LY3009120, a panRAF inhibitor, has significant anti-tumor activity in BRAF and KRAS mutant preclinical models of colorectal cancer.

Activating mutations in the KRAS and BRAF genes, leading to hyperactivation of the RAS/RAF/MAPK oncogenic signaling cascade, are common in patients with colorectal cancer (CRC). While selective BRAF inhibitors are efficacious in BRAFmut melanoma, they have limited efficacy in BRAFmut CRC patients. In a RASmut background, selective BRAF inhibitors are contraindicated due to paradoxical activation of the MAPK pathway through potentiation of CRAF kinase activity. A way to overcome such paradoxical activation is through concurrent inhibition of the kinase activity of both RAF isoforms. Here, we further examined the effects of LY3009120, a panRAF and RAF dimer inhibitor, in human models of CRC with various mutational backgrounds. We demonstrate that LY3009120 induced anti-proliferative effects in BRAFmut and KRASmut CRC cell lines through G1-cell cycle arrest. The anti-proliferative effects of LY3009120 in KRASmut CRC cell lines phenocopied molecular inhibition of RAF isoforms by simultaneous siRNA-mediated knockdown of ARAF, BRAF and CRAF. Additionally, LY3009120 displayed significant activity in in vivo BRAFmut and KRASmut CRC xenograft models. Examination of potential resistance to LY3009120 demonstrated RAF-independent ERK and AKT activation in the KRASmut CRC cell line HCT 116. These findings describe the preclinical activity of a panRAF inhibitor in a BRAFmut and KRASmut CRC setting.

Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays.

Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.

The novel IKK2 inhibitor LY2409881 potently synergizes with histone deacetylase inhibitors in preclinical models of lymphoma through the downregulation of NF-κB.

PURPOSE: To evaluate the pharmacologic activity of a novel inhibitor of IκB kinase ß (IKK2), LY2409881, in preclinical models of B- and T-cell lymphoma, as a single agent and in combination with histone deacetylase (HDAC) inhibitors. EXPERIMENTAL DESIGN: The in vitro activity of LY2409881 was determined using an ATP-based growth inhibition assay and flow cytometric assay of apoptosis in lymphoma cell lines. The in vivo activity of LY2409881 was determined using SCID-beige xenograft mouse model. The mechanism of action was determined using immunoblotting, immuofluorescence, and electrophoretic mobility shift assay. Synergy of LY2409881 with other drugs active in lymphoma was determined by calculating relative risk ratio (RRR) and combination index (CI). RESULTS: LY2409881 inhibited constitutively activated NF-κB, and caused concentration- and time-dependent growth inhibition and apoptosis in lymphoma cells. In models of diffuse large B-cell lymphoma (DLBCL), the cytotoxicity of LY2409881 correlated with the overall activation status of NF-κB, but not simply in a pattern predicted by the cell-of-origin classification of these cell lines. LY2409881 was safe to mice at three dose levels, 50, 100, and 200 mg/kg, all of which caused significant inhibition of tumor growth. LY2409881 suppressed the activity of the NF-κB subunit p65 in lymphoma cells treated by the HDAC inhibitor romidepsin, underlying a potential mechanism of the marked synergy observed of these two drugs. CONCLUSION: Collectively, these data strongly suggest that targeting the NF-κB pathway in combination with romidepsin could represent a novel and potent regimen for the treatment of B- and T-cell lymphoma.

Development and validation of a drug activity biomarker that shows target inhibition in cancer patients receiving enzastaurin, a novel protein kinase C-beta inhibitor.

PURPOSE: To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent. EXPERIMENTAL DESIGN: The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach. RESULTS: Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin. CONCLUSION: An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.

Increased localization and substrate activation of protein kinase C delta in lung epithelial cells following exposure to asbestos.

The protein kinase C (PKC) family consists of several isozymes whose substrates may be necessary for the regulation of key cellular events important in the pathogenesis of proliferative diseases. Asbestos is a carcinogen and fibroproliferative agent in lung that may cause cell signaling events through activation of PKC. Here we used a murine inhalation model of asbestos-induced inflammation and fibrosis to examine immunoreactivity of PKC delta and its substrate, phosphorylated-adducin (p-adducin), in cells of the lung. Moreover, we characterized PKC delta and p-adducin expression in a pulmonary epithelial cell line (C10) in both log versus confluent cells and in cells after mechanical wounding or crocidolite asbestos exposure. Both PKC delta and p-adducin were almost exclusively expressed in bronchiolar and alveolar type II (ATII) epithelial cells in lung sections and increased in these cell types after inhalation of asbestos by mice. Increases in membrane and nuclear localization of PKC delta were seen in log phase as compared to confluent C10 cells. Moreover, enhanced immunoreactivity of PKC delta was observed in epithelial cells expressing proliferating cell nuclear antigen (PCNA) after mechanical wounding or exposure to asbestos fibers. These studies show that activated PKC delta in pulmonary epithelial cells is a consequence of inhalation of asbestos and may be linked to the activation of cell proliferation.