MerTK Inhibitor Research Articles

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

Background Growth arrest-specific protein 6 signals through the TAM (TYRO-3-AXL-MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing αIIb β3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP-P2Y1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP-P2Y1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP-P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.

Abstract 1082: MerTK promotes resistance to irreversible EGFR TKIs by activation of the PI3K-AKT pathway in NSCLCs expressing wild-type EGFR

Abstract Lung cancer is the leading cause of cancer-related death with poor survival rates worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and 60% of these have overexpression of wild-type EGFR (wtEGFR), which portends a poor prognosis. In addition, in a recent clinical trial 6 of 12 lung cancer patients whose tumors acquired a resistance-conferring T790M EGFR mutation during treatment with an EGFR TKI reverted to wtEGFR after treatment with Rociletinib/CO-1686, an irreversible EGFR TKI that is selective for the T790M mutant. These data suggest that targeting wtEGFR may improve treatment outcomes; however, to date single agents that target wtEGFR have not been effective in large scale clinical trials and new ways to target wtEGFR in this context are needed. Our laboratory previously identified the MerTK receptor tyrosine kinase as a potential therapeutic target in NSCLC and developed MRX-2843 as a novel MerTK-selective small molecule tyrosine kinase inhibitor with favorable properties for clinical translation. Irrespective of driver oncogene status, treatment with a MerTK inhibitor yields potent anti-tumor effects in NSCLC cell culture models and blocks tumor growth in xenografts of the MerTK-dependent wtEGFR-expressing A549 cell line. In an attempt to more potently block A549 lung cancer cell proliferation, we screened a library of 378 kinase inhibitors that are in various stages of development and identified synergy between MRX-2843 and multiple irreversible EGFR TKIs, including CO-1686 and Osimertinib/AZD-9291. Further, we found that wtEGFR and MerTK were frequently co-expressed and co-immunoprecipitated with each other in NSCLC cancer cell lysates. Synergistic inhibition of cell expansion was observed in a spectrum of NSCLC cell lines with wtEGFR expression treated with the combination therapy, including H1299 (NRAS mutation), H157 (KRAS mutation), H3122 (ALK fusion), and Colo699 (FGFR1 overexpression). On a mechanistic level, combined treatment with 1μM CO-1686 and 100 nM MRX-2843 dramatically inhibited phosphorylation of both MerTK and EGFR and downstream signaling through the PI3K-AKT and MAPK-ERK pathways, while treatment with equivalent doses of either single agent did not efficiently inhibit MerTK, EGFR or downstream signaling. Additionally, CO-1686 mediated synergistic inhibition of A549 expansion in combination with a PI3K or AKT inhibitor, suggesting a role for PI3K-AKT activation downstream of MerTK contributing to the resistance of wtEGFR NSCLCs to irreversible EGFR TKIs. Taken together, our data provide rationale for a novel strategy for treatment of NSCLC with wtEGFR overexpression by combining MRX-2843 and an irreversible EGFR inhibitor. Citation Format: Dan Yan, Xiaodong Wang, Stephen V. Frye, Shelton H. Earp, Deborah DeRyckere, Douglas K. Graham. MerTK promotes resistance to irreversible EGFR TKIs by activation of the PI3K-AKT pathway in NSCLCs expressing wild-type EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1082. doi:10.1158/1538-7445.AM2017-1082

MERTK inhibition in the leukemia microenvironment promotes a therapeutic immune response to acute lymphoblastic leukemia

Abstract T cell immunotherapy is effective in some, but not all cancers, and targeting the immunosuppressive, innate immune tumor microenvironment is one key to further advancement. During efferocytosis, activation of MERTK tyrosine kinase receptor in innate immune cells suppresses proinflammatory cytokines, stimulates immunosuppressive cytokines and upregulates co-inhibitory ligands. MERTK has also been validated as a therapeutic target in the tumor microenvironment in solid tumor models. To investigate immunoregulatory roles for MERTK in leukemia, we utilized pharmacologic and genetic inhibition of MERTK in a MERTK-negative syngeneic murine model of acute lymphoblastic leukemia (ALL). Treatment with MRX-2843, a potent, orally bioavailable, small molecule MERTK inhibitor, significantly reduced leukemic burden and prolonged median survival in wildtype mice (38 days post-treatment vs. 24 days in vehicle-treated mice, p>0.01). Similarly, overall survival was increased more than four-fold in Mertk−/− mice transplanted with leukemia (>80 days vs. 21 days in wild-type mice, p>0.0001), suggesting that the observed therapeutic activity is immune-mediated. Indeed, treatment with MRX-2843 abrogated expression of the immune co-inhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages in the ALL microenvironment. Although T cells do not express MERTK, expression of the cognate PD-1 receptor was also reduced on CD8+ T cells and the frequency of FOXP3+CD4+ regulatory T cells was decreased in the leukemia microenvironment following MRX-2843 treatment. These data demonstrate several mechanisms by which MERTK can suppress anti-leukemia immunity and validate MERTK as a novel immunotherapeutic target in ALL.

MERTK Inhibition Induces Polyploidy and Promotes Cell Death and Cellular Senescence in Glioblastoma Multiforme.

BackgroundMER 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.MethodsCorrelations 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.ResultsDecreased 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.ConclusionsThe 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.

Abstract A168: Discovery of exquisitely selective MerTK inhibitors

Abstract MerTK is a receptor tyrosine kinase most prominently expressed in myeloid cells and known for its role in phagocytosis of early apoptotic cells and concomitant establishment of tolerogenic microenvironment. Hence, MerTK is thought to play a significant role in cancer immune evasion as, for example, a defining component of M2c-type tumor-associated macrophages (TAMs). Moreover, it has also been increasingly recognized that MerTK is overexpressed in a variety of malignant tumor cells, promoting the cancer cell survival, invasion, and treatment resistance. As such, it is anticipated that the cancer cell intrinsic and extrinsic effects might conspire to make MerTK a highly promising target for anticancer therapy. We describe herein an exquisitely selective series of MerTK inhibitors. A lead compound, XL1547, exhibited an IC50 of 1 nM against MerTK in an enzyme assay, with a greater than 300-fold selectivity over closely related AXL kinase (IC50 = 350 nM). In fact, MerTK was the only enzyme that was inhibited by XL1547 (> 99% at 100 nM) in the panel of 386 kinases (Reaction Biology Corp.; all others < 35%). X-ray crystal structure revealed a unique binding mode where the compound exploits a large back pocket next to the gatekeeper residue (known as a Type 1.5 binder). XL1547 potently inhibited autophosphorylation as well as the viability of engineered BaF3(CD8:MerTK) cells (GI50 = 230 nM) with little toxicity. Extensive medicinal chemistry efforts yielded a more advanced compound SA3686 with at least 10-fold improvement in potency. More importantly, it displayed a pharmacokinetic profile suitable for oral dosing. Indeed, SA3686 was highly efficacious in vivo in an allograft tumor model of subcutaneously implanted Ba/F3(CD8:MerTK) cells. [SA3686 is being tested in multiple other in vivo models at the time of abstract submission]. In summary, we have identified an essentially mono-selective MerTK inhibitor series that will help delineate the biology of MerTK-specific inhibition and hopefully validate the translational potential of MerTK as a therapeutic target for human diseases including cancer. Citation Format: Taeyoung Yoon, Joon-Ho Sheen, Seong Jin Park, Jin Kwan Kim, Hadong Kim, Eun-Jung Kim, Munjin Kwon, Hyung Ki Lee, Okyung Lee, Youngjee Jeong, Ki Moon Ryu, Miyeon Jang, Jae-Sung Hwang. Discovery of exquisitely selective MerTK inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A168.

UNC2025, a Small Molecule MerTK and Flt3 Tyrosine Kinase Inhibitor, Decreases Disease Burden, Prolongs Survival, and Promotes Sensitivity to Chemotherapy in Xenograft Models of Acute Leukemia

Acute myeloid leukemia (AML) and relapsed or refractory acute lymphoblastic leukemia (ALL) are extremely difficult to treat and new therapies are needed to improve survival and decrease toxicities associated with conventional therapy. The MerTK receptor tyrosine kinase is aberrantly expressed in 30% of pediatric B-ALL patient samples, 50% of pediatric T-ALL samples, and 80-90% of pediatric and adult AML samples and inhibition of MerTK using shRNA prolonged median survival time in ALL and AML xenograft models. These data validate MerTK as a potential therapeutic target in acute leukemias. In AML, internal tandem duplication (ITD) mutations of the FMS-like tyrosine kinase 3 (Flt3) receptor are observed in ~20-30% of adult and ~15% of pediatric AML patient samples and are associated with poor prognosis. Flt3 inhibitors have demonstrated therapeutic efficacy in clinical studies but resistance to current Flt3-targeted TKIs and unacceptable adverse side effects have been reported, indicating a need for new therapeutic agents directed against Flt3. Here we report a novel dual MerTK/Flt3-selective small molecule TKI as a potential therapy for acute leukemias with MerTK expression and/or activating Flt3 mutation. UNC2025 mediated potent inhibition of MerTK and Flt3 in enzymatic (Ki = 0.16 nM and 0.59 nM, respectively) and cell-based (IC50 = 2.7 nM and 14 nM, respectively) assays and had limited off-target activity against 300 other kinases. Treatment with 100-200 nM UNC2025 inhibited MerTK-dependent pro-survival signaling pathways, induced apoptosis in 40-90% of cells, and reduced colony-forming potential by 80-100% in leukemia cell line cultures. Similarly, a MerTK-expressing leukemic patient sample exhibited reduced MerTK-dependent pro-survival signaling, induction of apoptosis, and decreased colony formation in semi-solid medium in response to UNC2025. In contrast, a MerTK-negative leukemic patient sample did not respond to treatment, suggesting that UNC2025-mediated anti-leukemia effects are a consequence of MerTK inhibition. UNC2025 is orally bioavailable and inhibits MerTK phosphorylation/activation in leukemic blasts in the bone marrow. Treatment with UNC2025 resulted in a dose-dependent reduction in tumor burden and increased median survival from 27 days after inoculation with tumor cells to 70 days in an orthotopic B-ALL xenograft model of minimal residual disease (p < 0.0001) and from 27.5 to 45 days in a similar model of existent disease (p < 0.0001). In a MerTK-expressing patient-derived xenograft (PDX) model of AML, short-term treatment with UNC2025 resulted in a significant decrease in peripheral disease burden (9.3% blasts versus 35.6% for vehicle-treated animals, p = 0.0225) and both the fraction of splenic blasts (28.5% versus 75.7%, p = 0.0005) and spleen mass (170 mg versus 834 mg, p < 0.0001) were dramatically decreased. UNC2025 was even more effective in a FLT3-ITD mutant, MerTK-expressing AML PDX model. In this case, treatment with UNC2025 resulted in stable disease regression (0.24% peripheral blasts after 4 weeks of treatment versus 11.4% pre-treatment, p = 0.0094) and prolonged median survival from 16 days post-treatment to > 107 days (p = 0.0114). In addition, in the B-ALL xenograft model, treatment with UNC2025 and methotrexate, a chemotherapy currently in clinical use for treatment of pediatric ALL, resulted in reduced tumor burden and increased tumor-free survival relative to mice treated with either agent alone, suggesting that addition of UNC2025 to standard therapies may be a particularly effective treatment strategy and/or may allow for chemotherapy dose-reduction and decreased toxicity. The very high potency, relative selectivity, oral bioavailability, and target inhibition and therapeutic efficacy mediated by UNC2025 in murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of patients with acute leukemia. DisclosuresDeRyckere:Meryx, Inc.: Equity Ownership. Wang:University of North Carolina Chapel Hill: UNC2025 Patents & Royalties; Meryx, Inc.: Equity Ownership. Frye:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Earp:Meryx, Inc.: Equity Ownership; University of North Carolina Chapel Hill: UNC2025 Patents & Royalties. Graham:Merys, Inc.: Equity Ownership.

MRX2843, a Novel Dual MerTK-FLT3 Inhibitor with Activity Against Resistance-Conferring FLT3 Mutations in Acute Myeloid Leukemia

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<0.001). In soft agar or methylcellulose cultures, treatment of AML cell lines and patient samples with MRX2843 resulted in an 80-90% reduction in colony number.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<0.01).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<0.001) and in MOLM14:F691L (61±16% versus 12±1%, p<0.001). In contrast, both cell lines were resistant to treatment with AC220 at concentrations 20-fold higher than the inhibitory concentration in the parental line.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<0.01, Figure 1).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. [Display omitted] DisclosuresDeRyckere: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.

NT-38 * MerTK AS A TARGET IN GLIOBLASTOMA

Glioma-associated macrophages and microglia (GIM) are infiltrating immune cells that modulate the glioblastoma (GBM) micro-environment. Pharmacological targeting of GIM represents a promising therapeutic strategy. MerTK receptor tyrosine kinase triggers macrophage ingestion of apoptotic material and polarizes macrophages to an M2-like, immunosuppressive phenotype that promotes tumor growth. In addition, aberrant MerTK expression in GBM tumor cells can provide pro-survival, pro-invasion and chemo-resistance signals. We examined MerTK expression by double immunofluorescence (IF) in 40 human GBM. Both GFAP+ tumor cells and CD68+ GIM expressed MerTK. Quantification in 12 matched pairs of newly-diagnosed and recurrent GBM showed a 5.5-fold increase in MerTK/CD68+ macrophages (p = 0.002), but no consistent changes in MerTK/GFAP+ tumor cells upon recurrence. Next, we examined the efficacy of a novel UNC-developed small molecule MerTK inhibitor (MerTKI) in a genetically engineered orthotopic allograft model of GBM (TRP). GBM were established for 7 days upon stereotactic injection of luciferase-expressing TRP cells into syngeneic, immune-competent mice. Mice (N = 10-12/group) were then randomized to receive no treatment, daily MerTKI (65mg/kg p.o.), or MerTKI plus fractionated radiation (XRT, 5 Gy q.o.d x3). Median survival was 24, 22, and 41.5 days, respectively (p = 0.003), while historical survival of TRP allograft mice treated with XRT alone was 30 days. Bioluminescence imaging (BLI) showed a significant growth delay in MerTKI + XRT-treated mice (doubling time 14 versus 4-4.5 days, p < 0.0001). Two mice remain alive after 50 days of combination treatment and tumor growth remains stable with 90-99% reduction in pre-treatment BLI. Post-mortem histology and IF are pending. These results suggest that both human GBM tumor cells and GIM express MerTK and that MerTK+ GIM may increase upon disease recurrence. MerTK inhibition in an immune-competent pre-clinical model may potentiate XRT response, providing a rationale for future studies of novel MerTK inhibitors in the treatment of GBM patients.

352 UNC2025, a novel small molecule MerTK and Flt3 tyrosine kinase inhibitor, has therapeutic activity and promotes sensitivity to chemotherapy in animal models of acute leukemia

352 UNC2025, a novel small molecule MerTK and Flt3 tyrosine kinase inhibitor, has therapeutic activity and promotes sensitivity to chemotherapy in animal models of acute leukemia

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

Abstract The Mer Receptor Tyrosine Kinase (RTK) is overexpressed in hematologic and epithelial malignancies. Mer is not a proliferative driver but rather produces a cancer cell-intrinsic survival signal. In addition, tumor-associated macrophages (TAMs) express Mer, which upon binding ligand-associated with apoptotic material, triggers engulfment (effercytosis). The Mer signal also polarizes macrophages towards an M2-like, wound healing, anti-inflammatory state, calming the innate immune response when ingesting “self”. We have previously shown that syngeneic polyoma middle T (PyVmT) breast cancer cells demonstrate markedly attenuated growth when orthotopically implanted into Mer knock-out mice, with Mer-/- TAMs of PyVmT tumors producing M1-like inflammatory cytokines (JCI 123:3231, 2013). This result suggests that Mer inhibition could enhance innate anti-tumor responses, and toward that end, we examine the activity of first-in-class, orally bioavailable MerTK inhibitor on tumor growth in autochthonous murine tumor models. UNC MerTKi is 5-fold selective for Mer vs. Axl/Tyro3 and has favorable pharmacokinetics. Once daily, oral dosing inhibits the growth of Mer-expressing leukemia and NSCLC xenografts. UNC MerTKi was assessed in immune-competent, genetically engineered murine models (GEMMs) in the UNC Lineberger Mouse Phase 1 Unit (MP1U). After dose-finding studies in wild-type mice established an MTD, the inhibitor was given at 120 mpk/d in mouse chow, a dose which did not cause weight loss and produced a measurable effect (i.e. inhibition of second phase platelet aggregation, a known Mer pharmacodynamics marker). This dose did not exhibit single agent activity in a murine model of breast cancer (C3TAg), but exhibited pronounced single agent activity in RAS-driven, INK4a/Arf null melanoma GEMM (TRIA). The MP1U has previously reported the efficacy of 15 chemotherapeutic and/or targeted regimens in a large (&amp;gt;220) cohort of TRIA mice (CCR 18:5290, 2012). The overall response was 10% (partial responses and stable disease). There were no complete responses. A combination of MEK (AZD 6244) and PI3K/mTOR (BEZ235) inhibitors were the most active previous regimen (responses seen in 9/18 mice = 50%, with 0 CRs) with moderate toxicity. UNC MerTKi exhibited greater activity (6/8 mice = 75%, with 3 CRs) with mild, well-tolerated toxicity in the TRIA model. TRIA cell lines do not express Mer, suggesting that UNC MerTKi as a monotherapy may induce responses via Mer inhibition in TAMs and the tumor microenvironment, or via inhibition of Axl, Tyro or an unknown target. In summary, a potent and selective Mer inhibitor exhibited greater pre-clinical efficacy in a highly faithful model of RAS-mutant melanoma than any other drug tested to date, including several compounds that are FDA approved for use in metastatic melanoma. Citation Format: H Shelton Earp, David Darr, Albert Zimmermann, Kelly Clark, Norman E. Sharpless, Wolfgang Bergmeier, Weihe Zhang, Xiaodong Wang, Deborah DeRyckere, Stephen Frye, Douglas Graham. A small molecule Mer tyrosine kinase inhibitor (UNC MerTKi) effectively inhibits growth of murine melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 947. doi:10.1158/1538-7445.AM2014-947

Efferocytosis produces a prometastatic landscape during postpartum mammary gland involution.

Breast cancers that occur in women 2-5 years postpartum are more frequently diagnosed at metastatic stages and correlate with poorer outcomes compared with breast cancers diagnosed in young, premenopausal women. The molecular mechanisms underlying the malignant severity associated with postpartum breast cancers (ppBCs) are unclear but relate to stromal wound-healing events during postpartum involution, a dynamic process characterized by widespread cell death in milk-producing mammary epithelial cells (MECs). Using both spontaneous and allografted mammary tumors in fully immune-competent mice, we discovered that postpartum involution increases mammary tumor metastasis. Cell death was widespread, not only occurring in MECs but also in tumor epithelium. Dying tumor cells were cleared through receptor tyrosine kinase MerTK-dependent efferocytosis, which robustly induced the transcription of genes encoding wound-healing cytokines, including IL-4, IL-10, IL-13, and TGF-β. Animals lacking MerTK and animals treated with a MerTK inhibitor exhibited impaired efferocytosis in postpartum tumors, a reduction of M2-like macrophages but no change in total macrophage levels, decreased TGF-β expression, and a reduction of postpartum tumor metastasis that was similar to the metastasis frequencies observed in nulliparous mice. Moreover, TGF-β blockade reduced postpartum tumor metastasis. These data suggest that widespread cell death during postpartum involution triggers efferocytosis-induced wound-healing cytokines in the tumor microenvironment that promote metastatic tumor progression.