PKR, an interferon (IFN)-inducible protein kinase activated by double-stranded RNA, inhibits translation by phosphorylating the initiation factor eIF2alpha chain. We show that human IFN-gamma mRNA uses local activation of PKR in the cell to control its own translation yield. IFN-gamma mRNA activates PKR through a pseudoknot in its 5' untranslated region. Mutations that impair pseudoknot stability reduce the ability to activate PKR and strongly increase the translation efficiency of IFN-gamma mRNA. Nonphosphorylatable mutant eIF2alpha, knockout of PKR and PKR inhibitors 2-aminopurine, transdominant-negative PKR, or vaccinia E3L correspondingly enhances translation of IFN-gamma mRNA. The potential to form the pseudoknot is phylogenetically conserved. We propose that the RNA pseudoknot acts to adjust translation of IFN-gamma mRNA to the PKR level expressed in the cell.

Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and anti-apoptotic signaling from insulin-like growth factor 1 receptor (IGF1R), insulin receptor (IR) and other oncoproteins. IRS1 plays a central role in cancer cell proliferation, its expression is increased in many human malignancies and its up-regulation mediates resistance to anti-cancer drugs. IRS2 is associated with cancer cell motility and metastasis. Currently there are no anti-cancer agents that target IRS1/2. We present new IGF1R/IRS-targeted agents (NT compounds) that promote inhibitory Ser-phosphorylation and degradation of IRS1 and IRS2. Elimination of IRS1/2 results in long-term inhibition of IRS1/2-mediated signaling. The therapeutic significance of this inhibition in cancer cells was demonstrated while unraveling a novel mechanism of resistance to B-RAFV600E/K inhibitors. We found that IRS1 is up-regulated in PLX4032-resistant melanoma cells and in cell lines derived from patients whose tumors developed PLX4032 resistance. In both settings, NT compounds led to elimination of IRS proteins and evoked cell death. Treatment with NT compounds in vivo significantly inhibited the growth of PLX4032-resistant tumors, and displayed potent anti-tumor effects in ovarian and prostate cancers. Our findings offer preclinical proof of concept for IRS1/2 inhibitors as cancer therapeutics including in PLX4032-resistant melanoma. By the elimination of IRS proteins, such agents should prevent acquisition of resistance to mutated-B-RAF inhibitors and possibly restore drug sensitivity in resistant tumors.

BackgroundGlioblastoma multiforme (GBM) is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12–14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC]), a strong activator of apoptosis, selectively to cancer cells.Methods and FindingsPoly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF). EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and “bystander killing” of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors.ConclusionThe strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.