AWARD NUMBER: W81XWH-14-1-0609 TITLE: Studying the Immunomodulatory Effects of Small Molecule Ras-Inhibitors in Animal Models of Rheumatoid Arthritis

Ras-GTPases are molecular switches that regulate key cellular processes, such as proliferation, differentiation, apoptosis, and motility. In T cells, Ras-family GTPases (e.g. K/N-Ras) are crucial for proper TCR-dependent activation following antigen recognition. Defective Ras GTPases signaling has been associated with T cell anergy, and accordingly increased expression of active Ras was shown to reverse anergy and to restore IL-2 production. Importantly, T cells from patients with Rheumatoid Arthritis (RA) display augmented activation of the Ras/Raf/MEK/ERK1/2 signaling pathway, and accordingly overexpression of active K-RAS in normal CD4+ T cells has been shown to promote T cells reactivity to relevant autoantigen in RA. Thus, Ras GTPases appear to be a promising molecular target for inhibiting T cell activation in RA. Based on an innovative concept Kloog (the partnering PI) and colleagues discovered a potent non-toxic inhibitor of Ras, Farnesylthiosalicylic acid (FTS). This small molecule does not belong to the class of farnesyltransferase inhibitors (FTIs) that failed in clinical trials. It interferes with the interactions between Ras and distinct prenyl-binding chaperone proteins that are vital for the proper plasma membrane (PM) localization and signaling dynamics of RasGTPases, and indeed FTS dislodges the classical H/N/K-Ras GTPases from the PM and inhibits their effective downstream signaling. In multiple preclinical animal studies it has been shown that FTS effectively inhibited in vivo tumor growth of oncogenic K/N-Ras-dependent cancers. Thus, in collaboration with Concordia Pharmaceuticals Inc., FTS was developed into and oral drug, Salirasib®. The drug has been already tested in the clinic for the treatment of cancers with oncogenic mutations in KRAS and NRAS. No dose-limiting toxicities or major adverse events were reported during Salirasib® treatment, in phase I clinical trials of patients with advanced pancreatic cancer, hematolological malignancies (NCT00867230; M.D. Anderson Cancer Center, TX), and in phase II clinical study in non-small-cell lung cancer patients (NCT00531401; Memorial Sloan Kettering Cancer Center, NY). Thus, Salirasib® is the only available successful Ras GTPases inhibitor that reached Phase II clinical trials, and moreover received an orphan drug designation by the FDA for the treatment of pancreatic cancer. Importantly, we have extensively studied the effects of FTS and related derivatives (e.g., FTSAmide and FTS-methoxymethylester), in multiple pre-clinical animal models of autoimmune inflammation, such as: experimental autoimmune encephalomyelitis; Type-1 diabetes; colitis and others. More recently, our preliminary studies in the adjuvant-induced arthritis (AIA) rat model − a classical animal model for RA − imply that FTS attenuates disease manifestation, as assessed by: clinical scores; MRI imaging; histopathology; and serum levels of pro-inflammatory cytokines. Thus, our working hypothesis is that Salirasib® has a good potential to be a “silver bullet” drug for RA and other T cell-dependent autoimmune disorders. Our objectives are to test further this hypothesis in the AIA model as well as in another established animal model of RA, the collagen type-II induced arthritis (CIA) in DBA/1 (H-2q) mice. In parallel we will study in vitro, the effects of FTS and its different newer derivatives on a wide range of T cell functions and signaling networks implicated in RA. For the therapeutic treatment protocol, we will be administer FTS orally by gavage, once daily, starting immediately after disease initiation. Multiple modalities will be used to assess joint inflammation/damage and the immune response, as follows: arthritis clinical scores; MRI scans, micro-CT; histopathology examination by a blinded pathologist; serum cytokine profiles; T cell subset analysis (e.g., Foxp3+ Treg, Th1, Th17, etc.) by polychromatic flow cytometry. Additionally, we will analyzed the activation of different Ras downstream effectors and relevant cellular programs by Western blotting, Affymetrix GeneChip® whole-transcript arrays, and quantitative real-time PCR analysis. The proposed project is highly relevant to the FY13 PRMRP topic area of Rheumatoid Arthritis (RA). The short-term impact of our research will be an improved understanding of the role of Ras GTPases in shaping, tuning, and regulating the autoimmune T cell response, and the effects of Ras inhibitors on the pathogenesis of the inflammatory arthritis in two relevant preclinical models of RA. We envision that the long-term impact of our proposed research plan will be the introduction of a new class of synthetic drugs, orally available small molecule Rasinhibitors such as Salirasib®, to advance the clinical management of RA patients with conceivably fewer side effects and reduced healthcare system costs compared to biologic drugs.