Activation of dsRNA dependent protein kinase PKR in Karpas299 does not lead to cell death.

Activated double-stranded RNA (dsRNA)-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing them to die. We have recently shown that PKR can be selectively activated in cancer cells, by in situ generation of dsRNA following introduction of antisense RNA complementary to an RNA expressed specifically in the cancer cell. The feasibility of this approach was demonstrated using a glioblastoma line that overexpresses a truncated form of the EGFR. PKR and its signaling pathway are not restricted to a given cell line; therefore, in principle, this dsRNA killing approach can be applied to any cancer that expresses unique RNA sequences. Nonetheless, applying this approach to Karpas299 cells, from a T-cell non-Hodgkin's lymphoma that harbors the NPM/ALK translocation, did not result in cell death, implying that PKR signaling pathway is repressed in this cell line. Indeed, the phosphorylation of eIF2alpha by PKR was impaired in Karpas299 cells. Furthermore, levels of the cellular inhibitor p67 were elevated in these cells. Long antisense, as well as RNAi for p67, delivered into Karpas299 cells by adenoviruses, reduced p67 levels. The reduction in p67 levels led to increased phosphorylation of eIF2alpha, and an additive effect was achieved by coinfection with NPM/ALK-AS encoding adenoviruses. Infection with these adenoviruses, however, did not promote growth inhibition. These findings imply that anti-apoptotic mechanisms counteract PKR signaling in this T-cell non-Hodgkin's lymphoma.

A cellular screening assay to test the ability of PKR to induce cell death in mammalian cells.

Long double-stranded RNA (>30 bp), usually expressed in cells infected with RNA viruses, triggers antiviral responses that induce apoptosis of the infected cells. PKR can be selectively activated in glioblastoma cells by in situ generation of dsRNA following introduction of antisense RNA complementary to an RNA expressed specifically in these cells. Harnessing PKR for the selective killing of cancer cells is potentially a powerful strategy for treating cancer, but we were unable to induce apoptosis by this approach in a T cell lymphoma. We therefore established a cellular screening assay to test the ability of PKR to induce death in cell lines, especially those originating from human cancers. This "PKR killing screen" is based on the infection of cells with an adenoviral vector encoding GyrB-PKR, followed by coumermycin treatment. Cancers represented by cell lines in which PKR activation leads to cell death are good candidates for the dsRNA killing approach, using antisense to RNA molecules specifically expressed in these cells. The PKR killing screen may also serve as a tool for exploring PKR signaling and other related pathways, by identifying new cases in which PKR signaling is inhibited or impaired.

Tumor specific activation of PKR as a non-toxic modality of cancer treatment.

Over the past decade progress has been made in the development of therapies against cancer. Small molecules, mainly tyrosine kinase inhibitors (tyrphostins) like Gleevec, Iressa targeting CML and EGFR overexpressing tumors have entered the clinic, where a large number of other tyrphostins are at various stages of clinical development. In parallel a few antibodies like Herceptin targeting breast cancer overexpressing Her-2 and Rituxan targeting B cell malignancies are utilized in the clinic. In all these cases success is moderate and restricted to a narrow population of patients, except for Gleevec which is effective for a long duration for chronic CML. The cancer community agrees that this is actually a unique exception that proves the rule. Over the past few years a few modalities of cancer gene therapies have emerged. In this short review we shall summarize our efforts to develop methods to activate PKR selectively in cancer cells.

RNA molecules as anti-cancer agents.

The inhibition of cancer growth and progression is one of the major challenges facing modern medicine. Despite significant progress in the development of therapies against cancer, only in a few cases are these therapies effective. Because cancer is a complex disease, agents that target a single oncogenic pathway have low efficacy, in addition to allowing the emergence of drug resistance. There is a need for specific therapy, which can affect a broad range of cancers, with minimal side effects. Here we summarize several novel anti-cancer strategies that answer the above criteria. These strategies utilize the multiple anti-proliferative effects of double stranded RNA (dsRNA): (1) the classical antisense RNA hybridizes with its target mRNA, leading to reduced levels of a specific oncoprotein; (2) short dsRNA of specific sequence, known as small inhibitory RNA (siRNA), can selectively and efficiently inhibit expression of specific oncogenes, expressed in cancer cells but not in normal cells. Shutting down the expression of cancer-promoting genes by siRNA has proven to be an effective approach against several cancers; (3) long dsRNA, frequently expressed in cells infected with viruses, activates mechanisms that efficiently kill the infected cells, thereby preventing spread of the virus. The dsRNA killing strategy (DKS), involving the in situ generation of dsRNA of sufficient length to induce antiviral defenses specifically in cancer cells, is a novel strategy developed in our laboratory. DKS has the potential to be applicable to a wide range of cancers. Thus dsRNA-based anti-cancer strategies could be powerful tools for cancer treatment.