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Extracellular vesicles (EV) in the tumor microenvironment have emerged as crucial mediators that promote proliferation, metastasis, and chemoresistance. However, the role of circulating small EVs (csEV) in cancer progression remains poorly understood. In this study, we report that csEV facilitate cancer progression and determine its molecular mechanism. csEVs strongly promoted the migration of cancer cells via interaction with phosphatidylserine of csEVs. Among the three TAM receptors, TYRO3, AXL, and MerTK, TYRO3 mainly interacted with csEVs. csEV-mediated TYRO3 activation promoted migration and metastasis via the epithelial–mesenchymal transition and stimulation of RhoA in invasive cancer cells. Additionally, csEV–TYRO3 interaction induced YAP activation, which led to increased cell proliferation and chemoresistance. Combination treatment with gefitinib and KRCT-6j, a selective TYRO3 inhibitor, significantly reduced tumor volume in xenografts implanted with gefitinib-resistant non–small cell lung cancer cells. The results of this study show that TYRO3 activation by csEVs facilitates cancer cell migration and chemoresistance by activation of RhoA or YAP, indicating that the csEV/TYRO3 interaction may serve as a potential therapeutic target for aggressive cancers in the clinic. Significance: These findings demonstrate that circulating extracellular vesicles are a novel driver in migration and survival of aggressive cancer cells via TYRO3 activation.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti–PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti–PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti–PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti–PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti–PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. Significance: These findings show that pan-inhibition of TAM receptors in combination with anti–PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk–neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell–mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. Significance: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.

Background: Mutations in the NF2 gene, which codes for the tumor suppressor Merlin, is responsible for the development of all Neurofibromatosis Type 2 (NF2)-related tumors including schwannomas and meningiomas. These tumors can also occur spontaneously in non-NF2 patients. The only available treatments for this group of tumors are surgery and radiosurgery/radiotherapy. Therefore, in a search of effective drug-based treatments for NF2-mutated schwannomas and meningiomas, we are investigating TYRO3, AXL and MERTK (TAM) receptors as new therapeutic targets. Methods: The expression and activation of TAM receptors were investigated using human Merlin-deficient meningioma and schwannoma tissues compared to control by Western blotting. Infiltration of tumor associated macrophages and their correlation to TAM family receptors were investigated using immunofluorescence double staining and flow cytometry. To understand the role of TAM receptors in the pathogenesis of schwannomas and meningiomas, in vitro shRNA knock-down and pharmacological inhibition analysis were conducted using patient-derived schwannoma and meningioma cells. Results: The results revealed overexpression and hyper-activation of AXL, MERTK, TYRO3 in human Merlin-deficient meningiomas and schwannomas. Meningiomas and schwannomas show a varied level of infiltration by M2 macrophages (CD163+) and TAM receptors are expressed on these CD163+ macrophages in addition to the tumor cells. The expression of MERTK correlates to the level of infiltrated CD163+ macrophages. MERTK forms a complex with TYRO3 in both meningioma and schwannoma tissues, and its expression is mandatory to maintain AXL and TYRO3 levels in both cell types. MERTK and AXL contribute to the increased proliferation and survival of schwannoma and meningioma primary cells in vitro. Pathological proliferation and survival of both cell types were successfully reversed by AXL inhibitor BGB324 (BerGenBio, FDA approved for AML) and MERTK inhibitor UNC2025 (preclinical) in vitro, with UNC2025 being more effective. TYRO3 did not affect the proliferation of either schwannoma or meningioma primary cells. Conclusion: Our findings suggest that TAM receptors are aberrantly expressed and activated in human Merlin-deficient meningiomas and schwannomas. Infiltration of M2 macrophages may contribute to an overall increase in the expression of TAM receptors in these tumors. AXL and MERTK are important in schwannoma and meningioma pathogenesis and are potentially good therapeutic targets. MERTK inhibitor UNC2025 has the potential to be used as a common candidate for the treatment of NF2-related tumors. Citation Format: Foram Dave, Sylwia Ammoun, C Oliver Hanemann. TAM receptors as potential therapeutic targets in NF2-related schwannomas and meningiomas [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 941.

The TAM family of receptor tyrosine kinases (MERTK, AXL, and TYRO3) are expressed on DCs and macrophages, and they serve a critical role in efferocytosis, by which apoptotic bodies are cleared by engulfment. Their activation drives phagocytic activity concomitant to promoting an immuno-suppressive, tolerogenic state in DCs and the M2 polarization of macrophages. This dual signaling mechanism allows for the clearance of cells undergoing non-pathologic apoptotic turnover without inappropriately breaking self-tolerance and driving auto-immunity. Unlike most immuno-therapy targets, the KOs of the TAM kinases, PD-1, and CTLA-4 share common auto-immune disease phenotypes such as splenomegaly, immune infiltration of the liver, arthritis, and lupus. Furthermore, viruses have evolved to promote PD-1/CTLA-4 signaling to suppress the anti-viral immune response, and they similarly target the TAM receptors via apoptotic mimicry to reduce the inflammatory response. This confluence of immune phenotypes provided the biological rationale for the discovery of PF-5807/ARRY-067, a potent and selective inhibitor of MERTK/AXL as a DC-activating immuno-oncology therapeutic. Based on the IC50 for inhibition of phospho-MERTK/AXL in cell-based assays, our mouse PK and PK/PD studies determined that 50 mg/kg PF-5807 provided IC90 plasma coverage for 12 hours. This PK profile supported a twice-daily dosing schedule for maintaining continuous suppression of MERTK and AXL, which was used for mechanistic and efficacy studies in mouse models. To investigate DC and T cell responses to PF-5807, mice were adoptively-transferred with OT-I T cells and challenged with ovalbumin immunization; flow cytometry analysis indicated that PF-5807 treatment increased the fraction of activated, Class II MHC-positive DCs, as well as cross-primed IFNγ+ TNFα+ OT-I T cells. Also, we found that cytotoxic T lymphocytes were necessary for tumor control in the E0771 mouse syngeneic tumor model, as depletion of CD8+ T cells abrogated all activity by PF-5807 in inhibiting tumor growth. This anti-tumor activity was further demonstrated in the MC-38 and EMT6 tumor models, where single agent PF-5807 effected 57-88% TGI and a 13-38% cure rate, while combination with PD-1 blockade drove 99-100% TGI and a 75-100% cure rate. Finally, these complete responders rejected tumor re-challenge without further treatment, resulting in extended survival and durable cures that corroborate the development of immunologic memory. Therefore, we find that PF-5807 activates DCs to cross-prime CD8+ T cells, which are necessary for tumor control and are likely responsible for the establishment of tumor-specific memory T cells. PF-5807 is currently in an ongoing Phase I clinical trial in patients with advanced or metastatic solid tumors, with the intent to explore combination with anti-PD-1/anti-PD-L1 antibodies. Citation Format: Jim Wong, Jacqueline Harrison, Karyn Bouhana, Nhan Nguyen, Namir Shaabani, Vincent F. Vartabedian, LouAnn Cable, Robert Rieger, Lisa Pieti Opie, Shelley Allen, Adam Cook, Joshua Dahlke, Ronald Hinklin, David Chantry, Shannon Winski, John Teijaro. The potent and selective MERTK/AXL inhibitor PF-5807/ARRY-067 activates dendritic cells to cross-prime CD8+ T cells for anti-tumor activity [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 1735.

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.

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.

MERTK, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is a pleiotropic immune modulator that controls efferocytosis. Engagement of MERTK with its ligand GAS6, found anchored to phosphatidylserine exposed on the outer membrane of apoptotic cells, triggers MERTK phosphorylation and signaling events that culminate in the removal of apoptotic debris. Recent studies have highlighted the expression of MERTK on tumor-associated macrophages, and Mertk-deficient mice show reduced tumor cell growth accompanied by inflammatory cytokine production and alterations in macrophage activation. Thus, MERTK has emerged as a promising therapeutic target for augmenting innate antitumor immune responses. MERTK is also expressed in retinal pigmented epithelium (RPE) cells of the eye where it mediates phagocytosis of photoreceptor outer segment fragments. Mutations in MERTK that disrupt its expression or kinase activity lead to marked retinal degeneration and blindness in mice, rats, and humans. Due to known differences in blood-retinal permeability, we explored whether therapeutic antibodies targeting MERTK could inhibit macrophage-mediated efferocytosis and promote antitumor activity while sparing RPE toxicity. A diverse panel of high-affinity antibodies was developed to explore MERTK blockade in vitro and in vivo. Multiple antibodies disrupted MERTK-GAS6 binding and blocked human and murine macrophage-mediated efferocytosis. Two antibodies targeting distinct GAS6 binding epitopes were selected for further characterization. Both antibodies demonstrated antitumor activity in murine CT26 and MC38 syngeneic colorectal cancer models and led to alterations in immune cell-related gene expression. To investigate potential effects on RPE biology with MERTK antibodies, a multi-dose, 4-week cynomolgus monkey study with several in-life and post-mortem ophthalmologic endpoints was designed. While no abnormal ophthalmic or electroretinography (ERG) findings were detected, all animals treated with either MERTK antibody at all doses showed histological abnormalities of the retina, including vacuolation of the outer segments of photoreceptors, displacement of RPE cells, and single cell necrosis of the outer nuclear layer. These data suggest that inhibition of efferocytosis by antibody-mediated blockade of MERTK can promote immune activation and inhibit tumor growth in vivo; however, retinal toxicity consistent with histological observations made in Mertk mutant animals is an on-target effect. As several therapeutics that block MERTK function are currently in preclinical development, a thorough evaluation of retinal toxicity is warranted. Citation Format: Kerry F. White, Matthew Rausch, Jing Hua, Katherine H. Walsh, Christine E. Miller, Christopher C. Wells, Devapregasan Moodley, Benjamin H. Lee, Scott C. Chappel, Pamela M. Holland, Jonathan A. Hill. MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 558.

MERTK is a TAM family (TYRO-3, AXL, MERTK) receptor tyrosine kinase that is aberrantly expressed in >80% of primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by the small molecule tyrosine kinase inhibitor (TKI), MRX-2843, induced apoptosis in leukemia cell cultures and prolonged survival in mouse xenograft models of AML, but was not curative. In these models, treatment efficiently reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. To evaluate the role of the bone marrow stromal niche in resistance to MERTK inhibition by MRX-2843, Kasumi-1 and OCI-AML5 AML cell lines were cultured with the fibroblast-like Hs27 cell line and induction of apoptosis and cell death was measured by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with Hs27 cells significantly reduced AML cell death in response to MRX-2843 compared to leukemia cells alone (e.g. Kasumi-1 31.9% vs 61.2%, p1.5-fold in the presence or absence of stromal cells, including numerous MAPK pathway components - p38α, ERK1/2, EGFR, TOR, and HSP27. Changes in ERK1/2 phosphorylation (pERK) were confirmed by immunoblot in Kasumi-1 and OCI-AML5 cell cultures. In the absence of co-culture MRX-2843 potently inhibited pERK at doses as low as 100nM, while in co-culture even a 3-fold higher dose did not fully inhibit pERK, consistent with a potential role in resistance. To determine whether the observed upregulation and persistent activation of ERK1/2 has functional significance, Kasumi-1 cells were treated with MRX-2843 and the MEK1/2 TKI pimasertib in the presence or absence of stromal co-culture. Treatment with MRX-2843 or pimasertib alone did not significantly affect survival of AML cells in co-culture (31.3% MRX-2843, 28.1% pimasertib vs 22.7% vehicle, p=ns), but treatment with a combination of MRX-2843 and pimasertib resulted in significant induction of apoptosis compared to vehicle or single TKIs (56.7%, p Citation Format: Katherine A. Minson, Eleana Vasileiadi, Madeline G. Huey, Xiadong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3037.

Tumor growth and progression is influenced by the composition of the tumor microenvironment including host stromal cells and immune cells. In prostate cancer, a large proportion of the tumor volume is made up of macrophages, and high macrophage infiltrate correlates with poor prognosis. Macrophages can be polarized to various subtypes including M1 and M2 in response to stimuli in their environment. M1 macrophages have anti-tumor functions such as immune stimulation. M2 macrophages have pro-tumor functions such as promoting angiogenesis, extracellular matrix remodeling and immune suppression. The majority of prostate cancer tumor-associated macrophages exhibit M2-like characteristics. Thus, targeting M2 tumor-associated macrophages is a promising strategy for cancer therapy. M2 macrophages engage in efferocytosis, or phagocytosis of apoptotic cells. This process is an anti-inflammatory, immunosuppressive event that promotes tissue remodeling and tumor growth. The TAM receptors (Tyro3, Axl, MerTK) are a family of receptor tyrosine kinases whose role in efferocytosis has been well described in the literature. The TAM receptors bind phosphatidylserine on apoptotic cells using their ligands Gas6 and Protein S as bridging proteins. Following binding to phosphatidylserine, TAM receptor signaling induces a cytoskeleton rearrangement to engulf the apoptotic cell. Since efferocytosis is a tumor promoting process, we hypothesize targeting the TAM receptors on M2 macrophages may be a beneficial anti-cancer strategy. We are characterizing TAM receptor expression on M1 and M2 macrophages using two complementary in vitro methods: macrophage polarization of monocytes derived from healthy human donors and of the acute monocytic leukemia cell line THP-1. Cell surface markers of M1 macrophages (CD86) and M2 macrophages (CD163 and CD206) were measured to assess polarization by flow cytometry. We are currently further confirming the degree of polarization by measuring cytokine secretion and mRNA expression of M1- and M2-associated genes. We used flow cytometry analysis to assess TAM receptor expression on the in vitro polarized M1 and M2 macrophages. In the human monocyte derived model, we detected higher levels of expression of MerTK and Tyro3 on M2 macrophages in several biological replicates. We also found that M2 THP-1 macrophages express higher levels of MerTK and Tyro3 than M1 THP-1 macrophages, consistent with macrophages from the monocyte derived model. In conclusion, these data support the TAM receptors as a relevant therapeutic target for inhibiting M2 pro-tumor functions. Further work by qRT-PCR, western blot and immunofluorescence staining will be used to confirm higher levels of the MerTK and Tyro3 on M2 macrophages and further investigate expression of Axl on M1 and M2 macrophages. Citation Format: Kayla V. Myers, Kenneth J. Pienta. Targeting the TAM receptors on prostate cancer tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4556.

Introduction: MERTK is thought to play a role in cancer immune evasion mechanism based on M2 type macrophage-driven immunosuppression in tumor microenvironment (TME). Thus, MERTK is a highly promising target for anti-cancer therapy under high tumor associated macrophages (TAMs) condition. We evaluated the anti-tumor effects and immune reaction of combination or single MERTK inhibitor in two lung squamous patients derived xenograft (PDX) tumor engrafted Hu-CD34-NSG models. Methods: The aPD-1 sensitive (YHIM2004) and aPD-1 non-sensitive (YHIM2009) squamous cell carcinoma PDX models were selected from pre-test with pembrolizumab. The selected two models were composed four groups; control, pembrolizumab, MERTK inhibitor, and combination. We applied pembrolizumab as aPD-1 blockade (10 mpk, q2w, i.p. inj.). The dose of MERTK inhibitor was 50 mpk, q1d by orally. The treatment was started when tumor size reached 100 mm3. Whole exome sequencing (WES), microarray (mRNA), flow cytometry, and multispectral image analysis were performed on the tumors. To elucidate the involvement of MERTK inhibitor in tumor, the tumor mutation burden, tumor microenvironment and immune cell phenotype were deconvoluted. Results: The TMB of YHIM2004 and YHIM2009 was 3.23 and 2.64 (mutations/Mb) respectively. The PD-L1 and TIL level of YHIM2004 were relatively higher than YHIM2009. Both two models were observed high macrophage in tumor. The two PDX-models, YHIM2004 (aPD-1 sensitive), YHIM2009 (aPD-1 non-sensitive) were observed anti-tumor effects in MERTK inhibitor single treatment groups (TGI = 44.23%, 67.40% respectively, p < 0.05, t-test). Especially, YHIM2004 tumor was reduced after combination therapy more than single treatment. Of note, the NOG-PDX model demonstrated no anti-tumor effect in MERTK inhibitor monotherapy, suggesting that this drug involves anti-cancer immune responses. Flow cytometry data showed increased PD-1+ and granzyme B+ T cells (p Conclusion: We concluded that MERTK signaling involves regulation of anti-cancer immune responses. Our results suggest an anti-cancer therapeutics of MERTK selective inhibitor on aPD-1 sensitive or non-sensitive tumors under high TAM tumor microenvironment. Citation Format: Kyoung-Ho Pyo, Ha-Ni Cho, Chun-Feng Xin, Jae Seok Cho, Han Na Kang, Jiyeon Yun, Hye Ryun Kim, Byoung Chul Cho. Efficacy of MERTK inhibitor in combination with pembrolizumab in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4081.

MERTK, a receptor tyrosine kinase of the TYRO3/AXL/MERTK (TAM) family, is expressed in innate immune cells including macrophages, dendritic cells and NK cells and is overexpressed in a wide variety of cancers, including leukemia and many solid cancers. Activation of MERTK on cancer cells via ligand binding results in activation of several tumor-promoting signaling pathways, which stimulate proliferation, migration and angiogenesis, and decrease apoptosis and chemosensitivity. Furthermore, activation of MERTK on macrophages drives immune evasion through the promotion of an immune-suppressive M2 phenotype. Pre-clinical and clinical studies have shown promising emerging evidence of anti-tumor efficacy upon modulation of TAM receptor signaling. Herein, we report the pre-clinical characterization of RGX-019, a humanized IgG1 antibody with high affinity and specificity for human MERTK. Surface Plasmon Resonance, cell-based binding assays and competition ELISA demonstrate high affinity/avidity binding for RGX-019 to human MERTK. In contrast, binding to human AXL, human TYRO3, or murine MERTK was not detected. Binding of RGX-019 to the MERTK receptor triggered its rapid internalization and degradation from the surface of human cancer cells within 4 hours and prevented Gas6 induced phosphorylation of AKT, a downstream signal transduction pathway that promotes cell growth and survival. RGX-019 treatment of SKMel5 melanoma cells effectively inhibited colony formation. RGX-019-induced degradation of MERTK in in vitro differentiated human M2 macrophages reduced AKT activation and promoted a pro-inflammatory cytokine signature. Furthermore, a related surrogate murine MERTK antibody significantly inhibited growth of MDA-MB-231 breast cancer tumors, demonstrating single agent anti-tumor efficacy in vivo. These findings demonstrate that RGX-019 is a potent and selective inhibitor of MERTK signaling with a unique mechanism-of-action that potently disrupts MERTK signaling in both cancer cells and immune-suppressive macrophages via MERTK receptor degradation. This activity results in robust repression of cancer cell growth both in vitro and in vivo. Overall, these data support further development of RGX-019 as a cancer therapeutic. Citation Format: Shugaku Takeda, Celia Andreu-Agullo, Subhasree Sridhar, Nils Halberg, Ivo C. Lorenz, Sohail Tavazoie, Isabel Kurth, Masoud Tavazoie. Characterization of the anti-cancer and immunologic activity of RGX-019, a novel pre-clinical stage humanized monoclonal antibody targeting the MERTK receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-277.

Acute myeloid leukemia (AML) has a 5-year overall survival rate under 26% in adults and current cytotoxic chemotherapies are toxic, can cause long-term side effects and are often contraindicated for elderly patients. Targeted therapies may have reduced toxicity compared to chemotherapy; however, resistance to single-agents often develops and combination therapies may provide more durable responses. MERTK is aberrantly expressed in >80% of AML patient samples and is a promising therapeutic target. MRX-2843 is a novel small molecule dual MERTK and FMS-like tyrosine kinase 3 (FLT3) inhibitor that is currently in clinical trials. Here, we report synergistic anti-leukemia activity mediated by MRX-2843 and rho-associated, coiled-coil-containing protein kinases 1 and 2 (ROCK1/2) inhibitors. Both MERTK and ROCK1/2 have been implicated in actin/microtubule dynamics and cell cycle progression and previous publications demonstrated induction of apoptosis in AML cells in response to RNAi-mediated MERTK or ROCK1 inhibition. ROCK1/2 inhibitors RKI-1447 or GSK269962A synergized with sub-therapeutic doses of MRX-2843 to reduce cell density as indicated by decreased Presto Blue staining in cultures of 4 of 6 AML cell lines tested and had an additive effect in the remaining 2 cell lines. Similar interactions were observed between RKI-1447 and UNC3997, a MERTK-selective TKI with 15-fold weaker FLT3 activity. Flow cytometric analysis of cells stained with popro-1-iodide and propidium iodide (PI) dyes revealed synergistic induction of cell death in the KG-1, OCI-AML5, and NB4 cell lines and an additive effect in Kasumi-1 cultures. In addition, cells that survived treatment exhibited subsequent defects in expansion, even when they were cultured without inhibitor(s), and these effects were significantly more pronounced in cells treated with the combination therapy compared to single agents. Flow cytometric analysis of PI-stained permeabilized cells revealed an increased fraction of cells in G2/M phase in cultures treated with MRX-2843. This effect was more pronounced in cultures treated with the combination therapy and was accompanied by accumulation of a population with slightly reduced DNA content relative to untreated G2 cells, suggestive of a defect in late S-phase. Thus, the combination therapy mediates anti-leukemia activity by multiple mechanisms, including abrogation of cell cycle progression and induction of cell death. In addition, a preliminary analysis of The Cancer Genome Atlas database revealed significantly poorer overall survival in patients with higher levels of ROCK1 expression (p=0.00147, n=172). Together, these data suggest that combination therapies targeting MERTK and ROCK1/2 may be particularly effective for treatment of AML and support further studies to test the effects of this novel strategy in animal models. Citation Format: Dawn E. Barnes, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Combined MERTK and ROCK1/2 inhibition as a potential therapeutic strategy for AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2192A.

The beta-galactoside-binding protein galectin-3 (Gal-3) has been shown to influence the tumorigenic behaviour of cancer cells, and elevated serum levels of Gal-3 have been linked to tumor metastasis in several different cancers. Previously, Gal-3 was shown to be a ligand MerTK, a member of the TAM (Tyro3, Axl, MerTK) subfamily of receptor tyrosine kinases (RTKs) - these RTKs are implicated in tumorigenesis and/or enabling tumor chemoresistance in many different cancers. However, it is not known whether Gal-3 is also a ligand for the other two TAM RTKs, nor what signaling pathways it activates to regulate cancer cell functions. This study is aimed at determining the ligand properties of Gal-3 against Tyro3 and Axl RTKs in human cancer cells and the oncogenic signal pathways linked to them. The cultured human head and neck squamous cell carcinoma (HNSCC) cell line SCC-25 was determined by western blot and qPCR to express both Axl and Tyro3, but not MerTK. Recombinant Gal-3 and the TAM ligands Gas6 and ProS1 were added to serum-starved cells over a time-period of minutes and cells lysed to determine activation/phosphorylation of RTKs and intracellular signal mediators by western blotting. Longer incubations were performed to measure cell viability (MTS assay) and cell migration rate (scratch assay). All experiments were carried out a minimum three times. The known TAM ligands Gas6 and ProS1 stimulated TAM RTKs differentially in SCC-25 cells: Gas6 activated Axl strongly within minutes, with ProS1 having a weak effect. In contrast, ProS1 was the strongest ligand for Tyro3, activating it by fourfold, followed by Gal-3 at threefold and Gas6 at twofold. All three exogenous proteins also rapidly stimulated ERK1/2 phosphorylation by two-fold, whereas only Gas6 and Gal-3 stimulated Akt phosphorylation. All three proteins significantly maintained cell survival during acute induction of apoptosis by staurosporine. However, only Gas6 conferred a survival effect over long-term serum starvation-induced cell death. In addition, Gal-3 significantly stimulated cell migration during Axl blockade with the small molecule Axl inhibitor BGB324, further suggesting a role for Tyro3 in this effect. These results have shown that Gal-3 is a ligand for Tyro3 RTK, and that Gal-3 promotes cell survival and migration in HNSCC cells, coupled with activation of both the related signal pathways ERK and Akt. The data therefore reveal a new mechanism of Tyro3 activation; however, it remains to be determined whether Gal-3 interacts with the glycosyl chain on the RTK or else via other interfaces. Finally, cancer therapeutic strategies targeting Gal-3 action and/or cell surface glycosylation may be viable approaches in further research. Citation Format: Sassan Hafizi, Nour Al Kafri. Galectin-3 activates the receptor tyrosine kinase Tyro3 and stimulates ERK signaling, cell survival and migration in human head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2638.

The TAM receptors (Tyro3/Axl/MerTK) family of receptor tyrosine kinases (RTKs) are important negative regulators of innate immunity. TAM receptor activation in myeloid cells by its ligands Gas6 or Protein S (PROS) promotes phosphatidylserine-dependent efferocytosis of apoptotic cells, inducing a tolerogenic state and mediating resolution of inflammation. TAM-deficient mice exhibit phenotypes consistent with systemic inflammation and autoimmunity. Importantly, individual ablation of TAM receptors can confer tumor immunity, increased pro-inflammatory cytokines and tumor lymphocyte infiltration, leading to the proposal that TAM receptors act as checkpoints of innate immunity. We hypothesize that pharmacological targeting of this family of receptors with monoclonal antibodies (mAbs) may lead to a similar pro-inflammatory response and recapitulate the antitumor effects observed in TAM-deficient mice. From a panel of human anti-MerTK, Axl or Tyro3 mAbs derived from phage-display libraries or human IgG expressing mice we identified unique mAbs that markedly enhanced cytokine and chemokine release from primary human immune cells, alone or in the presence of inflammatory stimuli. Interestingly, the qualitative and quantitative pattern of cytokine response from dendritic cells was very similar using antibodies targeting the individual TAM receptors, and also similar to activation of dendritic cells using a CD40 agonist mAb, suggesting that TAM-targeting mAbs can promote immune activation. We identified surrogate mAbs targeting mouse TAM receptors that elicited similar responses in vivo, and demonstrated antitumor activity when dosed alone, or in combination with PD-1/L1 blockade in syngeneic tumor models. In addition, human MerTK transgenic, and TAM knockout mice have been generated and characterized in order to establish in vivo proof-of-concept with human TAM mAbs. Overall, pharmacological modulation of TAM receptors with mAbs enhances cytokine production in human and murine model systems consistent with the published role of TAMs as negative regulators of innate immunity. Mechanistic and proof-of-concept studies support further development of these mAbs as novel approaches to overcome these checkpoints of the innate immune response. Citation Format: Diego Alvarado, Laura Vitale, Mike Murphy, Thomas O'Neill, Andrew Proffitt, Jay Lillquist, Gwenda Ligon, Komal Patel, Anna Wasiuk, Jeff Weidlick, Jenifer Widger, Laura Mills-Chen, Andrea Crocker, Colleen Patterson, Russell A. Hammond, Li-Zhen He, Joel Goldstein, Lawrence J. Thomas, Henry C. Marsh, Tibor Keler, Richard Gedrich. Monoclonal antibodies targeting the TAM family of receptor tyrosine kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1555.

Triple-negative breast cancer (TNBC) and other molecularly heterogeneous malignancies present a significant clinical challenge due to a lack of high-frequency “driver” alterations amenable to therapeutic intervention. These cancers often exhibit genomic instability, resulting in chromosomal rearrangements that affect the structure and expression of protein-coding genes. However, identification of these rearrangements remains technically challenging. Using a newly developed approach that quantitatively predicts gene rearrangements in tumor-derived genetic material, we identified and characterized a novel oncogenic fusion involving the MER proto-oncogene tyrosine kinase (MERTK) and discovered a clinical occurrence and cell line model of the targetable FGFR3–TACC3 fusion in TNBC. Expanding our analysis to other malignancies, we identified a diverse array of novel and known hybrid transcripts, including rearrangements between noncoding regions and clinically relevant genes such as ALK, CSF1R, and CD274/PD-L1. The over 1,000 genetic alterations we identified highlight the importance of considering noncoding gene rearrangement partners, and the targetable gene fusions identified in TNBC demonstrate the need to advance gene fusion detection for molecularly heterogeneous cancers. Cancer Res; 76(16); 4850–60. ©2016 AACR.

Background: Early T-precursor acute lymphoblastic leukemia (ETP-ALL) is a subclass of T-cell ALL (T-ALL) accounting for 15% of pediatric T-ALL cases and characterized by an immature phenotype, resistance to therapy, and high rates of induction failure and relapse (Wood B et al., Blood 2009). MERTK receptor tyrosine kinase is not expressed in normal T cells but is ectopically expressed in 40-50% of T-ALLs, particularly those with an immature T-cell phenotype (Graham DK et al., Clin Cancer Res 2006), suggesting a role in ETP-ALL. One potential role is regulation of the anti-apoptotic protein B-cell lymphoma-2 (BCL-2). BCL-2 is specifically expressed in double-negative T-cell precursors and is preferentially expressed in ETP-ALL compared to T-ALL (Chonghaile et al., Cancer Discovery 2014). Moreover, ETP-ALL cells are dependent on BCL-2 for survival. Our previous studies demonstrated regulation of BCL-2 and BCL-2 family members downstream of MERTK in B-ALL and acute myeloid leukemia cells (Linger RM et al., Blood 2013; Lee-Sherick AB et al., Oncotarget 2015). This interplay between MERTK and BCL-2 and their association with an immature T-ALL phenotype suggest that combination therapies targeting these two proteins may be particularly effective to treat ETP-ALL. Methods: Publicly available mRNA expression data were used to assess MERTK and BCL-2 expression in T-ALL cell lines and patient samples. MERTK and BCL-2 protein expression were determined by immunoblot. ETP-ALL cell lines were cultured with vehicle or MRX-2843, a dual MERTK/FLT3 kinase inhibitor. MERTK protein was immunoprecipitated from cell lysates and phosphorylated and total proteins were assessed by immunoblot. Alternatively, cells were stained with PoPro-1-iodide and propidium iodide dyes and analyzed by flow cytometry to assess cell death. Orthotopic xenografts were established in NSGS mice using an ETP-ALL patient sample, and leukemia burden in peripheral blood (%hCD45+) was monitored by flow cytometry. After engraftment (1.86 +/- 0.43% peripheral blasts), mice were treated once daily with 75 mg/kg MRX-2843 or saline vehicle administered orally. Mice were euthanized when symptoms of advanced leukemia were evident and median survival was determined by Kaplan-Meier analysis. Results: MERTK mRNA was expressed at significantly higher levels in ETP-ALL cell lines and patient samples relative to other T-ALLs. Similarly, MERTK protein was ubiquitously expressed in ETP-ALL cell lines (n=2), ETP-ALL patient samples (n=2), and a near-ETP-ALL patient sample (n=1). In contrast, only 60% of other T-ALL cell lines (n=5) expressed MERTK. BCL-2 mRNA was expressed at significantly higher levels in ETP-ALL patient samples relative to other T-ALLs and BCL-2 protein was expressed in 2 of 2 ETP-ALL cell lines and 2 of 3 ETP-ALL and near-ETP-ALL patient samples. Treatment with MRX-2843 mediated a dose-dependent decrease in phosphorylated MERTK in ETP-ALL cells and induced dose-dependent cell death in the ETP-ALL cell lines PEER (43.2% vs 16% in vehicle-treated cultures, p Conclusions: MERTK and BCL-2 are preferentially expressed in ETP-ALL relative to T-ALL and MRX-2843, a dual MERTK/FLT3 kinase inhibitor, has robust therapeutic activity in cell culture and xenograft models of ETP-ALL. These data validate MRX-2843 as a novel agent with potential for clinical application in patients with ETP-ALL. In particular, combination therapy targeting MERTK and BCL-2 may be an effective therapeutic option for ETP-ALL. Citation Format: Ryan J. Summers, Katherine A. Minson, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. MERTK and BCL-2 as potential therapeutic targets in early T-precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A35.

Axl and MerTK are members of the TAM family (Tyro, Axl and MerTK) of receptor tyrosine kinases and have been reported to be altered in several types of cancer including Head and Neck Squamous Carcinoma (HNSCC). It is well known that aberrant expression or activity of Axl is associated with resistance to various therapies. Therefore, the translational potential of targeting AXL in HNSCC is worthy, however poorly defined. We recently identified an adaptive feedback mechanism of resistance to AXL-targeting agents mediated by upregulation of MerTK that reveals a direct role for MerTK in resistance to Axl inhibition. In this study, we investigated potential radiosensitization by dual targeting of Axl and MerTK in combination with radiation in human HNSCC cell lines and xenografts (SCC47 and TU138). Immunoblot analysis demonstrated that endogenous levels of Axl or MerTK expression were distinct between these two cell lines. SCC47 cells expressed higher endogenous levels of Axl compared to Tu138. Tu138 expressed higher levels of MerTK compared to SCC47 cells. Radiation induced Axl phosphorylation in SCC47 cells but not in Tu138 cells in vitro. Next, inhibitors of Axl (R428) and/or MerTK (UNC2025) were utilized in combination with single dose radiation in vitro. Clonogenic assays showed that the Axl inhibitor alone acted as a potent radiosensitizer in SCC47 cells. To better define Axl's role in radiation response in HNSCC, we developed HN30-Vector and HN30-Axl stable cells and exposed them to radiation. Phosphorylation of Axl was increased 1 hour post-radiation in Axl overexpressed HN30 compared to parental HN30-Vector which expresses low endogenous levels of Axl. We also found that the HN30-Axl cell line was associated with radioresistance by clonogenic assays. Tumor xenograft studies demonstrated that the combination of R428 and UNC2025 inhibitors improved the efficacy of radiation in Tu138 but not in SCC47. Interestingly, radiation therapy alone was more effective in SCC47 tumors than TU138 tumors. These results reveal that the co-targeting of Axl and MerTK enhanced tumor growth delay in TU138 xenograft mouse models in vivo. Mechanisms of radiosensitization are currently under investigation. Citation Format: Mahyar Abbariki, Mahmoud Toulany, Mari Iida, Alecia Morgan, Kwang Nickel, Nellie McDaniel, Grace Kang, Chunrong Li, Paul Harari, Deric Wheeler. Co-targeting of Axl and MerTK improves radiation response in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 846.

The combination of tumor targeted therapeutics with PD-L1 checkpoint blockade is being explored as a method to increase the clinical benefits of immunotherapy, and expand response to additional cancer types. Merestinib (Mer) is a kinase inhibitor targeting several oncokinases1 (including MET, MST1R, AXL, MERTK, and MKNK1/2) that can potentially modulate immune function, angiogenesis, as well as target the tumor 1-5. To determine the combinatorial potential with immunotherapy, the effects of Mer were evaluated in vitro on human T cells, PBMCs and murine tumor lines CT26 colon carcinoma (harbors KRASmt G12D expresses low Met/no p-Met/high Axl/p-Axl) and B16F10 melanoma (expressing high Met/pMet/peIF4E). Additionally, the anti-tumor effect of Mer was tested in vivo on established CT26 and B16F10 tumors compared to MET specific TKIs (savolitinib, PF4217903) alone or in combination with PD-L1 antibody (Ab) blockade. In vitro, Mer showed no significant effects on either T cells or PBMCs, but was able to inhibit downstream signaling in both CT26 and B16F10 showing activity on murine tumor cell lines. In vivo, daily Mer monotherapy (6, 12 or 24 mg/kg) showed significant anti-tumor effect at all doses in both CT26 and B16F10, that was not seen with either savolitinib or PF4217903. Concurrent combination of Mer (12 mg/kg) and anti-PD-L1 Ab (0.5 mg qw) in CT26 was found to have anti-tumor activity that was synergistic as compared to each single agent alone. While the effect of Mer monotherapy was lost when treatment ended, tumors continued to regress in the combination group even upon cessation of therapy. The combination was well tolerated and resulted in 90% complete responders compared to 30% with anti-PD-L1 Ab alone, 35 days after completing dosing. To test the ability to generate immunologic memory, complete responders were re-challenged with CT26 cells on the contralateral side. All mice in the combination group resisted re-challenge, showing that Mer/PD-L1 Ab combination was triggering immunologic memory. Although there was no significant change in intra-tumor immune cell populations between groups, combination therapy showed an enhanced and unique intra-tumor immune activation/inflammation gene expression signature compared to PD-L1 Ab monotherapy. The enhanced immune activation of the combination therapy, leading to synergistic anti-tumor efficacy, demonstrates that merestinib has the potential to augment immunotherapy while targeting the tumor directly. This preclinical data provides the rationale for the clinical investigation of merestinib in combination with checkpoint therapies targeting the PD-L1/PD1 axis (NCT02791334). 1 - Yan et al. Invest New Drugs 2013;31:833-44 2 - Balan et al. J Biol Chem 2015;290:8110-20 3 - Eyob et al. Cancer Discov 2013;3:751-60 4 - Lemke G. CSH Persp Biol 2013;5:a009076 5 - Piccirillo et al. Nat Immunol 2014;15:503-11 Citation Format: Sau-Chi Betty Yan, Victoria L. Peek, Jennifer R. Stephens, Um L. Um, Amaladas Nelusha, Colleen A. Burns, Kelly M. Credille, Thompson N. Doman, Scott W. Eastman, Beverly L. Falcon, Gerald E. Hall, Philip W. Iversen, Bruce W. Konicek, Jason R. Manro, Any T. Pappas, Julie A. Stewart, Michael B. Topper, Swee-Seong Wong, Michael Kalos, Ruslan D. Novosiadly, Richard A. Walgren, David Schaer. Combination of an oncokinase inhibitor merestinib with anti-PD-L1 results in enhanced immune mediated antitumor activity in CT26 murine tumor model [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 5590. doi:10.1158/1538-7445.AM2017-5590

In recent years, the TAM (TYRO3, AXL, and MER) family of receptor tyrosine kinases (RTKs) has emerged as attractive targets for oncology therapeutics. Under homeostatic conditions, the TAM RTKs are expressed by a number of immune cells where they play key roles in the negative regulation of the immune response. However, their expression on cancer cells has been specifically associated with epithelial-to-mesenchymal transition, an invasive phenotype, and more generally, with a poor prognostic outcome in human cancers. Here, we sought (1) to establish whether expression of the TAM was sufficient to drive cellular transformation, (2) to demonstrate that RXDX-106, a small molecule inhibitor of TAM RTKs, could inhibit tumors harboring activating TAM gene fusions, and furthermore, (3) to decipher how pharmacological inhibition of TAM signaling pathways both on cancer cells and immune cells would be beneficial, given their complex regulation and intimate relationship in the tumor microenvironment. TAM expression on cancer cells has emerged as one of the key mechanisms of resistance to targeted therapies, particularly to EGFR tyrosine kinase inhibitors (TKI). In addition, novel gene rearrangements (fusions) have been identified in the TCGA database for both Mertk (TMEM87B-Mertk) and Axl (Axl-MBIP) that retain the functionality of the tyrosine kinase domain, but have fusion partners that alter the expression profile or dimerization frequency, respectively. Here, we demonstrate both in vitro and in vivo that expression of either wild type receptors or TMEM87B-MER and AXL-MBIP fusion proteins is sufficient to drive oncogenic transformation by both ligand dependent and independent mechanisms. Treatment of these TAM-expressing cellular populations with RXDX-106 completely inhibits TAM phosphorylation and downstream signaling and, consistent with our signaling data, RXDX-106 completely inhibits cellular proliferation and viability at sub-nanomolar concentrations. Furthermore, we demonstrate that, in the co-culture of TAM-expressing cancer cells with immune cells such as macrophages, TAM ligands such as Gas6 are secreted to drive ligand dependent activation of TAM on cancer cells, leading to their survival and proliferation. In summary, our data suggest that TAM RTKs can act as traditional oncodrivers when activated either in a ligand dependent or ligand independent manner. In addition, we demonstrate that TAM fusions are tractable therapeutic targets and that patients with tumors harboring such molecular alterations may derive clinical benefit from RXDX-106. Finally, we show that RXDX-106 not only has the potential to inhibit cellular proliferation and survival on the cancer cell itself, but also affect the TAM-expressing tumor microenvironment to result in a global anti-cancer environment. Citation Format: Erin D. Lew, Elizabeth A. Tindall, Joanne Oh, Colin Walsh, Maria Barrera, Heather Ely, Amy Diliberto, Yumi Yokoyama, Gary Li, Amanda Albert. RXDX-106, a novel, selective and potent small molecule TAM (TYRO3, AXL, MER) inhibitor, demonstrates efficacy in TAM-driven tumors [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 4191. doi:10.1158/1538-7445.AM2017-4191

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

Introduction: MerTK, a TAM (Tyro3, Axl, MerTK) family RTK, is expressed on phagocytic myeloid and epithelial cells. Its normal function is to dampen innate immune responses to self-antigens. MerTK is an indirect phosphatidylserine (PtdSer) receptor: PtdSer-binding TAM ligands, Gas6 or Protein S, bridge interactions between MerTK and PtdSer externalized on apoptotic cells (ACs), resulting in AC internalization (efferocytosis). Ensuing MerTK signaling leads to anti-inflammatory M2 macrophage polarization, suppression of pro-inflammatory cytokine production, and a tolerogenic outcome. Tumors are rich in ACs and TAM ligands. Syngeneic tumors implanted in MerTK -/- mice exhibit impaired growth and metastasis compared with those implanted in WT mice, correlating with enhanced production of pro-inflammatory cytokines, splenocyte proliferation, and decreased IL-10. Moreover, MerTK aberrantly expressed on hematological and epithelial malignancies promotes survival and chemoresistance. Thus, pharmacological inhibition of MerTK may have clinical benefit by increasing availability of dead tumor cell antigens, blocking tumor induced immunosuppression, or directly promoting tumor cell survival. We have therefore developed small molecule inhibitors of MerTK. Methods: MerTK kinase activity was assayed using ADP-Glo. Cellular MerTK activity was stimulated in HUVEC or H1299 cells using anti-MerTK crosslinking and measured by immunoprecipitation followed by anti-phospho-MerTK blot, or by downstream phospho-Akt Ser 473 using HTRF. A high content assay was used to measure cell proliferation, DNA content and apoptosis. Immune effector assays were LPS-induced IL-23 production in human primary dendritic cells and anti-CD3/CD28-induced IL-2 production or IL-2 induced phospho-STAT5 in human primary T cells. Efferocytosis of CFSE-labeled apoptotic Jurkat cells by anti-CD14-labeled human primary macrophages was detected by flow cytometry. Results: Here we describe novel MerTK-selective and Mer-Axl small molecule inhibitors that potently block MerTK in biochemical assays. These compounds exhibit selectivity for TAM family members in an in vitro kinase panel. In cells, antibody-induced MerTK phosphorylation as well as downstream phosphorylation of Akt was inhibited by both compounds with EC50 Conclusions: We have discovered potent and novel small molecule inhibitors of MerTK that may have clinical benefit by both direct anti-tumor effects and by enhancing the anti-tumor immune response. Citation Format: Sacha J. Holland, Alex M. Owyang, Sothy Yi, Chi Young, Sylvia Braselmann, Roy Frances, Arthur Bagos, Ernest Tai, Stacey Siu, Gary Park, David Lau, Matt Duan, Rao Kolluri, Somasekhar Bhamidipati, Ihab Darwish, Matthew Duncton, Rajinder Singh, Esteban Masuda, Donald G. Payan. Small molecule inhibitors of the anti-inflammatory TAM receptor MerTK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4869.

The chicken anemia virus–derived protein apoptin induces apoptosis in a variety of human malignant and transformed cells but not in normal cells. However, the mechanisms through which apoptin achieves its selective killing effects are not well understood. We developed a lentiviral vector encoding a green fluorescent protein–apoptin fusion gene (LV-GFP-AP) that can efficiently deliver apoptin into hematopoietic cells. Apoptin selectively killed the human multiple myeloma cell lines MM1.R and MM1.S, and the leukemia cell lines K562, HL60, U937, KG1, and NB4. In contrast, normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays. In addition, dexamethasone-resistant MM1.R cells were found to be more susceptible to apoptin-induced cell death than the parental matched MM1.S cells. Death susceptibility correlated with increased phosphorylation and activation of the apoptin protein in MM1.R cells. Expression array profiling identified differential kinase profiles between MM1.R and MM1.S cells. Among these kinases, protein kinase Cβ (PKCβ) was found by immunoprecipitation and in vitro kinase studies to be a candidate kinase responsible for apoptin phosphorylation. Indeed, shRNA knockdown or drug-mediated inhibition of PKCβ significantly reduced apoptin phosphorylation. Furthermore, apoptin-mediated cell death proceeded through the upregulation of PKCβ, activation of caspase-9/3, cleavage of the PKCδ catalytic domain, and downregulation of the MERTK and AKT kinases. Collectively, these results elucidate a novel pathway for apoptin activation involving PKCβ and PKCδ. Further, they highlight the potential of apoptin and its cellular regulators to purge bone marrow used in autologous transplantation for multiple myeloma. Cancer Res; 70(18); 7242–52. ©2010 AACR.

Background: Mounting evidence supports MET as a target in urothelial carcinoma (UC) of the bladder. Activated MET can promote tumor growth and metastasis, as well as tumor angiogenesis by upregulating VEGF, and may play a role in UC pathogenesis. The effects of MET inhibitor cabozantinib on MET-driven UC cell growth, invasion and tumorigenicity were previously analyzed in biochemical, cell-based and animal models. Current known cabozantinib targets (MET, KDR, FLT3, KIT, RET, AXL and ROS) and possible targets based on structural similarity (MERTK, TYRO3, RON, RYK, CSF1R, ROR1, ROR2, ALK and LTK) were characterized in 13 UC-derived cell lines by determining the frequency and absolute level of mRNA expression, and ultimately, for targets where mRNA was present, encoded protein abundance, ligand expression and cabozantinib sensitivity. Methods: The effects and the targets of cabozantinib on human UC-derived cell lines were studied in vitro. Cells at 80% confluence were serum deprived 16h, then left untreated or treated with hepatocyte growth factor (HGF) and/or cabozantinib prior to analysis of MET, phospho- (p)MET, pAKT1, total AKT1, pMAPK1/2 and total MAPK1/2 by two-site immunoassay and/or immunoblotting. Cabozantinib effects on basal and HGF-induced UC cell invasion and proliferation were measured. Gene mRNA abundance (absolute copy number) of cabozantinib targets was measured using real time PCR from the extracted RNAs of 13 UC cell lines. Results: Basal pMET content was uniformly low, and was increased significantly by HGF; the latter was reversed by cabozantinib treatment. HGF-driven increases in pAKT1:AKT1 and pMAPK1/2:MAPK1/2 ratios in all cell lines were reversed by cabozantinib treatment, as were HGF-enhanced UC cell invasion, proliferation and tumorigenicity. UC cell lines displayed uniformly robust mRNA expression of MET, AXL, RYK, RON, CSF1R, ROR1 and ROR2, and frequent modest expression of RET and ROS, whereas KDR, ALK, KIT, LYK, TYRO3, MERTK and FLT3 showed little or no expression. MET mRNA and protein abundance were significantly positively correlated among these 13 UC cell lines. Conclusions: UC cell MET content was higher with higher UC grade. HGF was not expressed in any UC cell line tested, suggesting that this pathway is predominantly paracrine in vivo. Added HGF stimulated activation of MET and known effectors, and enhanced invasion, growth rate and anchorage-independent growth; cabozantinib effectively reversed these HGF-driven effects. Based on receptor expression, cabozantinib is most likely acting through MET, AXL and ROS, and potentially through RYK, ROR1, ROR2, and/or RET, in UC cells, all of which have been implicated in oncogenesis in other human malignancies. Note: This abstract was not presented at the meeting. Citation Format: Young H. Lee, Tiffany K. Wong, Andrea B. Apolo, Piyush K. Agarwal, Donald P. Bottaro. Oncogenic signaling by MET and other cabozantinib targets in cells derived from urothelial carcinoma of the bladder. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 140. doi:10.1158/1538-7445.AM2015-140

20% of all breast cancers occur in pre-menopausal women, a relatively under-investigated group of breast cancers. These studies addressed a subset of pre-menopausal breast cancers, those which are diagnosed during post-partum involution. Post-partum breast cancers (ppBCs) account for nearly 25% of all pre-menopausal breast cancers, are more frequently diagnosed at metastatic stages than other breast cancers in young women, and correlate with decreased disease-free survival, regardless of the woman's age at the time of diagnosis or the molecular breast cancer subtype. Increasing evidence shows that stromal remodeling events that occur during post-partum involution accelerate the malignant progression of breast cancers, although the mechanisms underlying this observation have remained poorly understood. Using transgenic mouse models of spontaneous breast cancer and allografted models of mammary tumors in immune-competent mice, we investigated pro-metastatic changes that occur during post-partum involution, recapitulating the findings that post-partum involution increases mammary tumor metastasis at least 10-fold. We investigated how physiological events that occur during post-partum involution, such as epithelial cell death, influx of macrophages, and local alterations in cytokines, might accelerate the metastasis of tumors within the post-partum mammary microenvironment. We discovered that a transient wave of widespread cell death occurs not only in normal mammary epithelial cells (MECs) during post-partum involution, but also within the mammary tumor epithelium. Like MECs in the normal mammary gland, these dying tumor cells are cleared from the tumor microenvironment by efferocytosis, the process by which macrophages or other phagocytes engulf dying cells. Efferocytosis by macrophages requires the receptor tyrosine kinase MerTK, and is followed by MerTK-mediated M2 macrophage polarization and production of pro-metastatic wound healing cytokines. Pharmacological inhibition of MerTK or genetic MerTK ablation impaired efferocytosis in the post-partum tumor microenvironment, blocked M2 macrophage polarization and dampened expression of pro-metastatic wound healing cytokines (IL-4 and TGF-β1). Further, blockade of TGFβ1 (but not IL-4) downstream of efferocytosis impaired post-partum metastasis. These data demonstrate for the first time that 1) efferocytosis occurs in the tumor microenvironment, 2.) efferocytosis promotes a pro-metastatic microenvironment, 3.) physiological induction of widespread epithelial cell death in the post-partum breast trigger the pro-metastatic stromal remodeling events of post-partum involution, and, 4.) targeted MerTK inhibition impairs efferocytosis-induced wound healing events that contribute to enhanced metastasis. Note: This abstract was not presented at the meeting. Citation Format: Rebecca S. Cook. Tumor wound healing in response to treatment-induced tumor cell death increases tumor metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2916. doi:10.1158/1538-7445.AM2015-2916

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.

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 (>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

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating human cancers, having a median survival of 6 months and a 5-year survival rate of 6%. It is the fourth leading cause of cancer deaths due to its resistance to conventional radiotherapy and chemotherapy and a lack of early detection. Alternatively, small molecule inhibitors have been emerging as a means of transitioning away from non-selective treatments towards a more personalized approach. Pancreatic cancer, having mutant K-RAS and TP53 in upwards of 90% of cases, appears to be a great candidate for molecular therapy due to this high genetic homogeneity among patients. TP53 is an inactivation mutation and not a candidate for inhibition. K-RAS is an over-activating mutation but unfortunately cannot currently be treated by small molecule inhibitors. However, its downstream pathways, which signal both growth and survival, can. The most predominant of these is the MAP kinase pathway. Several small molecules have been developed to target members of this pathway such as MEK 1/2 including U0126, PD0325901 and AZD6244. Often, however, these therapies are still ineffective and it would be immensely beneficial to be able to predict when MEK inhibition will be effective or should conversely be avoided. To discover such markers, we tested a panel of 18 pancreatic cancer cell lines for sensitivity to the MEK inhibitor AZD6244. After classifying each line's sensitivity, microarray and qPCR analysis found significantly different genes between the sensitive and resistant groups. Most notable were MERTK and MAPK8, which both were upregulated in resistant lines and showed some redundancy in the studies. These genes code for the Tyrosine-protein kinase Mer (MERTK) and c-Jun N-terminal kinase (JNK1) proteins respectively. Correlation studies showed both gene and protein expression levels to track with resistance to the small molecule inhibitor. To test whether these genes and the proteins they code for could serve as potential new therapy targets, shRNA studies were performed. Studies yielded a noticeable decrease in proliferation in both MAPK8 and MERTK knockdown, even being lethal in one case. While the results support the importance of these proteins for tumorgenecity, knockdown did not alter the cell lines’ sensitivity to MEK inhibition. Our findings suggest the use of MERTK and JNK1 as markers of MEK inhibition resistance and propose new target for future therapies. Citation Format: Jaymes Beech, Kim Kelly. JNK1 and MERTK are markers of MEK inhibitor resistance and new targets for therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5594. doi:10.1158/1538-7445.AM2013-5594

Background. Cytokines that promote wound healing and immune tolerance are profoundly upregulated during post-lactational involution of the breast and promote tumor growth and malignancy. This observation is supported by increasing evidence that post-lactational involution produces a highly malignant microenvironment contributing to poor outcome of pregnancy associated breast cancers (PABCs) which arise within 5 years of a full-term pregnancy. We have discovered that one mechanism driving cytokine modulation during post-lactational involution is efferocytosis, or the phagocytic clearance of apoptotic cells (ACs). Cell death is a prominent feature of the post-lactational breast, when up to 90% of the entire epithelial content dies and must be removed, making this an ideal model to study both apoptosis and AC clearance. Without efferocytosis, residual ACs undergo secondary necrosis, which stimulates acute inflammation and expression of Th1 cytokines. Thus, efferocytosis prevents secondary necrosis of dying cells, enhances immune tolerance and wound healing through production of Th2 cytokines. In healthy post-lactational breast tissue this is an optimal physiological response that promotes breast remodeling. However, in the context of breast cancer, efferocytosis may be pro-tumorigenic. Results. We have shown that post-lactational efferocytosis is necessary to re-establish breast tissue homeostasis after lactation, and for re-initiating successful lactation after future pregnancies. We have also demonstrated that during the earliest phases of post-lactational involution, the breast epithelium is the primary phagocytic resource in the breast, although it is clear that macrophages engulf dying cells during later stages of involution. We have identified the receptor tyrosine kinase MerTK as a necessary mediator of efferocytosis and subsequent cytokine modulation during post-lactational involution. Further, MerTK-mediated cytokine changes in the post-lactational mammary microenvironment promotes malignant progression of spontaneously forming PABC in a transgenic mouse model, as genetic targeting of MerTK decreased tumor growth and metastasis without altering tumor latency. Post-lactational tumors expressed abundant Th2 and wound healing cytokines as compared to tumors in nulliparous mice. However, loss of MerTK in post-lactational mammary tumors impaired the expression of Th2 and wound healing cytokines, while inducing the expression of Th1 cytokines. Conclusion. These results suggest that efferocytosis-mediated cytokine regulation is a key driver of malignant severity in pregnancy associated mammary tumors. These data are consistent with our previous demonstrations that MerTK in the mammary tumor microenvironment increases tumor malignancy. Targeted inhibition of MerTK may decrease efferocytosis-mediated cytokine modulation in breast tumors to limit pro-tumorigenic cytokine production and stimulate production of cytokines associated with anti-tumor immunity. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-01.