Several small molecules that inhibit the PI3K-Akt signaling pathway are in clinical development. In preclinical models bearing PTEN/PIK3CA mutations, however, single-agent Akt and dual PI3K/mTOR inhibitors only delay tumor growth and do not shrink existing tumors.25,26
Even when PI3K/Akt inhibition is effective in preclinical cancer models, apoptosis is not induced.27
Thus, single-agent PI3K pathway inhibitors often do not yield the expected responses in cancers that are supposed to be sensitive to these inhibitors.28
One potential reason for the limited efficacy of PI3K inhibition alone is the existence of compensatory or redundant pathways in tumor cells. For example, inhibition of mTORC1 leads to activation of the ERK and PI3K signaling pathways, showing increased Akt activity and downstream signals.29
Taken together, the evidence from the present and prior studies suggests that combining multiple pathway inhibitors might be more effective.
In this study, we developed a comprehensive method for identifying molecular targets whose inactivation could enhance the therapeutic efficacy of PI3K inhibitors, suggesting that combinations of PI3K inhibitors and agents targeting those molecules can act synergistically to improve therapeutic efficacy, thus, affirming that testing for synthetic lethality is useful for identifying new targets that would complement the effects of drugs of interest.30,31
Here, we used a retrovirus-based library that can target approximately one-third of human genes and a platform whereby we could systematically assess the loss-of-function phenotypes.32
Identifying the genes that are essential for cell survival may facilitate the discovery of compensatory signaling pathways that impair the effectiveness of PI3K pathway inhibitors clinically.33
Using a synthetic lethality screen, we confirmed the functional relevance of 15 cancer-related genes through the use of the glioma-related databases and by analyzing molecular pathways, showing that most, but not all, of the target genes can lead to activation of the ERK signaling pathway. Remarkably, recent studies showed that for many cancers, combined PI3K-Akt and Raf-MEK-ERK inhibition was required to effectively shut off PI3K/mTOR signaling and promote apoptosis through Bcl-2 family proteins.34
The genome-scale screening method used in our study has been reported to be powerful, because it can reveal interactions not only between gene products that have direct contact but also between those whose signaling occurs along the same or parallel pathways.35
An advantage of a large-scale shRNA library is that it is genome-wide and is robust for most if not all relevant targets. The key practical issues when performing RNA interference screening in mammalian cells are assay development, library selection, target validation, robust statistical algorithms, and functional validation experiments.36
We used retroviral vectors under optimized conditions so that cells are infected by only a single virion during retroviral transduction.37
Monitoring phenotypic changes simplified the subsequent identification of functional clones from the library and allowed us to rapidly screen millions of cells. This approach can be readily adapted to other cell types and phenotypic changes. Some siRNAs have “off-target” effects, and these effects are often the result of the RNAs being partially homologous to other transcripts. A sequence identity of as few as 11 or 12 nucleotides between an siRNA and an mRNA may be sufficient for interference to occur,38
indicating that cross-reactivity is a substantial problem. However, the shRNA library used in our study was designed to avoid off-target effects by minimizing the similarities between shRNAs and other transcripts.
We performed a target-specific screening with another PI3K/mTOR inhibitor, NVP-BEZ235, which confirmed the functional validity of the high-throughput screening, suggesting that our robust screening strategies facilitated the selection of high-confidence target genes that could be rationally combined with PI3K pathway inhibitors. In addition, the target validation was done using the shRNA approach to inactivate the individual target, and inactivation of the target gene showed an additive effect with PX-866 in growth inhibition.
The success of this large-scale screening approach is illustrated by our bioinformatics and molecular discovery that the target genes might be a significant contributor to the molecular mechanisms that underlie and regulate selective sensitivity of GBM cells to PI3K inhibitors. Our data strongly established the effectiveness of searching for synthetic lethality using an shRNA library with a specific molecular inhibitor to perform genome-wide screening of potential targets or pathways whose inactivation would synergize the antitumor effects of the drug. We expect that our results will not only advance knowledge of the interdependency among complex signaling pathways and their response to particular targeted therapies, but also increase the number of combination therapies for GBM.