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Supplementary Figures 1-6

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Myeloid cell receptor tyrosine kinases TYRO3, AXL, and MERTK and their ligands, GAS6 and PROTEIN S, physiologically suppress innate immune responses, including in the tumor microenvironment. Here, we showed that myeloid-derived suppressor cells (MDSC) dramatically upregulated TYRO3, AXL, and MERTK and their ligands [monocytic MDSCs (M-MDSC)>20-fold, polymorphonuclear MDSCs (PMN-MDSC)>15-fold] in tumor-bearing mice. MDSCs from tumor-bearing Mertk−/−, Axl−/−, and Tyro3−/− mice exhibited diminished suppressive enzymatic capabilities, displayed deficits in T-cell suppression, and migrated poorly to tumor-draining lymph nodes. In coimplantation experiments using TYRO3−/−, AXL−/−, and MERTK−/− MDSCs, we showed the absence of these RTKs reversed the protumorigenic properties of MDSCs in vivo. Consistent with these findings, in vivo pharmacologic TYRO3, AXL, and MERTK inhibition diminished MDSC suppressive capability, slowed tumor growth, increased CD8+ T-cell infiltration, and augmented anti–PD-1 checkpoint inhibitor immunotherapy. Mechanistically, MERTK regulated MDSC suppression and differentiation in part through regulation of STAT3 serine phosphorylation and nuclear localization. Analysis of metastatic melanoma patients demonstrated an enrichment of circulating MERTK+ and TYRO3+ M-MDSCs, PMN-MDSCs, and early-stage MDSCs (e-MDSC) relative to these MDSC populations in healthy controls. These studies demonstrated that TYRO3, AXL, and MERTK control MDSC functionality and serve as promising pharmacologic targets for regulating MDSC-mediated immune suppression in cancer patients.

Supplemental cell lines list

Evaluation of AXL inhibitor R428.

Supplemental Figure 2 shows impact of UNC2025 on colony forming potential.

Overexpression of TYRO3 does not confer resistance to AXL inhibition.

Supplemental Figure 4 shows TAM-family kinase expression in melanoma cell lines.

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

Evaluation of MERTK inhibitor UNC2025

Supplemental Figure 7 shows combined UNC2025 and vemurafenib treatment is tolerated in mice.

Supplemental Table 1: Concentration of UNC2025 in Mouse Serum and Tumors.

Inhibition of AXL upregulates MERTK expression and activity in additional cell culture models.

AXL and MERTK inhibitors mediate synergistic inhibition of tumor cell expression in vitro.

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

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

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

Supplemental Figures 6 shows efficacy of UNC2025 in a melanoma wild-type PDX mode.

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.

Supplemental Figure 3 shows impact of UNC2025 on cell viability.

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.

Supplemental Figure 3: pMERTK and MERTK are not Predictive of Sensitivity to UNC2025.

Supplemental Figure 1: Lack of Expression of Relevant Potential Off-Target Kinases.Sub-confluent cultures of the H2228, A549, Colo699, and H1299 NSCLC cell lines were lysed.

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

Supplemental Figure 2: UNC2025 Inhibits Survivin Expression in Various Cancer Types.

1uM CO-1686

Supplemental Tables 1-4. Table 1: pLKO shRNA clone information and antibody information. Table 2: Combination Indices to assess for synergism. Table 3: Quantitative 1H-, 31P- and 13C-NMR analysis of K562 CML cells treated with vehicle imatinib, oligomycin-A or the combination imatinib + oligomycin-A. Table 4: Complete blood counts and metabolic profiles after in vivo treatment with Oligomycin-A

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

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

Supplemental Table 1: Antibody Suppliers and Catalog Numbers Supplemental Table 2: Cell Cycle Profiles in Acute Leukemia Cell Lines Treated with UNC2025. Mean values, standard errors, and p values compared to vehicle treatment from 3 independent experiments are shown. (NS=Not Significant, *p

Supplemental Figures 1-8. Figure 1. Mitochondrial metabolism becomes essential for TKI-treated BCR-ABL+ leukemia cells. Figure 2: Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 3. Oligomycin-A sensitizes cells to BCR-ABL inhibition. Figure 4. Oligomycin-A sensitizes acute myeloid leukemia cells to TKI. Figure 5. Low nM concentrations of oligomycin-A do not perturb the TCA cycle. Figure 6. Oligomycin-A disrupts mitochondrial functions in leukemia cells. Figure 7. Low dose oligomycin-A synergizes with TKI to eliminate leukemia in vivo with no significant toxicity. Figure 8. Model for how TKI treatment of leukemias creates an altered metabolic state sensitive to mitochondrial perturbations.

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Supplemental Methods. This supplementary file includes additional methods for sample preparation, Seahorse analysis of oxygen consumption rates, reactive oxygen species measurements, flow cytometry analysis, NMR analysis, RT-PCR conditions, quizartinib synthesis information.

Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal–regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development. Mol Cancer Ther; 9(5); 1298–307. ©2010 AACR.

100nM MRX-2843

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.

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MERTK tyrosine kinase expression is upregulated upon monocyte to macrophage differentiation. This receptor tyrosine kinase enables macrophages to efficiently clear apoptotic cells to maintain tissue homeostasis. Activation of MERTK in macrophages during efferocytosis promotes an immunosuppressive phenotype, which is hijacked by tumors to inhibit anti-tumor immunity. Further, this immunosuppressive phenotype in MERTK expressing macrophages in the tumor microenvironment is reversed using MERTK selective inhibitors, suggesting that inhibiting MERTK expression or activity in the tumor microenvironment may be of therapeutic benefit in the treatment of cancer. In an attempt to further understand the mechanism of MERTK upregulation in macrophages, we treated the monocytic leukemia cell line THP1 with PMA to differentiate the cells from a monocytic morphology to an adherent macrophage-like morphology. Following differentiation of the THP-1 cells, MERTK upregulation was confirmed by western blot and flow cytometry. In a similar fashion, treatment with of murine bone marrow derived monocytic cells with PMA induced MERTK expression. Proteomics cytokine array analysis also revealed increased levels of multiple chemokines including MCP-1 and RANTES. Treatment of THP-1 cells with a pan STAT inhibitor in the presence of PMA abrogated the induction of MERTK expression, suggesting a critical role for STAT pathways in the regulation of MERTK expression during monocyte differentiation to macrophages. Specifically, the STAT3 pathway was found to be important in MERTK regulation, as treatment with a STAT3 selective inhibitor was sufficient to abrogate MERTK expression in the presence of PMA treatment. Single cell sequencing of the immune cells in the bone marrow demonstrated Stat3 expression in monocytic lineage cells. Furthermore, both CD11C+Ly6c+CD45+ population and MERTK+CD11C+Ly6c+CD45+ populations were lower in Stat3 -/- bone marrow cells treated with PMA relative to the untreated cells. Collectively, these data suggest that MERTK expression during macrophage maturation may be mediated by STAT3 activation. Previously published data have also demonstrated that STAT3 can be activated downstream of MERTK activation. Thus, we propose that MERTK and STAT3 form a positive feedback loop during macrophage maturation. Treatment with either a MERTK and/or STAT3 inhibitor may interfere with this feedback pathway, potentially reversing an immunosuppressive phenotype in the macrophages in the tumor microenvironment. Citation Format: Dan Yan, Justus M. Huelse, Swati Sharma Bhasin, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. Targeting a positive feedback loop of MERTK and STAT3 during macrophage differentiation may provide anti-tumor immune function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2335.

MERTK is a transmembrane receptor that belongs to the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases. The role of MERTK in cancer progression and resistance to therapies are reported in many cancer types, including non-small cell lung cancer (NSCLC). In canonical signaling, MERTK is localized at the cell membrane and mediates intracellular signal transduction pathways. Non-canonical signaling of receptor tyrosine kinases (RTKs) has been reported, including signaling which occurs when RTKs translocate to the nucleus. Our group previously reported nuclear localization of MERTK in leukemia cells and we now extend these findings to non-small cell lung cancer. We found exogenous MERTK localized to the nucleus in cells transfected with a plasmid expressing wild-type MERTK. Additionally, endogenous nuclear MERTK was phosphorylated, suggesting that MERTK may have a functional role in the nucleus. MERTK kinase inhibitor MRX-2843 modulates its nuclear translocation. Introducing point mutations to MERTK autophosphorylation sites diminished nuclear translocation in comparison to cells transfected with wild-type MERTK. Our analysis of proteins that may interact with MERTK to regulate nuclear localization and function demonstrated co-immunoprecipitation of nuclear MERTK and STAT1. Current studies are underway to further explore nuclear MERTK functions and how these roles may be modulated upon interaction with STAT1. Citation Format: K.M. Tanim, Deborah DeRyckere, Douglas K. Graham. Phosphorylation of MERTK is required for nuclear localization in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4991.

The TAM family receptor tyrosine kinases TYRO3, AXL and MERTK are potential therapeutic targets in a variety of cancers. In our previous studies, MERTK inhibition in the leukemia microenvironment significantly prolonged survival in a syngeneic B-cell acute leukemia (B-ALL) model, implicating MERTK as a promising immuno-oncology target. Strikingly, Mertk-/- mice were almost completely protected against leukemia. Here, we probed the mechanisms of anti-leukemia immunity mediated by MERTK inhibition and evaluated roles for TYRO3 and AXL in the leukemia microenvironment. Single cell RNA sequencing and flow cytometry studies revealed an increase in CD8α+ dendritic cells (DCs) with enhanced antigen-presentation capacity in naïve and leukemia-bearing Mertk-/- mice. These cells were also increased in leukemic wild type (WT) mice treated with the MERTK inhibitor MRX-2843 (currently in phase I/Ib clinical trials). Additionally, CD8+ T cells expressing high levels of the T cell exhaustion marker Tox were decreased in mice treated with MRX-2843, implicating a CD8α+ DC – T cell axis in the anti-leukemia immune response stimulated by MERTK inhibition. Indeed, combined depletion of CD8+ T cells and CD8α+ DCs completely abrogated the anti-leukemia response in Mertk-/- mice, while immunity remained partially intact in mice with selective depletion of CD8+ T cells. Similarly, protection from leukemia was abrogated in Mertk-/- scid mice, which lack functional B and T cells and have defects in DC function. These data demonstrate a critical immunosuppressive role for MERTK in DCs of the leukemia microenvironment. Similar to Mertk-/- mice, B-ALL growth was almost completely prevented in Tyro3-/- mice, while Axl-/- did not impact leukemogenesis, implicating TYRO3, but not AXL, as an additional immunotherapeutic target. However, in contrast to Mertk-/- mice, CD8α+ DCs with enhanced antigen-presentation capacity were not significantly increased in Tyro3-/- mice compared to WT, indicating differences in the underlying mechanisms by which MERTK and TYRO3 contribute to the immunosurveillance of leukemia cells. Indeed, in vivo depletion experiments confirmed differential roles for MERTK and TYRO3 in the leukemia microenvironment. In contrast to Mertk-/- mice, selective depletion of CD8+ T cells completely abrogated protection from leukemia in Tyro3-/- mice, indicating a mechanism less dependent on DCs. Together, these findings reveal novel and distinct mechanistic insights into the immunosuppressive roles for MERTK and TYRO3 in the leukemia microenvironment, demonstrate a critical role for MERTK in DC activity, and validate a similar mechanism mediated by MRX-2843. Thus, these studies provide strong rationale for development of MERTK and/or TYRO3-targeted immunotherapies for treatment of acute leukemia. Citation Format: Justus M Huelse, Swati S Bhasin, Kristen M Jacobsen, Beena E Thomas, Madison L Chimenti, Travon A Baxter, Xiaodong Wang, Stephen V Frye, Curtis J Henry, H. Shelton Earp, Manoj Bhasin, Deborah DeRyckere, Douglas K Graham. MERTK inhibition induces an anti-leukemia dendritic cell – T cell axis while TYRO3 inhibition protects by a separate mechanism [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A35.

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.

The TAM family receptor tyrosine kinases TYRO3, AXL and MERTK are potential therapeutic targets in a variety of cancers. In previous studies, inhibition of MERTK decreased PD-1 checkpoint proteins in the leukemia microenvironment and prolonged survival in a syngeneic BCR-ABL+/Arf-/- B-cell acute leukemia model, implicating MERTK as a promising immune-oncology target in leukemia. Strikingly, Mertk-/- mice were largely protected from leukemia. In our current studies, Tyro3-/- almost completely prevented development of leukemia, comparable to Mertk-/-, while Axl-/- mice died with similar timing to wild type (WT) mice (20-40 days). These data demonstrate differential roles for TAM kinases in the anti-leukemia immune response. Depletion studies were conducted to evaluate potential roles for T cells and dendritic cells (DCs) in anti-leukemia immunity in Mertk-/- mice. Selective depletion of CD8+ T cells abrogated protection from leukemia in Mertk-/- mice, but survival was still prolonged relative to WT. Thus, while CD8+ T cells were required for complete protection from leukemia, the anti-leukemia response remained partially intact even in the absence of CD8+ T cells, implicating an innate immune mechanism. Indeed, combined depletion of CD8+ T cell and CD8α+ DC subsets completely abrogated the anti-leukemic effects in Mertk-/- mice, revealing a critical immunosuppressive role for MERTK in DCs in the leukemia microenvironment. In contrast to Mertk-/- mice, selective depletion of CD8+ T cells completely abrogated protection from leukemia in Tyro3-/- mice, indicating a mechanism less dependent on DCs. Similarly, single cell RNA sequencing revealed CD8+ DCs with a more mature and antigen-presenting phenotype in Mertk-/- mice compared to WT, while antigen-presenting DCs were not increased in Tyro3-/- mice. Single cell sequencing data also suggest induction of an anti-leukemic DC - T cell axis in WT leukemic mice treated with the MERTK-selective inhibitor MRX-2843. DCs were nearly absent in leukemic bone marrow from saline-treated mice and were dramatically increased in response to treatment with MRX-2843. Treatment with MRX-2843 also decreased the incidence of CD8+ T cells expressing high levels of Tox, which has been associated with T cell exhaustion. These changes coincided with decreased leukemic blasts, even in the context of established disease.Together, our findings support a model whereby MERTK inhibition promotes DC function and CD8+ T cell activity, leading to anti-leukemia immunity. In contrast, anti-leukemia immunity in response to TYRO3 inhibition is less dependent on DCs. Differential roles for the TAM kinases in the leukemia microenvironment provide rationale for development of MERTK and/or TYRO3 targeted immunotherapies to treat acute leukemia. Citation Format: Justus M. Huelse, Swati S. Bhasin, Beena E. Thomas, Madison L. Chimenti, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. MERTK inhibition induces an anti-leukemia dendritic cell - T cell axis while TYRO3 inhibition protects through a separate mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 240.

While overall outcomes have improved for patients with acute leukemia, high-risk subsets including acute myeloid leukemia (AML) and relapsed/refractory early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) continue to have poor prognosis. New therapies are urgently needed. Both MERTK tyrosine kinase and the anti-apoptotic protein BCL-2 have been implicated as therapeutic targets in AML and ETP-ALL. We developed MRX-2843, a novel small molecule MERTK and FLT3 inhibitor currently in clinical trials in patients with leukemia. The BCL-2 inhibitor venetoclax is FDA-approved for treatment of AML and has clinical activity in relapsed/refractory T-ALL. Here, we investigated the impact of treatment with MRX-2843 in combination with BCL-2 inhibitors in preclinical models. Human AML and ETP-ALL cell lines were treated with MRX-2843 and/or a BCL-2 inhibitor for 48-72 hours and relative cell numbers were determined using CellTiter-Glo reagent. Synergy was assessed by mathematical modeling using the response additivity and fractional product methods. Combined treatment with MRX-2843 and venetoclax provided enhanced therapeutic efficacy compared to MRX-2843 or venetoclax alone. The interaction between drugs was dose-dependent and synergistic in AML cell lines. For instance, in KG-1 cells combined treatment with an IC50 concentration of MRX-2843 and an IC15 concentration of venetoclax reduced cell density by 88 ± 4.0% and the combination was significantly more effective than MRX-2843 or venetoclax alone (p < 0.0001, 2-way ANOVA). Moreover, the 88% reduction in cell density in cultures treated with the combination was significantly greater than the 58 ± 1.6% reduction expected for an additive interaction (p < 0.0001). Robust therapeutic activity and dramatic synergy were also observed in NOMO-1 and OCI-AML5 cell cultures treated with the combination and the interaction between drugs was additive or synergistic in Loucy ETP-ALL cells. Enhanced therapeutic efficacy and synergistic interactions were also observed in AML cell cultures treated with MRX-2843 and navitoclax, a BCL-2 and BCL-XL inhibitor, implicating BCL-2 inhibition as a mechanism of synergy. In a high-throughput screen, MRX-2843 mediated synergistic anti-leukemia activity in combination with venetoclax in all 7 AML and both ETP-ALL cell lines tested. Synergy was optimal when MRX-2843 and venetoclax were administered in a 1:20 ratio. Our data (i) implicate combined treatment with MRX-2843 and a BCL-2 inhibitor, such as venetoclax, as a promising new strategy for treatment of both AML and ETP-ALL, (ii) define optimized dosing strategies for MRX-2843 and venetoclax combination therapy, and (iii) support further evaluation of MRX-2843 in combination with venetoclax in murine models and potentially in upcoming clinical trials. Citation Format: Aashis Thapa, Juhi Jain, Ryan J. Summers, James M. Kelvin, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Erik C. Dreaden, Deborah DeRyckere, Douglas K. Graham. MRX-2843, a dual MERTK and FLT3 inhibitor, mediates synergistic anti-leukemia activity in combination with BCL-2 inhibitors in acute myeloid leukemia and early T-cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3339.

Ewing sarcoma (EWS) is the second most common pediatric bone tumor, and outcomes remain poor in patients with advanced or relapsed disease. In addition, current treatments rely on multi-modal therapy that has significant short- and long-term side effects. New, less toxic and more effective treatments are urgently needed. One potential therapeutic target is the receptor tyrosine kinase MERTK. MERTK is overexpressed in numerous cancers where it promotes tumor cell survival, metastasis, and resistance to cytotoxic and targeted therapies. We demonstrated expression and phosphorylation of MERTK protein in 5 of 5 EWS family cell lines tested. Stimulation with the MERTK ligand GAS6 resulted in activation of downstream oncogenic signaling pathways, including JAK/STAT and MAPK/ERK. Moreover, publicly available data from CRISPR-based library screens suggest that EWS cell lines are particularly dependent on MERTK. Thus, therapeutic strategies targeting MERTK may be particularly effective for treatment of EWS. To explore the therapeutic impact of MERTK inhibition in various cancers, we developed MRX-2843, a first-in-class MERTK-selective tyrosine kinase inhibitor that is currently in clinical trials. Treatment with MRX-2843 decreased phosphorylation of MERTK in a dose dependent manner, with an IC50 of 123 nM (95% CI; 53-180 nM) in A673 cells, and reduced downstream STAT6 signaling. In addition, MRX-2843 had potent anti-tumor activity against all 5 EWS cell lines, leading to reduced expansion and decreased cell density in culture with IC50 values ranging from 178 - 297 nM. Of note, inhibition of MERTK phosphorylation correlated with anti-tumor activity in both the A673 and TC106 cell lines, implicating MERTK inhibition as a mechanism of MRX-2843 anti-tumor activity. Together these data validate MERTK as a promising therapeutic target in EWS and support development of MRX-2843 for treatment of EWS, with potential to directly inform and enable a clinical trial in pediatric patients and, ultimately, to improve both outcomes and quality of life for patients with this disease. Citation Format: Sherri K. Smart, Tsz Y. Yeung, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Douglas K. Graham, Deborah DeRyckere. MERTK is a potential therapeutic target in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3990.

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

Osimertinib is currently the preferred treatment for EGFR-mutated non-small cell lung cancer (NSCLC) patients due to its superior therapeutic efficacy and prolonged overall survival compared to earlier generation EGFR tyrosine kinase inhibitors, but durable responses to osimertinib treatment are rare due to acquired drug resistance. Thus, there is an urgent need for novel strategies to treat osimertinib-resistant NSCLC. Recently, we found that treatment with MRX-2843, a novel MERTK-selective kinase inhibitor currently in Phase I clinical trials, resulted in dose-dependent inhibition of cell expansion and colony formation in an osimertinib-resistant (osiR) H4006 derivative cell line. An unbiased screen of 378 kinase inhibitors was carried out to identify compounds that synergized with MRX-2843 to inhibit expansion of an osiR derivative of the EGFR-mutated H4011 cell line. Treatment with 1µM PIM kinase inhibitor SGI-1776 or 100nM MRX-2843 alone reduced cell density by 5±3% and 44±7%, respectively, while treatment with MRX-2843 and SGI-1776 combined mediated an 82±0.4% decrease. Synergy was also observed in H4006 osiR and H1650 osiR derivative cell lines. Furthermore, treatment with PIM447, a structurally distinct PIM kinase inhibitor, and MRX-2843 decreased cell expansion more effectively than either agent alone. Mechanistically, treatment with a PIM kinase inhibitor in combination with MRX-2843 decreased downstream PI3K-AKT and MAPK-ERK signaling more effectively than single agents. Additionally, combined treatment with MRX-2843 and SGI-1776 prevented colony formation, while single agents had limited effect. In all, these data indicate that combining MRX-2843 and a PIM TKI may control osimertinib resistant tumor growth, providing a potential treatment strategy for osimertinib resistant EGFR-mutated NSCLC patients for whom the choices are still limited. Citation Format: Dan Yan, Zikang Tan, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, III, Deborah DeRyckere, Douglas K. Graham. A novel strategy to cope with osimertinib resistance in non-small cell lung cancer by treatment with a PIM kinase inhibitor in combination with a MERTK-selective kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1109.

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

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.

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.