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RGX-202, a small-molecule creatine transporter SLC6A8 inhibitor, suppresses colorectal cancer and modulates human creatine levels.

Background: Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide with more than 140,000 patients diagnosed and nearly 50,000 deaths annually in the U.S. alone. Roughly 60% of patients present with locally advanced or distant metastatic disease, with the liver being a primary site of metastatic colonization. Creatine metabolism has been implicated in colon cancer progression and metastatic colonization of the liver. Metastatic colon cancer cells upregulate and release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high-energy metabolite phosphocreatine. Phosphocreatine is imported via the creatine transporter SLC6a8. Intracellular phosphocreatine can be converted to ATP to fuel the survival of metastatic cancer cells within the hypoxic hepatic microenvironment. Consistent with this finding, genetic depletion of SLC6a8 in colon and pancreatic cancer cell lines significantly reduced liver colonization in mouse xenograft models. Results: We herein demonstrate that the novel small molecule RGX-202 is a robust inhibitor of creatine uptake in cancer cells, both in vitro and in vivo. Oral administration of RGX-202 induced apoptosis of colon cancer cells in vivo, and significantly suppressed colon cancer liver metastatic colonization and primary tumor growth, both in KRAS wild-type and KRAS mutant colon cancer cell lines as well as in human PDX mouse models. Using genetic studies, these effects were found to be dependent on tumoral expression of SLC6a8. In addition, combination treatment of the CT26 syngeneic colon cancer mouse model with 5-FU resulted in synergistic antitumor activity, with complete tumor regressions observed in more than 40% of treated mice. Similarly, combination treatment of the KPC syngeneic mouse model with gemcitabine significantly reduced the growth of primary pancreatic tumors. Definitive 28-day GLP toxicology and pharmacokinetics studies of RGX-202 are currently ongoing. Preliminary observations suggest good tolerability in several animal species with a favorable pharmacokinetic profile, including bioavailability. Conclusion: These results strongly support clinical development of RGX-202 in patients with gastrointestinal cancers, including colorectal and pancreatic cancer, both as monotherapy and in combination with standard-of-care treatment. Citation Format: Isabel Kurth, Celia Andreu, Shugaku Takeda, Helen Tian, Foster Gonsalves, Katya Leites, Subhasree Sridhar, Jia Min Loo, Rob Busby, Sohail Tavazoie, Masoud Tavazoie. RGX-202, a first-in-class small-molecule inhibitor of the creatine transporter SLC6a8, is a robust suppressor of cancer growth and metastatic progression [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 5863.

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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.