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.

The optimized LPEI-PEG2 kDa-EGF conjugate displays reduced chemical complexity and efficient poly(I:C)-mediated killing of EGFR overexpressing tumors in vitro and in vivo.

Summary Phage display has identified the dodecapeptide YHWYGYTPQNVI (GE11) as a ligand that binds to the EGFR but does not activate the receptor. Here we compare the EGFR binding affinities of GE11, EGF and their polyethyleneimine-polyethyleneglycol (PEI-PEG) conjugates. We find that although GE11 by itself does not exhibit measurable affinity to the EGFR, tethering it to PEI-PEG increases its affinity markedly, and complex formation with PolyIC further enhances the affinity to the sub-micromolar range. PolyIC/PPGE11 has a similar strong antitumor effect against EGFR over-expressing tumors in vitro and in vivo, as PolyIC/Polyethyleneimine-polyetheleneglycol-EGF (PolyIC/PP-EGF). Absence of EGFR activation, as previously shown by us (see text) and easier production of GE11 and GE11 conjugates, confer PolyIC/PPGE11 a significant advantage over similar EGF-based polyplexes as a potential therapy of EGFR over-expressing tumors.

Activated double-stranded RNA (dsRNA-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. Here we investigate whether selective activation of PKR can be used to kill cancer cells that express mutated genes containing deletions or chromosomal translocations. We show that antisense (AS) RNA complementary to fragments flanking the deletion or translocation can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death following hybridization with mutated but not wild-type mRNA. Using the U87MG Delta EGFR cell line, which expresses a truncated form of epidermal growth factor receptor (EGFR), Delta(2-7) EGFR, we found that expression of a 39-nucleotide (nt) AS RNA complementary to the unique exon 1 to 8 junction caused selective death of cells harboring the truncated EGFR both in vitro and in vivo but did not affect cells expressing wild-type EGFR. A lentiviral vector expressing the 39-nt AS sequence strongly inhibited glioblastoma growth in mouse brain when injected after tumor cell implantation. This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.

Purpose: The cause of most cancer deaths is incurable dissemination of cancer cells into vital organs. Current systemic therapies for disseminated cancers provide limited efficacy and are often accompanied by toxic side effects. We have recently shown that local application of epidermal growth factor receptor (EGFR)-targeted polyinosine-cytosine (polyIC) eradicates preestablished EGFR-overexpressing tumors. Here we show for the first time the high efficiency of systemic application of polyIC/melittin-polyethyleneimine-polyethyleneglycol-EGF (polyIC/MPPE) in combination with human immune cells.Experimental design: Cancer-targeted activation of immune cells was examined in vitro and in vivo following transfection with polyIC/MPPE. The therapeutic efficiency of the strategy was then examined on disseminated EGFR-overexpressing tumors grown in severe combined immunodeficient (SCID) mice.Results: Intravenous delivery of polyIC/MPPE followed by intraperitoneal injection of peripheral blood mononuclear cells induced the complete cure of SCID mice with preestablished disseminated EGFRoverexpressing tumors, with no adverse toxic effects. The immune cells and the cytokines they produce are localized to the tumor site of the treated animal and contribute decisively to the demise of the tumor cells. The immune system homes to the tumors, due to the chemokines produced by the internalized polyIC.Conclusion: The EGFR-homing vector loaded with polyIC can be used to treat and possibly cure patients with disseminated EGFR-overexpressing tumors. The possibility of adopting this strategy to treat other tumors that express a protein capable of ligand induced internalization is discussed.

The RTK/Ras/Raf cascade is overactive in cancers. Its targets are the MAP kinases Erks, but Erks are not mutated in cancers. An active Erk, Erk1(R84S), is an oncoprotein. Further, Erk1(R84S) and Erk2(R65S) autophosphorylate the TEY motif and Thr-207/Thr-188. Erk2(R65S) autophosphorylates Thr-188 when dually mutated in the TEY.