Activation of protein kinase CB (PKCB) has been repeatedly implicated in tumor-induced angiogenesis. The PKCB-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCB has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3B ser9 , ribosomal protein S6 S240/244 , and AKT Thr308 . Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3B in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3B phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3B phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3B phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

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Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme that catalyzes the initial and rate-limiting step of tryptophan catabolism resulting in the local depletion of tryptophan and the concomitant production of kynurenine, both of which are immunosuppressive. Targeting IDO1 in combination with PD-1/PD-L1-targeted antibodies has shown promise in early phase clinical trials in several cancers and strongly suggests that, in some patients, IDO1 expression restrains PD-1/PD-L1-targeted checkpoint therapies. While some cancers extrinsically express IDO1 in response to IFN-γ produced from an ongoing, yet ineffective immune response, others select for the intrinsic expression of IDO1, independent of an immune response. We identified several cancer cell lines that intrinsically expressed either IDO1 or the related isozyme TDO2. Using these cell lines, we discovered LY3381916, a potent and selective inhibitor of cell-based IDO1 activity (IDO1 7 nM; TDO2 >20 µM). Using a variety of techniques, we demonstrated that LY3381916 binds to newly synthesized apo-IDO1 lacking heme, but does not inhibit mature heme-bound IDO1. Protein x-ray crystallography confirmed that LY3381916 binds to apo-IDO1 where it occupies the heme-binding pocket of IDO1. As a result of this novel mechanism of action, substantial inhibition of IDO1 in tumors requires the turn-over of mature heme-bound IDO1. Modeling of the pre-clinical PK/PD relationship suggests QD dosing of LY3381916 will maintain greater than 90% inhibition over 24 hours. In addition, due to the favorable properties of the drug, significant central nervous system (CNS) penetration has been measured for LY3381916 (rodent kp,uu 0.26). Kynurenine-mediated agonism of the aryl hydrocarbon receptor (AHR) is immunosuppressive in the tumor microenvironment. Inhibition of IDO1 and the subsequent reduction of kynurenine can relieve this immunosuppression. However, several heme-binding IDO1 inhibitors have been shown to replace kynurenine as an AHR agonist potentially limiting their ability to relieve this IDO1-dependent immunosuppressive mechanism. LY3381916 shows no confounding agonism of AHR up to 100 µM. Additionally, we characterized LY3381916 in pre-clinical tumor models and demonstrated that it was able to enhance LY3300054, anti-PD-L1 antibody (LY3300054) activity, which was associated with an enhanced T cell response. Based on these characteristics, LY3381916 is currently being investigated in a Phase I clinical trial. These data suggest further development of LY3381916 may be warranted. Citation Format: Frank C. Dorsey, Karim A. Benhadji, Lillian L. Sams, Debra A. Young, John F. Schindler, Karen L. Huss, Alexander Nikolayev, Carmine Carpenito, David Clawson, Bonita Jones, Andrew L. Faber, James E. Thomas, Steven A. Haney, Gaiying Zhao, William T. McMillen, Tod Smeal, Daniel J. Sall, Michael D. Kalos, Sandaruwan Geeganage, James R. Henry. Identification and characterization of the IDO1 inhibitor LY3381916 [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 5245.

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