RON Phosphorylation Research Articles

Abstract 3948: Development of bone-targeted Ron inhibitors to treat osseous metastases from breast cancers

Abstract Purpose: About 500,000 people each year worldwide die from metastatic breast cancer. More than 70% of these metastatic breast cancer patients have bone metastases and median survival time from diagnosis of bone metastasis is only 2-3 years. As breast cancer bone metastases are primarily osteolytic, patients suffer from significant morbidities, including severe pain, pathological fractures, nerve compression, and hypercalcemia. Previously, it was shown that RON receptor tyrosine kinase mediates breast cancer outgrowth at metastatic sites, promotes osteolytic bone destruction, and induces systemic immune suppression. We hypothesized that inhibition of RON in specialized resident macrophages and/or osteoclasts will eliminate metastasis through dual effects of blocking osteolysis and enhancing anti-tumor immunity. In this study, we have developed several bis(phosphonate) or phosphate conjugates of BMS-777607 and LY2801653, and report their accumulation in the bone microenvironment along with effects on osteolysis and tumor burden. Methods: We have utilized novel synthetic schemes for synthesis of the drug conjugates. In vitro kinase assays were performed using ADP-Glo RON kinase assay kit (Promega). Hydroxyapatite binding assay was used to determine binding of drug conjugates to the bone mineral. Changes in phosphorylation of RON was demonstrated by Western blotting. Plasma pharmacokinetics and bone accumulation of lead drug conjugates in mice was assessed by mass-spectrometry. An intratibial bone metastasis model in which MSP-expressing MMTV-PyMT tumor cells are injected into the tibia of mice was used to assess the effect of treatment with lead drug conjugates. Results: Seven novel drug conjugates were synthesized and inhibited RON kinase activity with an IC50 of 0.060-2.4 µM. Three drug conjugates demonstrated better hydroxyapatite binding when compared with BMS-777607. In agreement with the in vitro kinase activity, a variable decrease in the phosphorylation of RON was observed in cells treated with the drug conjugates. ZB-28, a monophosphonate derivative of BMS-777607, showed higher accumulation in the bone when compared with BMS-777607 but failed to block tumor-mediated bone osteolysis in an intratibial bone metastasis mouse model. Currently, a cleavable phosphate conjugate of BMS-777607 (ZB-32) is undergoing testing in the same mouse model. We have confirmed that ZB-32 and its hydrolyzed product ZB-33 accumulate in the bone to a significantly higher level than BMS-777607. These results along with preliminary ADME will be presented. Conclusion: Targeting RON kinase in the bone microenvironment is a promising approach to alleviate morbidities observed in breast cancer patients with bone metastases. We present novel bone-targeting conjugates of BMS-777607 and highlight the potential for clinical development. Citation Format: Trason Thode, Jaime Fornetti, Ryan Rodriguez del Villar, Alexis Weston, Serina Ng, Srikanta Dana, Raffaella Soldi, Mohan Kaadige, Hariprasad Vankayalapati, Srinivas Kasibhatla, Alana Welm, Sunil Sharma. Development of bone-targeted Ron inhibitors to treat osseous metastases from breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3948.

EGFR transactivates RON to drive oncogenic crosstalk.

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation - arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.

RON Kinase Is a Novel Therapeutic Target for Philadelphia-Negative Myeloproliferative Neoplasms

The Philadelphia-chromosome negative myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by the proliferation of one or more myeloid lineage compartments. Activation of JAK/STAT signaling is a major driver of all Ph-negative MPNs. During disease progression, MPN patients experience increased pro-inflammatory cytokine secretion, leading to remodeling of the bone marrow microenvironment and subsequent fibrosis. The JAK inhibitor ruxolitinib is an approved targeted therapy for MPN patients and has shown promise in its ability to reduce splenomegaly and the cytokine storm observed in patients. However, JAK inhibitors alone are not sufficient to reduce bone marrow fibrosis or to eliminate the JAK2-mutated clone. Furthermore, JAK inhibitor persistence, or reactivation of JAK/STAT signaling upon chronic JAK inhibitor treatment, has been observed in both MPN mouse models and MPN patients. Therefore, there is an urgent need for new treatment options in MPN. The tyrosine kinase RON, a member of the MET kinase family, has well-characterized roles in erythroblast proliferation and pro-inflammatory cytokine production. RON can be phosphorylated by JAK2 to stimulate erythroblast proliferation. However, the role of RON in MPN pathogenesis is unknown.We found that the ALK/MET/RON/ROS1 inhibitor crizotinib inhibited colony formation by MPN patient CD34+ cells, regardless of their disease subtype, mutation status, or JAK2 inhibitor treatment history (Figure 1A). To determine whether this is due to inhibition of the JAK/STAT signaling pathway, we performed phospho-flow cytometry of STAT3 and STAT5 in myelofibrosis patient erythroblasts treated with crizotinib ex vivo as well as Western blot analysis in the JAK2-mutated cell lines SET2 and HEL. We found that crizotinib inhibits the phosphorylation of JAK2, STAT3, and STAT5 (Figure 1B). Since crizotinib has not been reported to directly inhibit JAK2, we asked whether these effects of crizotinib in MPN cells could be explained by RON inhibition. Consistent with this hypothesis, we observed that shRNA knockdown of multiple RON isoforms also decreases the phosphorylation of JAK2, STAT5, and STAT3 in HEL cells (Figure 1C-D).To determine whether crizotinib can alter the MPN disease course in vivo, we tested crizotinib by oral gavage in the MPLW515L bone marrow transplant murine model of myelofibrosis at 100mg/kg daily for 2 weeks. We showed that crizotinib decreased the disease burden of MPL-W515L mice, as evidenced by decreased spleen and liver weights (Figure 1E). To determine the effects of RON genetic deletion on MPN pathogenesis, we tested whether genetic deletion of Stk (mouse gene for RON) impairs disease progression in the JAK2V617F bone marrow transplant MPN model by transplanting Stk-/- c-Kit+ bone marrow cells transduced with the JAK2V617F-GFP retrovirus into lethally irradiated recipients. We observed a significant delay in disease onset in Stk-/- transplant recipients compared to WT controls (Figure 1F). However, we found that Stk-/- mice have normal numbers of hematopoietic stem and progenitor cells, and normal bone marrow myeloid colony forming capacity, suggesting that RON is a safe therapeutic target.To determine whether RON plays a role in the JAK inhibitor persistence phenotype, we generated persistent cells by treating SET2 cells with increasing doses of ruxolitinib over 8 weeks, and confirmed persistent proliferation and JAK/STAT activation. Interestingly, we found that RON phosphorylation is enhanced in JAK inhibitor persistent cells, and that dual inhibition of RON and JAK2 overcomes JAK inhibitor persistence in SET2 cells (Figure 1G-H), suggesting that RON may potentiate the JAK2 persistence phenotype in response to ruxolitinib. Importantly, we showed by immunoprecipitation that phospho-RON and phospho-JAK2 physically interact in JAK inhibitor persistent SET2 cells, and that this interaction is disrupted by crizotinib (Figure 1I). In summary, our data demonstrate that RON kinase is a novel mediator of JAK/STAT signaling in MPNs, and that it plays a particularly important role in JAK inhibitor persistence. Our work suggests that therapeutic strategies to inhibit RON, such as crizotinib, should be investigated in MPN patients. [Display omitted] DisclosuresHalmos: Guardant Health: Membership on an entity's Board of Directors or advisory committees; Apollomics: Membership on an entity's Board of Directors or advisory committees; TPT: Membership on an entity's Board of Directors or advisory committees; Eli-Lilly: Research Funding; Advaxis: Research Funding; Blueprint: Research Funding; Elevation: Research Funding; Mirati: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra-Zeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Gritsman: iOnctura: Research Funding.

Abstract 3451: Non-genetic RTK-mediated cetuximab resistance in colorectal cancer offers multiple targets for therapeutic intervention

Abstract Using 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives (CC, SC, CC-CR), we previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to anti-EGFR monoclonal antibody, cetuximab. CC cells are sensitive to cetuximab, while SC and CC-CR cells are resistant in 3D. Both de novo and acquired modes of cetuximab resistance in SC and CC-CR, respectively, could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. In fact, we now show that the cetuximab/crizotinib combination leads to synergistic growth inhibition in both SC and CC-CR. In addition to steady-state upregulation of MET and RON phosphorylation in SC cells, we also observe upregulation of ERBB3 phosphorylation after cetuximab addition, indicating a need for monitoring RTK activity during treatment. We have also identified a set of KRAS wild-type human CRC patient-derived tumor xenografts (PDXs) that show increased MET/RON phosphorylation. As noted previously, addition of the MET ligand (HGF) and ERBB3 ligand (NRG1) induced cetuximab resistance in CC cells and HGF-induced cetuximab resistance was overcome by crizotinib addition. We now show that MET/RON-dependent cetuximab resistance is due to autocrine production of their cognate ligands HGF/HGFL, respectively. For these studies, we employed selective inhibitors of the proteases (HGFA, Matriptase, and Hepsin) that mediate processing and maturation of HGF/HGFL; these inhibitors (ZFH7116 and VD2173) were able to overcome cetuximab resistance in SC and CC-CR cells. Thus, RTK ligand processing is an additional therapeutic target to counter cetuximab resistance in CRC. Citation Format: Ramona Graves-Deal, Galina Bogatcheva, Vishnu Damalanka, Lidija Klampfer, James W. Janetka, Robert J. Coffey, Bhuminder Singh. Non-genetic RTK-mediated cetuximab resistance in colorectal cancer offers multiple targets for therapeutic intervention [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 3451.

Abstract 4855: AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases

Abstract Introduction: CD44v6, a member of the CD44 family, is an essential co-receptor for c-MET, RON and VEGFR-2. The receptor tyrosine kinases (RTKs) c-MET and RON, are key players in tumorigenic pathways, are activated by the ligands HGF and MSP, respectively and homo- or heterodimerize with each other or can be autoactivated due to amplification. The RTK VEGFR-2 is critical for angiogenesis in solid epithelial tumors. The peptidergic inhibitor AMC303, which allosterically and selectively binds to CD44v6, was investigated for its inhibitory effects on ligand-induced and autophosphorylated c-MET activation, and its impact on tumor growth and metastases in vivo. Methods: In vitro, tumor cells were incubated with AMC303 for 30 min prior to induction with an RTK-ligand. Analysis of RTK activation was carried out by Western blotting. For in vivo xenotransplantation human pancreatic tumor cells (L3.6pl) were orthotopically injected. Animals were treated with AMC303 i.p. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks to address dose-dependency, the optimal treatment schedule was investigated with one, three or five times weekly administrations. Regression of metastases was investigated at 1 mg/kg QOD. Survival was evaluated with a dose of 1 mg/kg QOD. Immunohistochemical (IHC) analysis was performed for cleaved caspase 6, smooth muscle actin and CD31. Reduction of vessel permeability was assessed in Balb/C mice with the Miles assay and quantification of intra-tumoral FITC dextran after i.v. injection. Results: In vitro, AMC303 inhibited c-MET, VEGFR2 and RON phosphorylation induced by their respective ligands. Notably, AMC303 inhibited c-MET activation in cells with enhanced c-MET signaling caused by the exon 14 skipping mutation, but not c-MET auto-phosphorylation due to gene amplification. In vivo, AMC303 treatment attenuated primary xenograft tumor growth and metastasis and significantly prolonged survival. IHC on treated xenograft-tumors revealed an increase in apoptosis and necrosis along with a reduction in myofibroblast infiltration, angiogenesis and vessel permeability. Pharmacokinetic properties after i.p. administration of AMC303 revealed an terminal half-life of 6h. Administration schedules of AMC303 (1mg/kg) given one, three or five times per week had no significant impact on tumor growth attenuation. AMC303 treatment could be paused for 10 days without incurring significant tumor regrowth. Conclusions: AMC303 inhibits c-MET in vitro in a ligand-dependent fashion and in vivo significantly improved overall survival in an orthotopic pancreatic xenograft mouse tumor model. AMC303 effectively attenuated both primary and metastatic tumor growth by increasing apoptosis, reduction of angiogenesis and vascular normalization. These findings demonstrate the benefit of targeting multiple oncogenic RTK signaling pathways by AMC303. Citation Format: Tina Heumann, Vanessa Al-Rawi, Thorsten Läufer, Martin Augsten, Alexandra Matzke-Ogi, Oliver Coutelle. AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic 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 4855.

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.

Differential Response of MET inhibition by Glesatinib (MGCD265) and Sitravatinib (MGCD516) in Non‐small Cell Lung Cancer and Malignant Mesothelioma

Aberrant activation of the receptor tyrosine kinase (RTK) MET is frequently involved in malignant transformation and inhibiting its activity in dependent cancers is associated with improved clinical outcomes. Glesatinib (MGCD265) and sitravatinib (MGCD516) are next generation MET RTK inhibitors (RTKIs) and while glesatinib also targets the AXL RTK, sitravatinib inhibits a closely related spectrum of RTKs. The efficacy of these compounds in MET‐dependent non‐small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM) cell lines that differed with respect to MET gene expression was evaluated. Both drugs inhibited cell growth and migratory activity with similar potency in NSCLC H1993 cells with clustered MET amplification and A459 cells with ‘wild type’ MET. Also, MPM cell lines H2373 and H2461 with elevated MET expression showed comparable levels of sensitivity to either drug. Kinomics analysis of the affected pathways suggested that both drugs acted similarly, yet distinct signaling was affected when different cell lines were tested. In particular, cellular pathways associated with transformation were inhibited whereas those promoting cell death were activated upon drug treatment. Furthermore, phosphorylation of RON, which interacts with MET, and the downstream signaling molecules AKT and p44/42MAPK were also inhibited in response to hepatocyte growth factor (HGF) stimulation. Importantly, in a pre‐clinical in vivo xenotransplant mouse model, both drugs caused significant tumor growth inhibition. Our data suggest that discerning the expression and activation status of MET and its associated signaling pathways may help guide treatment decisions concerning targeted therapies for NSCLC and MPM.Support or Funding InformationThe National Cancer Institute of the National Institutes of Health under award number P30CA033572 and Mirati Therapeutics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Study of RON mediated invasion of Raji cell line and drug-target effects

To study the proto-oncogene RON mediated aggression of Raji cells and the inhibitory effects by monoclonal antibody Zt/f2 (2f2). The effects of RON ligand macrophage stimulating protein (MSP) (2.0 nmol/L) and inhibitory Zt/f2 (2F2) (2.0 nmol/L) antibody on proliferation of RON positive Raji cells after treatment for 24 and72 hours were detected by MTT method, colony formation units (CFU) of Raji cells by methylcellulose semi solid culture, Raji cells apoptosis and cell cycle analysis by AnnexinV/PI double staining, expression of RON, apoptosis-related proteins, and cyclins by Western blot. (1)Compared with the cell viability (1.0) and counts of CFU (103.6±7.0) in control group, Raji cells after MSP treatment had better viability (1.35±0.20) and CFU counts (133.7±10.4) (P<0.05), but worse viability (0.68±0.11) and CFU counts (66.3±6.1) after Zt/f2 (2F2) treatment (P<0.05). (2)Percentage of Raji cells apoptosis after Zt/f2 (2F2) antibody treatment (12.16±2.33)% was significantly increased than the control (2.89±1.03)% (P<0.05). The percentage of Raji cells arrested in G0/G1 phase was increased after Zt/f2 (2F2) antibody treatment as compared to the control [ (54.96 ±3.70)% vs (39.10±2.30)%, (P<0.05) ]. (3) High-level of RON phosphorylation and β-catenin expression activated by MSP could be inhibited significantly by Zt/f2 (2F2), which also up-regulated the expression of caspase-3, caspase-8, caspase-9 and PARP and down-regulated anti-apoptotic MCL-1 gene and inhibitor of apoptosis protein XIAP expression, accompanied with G1 phase protein changes accordingly. MSP could aggravate Raji cells proliferation. Inversely, Zt/f2 (2F2) could inhibit proliferation and induce apoptosis by inhibition of RON phosphorylation and up-regulation of apoptosis related proteins.

Abstract 3019: Alternative transcription of the RON tyrosine kinase receptor in human pancreatic adenocarcinoma generates active protein isoforms.

Abstract Introduction: The RON tyrosine kinase receptor is overexpressed in the majority of pancreatic duct adenocarcinomas (PDAC) and has been shown to promote pancreatic cancer cell migration, invasion and apoptotic resistance. The RON gene can be alternatively transcribed to produce protein isoforms which have variable activities. RON isoforms have been described previously in colon, breast, gastric, and lung cancer specimens and have been shown to be oncogenic. Several therapies directed against RON are in preclinical and Phase 1 trials, therefore determining biological significance of RON isoforms is of great importance. Here we report their identification in human PDAC and investigate their importance using pancreatic cancer cell lines created to overexpress the isoforms. Methods: Patient derived PDAC xenografts (PDXS) were established in the pancreata of NSG mice. Low passage (P4 or less) xenograft tumors were used to generate RNA and protein lysates for RT-PCR and Western blot analysis. Primers specific for exons 4-6 and 10-12 were created to assess alternative transcription and deletion of these exons. Results: 19 PDX's were analyzed and alternative transcription of the RON gene was identified in all but 1 tumor. Deletions of exons 5, 6, and 11 were common and resulted in transcripts for RONΔ165 (which was as common as the wild type transcript), RONΔ160, RONΔ155, and RONΔ90 isoforms. The RONΔ55 isoform (short-form RON, is created by transcription from an alternative start in exon 11 resulting in a truncated protein where the extra-cellular domain is deleted) was present in 15 of 19 tumors. Western blot analysis and Immunoprecipitation (IP) was used to confirm protein expression and determine RON activity. 7 of 9 fresh tumor samples showed RON phosphorylation. Protein isoforms were purified by IP with RON 8 antibody and then resolved using gel electrophoresis. Mass spectrometry identified the presence of RONΔ165, RON Δ170, and RONe5|6in isoforms with RONΔ165 being the most abundant. We further tested our hypothesis that the RONΔ55 isoform is active by tagging it with green fluorescent protein (GFP) and transfecting the fusion protein into 3 cell lines (Mia-Paca 2, BxPC3, and HPDE6/E7). Analysis of the novel cell lines showed constitutive phosphorylation of Δ55 and increased phosphorylation of ERK and AKT indicating enhanced signaling via the MAPK and PI3K pathways. Conclusions: Our findings show for the first time that RON is alternatively transcribed and produces biologically active isoforms in tissue derived from human pancreatic duct adenocarcinoma. We have confirmed the presence of these isoforms at the protein level and found that RONΔ165 is abundant. Further work to understand the biologic significance of these isoforms is required to guide future drug development and to validate RON as a rational target in the treatment of pancreatic cancer. Citation Format: Jeffery Chakedis, Randall French, Michele Babicky, Megan Harper, Dawn Jaquish, Evangeline Mose, Andrew Lowy. Alternative transcription of the RON tyrosine kinase receptor in human pancreatic adenocarcinoma generates active protein isoforms. [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 3019. doi:10.1158/1538-7445.AM2013-3019

Abstract 2915: Discovery and characterization of OSI-296, a dual inhibitor of cMET and RON kinases

Abstract cMET and RON are receptor tyrosine kinases of the MET proto-oncogene family that are activated by their respective ligands HGF and MSP. Signaling through the cMET/HGF system can be deregulated in cancer by HGF-dependent autocrine activation, gene amplification, and/or the presence of activating mutations, among others, while for RON, constitutively active variants generated by alternative splicing or methylation-dependent promoter usage [short-form RON (sfRON)] have been identified. Approaches to abrogate aberrant cMET and RON signaling that have led to agents in clinical trials include inhibiting their kinase function with small molecules. We report here the discovery and characterization of OSI-296, a dual inhibitor of cMET and RON. The compound exhibited selectivity in a panel of 96 kinases with potent activity against cMET, including common Y1230 mutants, and RON. OSI-296 blocked cMET autophosphorylation in MKN45 cells, resulting in dose-dependent inhibition of downstream ERK, AKT, and STAT3 phosphorylation. It also showed potent cellular activity in ELISA-format sfRON and caRON cell mechanistic assays that we developed, resulting in dose-dependent inhibition of downstream ERK and AKT phosphorylation. OSI-296 showed a PK profile in rodents suitable for oral dosing with &amp;gt;70% bioavailability. In multiple xenografts models (cMET: MKN45, SNU-5, U87MG; RON: caRON), significant tumor growth inhibition was observed upon oral dosing with regression at higher doses. OSI-296 was very well tolerated with little body weight loss and no adverse effects even at the highest tested dose of 300 mg/kg p.o. qdx14. Solid PK/PD/TGI correlations have been established wherein &amp;gt;90% inhibition of cMET or RON phosphorylation sustained over 24 h by OSI-296 translated to 100% TGI. In summary, OSI-296 was shown to be a well tolerated, dual inhibitor of cMET and RON with in vivo activity in mouse xenografts models for both targets upon oral dosing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2915. doi:1538-7445.AM2012-2915

IGF1-R signals through the RON receptor to mediate pancreatic cancer cell migration

The RON receptor tyrosine kinase (RTK) is overexpressed in the majority of pancreatic cancers, yet its role in pancreatic cancer cell biology remains to be clarified. Recent work in childhood sarcoma identified RON as a mediator of resistance to insulin-like growth factor receptor (IGF1-R)-directed therapy. To better understand RON function in pancreatic cancer cells, we sought to identify novel RON interactants. Using multidimensional protein identification analysis, IGF-1R was identified and confirmed to interact with RON in pancreatic cancer cell lines. IGF-1 induces rapid phosphorylation of RON, but RON signaling did not activate IGF-1R indicating unidirectional signaling between these RTKs. We next demonstrate that IGF-1 induces pancreatic cancer cell migration that is RON dependent, as inhibition of RON signaling by either shRNA-mediated RON knockdown or by a RON kinase inhibitor abrogated IGF-1 induced wound closure in a scratch assay. In pancreatic cancer cells, unlike childhood sarcoma, STAT-3, rather than RPS6, is activated in response to IGF-1, in a RON-dependent manner. The current study defines a novel interaction between RON and IGF-1R and taken together, these two studies demonstrate that RON is an important mediator of IGF1-R signaling and that this finding is consistent in both human epithelial and mesenchymal cancers. These findings demand additional investigation to determine if IGF-1R independent RON activation is associated with resistance to IGF-1R-directed therapies in vivo and to identify suitable biomarkers of activated RON signaling.

Ron Kinase Transphosphorylation Sustains MET Oncogene Addiction

Receptors for the scatter factors HGF and MSP that are encoded by the MET and RON oncogenes are key players in invasive growth. Receptor cross-talk between Met and Ron occurs. Amplification of the MET oncogene results in kinase activation, deregulated expression of an invasive growth phenotype, and addiction to MET oncogene signaling (i.e., dependency on sustained Met signaling for survival and proliferation). Here we show that cancer cells addicted to MET also display constitutive activation of the Ron kinase. In human cancer cell lines coexpressing the 2 oncogenes, Ron is specifically transphosphorylated by activated Met. In contrast, Ron phosphorylation is not triggered in cells harboring constitutively active kinase receptors other than Met, including Egfr or Her2. Furthermore, Ron phosphorylation is suppressed by Met-specific kinase inhibitors (PHA-665752 or JNJ-38877605). Last, Ron phosphorylation is quenched by reducing cell surface expression of Met proteins by antibody-induced shedding. In MET-addicted cancer cells, short hairpin RNA-mediated silencing of RON expression resulted in decreased proliferation and clonogenic activity in vitro and tumorigenicity in vivo. Our findings establish that oncogene addiction to MET involves Ron transactivation, pointing to Ron kinase as a target for combinatorial cancer therapy.

Combining RON And EGFR Kinase Directed Therapy Markedly Inhibits Orthotopic Pancreatic Cancer Growth

Introduction: The RON receptor tyrosine kinase is overexpressed in pancreatic cancer and mediates apoptotic resistance. We have recently demonstrated that RON inhibition can sensitize pancreatic cancer cells to the effects of Gemcitabine and that resistance to RON inhibition may be mediated by activation of EGFR. A previous study demonstrated crosstalk between RON and EGFR in 3T3 cells. The goals of this study were: 1) To determine if RON and EGFR crosstalk occurs in pancreatic cancer cells and 2) To determine the effects of combined inhibition of RON and EGFR signaling on orthotopic pancreatic cancer growth. Methods: BxPC3 and FG pancreatic cancer cell lines were cultured in RPMI/DMEM media with 10% FBS, serum starved overnight, and stimulated with MSP, the ligand for RON, or EGF over a 5-60 minute time course and compared with null and EGF/MSP stimulated controls. Cell lysates were used for RON immunoprecipitation and/or subjected to western blot analysis for phospho-Tyr or phospho-EGFR. Orthotopic xenografts were developed by injecting 1×106 FG-mCherry cells in 20 μL of DMEM + Growth Factor Reduced Matrigel into the pancreas of 8 week old nu/nu mice using a 28-guage needle. Seven days post-implantation, treatment was started with either PBS, RON8 (a human monoclonal antibody that inhibits RON), RON8+Erlotinib, or RON8+Erlotinib+Gemcitabine. Mice were sacrificed after 27 days post-implantation and tumor volume and weight were measured. Results: Stimulation with MSP resulted in increased EGFR phosphorylation after 60 minutes in BxPC3 pancreatic cancer cells. Similarly, stimulation with EGF resulted in increased RON phosphorylation after 30-60 minutes in both FG and BxPC3 cell lines, thereby demonstrating bidirectional crosstalk between these two receptors. Combination therapy directed at inhibition of both RON and EGFR kinase activity in orthotopic xenografts resulted in significantly decreased tumor volume and weight when compared with untreated controls. Conclusions: There is interaction between RON and EGFR and there is bidirectional crosstalk between the two receptors in pancreatic cancer cells. Combined RON and EGFR inhibition demonstrated a significant decrease in tumor growth in this orthotopic model of pancreatic cancer. Therefore, combined therapy targeting both RON and EGFR is a strategy worthy of further investigation.

Inhibition of MSP-RON signaling pathway in cancer cells by a novel soluble form of RON comprising the entire sema sequence

The RON receptor tyrosine kinase and its ligand macrophage stimulating protein (MSP) play a role in epithelial tumorigenesis. We report here a novel RON variant that antagonizes the RON-MSP pathway in various cancer cells. The variant is an 85 kDa soluble protein from an mRNA transcript with an insertion of 49 nucleotides between exons 5 and 6. The insertion created a stop codon leading to the formation of a RON variant consisting of the entire 35 kDa alpha-chain and a 45 kDa partial extracellular beta-chain. The protein was featured by a sema domain, a hinge motif and a portion of the first IPT unit (designated as RONDelta85). RONDelta85 binds directly to MSP, forms MSP-RONDelta85 complex, and inhibits RON phosphorylation. RONDelta85 disrupts RON or RONDelta160 dimerization, prevents their phosphorylation, and attenuates downstream signaling events. The action of RONDelta85 is specific to RON and has no effect on MET and EGFR. In colon and pancreatic cancer cells, RONDelta85 inhibits spontaneous or MSP-induced Erk1/2 and AKT phosphorylation, which results in impaired cell proliferation and colony formation. RONDelta85 also inhibits spontaneous and MSP-induced cell migration. We conclude that RONDelta85 is an antagonist to the MSP-RON pathway, which has potential for regulating RON/RON160-mediated tumorigenic activities.

Abstract B240: Inhibition of MSP-RON signaling pathway in cancer cells by a novel soluble form of RON variant containing the complete sema sequences

Abstract The RON receptor tyrosine kinase and its ligand macrophage stimulating protein (MSP) play a role in epithelial tumorigenesis. We report here a novel RON variant that antagonizes the RON-MSP pathway in various cancer cells. The variant was an 85 kDa soluble protein from a mRNA transcript with an insertion of 49 nucleotides between exons 5 and 6. The insertion created a stop code leading to a RON variant composed of a full 35kDa α-chain and a 45 kDa partial extracellular β-chain. The protein was featured by a sema domain, a hinge and a portion of the first IPT unit (designated as RON 85). RON 85 directly bound to the MSP, formed the MSP-RON 85 complex, and inhibited RON phosphorylation. RON 85 disrupted RON or RON 160 dimerization, prevented their phosphorylation, and attenuated their downstream signaling events. The action of RON 85 was specific to RON and had no effect on MET and EGFR. In colon and pancreatic cancer cells, RON 85 inhibited spontaneous or MSP-induced Erk1/2 and AKT phosphorylation, which results in impaired cell proliferation and colony formation. RON 85 also inhibited spontaneous or MSP-induced cell migration. We concluded that RON 85 is an antagonist to the MSP-RON pathway with therapeutic potentials for controlling tumorigenic phenotypes of cancer cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B240.

Abstract B130: Monoclonal antibody (mAb)-induced down-regulation of the RON receptor tyrosine kinase impairs malignant phenotypes of colon cancer cells

Abstract Targeting receptor tyrosine kinases (RTK) with specific mAbs disrupts oncogenic signals and diminishes tumorigenic activities. The RON receptor tyrosine kinase is a member of the MET proto-oncogene family and contributes to motile/invasive phenotypes of colon cancer cells. Here, we studied mAb-induced down-regulation of RON in modulating tumorigenic phenotypes of colon cancer cells. Three mAbs (Zt/g4, Zt/c9 and Zt/f2), recognizing different epitopes in the RON extracellular sequences, were used in various in vitro experiments. Colon cancer SW620 and other cells were used as the model. Treatment of SW620 cells individual mAbs results in transient RON phosphorylation within minutes. However, persistent treatment of cells with Zt/g4 or other mAb causes the down-regulation RON expression as evident by Western blot and cell surface fluorescent analyses. The effect of mAb was in both concentration and time-dependent manners. These activities were not related to antigenic epitopes recognized by individual mAb but highly specific to RON because MET or EGFR expression was not affected. Down regulation of RON impairs intracellular signaling events. Phosphorylation of Erk1/2 and AKT was dramatically reduced after Zt/g4 and other mAb treatment, indicating that the anti-RON mAb has the ability to disrupt downstream signal cascades. Functional studies showed that treatment of SW620 cells with anti-RON mAb affects tumor cell growth in soft agar. It also increases the sensitivity of colon cancer SW620 cells towards chemoagent gemcitabine. In this case, the cytotoxicity of SW620 cells treated with mAbs was significantly increased with reduced IC50 of gemcitabine. In conclusion, our results demonstrated that the persistent treatment of cancer cells with antibodies specific to the RON extracellular domains results in down-regulation of RON expression in colon cancer cells. The reduced RON expression is accompanied with the impaired signaling events and diminished tumorigenic activities. Thus, anti-RON mAb directed targeting could have therapeutic implication for controlling malignant phenotypes of cancer cells (*supported in part by NIH R01 grant Ca91980 and Amarillo Area Foundation of Texas) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B130.

Agonistic monoclonal antibodies potentiate tumorigenic and invasive activities of splicing variant of the RON receptor tyrosine kinase

Ligand-dependent or independent activation of the RON receptor tyrosine kinase is essential in transducing invasive signals leading to increased tumorigenic activities. In this study, we characterized two monoclonal antibodies (mAbs) specific to the extracellular domains of human RON and studied their agonistic effect on tumorigenic activities mediated by oncogenic variant RON?160. The mAb Zt/g4 and Zt/c1 are specific to human RON. They bind to RON with high affinities and recognized different epitopes on the RON extracellular domain. Because of their reactivity with native RON, Zt/g4 and Zt/c1 are useful in various applications such as immunoprecipitation, immunofluorescent analysis, and immunohistochemical staining. Functional studies revealed that Zt/g4 and Zt/c1 are capable of inducing RON phosphorylation which activates signaling proteins such as Erk1/2 and Akt. In NIH3T3 cells expressing RON?160, both mAbs significantly enhanced RON?160-mediated tumorigenic activities including cell proliferation, focus formation, and anchorage-independent growth. Cell shape changes with increased motile and invasive activities were also observed. Studies in vivo further demonstrated that Zt/g4 and Zt/c1 increase RON?160-mediated tumor growth in nude mice with a shortened time of onset and enlarged tumor volume. Thus, by recognizing specific epitopes on the RON extracellular domains, Zt/g4 and Zt/c1 have abilities to elicit a full array of RONmediated responses. These mAbs will be useful in studying mechanisms underlying RON activation which lead to increased tumorigenic activities.

Macrophage-stimulating protein is produced by tubular cells and activates mesangial cells.

Until now, hepatocytes have been the only known cell source of macrophage-stimulating protein (MSP), and tissue macrophages have been the cells on which the biologic effects of MSP have been proved. To extend the understanding of the biologic meaning of MSP, it was investigated whether MSP operates in the kidney. MSP protein was evaluated by Western blot in supernatant of cultured human tubular cells (HK2) and human mesangial cells (HMC). MSP mRNA was investigated in HK2 by reverse transcription-polymerase chain reaction (RT-PCR). The expression of the MSP receptor, RON, was evaluated in HMC and HK2 by Western blot. RON mRNA was investigated in HMC by RT-PCR. The expression of MSP and RON in normal human renal tissue was studied by immunohistochemistry. HMC were stimulated with recombinant MSP (rMSP) and HK2 supernatant to study cell growth, migration, and the capacity to invade an artificial collagen matrix and synthesize interleukin-6 (IL-6). HK2 produced MSP and expressed RON in a form that was phosphorylated by rMSP. HMC expressed RON but did not produce MSP. MSP in HK2 supernatant and rMSP induced in HMC phosphorylation of RON, growth, migration, invasion, and IL-6 synthesis. In normal human kidney, tubules expressed MSP and RON. These results indicate a novel field of operation for MSP and suggest a pathogenic role of the MSP/RON system in renal disease. In fact, MSP released by tubular cells may recruit monocytes/macrophages in inflammatory tubulointerstitial disorders. In addition, MSP either circulating or as paracrine product may sustain glomerular mesangioproliferative disease.

Cross-talk between RON receptor tyrosine kinase and other transmembrane receptors.

RON is a transmembrane receptor tyrosine kinase that mediates biological activities of Macrophage Stimulating Protein (MSP). MSP is a multifunctional factor regulating cell adhesion, motility, growth and survival. MSP binding to RON causes receptor tyrosine phosphorylation leading to up-regulation of RON catalytic activity and subsequent activation of downstream signaling molecules. Recent studies show that RON is spatially and functionally associated with other transmembrane molecules including adhesion receptors integrins and cadherins, and cytokine and growth factor receptors IL-3 betac, EPOR and MET. For example, MSP-induced cell shape change is mediated via RON-activated IL-3 betac receptor. Activation of integrins causes MSP-independent RON phosphorylation, and the integrin/RON collaboration regulates cell survival. Thus, RON can be activated without MSP by ligand stimulation of RON-associated receptors, and MSP-activated RON can cause ligand-independent activation of RON-associated receptors. As a result of the receptor cross-activation RON-specific pathways become a part of a signal transduction network of other receptors, and conversely signaling pathways activated by other receptors can be used by RON. This receptor collaboration extends the spectrum of cellular responses generated by MSP and by putative ligands of RON-associated receptors. However signaling pathways involved in the receptor cross-talk and underlying activation mechanisms remain to be investigated. The purpose of this review is to summarize data and to discuss a role of cross-talk between RON and other transmembrane receptors.

Integrin-mediated RON Growth Factor Receptor Phosphorylation Requires Tyrosine Kinase Activity of Both the Receptor and c-Src

Cooperation between integrins and growth factor receptors plays an important role in the regulation of cell growth, differentiation, and survival. The function of growth factor receptor tyrosine kinases (RTKs) can be regulated by cell adhesion to extracellular matrix (ECM) even in the absence of ligand. We investigated the pathway involved in integrin-mediated RTK activation, using RON, the receptor for macrophage-stimulating protein. Adhesion of RON-expressing epithelial cells to ECM caused phosphorylation of RON, which depended on the kinase activity of both RON itself and c-Src. This conclusion is based on these observations: 1) ECM-induced RON phosphorylation was inhibited in cells expressing kinase-inactive c-Src; 2) active c-Src could phosphorylate immunoprecipitated RON from ECM-stimulated cells but not from unstimulated cells; and 3) ECM did not cause RON phosphorylation in cells expressing kinase-dead RON, nor could active c-Src phosphorylate RON immunoprecipitated from these cells. The data fit a pathway in which ECM-induced integrin aggregation causes both c-Src activation and RON oligomerization followed by RON kinase-dependent autophosphorylation; this results in RON becoming a target for activated c-Src, which phosphorylates additional tyrosines on RON. Integrin-induced epidermal growth factor receptor (EGFR) phosphorylation also depended on both EGFR and c-Src kinase activities. This sequence appears to be a general pathway for integrin-dependent growth factor RTK activation.