The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF-and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAFand KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).

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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

The RAS/MAPK pathway is dysregulated in approximately 30% of human cancers, and the extracellular-signal-regulated kinases (ERK1 and ERK2) serves as key central nodes within this pathway. The feasibility and clinical impact of targeting the RAS/MAPK pathway has been demonstrated by the therapeutic success of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma. However, resistance develops frequently through reactivation of the pathway. Therefore, simultaneous targeting of multiple effectors such as RAF, MEK and ERK in this pathway, offers a potential for enhanced efficacy while delaying and overcoming resistance. LY3214996 is a highly selective inhibitor of ERK1 and ERK2, with IC50 of 5 nM for both enzymes in biochemical assays. It potently inhibits cellular phospho-RSK1 in BRAF and RAS mutant cancer cell lines. In an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation, tumor cells with MAPK pathway alterations including BRAF, NRAS or KRAS mutation are generally sensitivity to LY3214996. In tumor xenograft models, LY3214996 inhibits PD biomarker phospho-p90RSK1 in tumors and the PD effects are correlated with compound exposures and anti-tumor activities. LY3214996 shows either similar or superior anti-tumor activity as compared to other published ERK inhibitors in BRAF or RAS mutant cell lines and xenograft models. Oral administration of single-agent LY3214996 significantly inhibits tumor growth in vivo and is well tolerated in BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal, lung and pancreatic cancer xenografts or PDX models. Therefore, LY3214996 can be tailored for treatment of cancers with MAPK pathway alteration. In addition, LY3214996 has anti-tumor activity in a vemurafenib-resistant A375 melanoma xenograft model due to MAPK reactivation, may have potential for treatment of melanoma patients who have failed BRAF therapies. More importantly, LY3214996 can be combined with investigational and approved agents in preclinical models, particularly KRAS mutant models. Combination treatment of LY3214996 and CDK4/6 inhibitor abemaciclib was well tolerated and results in potent tumor growth inhibition or regression in multiple in vivo cancer models, including KRAS mutant colorectal and non-small cell lung cancers. Here, we first report the preclinical characterization of LY3214996, a novel small molecule ERK1/2 inhibitor currently in Phase I clinical trials in patients with advanced and metastatic cancers (NCT02857270). Citation Format: Shripad V. Bhagwat, William T. McMillen, Shufen Cai, Baohui Zhao, Matthew Whitesell, Lisa Kindler, Robert S. Flack, Wenjuan Wu, Karen Huss, Bryan Anderson, Xiu-Juan Yuan, Susan Jaken, Denis McCann, Brian Mathes, Andrew J. Dropsey, Jason Manro, Jennie Walgren, Eunice Yuen, Xueqian Gong, Michael J. Rodriguez, Jianping Huang, Ramon V. Tiu, Sajan Joseph, Sheng-Bin Peng. Discovery of LY3214996, a selective and novel ERK1/2 inhibitor with potent antitumor activities in cancer models with MAPK pathway alterations [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 4973. doi:10.1158/1538-7445.AM2017-4973

Therapeutic strategies focused on kinase inhibition rely heavily on surrogate measures of kinase inhibition obtained from in vitro assay systems. There is a need to develop methodology that will facilitate measurement of kinase inhibitor activity or specificity in tissue samples from whole animals treated with these compounds. Many of the current methods are limited by the use of antibodies, many of which do not cross-react between several species. The proteomics approach described herein has the potential to reveal novel tissue substrates, potential new pathway interconnections, and inhibitor specificity by monitoring differences in protein phosphorylation. We used the protein kinase inhibitor H89 (N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide) as a tool to determine whether differential profiling of tissue phosphoproteins can be used to detect treatment-related effects of a protein kinase A (PKA) inhibitor in vivo. With a combination of phosphoprotein column enrichment, high-throughput two-dimensional gel electrophoresis, differential gel staining with Pro-Q Diamond/ SYPRO Ruby, statistical analysis, and matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis, we were able to show clear differences between the phosphoprotein profiles of rat liver protein extract from control and treated animals. Moreover, several proteins that show a potential change in phosphorylation were previously identified as PKA substrates or have putative PKA phosphorylation sites. The data presented support the use of differential proteomic methods to measure effects of kinase inhibitor treatment on protein phosphorylation in vivo.Determining the effects of kinase inhibitors on protein substrates in vivo is of central importance relative to inferences about inhibitor specificity and mechanisms of observed biological effects. In addition, in vivo studies are best suited to represent metabolism and other physiological parameters likely to influence kinase inhibitor activity at the organ level.