The transforming growth factor β (TGFβ) signaling pathway is a pleiotropic cellular pathway that plays a critical role in cancer. In fact, aggressive tumors are typically associated with high ligand levels and thus associated with poor prognosis in various tumor types. Cancer cells use autocrine and paracrine TGFβ signaling to modulate tumor cells and the tumor microenvironment leading to a highly invasive and metastatic phenotype, inducing and increasing tumor vascularization, modulating the extracellular matrix in the stroma, and inhibiting immune surveillance and antitumor immunity. Clinical studies with galunisertib (aka LY2157299 monohydrate), a small molecule inhibitor targeting the TGFβ pathway, have provided proof of concept data supporting the role of TGFβ in cancer and the utility of targeting the TGFβ pathway. Here we describe the identification of LY3200882, a next generation small molecule inhibitor of TGF-β receptor type 1 (TGFβRI). The molecule is a potent, highly selective inhibitor of TGFβRI embodied in a structural platform with a synthetically scalable route. It is an ATP competitive inhibitor of the serine-threonine kinase domain of TGFβRI. Mechanism of action studies reveal revealed that LY3200882 inhibits various pro-tumorigenic activities. LY3200882 potently inhibits TGFβ mediated SMAD phosphorylation in vitro in tumor and immune cells and in vivo in subcutaneous tumors in a dose dependent fashion. In preclinical tumor models, LY3200882 showed potent anti-tumor activity in the orthotopic 4T1-LP model of triple negative breast cancer and this activity correlated with enhanced tumor infiltrating lymphocytes in the tumor microenvironment. Durable tumor regressions in the orthotopic 4T1-LP model were observed and rechallenge of congenic tumors resulted in complete rejection in all mice. In in vitro immune suppression assays, LY3200882 has shown the ability to rescue TGFβ1 suppressed or T regulatory cell suppressed naïve T cell activity and restore proliferation. Therefore, LY3200882 shows promising activity as an immune modulatory agent. In addition, LY3200882 has shown anti-metastatic activity in vitro in migration assays as well as in vivo in an experimental metastasis tumor model (intravenous EMT6-LM2 model of triple negative breast cancer). Finally, LY3200882 shows combinatorial anti-tumor benefits with checkpoint inhibition (anti-PD-L1) in the syngeneic CT26 model. In conclusion, we have developed a novel potent and highly selective small molecule inhibitor of TGFβRI for the treatment of cancer. Citation Format: Huaxing Pei, Saravanan Parthasarathy, Sajan Joseph, William McMillen, Xiaohong Xu, Stephen Castaneda, Ivan Inigo, Karen Britt, Bryan Anderson, Gaiying Zhao, Scott Sawyer, Douglas Beight, Talbi Kaoudi, Chandrasekar Iyer, Huimin Bian, Amy Pappas, David Surguladze, David Schaer, Karim Benhadji, Michael Kalos, Kyla Driscoll. LY3200882, a novel, highly selective TGFβRI small molecule inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 955. doi:10.1158/1538-7445.AM2017-955

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Under normal conditions MET receptor activation, internalization, and degradation is a highly controlled process regulated by the binding of its ligand, HGF. In many cancers however, MET becomes highly dysregulated, and MET overexpression, with or without gene amplification, is associated with a poorer prognosis. Aberrant activation of MET can also occur through mutation, or increased autocrine or paracrine HGF production. MET activation leads to increased oncogenic activities including cell migration, proliferation, invasion, survival, tumor neovascularization and decreased apoptosis. Several inhibitors of MET, including type I and type II kinase inhibitors, are currently being investigated in clinical trials. LY2801653 is a type II MET kinase inhibitor being investigated in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). In addition to MET, LY2801653 also inhibits ROS1 kinase, and the serine/threonine kinases MKNK1/2, with cell-based IC50 of 23nM and 7nM respectively. ROS1 is an orphan receptor that can be constitutively activated as a fusion protein either by genomic microdeletion or translocation. ROS1 fusion proteins are found in glioblastoma, NSCLC and cholangiocarcinoma. MKNK1/2 are kinases downstream of ERK and p38 and are capable of regulating translation through the direct phosphorylation of eIF4E. Increased MKNK1/2-dependent phosphorylation of the translation factor eIF4E is observed in head and neck squamous cell carcinoma and is correlated with a poor prognosis. This study evaluated the relative inhibitory effects of LY2801653, Crizotinib, XL184, XL880, and PF4217903 in a variety of cell lines against MET, ROS1, MKNK1/2, ERK, and other downstream molecules. In HCC78, a ROS1-fusion protein bearing cell line, and in NIH3T3 cells stably transfected with the FIG-ROS(s) fusion, LY2801653, XL184 and XL880 were more potent inhibitors of ROS1 compared with Crizotinib. Moreover, LY2801653 and Crizotinib were both efficacious in NIH3T3 FIG-ROS(s) xenografts, although LY2801653 was more potent in reducing p-ROS1 in these tumors than Crizotinib. In NCI-H2122 and Hs746T cells (mutated MET), A2780 cells (MET-negative), KP-4 cells (MET autocrine), H441 cells (HGF-independent; MET positive), HCT116 (mutated KRAS), and the cholangiocarcinoma cell lines EGI-1 and TFK-1, LY2801653 demonstrated more potent inhibitory activity against MKNK1/2 versus treatment with XL184 and XL880, while Crizotinib had no observed inhibitory activity against MKNK1/2. LY2801653 also showed inhibitory activity against MKNK1/2 in MET-amplified MKN45 cells. These findings suggests there may be clinical merits to testing LY2801653 in tumor types associated with increased MKNK1/2 activity or ROS1 fusion proteins alone, or in conjunction with aberrant MET activity, such as colorectal carcinoma, head and neck squamous cell carcinoma, and cholangiocarcinoma. Citation Format: Sau-Chi B. Yan, Megan N. Thobe, Bruce W. Konicek, Victoria L. Peek, Suzane L. Um, Richard A. Walgren, Huaxing Pei, Timothy P. Burkholder, Jeremy R. Graff. LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2. [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 3042. doi:10.1158/1538-7445.AM2013-3042