Aurora A-Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy.

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Therapeutic inhibition of MAP kinase interacting kinase blocks eukaryotic initiation factor 4E phosphorylation and suppresses outgrowth of experimental lung metastases.

Activation of the translation initiation factor 4E (eIF4E) promotes malignant transformation and metastasis. Signaling through the AKT-mTOR pathway activates eIF4E by phosphorylating the inhibitory 4E binding proteins (4E-BP). This liberates eIF4E and allows binding to eIF4G. eIF4E can then be phosphorylated at serine 209 by the MAPK-interacting kinases (Mnk), which also interact with eIF4G. Although dispensable for normal development, Mnk function and eIF4E phosphorylation promote cellular proliferation and survival and are critical for malignant transformation. Accordingly, Mnk inhibition may serve as an attractive cancer therapy. We now report the identification of a potent, selective and orally bioavailable Mnk inhibitor that effectively blocks 4E phosphorylation both in vitro and in vivo. In cultured cancer cell lines, Mnk inhibitor treatment induces apoptosis and suppresses proliferation and soft agar colonization. Importantly, a single, orally administered dose of this Mnk inhibitor substantially suppresses eIF4E phosphorylation for at least 4 hours in human xenograft tumor tissue and mouse liver tissue. Moreover, oral dosing with the Mnk inhibitor significantly suppresses outgrowth of experimental B16 melanoma pulmonary metastases as well as growth of subcutaneous HCT116 colon carcinoma xenograft tumors, without affecting body weight. These findings offer the first description of a novel, orally bioavailable MNK inhibitor and the first preclinical proof-of-concept that MNK inhibition may provide a tractable cancer therapeutic approach.

eIF4E activation is commonly elevated in advanced human prostate cancers and significantly related to reduced patient survival.

Elevated eukaryotic translation initiation factor 4E (eIF4E) function induces malignancy in experimental models by selectively enhancing translation of key malignancy-related mRNAs (c-myc and BCL-2). eIF4E activation may reflect increased eIF4E expression or phosphorylation of its inhibitory binding proteins (4E-BP). By immunohistochemical analyses of 148 tissues from 89 prostate cancer patients, we now show that both eIF4E expression and 4E-BP1 phosphorylation (p4E-BP1) are increased significantly, particularly in advanced prostate cancer versus benign prostatic hyperplasia tissues. Further, increased eIF4E and p4E-BP1 levels are significantly related to reduced patient survival, whereas uniform 4E-BP1 expression is significantly related to better patient survival. Both immunohistochemistry and Western blotting reveal that elevated eIF4E and p4E-BP1 are evident in the same prostate cancer tissues. In two distinct prostate cancer cell models, the progression to androgen independence also involves increased eIF4E activation. In these prostate cancer cells, reducing eIF4E expression with an eIF4E-specific antisense oligonucleotide currently in phase I clinical trials robustly induces apoptosis, regardless of cell cycle phase, and reduces expression of the eIF4E-regulated proteins BCL-2 and c-myc. Collectively, these data implicate eIF4E activation in prostate cancer and suggest that targeting eIF4E may be attractive for prostate cancer therapy.

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity.

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.

A 15-ketosterol is a liver X receptor ligand that suppresses sterol-responsive element binding protein-2 activity.

Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-alpha and -beta (LXRalpha and LXRbeta). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.

Cyclic AMP-independent activation of CYP3A4 gene expression by forskolin.

Forskolin and cAMP have been shown to have paradoxical effects in the regulation of expression levels of mRNA of cytochrome P450 3A (CYP3A) family members. We demonstrate in this study that forskolin upregulated the promoter for CYP3A4 independent of its ability to increase cAMP levels. This activity was explained showing forskolin directly activated the pregnane-X-receptor, a known regulator of CYP3A genes.

A natural product ligand of the oxysterol receptor, liver X receptor.

Natural products have been identified as ligands for a number of members of the nuclear hormone receptor (NHR) superfamily. Often these natural products are used as dietary supplements to treat myriad ailments ranging from perimenopausal hot flashes to hypercholesterolemia and reduced cognitive function. Examples of some natural product ligands for NHRs include genestein (estrogen receptors NR3A1 and NR3A2), guggulsterone (farnesoid X receptor NR1H4), and St. John's wort (pregnane X receptor, NR1I2). In this study, we identified the first nonoxysterol natural product that functions as a ligand for the liver X receptor (LXRalpha and LXRbeta; NR1H3, NR1H2), a NHR that acts as the receptor for oxysterols and plays a key role in regulation of cholesterol metabolism and transport as well as glucose metabolism. We show that paxilline, a fungal metabolite, is an efficacious agonist of both LXRalpha and LXRbeta in biochemical and in vitro cell-based assays. Paxilline binds directly to both receptors and is an activator of LXR-dependent transcription in cell-based reporter assays. We also demonstrate that paxilline binding to the receptors results in efficient activation of transcription of two physiological LXR target genes, ABCA1 and SREBP. The discovery of paxilline, the first reported nonoxysterol natural product ligand of the LXRs, may provide insight into the mechanism of ligand recognition by these receptors and reaffirms the utility of examining natural product libraries for identifying novel NHR ligands.