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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In work performed by a number of laboratories, it has become quite clear that the oral administration of autoantigens exerts a profoundly suppressive effect on the development and long-term clinical course of autoimmune disease. Specific peptide sequences derived from the autoantigens are similarly suppressive. An interesting sidelight to emerge from specificity studies is that oral administration of a self-protein or peptide sequence (i.e., rat MBP peptide administered to a rat) is markedly less tolerogenic than oral administration of a non-self or even closely related sequence (guinea pig MBP peptide administered to a rat). The dose of oral antigen is now known to play a critical role in determination of the mechanism of oral tolerance, with low doses of antigen causing active suppression with concomitant release of TGFbeta1. Studies outlined here suggest that oral administration of higher antigen doses (e.g., 20 mg MBP to rats or mice) results in deletion of specific antigen-reactive T lymphocytes. This conclusion stems from the fact that injections of IL-2 could not reverse high-dose tolerance while reversing low-dose oral tolerance. Moreover, feeding MBP to MBP-TCR transgenic mice caused trafficking of transgenic cells to the intestine followed by a profound depletion of transgene-positive cells and reduction in proliferative function in all peripheral lymphoid organs. Oral tolerance has proven to be of therapeutic benefit in other animal models of autoimmune disease as well, including uveitis, collagen-induced arthritis, adjuvant arthritis, thyroiditis, myasthenia gravis, and diabetes. Initial human trials in multiple sclerosis, rheumatoid arthritis, and uveitis show promising results.

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Interference with DNA damage checkpoints has been demonstrated preclinically to be a highly effective means of increasing the cytotoxicity of a number of DNA-damaging cancer therapies. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA may proceed into premature mitosis followed by cell death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 functionality leads to abrogation of DNA damage checkpoints and has been shown preclinically to enhance the activity of many standard of care chemotherapeutic agents. LY2603618 is a potent and selective small molecule inhibitor of Chk1 protein kinase activity in vitro (IC(50) = 7 nM) and the first selective Chk1 inhibitor to enter clinical cancer trials. Treatment of cells with LY2603618 produced a cellular phenotype similar to that reported for depletion of Chk1 by RNAi. Inhibition of intracellular Chk1 by LY2603618 results in impaired DNA synthesis, elevated H2A.X phosphorylation indicative of DNA damage and premature entry into mitosis. When HeLa cells were exposed to doxorubicin to induce a G2/M checkpoint arrest, subsequent treatment with LY2603618 released the checkpoint, resulting in cells entering into metaphase with poorly condensed chromosomes. Consistent with abrogation of the Chk1 and p53-dependent G2/M checkpoint, mutant TP53 HT-29 colon cancer cells were more sensitive to gemcitabine when also treated with LY2603618, while wild-type TP53 HCT116 cells were not sensitized by LY2603618 to gemcitabine. Treatment of Calu-6 human mutant TP53 lung cancer cell xenografts with gemcitabine resulted in a stimulation of Chk1 kinase activity that was inhibited by co-administration of LY2603618. By all criteria, LY2603618 is a highly effective inhibitor of multiple aspects of Chk1 biology.