RON Signaling Research Articles

Lipid profiling of RON and DEK-dependent signaling in breast cancer guides discovery of gene networks predictive of poor outcomes.

Recurrent and metastatic breast cancer is frequently treatment resistant. A wealth of evidence suggests that reprogrammed lipid metabolism supports cancer recurrence. Overexpression of the RON and DEK oncoproteins in breast cancer is associated with poor outcome. Both proteins promote cancer metastasis in laboratory models, but their influence on lipid metabolite levels remain unknown. To measure RON- and DEK-dependent steady-state lipid metabolite levels, a nuclear magnetic resonance (NMR)-based approach was utilized. The observed differences identified a lipid metabolism-related gene expression signature that is prognostic of overall survival (OS), distant metastasis-free survival (DMFS), post-progression survival (PPS), and recurrence-free survival (RFS) in patients with breast cancer. RON loss led to decreased cholesterol and sphingomyelin levels, whereas DEK loss increased total fatty acid levels and decreased free glycerol levels. Lipid-related genes were then queried to define a signature that predicts poor outcomes for patients with breast cancer patients. Taken together, RON and DEK differentially regulate lipid metabolism in a manner that predicts and may promote breast cancer metastasis and recurrence.

The MET Family of Receptor Tyrosine Kinases Promotes a Shift to Pro-Tumor Metabolism.

The development and growth of cancer is fundamentally dependent on pro-tumor changes in metabolism. Cancer cells generally shift away from oxidative phosphorylation as the primary source of energy and rely more heavily on glycolysis. Receptor tyrosine kinases (RTKs) are a type of receptor that is implicated in this shift to pro-tumor metabolism. RTKs are important drivers of cancer growth and metastasis. One such family of RTKs is the MET family, which consists of MET and RON (MST1R). The overexpression of either MET or RON has been associated with worse cancer patient prognosis in a variety of tumor types. Both MET and RON signaling promote increased glycolysis by upregulating the expression of key glycolytic enzymes via increased MYC transcription factor activity. Additionally, both MET and RON signaling promote increased cholesterol biosynthesis downstream of glycolysis by upregulating the expression of SREBP2-induced cholesterol biosynthesis enzymes via CTTNB1. These changes in metabolism, driven by RTK activity, provide potential targets in limiting tumor growth and metastasis via pharmacological inhibition or modifications in diet. This review summarizes pro-tumor changes in metabolism driven by the MET family of RTKs. In doing so, we will offer our unique perspective on metabolic pathways that drive worse patient prognosis and provide suggestions for future study.

Identification of protein biomarkers associated with congenital diaphragmatic hernia in human amniotic fluid

Congenital diaphragmatic hernia (CDH) is a severe birth defect frequently associated with pulmonary hypoplasia, pulmonary hypertension, and heart failure. Since amniotic fluid comprises proteins of both fetal and maternal origin, its analysis could provide insights on mechanisms underlying CDH and provide biomarkers for early diagnosis, severity of pulmonary changes and treatment response. The study objective was to identify proteomic changes in amniotic fluid consistently associated with CDH. Amniotic fluid was obtained at term (37–39 weeks) from women with normal pregnancies (n = 5) or carrying fetuses with CDH (n = 5). After immuno-depletion of the highest abundance proteins, off-line fractionation and high-resolution tandem mass spectrometry were performed and quantitative differences between the proteomes of the groups were determined. Of 1036 proteins identified, 218 were differentially abundant. Bioinformatics analysis showed significant changes in GP6 signaling, in the MSP–RON signaling in macrophages pathway and in networks associated with cardiovascular system development and function, connective tissue disorders and dermatological conditions. Differences in selected proteins, namely pulmonary surfactant protein B, osteopontin, kallikrein 5 and galectin-3 were validated by orthogonal testing using ELISA in larger cohorts and showed statistically significant differences aiding in the diagnosis and prediction of CDH. The findings provide potential tools for clinical management of CDH.

Conformation specific antagonistic high affinity antibodies to the RON receptor kinase for imaging and therapy

The RON receptor tyrosine kinase is an exceptionally interesting target in oncology and immunology. It is not only overexpressed in a wide variety of tumors but also has been shown to be expressed on myeloid cells associated with tumor infiltration, where it serves to dampen tumour immune responses and reduce the efficacy of anti-CTLA4 therapy. Potent and selective inhibitory antibodies to RON might therefore both inhibit tumor cell growth and stimulate immune rejection of tumors. We derived cloned and sequenced a new panel of exceptionally avid anti-RON antibodies with picomolar binding affinities that inhibit MSP-induced RON signaling and show remarkable potency in antibody dependent cellular cytotoxicity. Antibody specificity was validated by cloning the antibody genes and creating recombinant antibodies and by the use of RON knock out cell lines. When radiolabeled with 89-Zirconium, the new antibodies 3F8 and 10G1 allow effective immuno-positron emission tomography (immunoPET) imaging of RON-expressing tumors and recognize universally exposed RON epitopes at the cell surface. The 10G1 was further developed into a novel bispecific T cell engager with a 15 pM EC50 in cytotoxic T cell killing assays.

Prostate tumor RON receptor signaling mediates macrophage recruitment to drive androgen deprivation therapy resistance through Gas6-mediated Axl and RON signaling.

Androgen deprivation therapy (ADT), or chemical castration, is the first-line therapy for prostate cancer; however, resistance leaves few treatment options. Prostatic tumor-associated macrophages (TAMs) have been shown to promote prostate cancer growth and are abundant in castration-resistant prostate cancer (CRPC), suggesting a role in promoting CRPC. We recently showed a tumor cell-intrinsic mechanism by which RON promotes CRPC. Given previous reports that RON alters prostate cancer cell chemokine productionand RON-overexpressing tumors alter macrophage function, we hypothesized that a macrophage-dependent mechanism regulated by tumor cell intrinsic RON also promotes CRPC. Using RON-modulated genetically engineered mouse models (GEMMs) and GEMM-derived cell lines and co-cultures with bone marrow-derived macrophages, we show functional and molecular characteristics of signaling pathways in supporting CRPC. Further, we used an unbiased phosphokinase array to identify pathway interactions regulated by RON. Finally, using human prostate cancer cell lines and prostate cancer patient data sets, we show the relevance of our findings to human prostate cancer. Studies herein show that macrophages recruited into the prostate tumor microenvironment (TME) serve as a source for Gas6 secretion which serves to further enhance RON and Axl receptor activation in prostate tumor cells thereby driving CRPC. Further, we show targeting RON and macrophages in a murine model promotes CRPC sensitization to ADT. We discovered a novel role for the RON receptor in prostate cancer cells in promoting CRPC through the recruitment of macrophages into the prostate TME. Macrophage-targeting agents in combination with RON/Axl inhibition are likely to provide clinical benefits for patients with CRPC.

RON (MST1R) and HGFL (MST1) Co-Overexpression Supports Breast Tumorigenesis through Autocrine and Paracrine Cellular Crosstalk.

Simple SummaryRON is a protein that sits on the surface of a cell and passes signals from the outside-in when encountering the HGFL protein. In cancer, the RON protein is often found at high levels leading to too many signals being passed. Given the close proximity of RON and HGFL in the human genome, we hypothesized that the two proteins are overproduced together in cancer cells. Other cells that interact with cancer cells, such as macrophages, also express RON and can respond to HGFL produced by cancer cells, creating a diverse response to overly abundant RON and HGFL. In this study, we evaluated how RON and HGFL in cancer cells affect cancer growth and progression and their response to other cells, and examined explanations for increases in RON and HGFL abundance in breast cancer.Background: Aberrant RON signaling is present in numerous cancers including breast cancer. Evidence suggests that the ligand, hepatocyte growth factor-like (HGFL), is also overexpressed in breast cancer. RON (MST1R) and HGFL (MST1) genes are located on human chromosome 3 and mouse chromosome 9 respectively and are found near each other in both species. Based on co-expression patterns, we posited that RON and HGFL are co-regulated and that coordinate upregulation drives aggressive tumorigenesis. Methods: Mouse models were used to establish the functional significance of RON and HGFL co-overexpression on the activation of tumor cells and tumor-associated macrophages in breast cancer. TCGA and METABRIC gene expression and alteration data were used to query the relationships between MST1R and MST1 in breast cancer. Results: In tumor models, physiologic sources of HGFL modestly improve Arginase-1+ (M2) macrophage recruitment to the tumor proper. Tumor-cell produced HGFL functions in autocrine to sustain tumor cell RON activation and MAPK-dependent secretion of chemotactic factors and in paracrine to activate RON on macrophages and to promote breast cancer stem cell self-renewal. In silico analyses support that RON and HGFL are co-expressed across virtually all cancer types including breast cancer and that common genomic alterations do not appear to be drivers of RON/HGFL co-overexpression. Conclusions: Co-overexpression of RON and HGFL in breast cancer cells (augmented by physiologic sources of HGFL) promotes tumorigenesis through autocrine-mediated RON activation/RON-dependent secretome changes and paracrine activation of macrophage RON to promote breast cancer stem cell self-renewal.

Macrophage-mediated RON signaling supports breast cancer growth and progression through modulation of IL-35.

Tumor associated macrophages (TAMs) play a major role in regulating mammary tumor growth and in directing the responses of tumor infiltrating leukocytes in the microenvironment. However, macrophage-specific mechanisms regulating the interactions of macrophages with tumor cells and other leukocytes that support tumor progression have not been extensively studied. In this study, we show that the activation of the RON receptor tyrosine kinase signaling pathway specifically in macrophages supports breast cancer growth and metastasis. Using clinically relevant murine models of breast cancer, we demonstrate that loss of macrophage RON expression results in decreases in mammary tumor cell proliferation, survival, cancer stem cell self-renewal, and metastasis. Macrophage RON signaling modulates these phenotypes via direct effects on the tumor proper and indirectly by regulating leukocyte recruitment including macrophages, T-cells, and B-cells in the mammary tumor microenvironment. We further show that macrophage RON expression regulates the macrophage secretome including IL-35 and other immunosuppressive factors. Overall, our studies implicate activation of RON signaling in macrophages as a key player in supporting a thriving mammary pro-tumor microenvironment through novel mechanisms including the augmentation of tumor cell properties through IL-35.

RON in hepatobiliary and pancreatic cancers: Pathogenesis and potential therapeutic targets.

The receptor protein tyrosine kinase RON belongs to the c-MET proto-oncogene family. Research has shown that RON has a role in cancer pathogenesis, which places RON on the frontline of the development of novel cancer therapeutic strategies. Hepatobiliary and pancreatic (HBP) cancers have a poor prognosis, being reported as having higher rates of cancer-related death. Therefore, to combat these malignant diseases, the mechanism underlying the aberrant expression and signaling of RON in HBP cancer pathogenesis, and the development of RON as a drug target for therapeutic intervention should be investigated. Abnormal RON expression and signaling have been identified in HBP cancers, and also act as tumorigenic determinants for HBP cancer malignant behaviors. In addition, RON is emerging as an important mediator of the clinical prognosis of HBP cancers. Thus, not only is RON significant in HBP cancers, but also RON-targeted therapeutics could be developed to treat these cancers, for example, therapeutic monoclonal antibodies and small-molecule inhibitors. Among them, antibody-drug conjugates have become increasingly popular in current research and their potential as novel anti-cancer biotherapeutics will be determined in future clinical trials.

Evidence for 2-Methoxyestradiol-Mediated Inhibition of Receptor Tyrosine Kinase RON in the Management of Prostate Cancer

2-Methoxyestradiol (2-ME2) possesses anti-tumorigenic activities in multiple tumor models with acceptable tolerability profile in humans. Incomplete understanding of the mechanism has hindered its development as an anti-tumorigenic compound. We have identified for the first-time macrophage stimulatory protein 1 receptor (MST1R) as a potential target of 2-ME2 in prostate cancer cells. Human tissue validation studies show that MST1R (a.k.a RON) protein levels are significantly elevated in prostate cancer tissues compared to adjacent normal/benign glands. Serum levels of macrophage stimulatory protein (MSP), a ligand for RON, is not only associated with the risk of disease recurrence, but also significantly elevated in samples from African American patients. 2-ME2 treatment inhibited mechanical properties such as adhesion and elasticity that are associated with epithelial mesenchymal transition by downregulating mRNA expression and protein levels of MST1R in prostate cancer cell lines. Intervention with 2-ME2 significantly reduced tumor burden in mice. Notably, global metabolomic profiling studies identified significantly higher circulating levels of bile acids in castrated animals that were decreased with 2-ME2 intervention. In summary, findings presented in this manuscript identified MSP as a potential marker for predicting biochemical recurrence and suggest repurposing 2-ME2 to target RON signaling may be a potential therapeutic modality for prostate cancer.

Tumor cell intrinsic RON signaling suppresses innate immune responses in breast cancer through inhibition of IRAK4 signaling.

Increasing evidence suggests that cancer cells require both alterations in intrinsic cellular processes and the tumor microenvironment for tumor establishment, growth, and progression to metastatic disease. Despite this, knowledge of tumor-cell intrinsic molecular mechanisms controlling both tumor cell processes as well as the tumor microenvironment is limited. In this study, we provide evidence demonstrating the novel role of RON signaling in regulating breast cancer initiation, progression, and metastasis through modulation of tumor cell intrinsic processes and the tumor microenvironment. Using clinically relevant models of breast cancer, we show that RON signaling in the mammary epithelial tumor cells promotes tumor cell survival and proliferation as well as an immunopermissive microenvironment associated with decreased M1 macrophage, natural killer (NK) cell, and CD8+ T cell recruitment. Moreover, we demonstrate that RON signaling supports these phenotypes through novel mechanisms involving suppression of IRAK4 signaling and inhibition of type I Interferons. Our studies indicate that activation of RON signaling within breast cancer cells promotes tumor cell intrinsic growth and immune evasion which support breast cancer progression and highlight the role of targeting RON signaling as a potential therapeutic strategy against breast cancer.

Metabolic Profiling Reveals Aggravated Non-Alcoholic Steatohepatitis in High-Fat High-Cholesterol Diet-Fed Apolipoprotein E-Deficient Mice Lacking Ron Receptor Signaling.

Non-alcoholic steatohepatitis (NASH) represents the progressive sub-disease of non-alcoholic fatty liver disease that causes chronic liver injury initiated and sustained by steatosis and necroinflammation. The Ron receptor is a tyrosine kinase of the Met proto-oncogene family that potentially has a beneficial role in adipose and liver-specific inflammatory responses, as well as glucose and lipid metabolism. Since its discovery two decades ago, the Ron receptor has been extensively investigated for its differential roles on inflammation and cancer. Previously, we showed that Ron expression on tissue-resident macrophages limits inflammatory macrophage activation and promotes a repair phenotype, which can retard the progression of NASH in a diet-induced mouse model. However, the metabolic consequences of Ron activation have not previously been investigated. Here, we explored the effects of Ron receptor activation on major metabolic pathways that underlie the development and progression of NASH. Mice lacking apolipoprotein E (ApoE KO) and double knockout (DKO) mice that lack ApoE and Ron were maintained on a high-fat high-cholesterol diet for 18 weeks. We observed that, in DKO mice, the loss of ligand-dependent Ron signaling aggravated key pathological features in steatohepatitis, including steatosis, inflammation, oxidation stress, and hepatocyte damage. Transcriptional programs positively regulating fatty acid (FA) synthesis and uptake were upregulated in the absence of Ron receptor signaling, whereas lipid disposal pathways were downregulated. Consistent with the deregulation of lipid metabolism pathways, the DKO animals exhibited increased accumulation of FAs in the liver and decreased level of bile acids. Altogether, ligand-dependent Ron receptor activation provides protection from the deregulation of major metabolic pathways that initiate and aggravate non-alcoholic steatohepatitis.

Prostate Epithelial RON Signaling Promotes M2 Macrophage Activation to Drive Prostate Tumor Growth and Progression.

Effective treatment of advanced prostate cancer persists as a significant clinical need as only 30% of patients with distant disease survive to 5 years after diagnosis. Targeting signaling and tumor cell-immune cell interactions in the tumor microenvironment has led to the development of powerful immunotherapeutic agents, however, the prostate tumor milieu remains impermeable to these strategies highlighting the need for novel therapeutic targets. In this study, we provide compelling evidence to support the role of the RON receptor tyrosine kinase as a major regulator of macrophages in the prostate tumor microenvironment. We show that loss of RON selectively in prostate epithelial cells leads to significantly reduced prostate tumor growth and metastasis and is associated with increased intratumor infiltration of macrophages. We further demonstrate that prostate epithelial RON loss induces transcriptional reprogramming of macrophages to support expression of classical M1 markers and suppress expression of alternative M2 markers. Interestingly, our results show epithelial RON activation drives upregulation of RON expression in macrophages as a positive feed-forward mechanism to support prostate tumor growth. Using 3D coculture assays, we provide additional evidence that epithelial RON expression coordinates interactions between prostate tumor cells and macrophages to promote macrophage-mediated tumor cell growth. Taken together, our results suggest that RON receptor signaling in prostate tumor cells directs the functions of macrophages in the prostate tumor microenvironment to promote prostate cancer. IMPLICATIONS: Epithelial RON is a novel immunotherapeutic target that is responsible for directing the macrophage antitumor immune response to support prostate tumor growth and progression.

Abstract A69: Elucidating the cell type-specific mechanisms of the Ron receptor in the prostate tumor microenvironment

Abstract The incidence of advanced prostate cancer (PCa) in American men has grown by 72% within the last decade, yet current treatments fail to effectively treat advanced disease. We study the Ron receptor tyrosine kinase as a novel target for PCa treatment. Ron is primarily expressed in macrophages and epithelial cells and is found overexpressed in PCa. Ron expression increases with disease severity in humans and mouse models, suggesting Ron drives progression to advanced PCa. Our laboratory has shown that loss of Ron signaling in prostate epithelial cells or in myeloid cells markedly reduces prostate tumor growth and modulates the tumor microenvironment, particularly the functions of macrophages and CD8+ T cells. Thus, we hypothesize that the Ron receptor utilizes cell type-specific mechanisms to suppress the antitumor immune response. However, our previous studies were limited because they were performed separately in different orthotopic models. Therefore, we employed the well-established Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) PCa mouse model to study loss of Ron signaling in either cell type in a single autochthonous model. We used Cre-Lox technology to generate TRAMP mice containing conditional deletion of Ron signaling in prostate epithelial cells or in myeloid cells. At 30 weeks of age, prostate tumors are harvested and analyzed by weight, immunohistochemical staining, quantitative PCR, Western blot, and flow cytometry. To assess the effects of Ron modulation in each cell type and interrogate the interaction between tumor cells and macrophages, we perform in vitro co-culture assays utilizing an established prostate cancer epithelial cell line derived from the TRAMP model and bone marrow-derived macrophages. We observe that Ron signaling in either cell type is critical for prostate tumor growth in TRAMP mice. Furthermore, loss of Ron in either cell type leads to increased macrophage and CD8+ T-cell tumor infiltration, and our in vitro data show that loss of Ron in either cell type suppresses antitumor M1 macrophage activation and promotes tumor-supporting M2 macrophage activation. Additionally, preliminary data suggest loss of Ron in either cell type leads to increased activation of CD8+ tumor-infiltrating lymphocytes. Overall, our data suggest the Ron receptor coordinates immunosuppressive mechanisms in both the malignant tumor cells and the host macrophages to dampen antitumor immunity and promote prostate cancer. These studies implicate Ron targeting as a powerful therapeutic tool with broad utility for understanding and refining therapeutic strategies to treat advanced PCa. Citation Format: Camille Sullivan, Nicholas E. Brown, Susan E. Waltz. Elucidating the cell type-specific mechanisms of the Ron receptor in the prostate tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A69.

OPN promotes the aggressiveness of non-small-cell lung cancer cells through the activation of the RON tyrosine kinase

Osteopontin (OPN) is identified as a diagnostic and prognostic biomarker of tumor progression and metastasis. In non-small-cell lung cancer (NSCLC), the functions of OPN have not been well characterized. The current study sought to investigate the clinical implications of OPN expression in NSCLC and the role of OPN in the aggressiveness of the lung cancer cells. Using a proteomics approach, we identified that phospho-RON (p-RON) was one of the most remarkably up-regulated proteins in OPN-overexpressing cells. The levels of OPN and RON transcripts were unveiled as independent prognostic indicators of survival in NSCLC (p = 0.001). Higher levels of OPN, RON and p-RON proteins were observed in tumor tissues. Knock down of the OPN gene suppressed the migration and invasion abilities of the A549 lung cancer cells which endogenously expresses OPN. While ectopic expression of OPN in the SK-MES-1 lung cancer cells increased levels of cellular invasion and migration. In addition, these changes were accompanied by a phosphorylated activation of RON. Small-molecule inhibition of RON or siRNA silencing of RON significantly reduced OPN-induced migration and invasion of lung cancer cells and had an inhibitory effect on the OPN-mediated cell epithelial-mesenchymal transition. Our study suggests that in NSCLC, the aberrant expression of OPN can be considered as an independent survival indicator and is associated with disease progression. OPN plays a crucial role in promoting migration and invasion properties of lung cancer cells through its phosphorylation activation of the RON signaling pathway, implying its potential as a therapeutic target in the treatment of NSCLC.

MTORC1 is a key mediator of RON-dependent breast cancer metastasis with therapeutic potential

Metastasis is the biggest challenge in treating breast cancer, and it kills >40,000 breast cancer patients annually in the US. Aberrant expression of the RON receptor tyrosine kinase in breast tumors correlates with poor prognosis and has been shown to promote metastasis. However, the molecular mechanisms that govern how RON promotes metastasis, and how to block it, are still largely unknown. We sought to determine critical effectors of RON using a combination of mutational and pharmacologic strategies. High-throughput proteomic analysis of breast cancer cells upon activation of RON showed robust phosphorylation of ribosomal protein S6. Further analysis revealed that RON strongly signals through mTORC1/p70S6K, which is mediated predominantly by the PI3K pathway. A targeted mutation approach to modulate RON signaling validated the importance of PI3K/mTORC1 pathway for spontaneous metastasis in vivo. Finally, inhibition of mTORC1 with an FDA-approved drug, everolimus, resulted in transient shrinkage of established RON-dependent metastases, and combined blockade of mTORC1 and RON delayed progression. These studies have identified a key downstream mediator of RON-dependent metastasis in breast cancer cells and revealed that inhibition of mTORC1, or combined inhibition of mTORC1 and RON, may be effective for treatment of metastatic breast cancers with elevated expression of RON.

Identification of the splice variants of Recepteur d'Origine nantais (RON) in lung cancer cell lines

RON receptor tyrosine kinase is a transmembrane protein directly involved in suppression of inflammation and its aberrant expression linked to cancers and metastasis. Efforts to block deregulated RON signaling in tumors using small molecule kinase inhibitors or antibodies have been complicated by the presence of unknown number/types of isoforms of RON, which, despite being structurally similar, localize differently and mediate varied functions. Current study was designed to identify the splice variants of RON transcripts formed by skipping of sequences between exons 9 and 14 for better understanding of isoform specific RON signaling in cancers. PCR amplification and bi-directional sequencing of a 901 bp cDNA sequence located between exons 9 to 14 of RON from lung cancer cell lines revealed the presence of two splicing variants formed by skipping of exons 11 and 11–13. Each of these transcripts was found in more than one cell line. Expressed sequence tag (EST) database search indicated that the splicing variant lacking exons 11–13 was a novel one. Here we conclude that the splice variants of RON lacking exon 11 and exons 11–13 were detected in several lung cancer cell lines. Novel variant formed by skipping exons 11–13, the sequence of which code for transmembrane region, is predicted to code for a truncated isoform that may be secreted out. Tumors may antagonize the ligand dependent anti-inflammatory function of wild-type RON by secreting out the ligand binding isoforms.

Abstract A30: Ron kinase inhibition to improve immunotherapy for breast cancer metastasis

Abstract Metastasis is the cause of death for nearly all types of cancer, including breast cancer. An exciting new area of research in metastatic breast cancer centers on immune therapy. Although new immune checkpoint blockade therapies have provided benefit for a fraction of patients tested so far, the majority of patients still do not respond to these drugs. A better understanding of how the immune system can be harnessed against metastatic breast cancer is required in order to improve patient outcomes in this area. We previously discovered that macrophage Ron receptor tyrosine kinase promotes breast cancer metastasis by inhibiting CD8+ cytotoxic T lymphocyte activity. We hypothesized that dual blockade of Ron activity and existing immune checkpoint molecules would unleash a more effective CD8+ T cell response to control or eliminate metastatic breast cancer. Our strategy would simultaneously disable tumor-mediated immune evasion mechanisms on both the innate and adaptive immune systems. To test the potential of combination therapy, the Ron inhibitor BMS-777607 and/or the immune checkpoint blocking agents anti-PD1 or anti-CTLA4, were administered in our mouse mammary tumor model to examine the effects on breast tumor progression. To examine Ron-specific effects of BMS-777607, we also examined the effect of genetic deletion of host Ron signaling activity in combination with immunotherapy. The anti-tumor immune response was comprehensively examined by multi-color flow cytometry and immunohistochemical staining for tumor-infiltrating lymphocytes (TILs). Our data suggest that the combination of Ron inhibition with immune checkpoint blockade may be an effective therapy in breast cancer. Citation Format: Shu Chin Alicia Lai, Atakan Ekiz, Alana Welm. Ron kinase inhibition to improve immunotherapy for breast cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A30.

Tumor Cell Autonomous RON Receptor Expression Promotes Prostate Cancer Growth Under Conditions of Androgen Deprivation

Current treatment strategies provide minimal results for patients with castration-resistant prostate cancer (CRPC). Attempts to target the androgen receptor have shown promise, but resistance ultimately develops, often due to androgen receptor reactivation. Understanding mechanisms of resistance, including androgen receptor reactivation, is crucial for development of more efficacious CRPC therapies. Here, we report that the RON receptor tyrosine kinase is highly expressed in the majority of human hormone-refractory prostate cancers. Further, we show that exogenous expression of RON in human and murine prostate cancer cells circumvents sensitivity to androgen deprivation and promotes prostate cancer cell growth in both in vivo and in vitro settings. Conversely, RON loss induces sensitivity of CRPC cells to androgen deprivation. Mechanistically, we demonstrate that RON overexpression leads to activation of multiple oncogenic transcription factors (namely, β-catenin and NF-κB), which are sufficient to drive androgen receptor nuclear localization and activation of AR responsive genes under conditions of androgen deprivation and support castration-resistant growth. In total, this study demonstrates the functional significance of RON during prostate cancer progression and provides a strong rationale for targeting RON signaling in prostate cancer as a means to limit resistance to androgen deprivation therapy.

Abstract 3960: The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes

Abstract Introduction: In cancer patients, the development of metastases is associated with a poor clinical prognosis. Metastatic dissemination requires cancer cells to detach from the primary tumor and invade adjacent or distant tissue. This process involves epithelial-to-mesenchymal transition (EMT), whereby tumor cells lose cell-cell adhesion mediated by E-cadherin repression. The CD44 family of transmembrane glycoproteins comprises alternatively spliced variants that are involved in many cellular processes. The isoform CD44v6 has been shown to play a major role in tumor growth and metastasis. CD44v6 acts as a co-receptor for the receptor tyrosine kinases (RTKs) c-MET, RON and VEGFR-2, and is preferentially expressed in a variety of epithelial cancers. We have previously shown that the co-receptor function of CD44v6 for c-MET is the driving force for metastasis. c-Met and RON facilitate metastasis through MAPK signaling and VEGFR-2 plays a role in angiogenesis. AMC303, an allosteric and selective peptide inhibitor of CD44v6, prevents phosphorylation of the CD44v6 dependent RTKs and respective cellular processes. Methods: The effect of AMC303 on cancer cell function in response to the growth factors HGF, MSP and VEGF was studied in various assays. Cell migration and invasion was analyzed in scratch assays and the Boyden Chamber. Morphological changes of EMT were monitored using microscopy in scattering assays in the presence of growth factors. EMT markers were analyzed by Western blot and immunohistochemistry. Tube formation was microscopically analyzed. Results: AMC303 dose dependently and at low nanomolar concentrations (IC50 10 - 20nM) prevents HGF- or VEGF-induced activation of c-MET/VEGFR-2 in various cancer cells of pancreatic, lung and colonic origin or endothelial cells, which cannot be overcome by excess HGF and VEGF. HGF- and MSP-dependent cell migration, invasion and wound closure in scratch assays was prevented by AMC303 using different cancer cell lines. AMC303 had a strong inhibitory effect on HGF- and MSP- induced cell scattering, a morphological feature of EMT. Consistent with this, AMC303 inhibited the downregulation of HGF induced EMT markers (E-cadherin). In primary endothelial cells, AMC303 blocked activation of VEGFR-2 and VEGF-A induced formation of a tubular network. Conclusions: Phosphorylation of CD44v6 dependent RTKs is inhibited in primary endothelial cells and different epithelial tumor cells by binding of AMC303 to the extracellular domaine of CD44v6. This novel mode of action results in strong anti-tumor and anti-metastatic effects of AMC303 involving inhibition of migration and invasion of tumor cells and prevention of EMT. AMC303 is currently tested in a First-in-Human Phase I/Ib study in cancer patients with solid epithelial tumors. Citation Format: Thorsten Läufer, Martin Augsten, Vanessa Al-Rawi, Birgit Hotz, Yvonne Heneka, Oliver Coutelle, Alexandra Matzke-Ogi. The allosteric inhibitor of CD44v6 AMC303 blocks c-MET, Ron and VEGFR-2 dependent signaling and cellular processes [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 3960.

Abstract 5514: Loss of hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinomas promotes RON receptor phosphorylation and increased sensitivity to crizotinib

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the top cause of cancer-related mortality. We have identified a role for the tumor suppressor hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinoma. HAI-1 loss results in unregulated downstream MET/RON tyrosine kinase receptor signaling. We hypothesized that HAI-1 is lost in human lung adenocarcinomas and could serve as a biomarker for therapy with crizotinib, a tyrosine kinase inhibitor (TKI) with activity against both MET and RON. We also hypothesized that unregulated RON signaling in the tumor microenvironment, via HAI-1 loss, may promote tumor metastasis by skewing tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. Methods: Immunohistochemical staining with HAI-1 antibody (1N7) was performed on human tissue microarray containing 20 normal and 20 lung tumor specimens. Intensity grading (0, 1+, 2+, 3+) was performed, and percent cell expression was calculated for each group. For all in vitro assays, H358 lung adenocarcinoma cells, which express high levels of endogenous HAI-1, were stably transfected with mock shRNA or HAI-1 shRNA. Cell protein expression and phosphorylation was assessed via western blot. Cell viability after exposure to crizotinib was assessed by trypan blue staining. For flow cytometric analysis of macrophage phenotype, undifferentiated (M0) macrophages were exposed to conditioned medium from transfected H358 cells and macrophage phenotype was assessed by flow for surface markers CD68 (M0), CCR7 (M1), and CD206 (M2). Results: HAI-1 expression is significantly decreased in human lung adenocarcinoma compared to normal lung with overall HAI-1 IHC positivity ~20% compared to ~60% respectively. HAI-1 knockdown in H358 cells caused increased phosphorylation of RON and increased sensitivity to crizotinib. Culturing M0 macrophages in conditioned media from H358 shHAI-1 knockdown cells caused a decrease in the CCR7 positive M1 fraction from 8.4% to 1.9%, and an increase in the CD206 positive M2 fraction from 0.77% to 2.09%. Conclusion: We have demonstrated that loss of HAI-1 occurs in human lung adenocarcinoma consistent with previous studies in human NSCLC cell lines. Furthermore, we showed significantly increased RON signaling activity after HAI-1 knockdown. We have also demonstrated that HAI-1 loss can skew tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. We have shown that loss of HAI-1 in vitro can increase sensitivity to crizotinib, suggesting that loss of HAI-1 may predict for tumor sensitivity to MET/RON inhibition. Our findings show that dysregulation of the HAI-1/MET/RON pathway exerts effects both on lung tumor cells as well as TAMs, making this pathway a potentially powerful therapeutic target. Citation Format: Austin J. McHenry, Gautam Sondarva, Vijayalakshmi Ananthanarayanan, Ashley Hess, Patricia E. Simms, Alhareth Alsayed, Hiroaki Kataoka, Jan Marusarz, Sandeep Kumar, Ravi Salgia, Arkadiusz Dudek, Stanley Borowicz, Ajay Rana. Loss of hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinomas promotes RON receptor phosphorylation and increased sensitivity to crizotinib [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 5514.