor growth. Citation Format: Dan Yan, Justus Huelse, Rebecca Parker, Zikang Tan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK drives residual tumor growth in EGFR-mutated non-small cell lung cancer cells treated with osimertinib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1882.

The original version of this Article omitted the following from the Acknowledgements: This work was supported by the Luke's Army Pediatric Cancer Research Fund St. Baldrick's Scholar Award. This has now been corrected in both the PDF and HTML versions of the Article.

The TAM (TYRO3, AXL, MERTK) family receptor tyrosine kinases (RTK) play an important role in promoting growth, survival, and metastatic spread of several tumor types. AXL and MERTK are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non–small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. AXL is the most well-characterized TAM receptor and mediates resistance to both conventional and targeted cancer therapies. AXL is highly expressed in aggressive tumor types, and patients with cancer are currently being enrolled in clinical trials testing AXL inhibitors. In this study, we analyzed the effects of AXL inhibition using a small-molecule AXL inhibitor, a monoclonal antibody (mAb), and siRNA in HNSCC, TNBC, and NSCLC preclinical models. Anti-AXL–targeting strategies had limited efficacy across these different models that, our data suggest, could be attributed to upregulation of MERTK. MERTK expression was increased in cell lines and patient-derived xenografts treated with AXL inhibitors and inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibition. Dual targeting of AXL and MERTK led to a more potent blockade of downstream signaling, synergistic inhibition of tumor cell expansion in culture, and reduced tumor growth in vivo. Furthermore, ectopic overexpression of MERTK in AXL inhibitor–sensitive models resulted in resistance to AXL-targeting strategies. These observations suggest that therapeutic strategies cotargeting both AXL and MERTK could be highly beneficial in a variety of tumor types where both receptors are expressed, leading to improved survival for patients with lethal malignancies. Mol Cancer Ther; 17(11); 2297–308. ©2018 AACR.

Treatment of non–small cell lung cancer (NSCLC) has been transformed by targeted therapies directed against molecular aberrations specifically activated within an individual patient's tumor. However, such therapies are currently only available against a small number of such aberrations, and new targets and therapeutics are needed. Our laboratory has previously identified the MERTK receptor tyrosine kinase (RTK) as a potential drug target in multiple cancer types, including NSCLC. We have recently developed UNC2025—the first-in-class small molecule inhibitor targeting MERTK with pharmacokinetic properties sufficient for clinical translation. Here, we utilize this compound to further validate the important emerging biologic functions of MERTK in lung cancer pathogenesis, to establish that MERTK can be effectively targeted by a clinically translatable agent, and to demonstrate that inhibition of MERTK is a valid treatment strategy in a wide variety of NSCLC lines independent of their driver oncogene status, including in lines with an EGFR mutation, a KRAS/NRAS mutation, an RTK fusion, or another or unknown driver oncogene. Biochemically, we report the selectivity of UNC2025 for MERTK, and its inhibition of oncogenic downstream signaling. Functionally, we demonstrate that UNC2025 induces apoptosis of MERTK-dependent NSCLC cell lines, while decreasing colony formation in vitro and tumor xenograft growth in vivo in murine models. These findings provide further evidence for the importance of MERTK in NSCLC, and demonstrate that MERTK inhibition by UNC2025 is a feasible, clinically relevant treatment strategy in a wide variety of NSCLC subtypes, which warrants further investigation in clinical trials. Mol Cancer Ther; 14(9); 2014–22. ©2015 AACR.

MERTK is a receptor tyrosine kinase that is aberrantly expressed in a number of cancers and is a member of the TAM family of RTKs, along with TYRO3 and AXL. Recent studies have elucidated a role for MERTK in a variety of oncogenic processes including cancer cell growth, proliferation, migration, invasion, apoptosis, survival and transcriptional regulation. In addition, MERTK also functions to suppress anti-tumour immunity. These functions are mediated by MERTK signalling through classical oncogenic pathways such as MAPK/ERK, PI3K/AKT and JAK/STAT. Transcription factors, such as NF-κB, as well as a number of apoptotic proteins have also been identified as key downstream signalling molecules. Multiple therapies that specifically target MERTK are in development. Clinical application of translational agents targeting MERTK has the potential to enhance outcomes for patients with a variety of types of cancer. Key Concepts MERTK is a member of the TAM family (Tyro-3, Axl and MerTK) of receptor tyrosine kinases. MERTK is activated by ligand-induced dimerisation and subsequent auto-phosphorylation of the kinase domain. MERTK regulates known oncogenic and pro-survival signalling pathways, including MAPK/ERK, PI3K/AKT, JAK/STAT, NF-κB and other transcription factors, and numerous apoptotic proteins. The outcome of physiologic MERTK signalling depends on which ligand stimulates the receptor and the function of MERTK in that cell type. MERTK is aberrantly expressed in many solid tumours and haematological malignancies. MERTK plays roles in cancer cell survival, proliferation, migration, invasion, apoptosis and transcriptional regulation. MERTK plays a role in immune modulation of cancer cells. Specific inhibition of MERTK is a promising therapeutic strategy. Keywords: MERTK; RTK; cancer; TAM family; GAS6; oncogene; MAPK; PI3K; apoptosis

Purpose: Although tyrosine kinase inhibitors (TKI) can be effective therapies for leukemia, they fail to fully eliminate leukemic cells and achieve durable remissions for many patients with advanced BCR-ABL+ leukemias or acute myelogenous leukemia (AML). Through a large-scale synthetic lethal RNAi screen, we identified pyruvate dehydrogenase, the limiting enzyme for pyruvate entry into the mitochondrial tricarboxylic acid cycle, as critical for the survival of chronic myelogenous leukemia (CML) cells upon BCR-ABL inhibition. Here, we examined the role of mitochondrial metabolism in the survival of Ph+ leukemia and AML upon TK inhibition. Experimental Design: Ph+ cancer cell lines, AML cell lines, leukemia xenografts, cord blood, and patient samples were examined. Results: We showed that the mitochondrial ATP-synthase inhibitor oligomycin-A greatly sensitized leukemia cells to TKI in vitro. Surprisingly, oligomycin-A sensitized leukemia cells to BCR-ABL inhibition at concentrations of 100- to 1,000-fold below those required for inhibition of respiration. Oligomycin-A treatment rapidly led to mitochondrial membrane depolarization and reduced ATP levels, and promoted superoxide production and leukemia cell apoptosis when combined with TKI. Importantly, oligomycin-A enhanced elimination of BCR-ABL+ leukemia cells by TKI in a mouse model and in primary blast crisis CML samples. Moreover, oligomycin-A also greatly potentiated the elimination of FLT3-dependent AML cells when combined with an FLT3 TKI, both in vitro and in vivo. Conclusions: TKI therapy in leukemia cells creates a novel metabolic state that is highly sensitive to particular mitochondrial perturbations. Targeting mitochondrial metabolism as an adjuvant therapy could therefore improve therapeutic responses to TKI for patients with BCR-ABL+ and FLT3ITD leukemias. Clin Cancer Res; 21(6); 1360–72. ©2014 AACR.

Mer and Flt3 receptor tyrosine kinases have been implicated as therapeutic targets in acute myeloid leukemia (AML). In this manuscript we describe UNC1666, a novel ATP-competitive small molecule tyrosine kinase inhibitor, which potently diminishes Mer and Flt3 phosphorylation in AML. Treatment with UNC1666 mediated biochemical and functional effects in AML cell lines expressing Mer or Flt3 internal tandem duplication (ITD), including decreased phosphorylation of Mer, Flt3 and downstream effectors Stat, Akt and Erk, induction of apoptosis in up to 98% of cells, and reduction of colony formation by greater than 90%, compared to treatment with vehicle. These effects were dose-dependent, with inhibition of downstream signaling and functional effects correlating with the degree of Mer or Flt3 kinase inhibition. Treatment of primary AML patient samples expressing Mer and/or Flt3-ITD with UNC1666 also inhibited Mer and Flt3 intracellular signaling, induced apoptosis, and inhibited colony formation. In summary, UNC1666 is a novel potent small molecule tyrosine kinase inhibitor that decreases oncogenic signaling and myeloblast survival, thereby validating dual Mer/Flt3 inhibition as an attractive treatment strategy for AML.

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.

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); 5275–80. ©2013 AACR.

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

FMS-like tyrosine kinase 3–targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD–mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.

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.

Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway-involving AKT, CREB, Bcl-xL, survivin, and Bcl-2-downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that short hairpin RNA (shRNA) knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl have complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.

The TAM family of receptor tyrosine kinases (RTKs) has been discovered to play a predominant role in promoting the growth, survival, and metastatic spread of several tumor types. AXL and MERTK are two TAM family RTKs that are overexpressed in head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and non-small cell lung cancer (NSCLC), malignancies that are highly metastatic and lethal. The AXL receptor is the most well-characterized TAM receptor and has been found to mediate resistance to both conventional and targeted cancer therapies. Since AXL is overexpressed in aggressive tumor types, cancer patients are currently being enrolled in clinical trials testing AXL inhibitors. In the current study, we analyzed the efficacy of AXL inhibitors—both small molecule and monoclonal antibody therapy—in HNSCC, TNBC, and NSCLC preclinical models. We observed limited efficacy of anti-AXL targeting strategies across these different models, which was attributed to the upregulation of MERTK. MERTK was robustly overexpressed in cell lines and patient-derived xenografts treated with AXL inhibitors. Inhibition of MERTK sensitized HNSCC, TNBC, and NSCLC preclinical models to AXL inhibitors, leading to a more potent blockade of downstream signaling, decreased expansion of tumor cells in culture, and reduced tumor growth in vivo. Furthermore, ectopic overexpression of MERTK in AXL inhibitor-sensitive models resulted in resistance to AXL-targeting strategies. These results suggest that cotargeting both AXL and MERTK may be highly beneficial in a variety of tumor types where both receptors are expressed and may therefore prolong antitumor effects and improve the survival of patients with lethal malignancies. Citation Format: Nellie K. McDaniel, Christopher T. Cummings, Toni M. Brand, Mari Iida, Justus Hulse, Hannah E. Pearson, Rachel A. Orbuch, Olivia J. Ondracek, Kurtis D. Davies, Parkash Gill, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Randall J. Kimple, Paul M. Harari, Deborah DeRyckere, Douglas K. Graham, Deric L. Wheeler. MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A140.

Acute lymphoblastic leukemia (ALL) is currently treated with an intense regimen of chemotherapy yielding cure rates near 80%. However, additional changes using available drugs are unlikely to provide significant improvement in survival. New therapies are warranted given the risk of severe therapy-associated toxicities including infertility, organ damage, and secondary malignancy. Here, we report ectopic expression of the receptor tyrosine kinase Mer in pediatric B-cell ALL. Inhibition of Mer prevented Erk 1/2 activation, increased the sensitivity of B-ALL cells to cytotoxic agents in vitro by promoting apoptosis, and delayed disease onset in a mouse model of leukemia. In addition, we discovered cross-talk between the Mer and mammalian target of rapamycin (mTOR) signaling pathways. Our results identify Mer as a novel therapeutic target in ALL and suggest that inhibitors of Mer will interact synergistically with currently used therapies. This strategy may allow for dose reduction resulting in decreased toxicity and increased survival rates. Mer is aberrantly expressed in numerous other malignancies suggesting that this approach may have broad applications.

The TYRO3, AXL (also known as UFO) and MERTK (TAM) family of receptor tyrosine kinases (RTKs) are aberrantly expressed in multiple haematological and epithelial malignancies. Rather than functioning as oncogenic drivers, their induction in tumour cells predominately promotes survival, chemoresistance and motility. The unique mode of maximal activation of this RTK family requires an extracellular lipid–protein complex. For example, the protein ligand, growth arrest-specific protein 6 (GAS6), binds to phosphatidylserine (PtdSer) that is externalized on apoptotic cell membranes, which activates MERTK on macrophages. This triggers engulfment of apoptotic material and subsequent anti-inflammatory macrophage polarization. In tumours, autocrine and paracrine ligands and apoptotic cells are abundant, which provide a survival signal to the tumour cell and favour an anti-inflammatory, immunosuppressive microenvironment. Thus, TAM kinase inhibition could stimulate antitumour immunity, reduce tumour cell survival, enhance chemosensitivity and diminish metastatic potential.

Negative selection is the process whereby immature thymocytes expressing TCRs with high affinity for self-peptide:MHC complexes are induced to undergo apoptosis. The transcriptional events that occur as a result of TCR signaling during negative selection are not well-characterized. Using oligonucleotide arrays, we have identified 33 genes that exhibit changes in RNA levels in CD4+CD8+ thymocytes during negative selection in vivo. Of 18 genes that have been further characterized, 13 are regulated in response to stimulation with Ag or anti-CD3 and anti-CD28 Abs ex vivo, indicating that these genes are regulated independently of activation of the peripheral immune system. These data also support the idea that anti-CD3/CD28-mediated thymocyte apoptosis is a valid model for negative selection in vivo. A detailed examination of the regulation of many of the identified genes in response to treatment with dexamethasone or gamma-radiation or in response to anti-CD3/anti-CD28 stimulation in the presence of pharmacological inhibitors of mitogen-activated protein kinase kinase kinase 1, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, calcineurin, and cyclin-dependent kinase 2 has facilitated the elucidation of a map of the transcriptional events that occur downstream of the TCR. These studies support a model whereby similar signal transduction pathways are activated by stimuli that induce positive and negative selection and are consistent with the idea that the balance between opposing proapoptotic and antiapoptotic pathways determines cell fate. The data presented in this study also suggest that calcineurin functions to amplify TCR signals by promoting sustained increases in the levels of specific transcripts.

// Christopher T. Cummings 1,* , Rachel M.A. Linger 1,4,* , Rebecca A. Cohen 1 , Susan Sather 1 , Gregory D. Kirkpatrick 1 , Kurtis D. Davies 1 , Deborah DeRyckere 1 , H. Shelton Earp 2,3 and Douglas K. Graham 1 1 Department of Pediatrics, Section of Hematology, Oncology, and Bone Marrow Transplantation, University of Colorado Anschutz Medical Campus, Aurora, CO, USA 2 Department of Medicine, UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA 3 Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 4 Department of Biomedical Sciences, Rocky Vista University College of Osteopathic Medicine, Parker, CO, USA * These authors contributed equally to this work Correspondence: Douglas K. Graham, email: // Keywords : MER, NSCLC, Monoclonal Antibody, Chemosensitivity, Targeted Therapy Received : May 19, 2014 Accepted : June 24, 2014 Published : June 26, 2014 Abstract 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.

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.

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.

Cdc6/Cdc18 is a conserved and essential component of prereplication complexes. The 2.0 A crystal structure of an archaeal Cdc6 ortholog, in conjunction with a mutational analysis of the homologous Cdc18 protein from Schizosaccharomyces pombe , reveals novel aspects of Cdc6/Cdc18 function. Two domains of Cdc6 form an AAA + -type nucleotide binding fold that is observed bound to Mg•ADP. A third domain adopts a winged-helix fold similar to known DNA binding modules. Sequence comparisons show that the winged-helix domain is conserved in Orc1, and mutagenesis data demonstrate that this region of Cdc6/Cdc18 is required for function in vivo. Additional mutational analyses suggest that nucleotide binding and/or hydrolysis by Cdc6/Cdc18 is required not only for progression through S phase, but also for maintenance of checkpoint control during S phase.

The fission yeast cdc18+ gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18+ gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C–2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and

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.

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.

MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK–/– mice. Transplantation of MerTK–/– bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b+ cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK–/– leukocytes exhibited lower tumor cell–induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8+ T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK–/– mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8+ T lymphocyte depletion restored tumor growth in MerTK–/– mice. These data demonstrate that MerTK signaling in tumor-associated CD11b+ leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.

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

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.

Background We investigated the utility of bioluminescence imaging (BLI) using firefly luciferase in monoclonal and polyclonal populations of leukemia cells in vitro and in vivo. Methods Monoclonal and polyclonal human lymphoid and myeloid leukemia cell lines transduced with firefly luciferase were used for BLI. Results Kinetics and dynamics of bioluminescence signal were cell line dependent. Luciferase expression decreased significantly over time in polyclonal leukemia cells in vitro. Transplantation of polyclonal luciferase-tagged cells in mice resulted in inconsistent signal intensity. After selection of monoclonal cell populations, luciferase activity was stable, equal kinetic and dynamic of bioluminescence intensity and strong correlation between cell number and light emission in vitro were observed. We obtained an equal development of leukemia burden detected by luciferase activity in NOD-scid-gamma mice after transplantation of monoclonal populations. Conclusion The use of monoclonal leukemia cells selected for stable and equal luciferase activity is recommended for experiments in vitro and xenograft mouse models. The findings are highly significant for bioluminescence imaging focused on pre-clinical drug development.

Mer tyrosine kinase is ectopically expressed in acute lymphoblastic leukemia and associated with enhanced chemoresistance and disease progression. While such effects are generally ascribed to increased engagement of oncogenic pathways downstream of Mer stimulation by its ligand, Gas6, Mer has not been characterized beyond the scope of its signaling activity. The present study explores Mer behavior following prolonged exposure to Gas6, a context similar to the Gas6-enriched microenvironment of the bone marrow, where a steady supply of ligand facilitates continuous engagement of Mer and likely sustains the presence of leukemic cells. Long-term Gas6 exposure induced production of a partially N-glycosylated form of Mer from newly synthesized stores of protein. Preferential expression of the partial Mer glycoform was associated with diminished levels of Mer on the cell surface and altered Mer localization within the nuclear-soluble and chromatin-bound fractions. The presence of Mer in the nucleus is a novel finding for this receptor, and the glycoform-specific preferences observed in each nuclear compartment suggest that glycosylation may influence Mer function within particular subcellular locales. Previous studies have established Mer as an attractive cancer biologic target, and understanding the complexity of its activity has important implications for potential strategies of Mer inhibition in leukemia therapy. Our results identify several novel features of Mer that expand the breadth of its functions and impact the development of therapeutic modalities designed to target Mer.

Vandetanib is an orally active small molecule tyrosine kinase inhibitor (TKI) with activity against several pathways implicated in malignancy including the vascular endothelial growth factor receptor pathway, the epidermal growth factor receptor pathway, the platelet derived growth factor receptor β pathway, and REarranged during Transfection pathway. To determine if vandetanib-mediated inhibition of receptor tyrosine kinases is a potential therapeutic strategy for pediatric acute leukemia, these studies aimed to characterize the activity of vandetanib against acute leukemia in vitro. Treatment of leukemia cell lines with vandetanib resulted in a dose-dependent decrease in proliferation and survival. Vandetanib’s anti-leukemic activity appeared mediated by multiple mechanisms including accumulation in G1 phase at lower concentrations and apoptosis at higher concentrations. Alterations in cell surface markers also occurred with vandetanib treatment, suggesting induction of differentiation. In combination with DNA damaging agents (etoposide and doxorubicin) vandetanib demonstrated synergistic induction of cell death. However in combination with the anti-metabolite methotrexate, vandetanib had an antagonistic effect on cell death. Although several targets of vandetanib are expressed on acute leukemia cell lines, expression of vandetanib targets did not predict vandetanib sensitivity and alone are therefore not likely candidate biomarkers in patients with acute leukemia. Interactions between vandetanib and standard chemotherapy agents in vitro may help guide choice of combination regimens for further evaluation in the clinical setting for patients with relapsed/refractory acute leukemia. Taken together, these preclinical data support clinical evaluation of vandetanib, in combination with cytotoxic chemotherapy, for pediatric leukemia.

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

Internal tandem duplication mutations of the FLT3 tyrosine kinase (FLT3-ITDs) are present in 15-30% of cases of pediatric and adult acute myeloid leukemias (AML), and are known to portend a poor prognosis. Over the past decade small molecule inhibitors targeting FLT3 have entered clinical trials. Although initial responses have been observed, patients typically develop resistance to current FLT3 inhibitors during the first year of therapy via acquisition of or selection for point mutations in the FLT3 kinase domain at amino acid F691 or D835. Although FLT3 is the most commonly targeted protein in AML, other candidates for pharmacologic inhibition have been identified. Ectopic expression of MerTK, a receptor tyrosine kinase, has been identified in 80-100% of primary AML patient samples, and previous data demonstrate anti-leukemic effects in response to shRNA-mediated MerTK inhibition. Here we describe MRX2843, a novel small molecule inhibitor of MerTK and FLT3 with activity against FLT3 point mutations and therapeutic efficacy in mouse xenograft models of AC220-resistant AML. MRX2843 potently inhibits MerTK and FLT3 with enzymatic IC50 values of 1.3 and 0.64nM, respectively. Treatment of two AML cell lines that express a FLT3-ITD mutation (MV4;11 has low MerTK expression; MOLM-14 does not express MerTK), and two AML cell lines that express MerTK but do not have activating mutations in FLT3 (U937, Kasumi-1) with 25-300nM MRX2843 abrogated activation of intracellular signaling pathways downstream of FLT3 and MerTK, including AKT and ERK1/2. In addition, 72 hour treatment with MRX2843 led to induction of apoptosis in AML cell lines compared to vehicle-treated controls, as determined by flow cytometic analysis after staining with YO-PRO-1 iodide and propidium iodide. For example, in MOLM-14 cultures, treatment with MRX2843 resulted induction of apoptosis in 84±2% of cells, compared to 5±2% after vehicle treatment (p To determine the effects of MRX2843 treatment in vivo, a patient-derived murine xenograft model was established by intravenous injection of primary AML patient blasts that express both MerTK and FLT3-ITD into NOD-SCID-gamma (NSG) mice. Engraftment was monitored by flow cytometric determination of peripheral blast count. When ~10% peripheral blasts were detected, mice were randomized to treatment with 50mg/kg MRX2843 or vehicle (saline) once daily by oral gavage. Treatment with MRX2843 significantly prolonged survival (median survival of 96 days after MRX2843 treatment versus 16 days in control mice, n=4 per group, p Two derivatives of the human FLT3-ITD AML cell line MOLM-14, which acquired either the D835Y (activation loop) or F691L (gatekeeper) mutation after selection in escalating doses of the FLT3 inhibitor AC220, were used to test the activity of MRX2843 against clinically relevant FLT3 point mutations. Treatment with MRX2843 resulted in a significant reduction in the number of viable cells after 48 hours of culture in MOLM-14, MOLM-14:D834Y, and MOLM-14:F691L with IC50 values of 17nM, 20nM, and 30nM, respectively. In both mutant cell lines, MRX2843 potently inhibited phosphorylation of FLT3 and abrogated activation of downstream intracellular signaling molecules. Treatment with MRX2843 induced cell death in MOLM-14:D835Y (80±7% versus 10±1%, p A murine model of AC220-resistant AML was developed by intravenous injection of MOLM-14:D835Y cells into NSG mice. Daily treatment with saline, 10mg/kg AC220, or 50mg/kg MRX2843 was administered by oral gavage beginning 4 days after transplant. Treatment with MRX2843 significantly prolonged survival when compared to mice treated with saline or AC220 with median survival of 103 days, 33 days, and 48 days, respectively (n=5 per group, p In summary, MRX2843 is a novel MerTK and FLT3 inhibitor that retains activity against clinically relevant, resistance-conferring FLT3 point mutations and prolongs survival in murine models of AML. These data support further development of MRX2843 and advancement to early phase clinical trials. Disclosures DeRyckere: University of Colorado - Denver: targeting of the Mer tyrosine kinase as cancer therapy Patents & Royalties; Meryx, Inc: Equity Ownership. Wang:University of North Carolina Chapel Hill: MRX-2843 Patents & Royalties; Meryx, Inc: Equity Ownership. Frye:University of North Carolina Chapel Hill: MRX-2843 Patents & Royalties; Meryx, Inc: Equity Ownership. Earp:University of North Carolina Chapel Hill: MRX-2843 Patents & Royalties; University of North Carolina Chapel Hill: targeting of the Mer tyrosine kinase as cancer therapy Patents & Royalties; Meryx, Inc: Equity Ownership. Graham:University of Colorado - Denver: MRX-2843 Patents & Royalties; University of Colorado - Denver: targeting of the Mer tyrosine kinase as cancer therapy Patents & Royalties; Meryx, Inc: Equity Ownership.

Acute myeloid leukemia (AML) and relapsed or refractory acute lymphoblastic leukemia (ALL) continue to be extremely difficult to treat and unacceptably low overall survival rates mandate the exploration of new potential therapies to ameliorate poor clinical response to conventional therapy. Abnormal tyrosine kinase activation has been associated with poor prognosis in patients with AML and ALL, providing strategic targets for novel therapy development. The MerTK receptor tyrosine kinase is overexpressed in a majority of leukemia cell lines and pediatric patient samples at the time of diagnosis and expression is often upregulated at relapse. In contrast, normal bone marrow myeloid and lymphoid precursors express little or no MerTK. Stimulation of leukemia cell lines with Gas6, a MerTK ligand, activates prosurvival and proliferative signaling pathways known to contribute to leukemogenesis, including ERK1/2, p38, MSK1, CREB, ATF1, AKT and STAT6. Inhibition of MerTK using shRNA significantly increased myeloblast apoptosis in response to serum starvation in the Nomo-1 and Kasumi cell lines and decreased colony forming potential. Importantly, these phenotypes were reversed by exogenous expression of wild-type MerTK protein but not by expression of a mutant MerTK that lacks kinase activity, indicating that oncogenic roles for MerTK are mediated its kinase activity. Additionally, NOD-scid-gamma mice transplanted with Nomo-1 myeloblasts or 697 lymphoblasts with reduced levels of MerTK have a significantly prolonged median survival time compared to mice transplanted with control cell lines. These data validate Mer as a potential therapeutic target in acute leukemias. We have recently developed first-in-class MerTK-selective small molecule tyrosine kinase inhibitors (MerTKIs). Treatment with MerTKI phenocopied the effects of shRNA, including inhibition of prosurvival signaling through ERK1/2, Akt, and STAT6. Flow cytometric analysis of myeloblast cell lines following treatment with MerTKI and staining with YoPro-1-iodide and propidium iodide dyes revealed induction of apoptosis in Mer-positive leukemia cell lines (mean of 66±10% apoptotic and dead cells verses 20±10% after treatment with vehicle, p Citation Format: Douglas K. Graham, Deborah DeRyckere, Alisa Lee-Sherick, Amanda A. Hill, Katherine Minson, Wnag Xiaodong, Weihe Zhang, Jing Liu, Stephen Frye, H. Shelton Earp. MerTK receptor tyrosine kinase is a therapeutic target in pediatric acute leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A37.

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

Tumor-associated macrophages (TAMs) and other tumor-infiltrating myeloid-derived cells are known to support tumor growth, proliferation and metastasis. Increased numbers of TAMs are also associated with poor prognosis in many solid tumors, including breast cancer. Previous studies have shown that targeting TAMs, either by re-programming or depletion, is an effective method to reduce solid tumor growth. MER, a receptor tyrosine kinase, is expressed on macrophages. MER-dependent signaling upon recognition and ingestion of apoptotic material (efferocytosis) suppresses proinflammatory cytokine production and increases secretion of immunosuppressive, wound healing cytokines. Recently we have identified MER as a potential therapeutic target in the tumor microenvironment utilizing polyoma middle T (PyVmT) orthotopic and transgenic models of breast cancer in Mertk-/- mice. Genetic deletion of MER in the tumor microenvironment led to a reduction in tumor growth, as well as increased proinflammatory cytokines and decreased IL-10 production. To further investigate the utility of MER inhibition in the tumor microenvironment as a therapeutic strategy, the efficacy of a first in class MER-selective, orally bioavailable, small molecule tyrosine kinase inhibitor (UNC TKI) was evaluated in immunocompetent C57Bl/6 mice implanted orthotopically with PyVmT mammary gland tumor cells. These PyVmT tumors cells do not express MER, AXL or TYRO3. Treatment with UNC TKI inhibited phosphorylation of MER in mouse macrophages in vitro, but did not affect survival of macrophages or MER-negative PyVmT tumor cells. However, after four weeks of daily treatment with UNC TKI, primary tumor growth was reduced two-fold compared to vehicle-treated tumor bearing mice. Serum IL-10 and IL-4 levels were reduced by 20% and 30%, respectively, in UNC TKI treated tumor-bearing mice compared to vehicle-treated tumor-bearing mice. Taken together, these data suggest that MER inhibition in the tumor microenvironment reduces tumor growth by altering the immunosuppressive environment and stimulating anti-tumor immunity. Moreover, these data validate UNC TKI as a promising strategy for immune-mediated treatment of breast cancer. Citation Format: Kristen M. Jacobsen, Deborah DeRyckere, Weihe Zhang, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Douglas K. Graham. Targeted inhibition of MER tyrosine kinase in the tumor microenvironment decreases tumor growth in a mouse model of breast 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 3637. doi:10.1158/1538-7445.AM2014-3637