Screening Identifies Mechanisms of Drug Resistance to PI3K Inhibitors

Screening Identifies Mechanisms of Drug Resistance to PI3K Inhibitors

One of the most important focuses in personalized oncology therapeutics is on the ability to understand mechanisms of treatment resistance, with the aim of selecting more effective treatments. Underlying this is the need to identify tumor mutations that are inherently resistant to a specific treatment modality. The complexity of the pathways and networks of pathways in cancer makes it necessary to test a large number of combinations of gene-drug interactions; in the future, genomic methods combined with clinical investigation should greatly improve patient treatment outcomes. 

Phosphoinositide 3-kinases

In an online-first article in Nature Chemical Biology (DOI: 10.1038/nchembio.695), Sebastian Nijman of the CeMM–Research Center for Molecular Medicine of the Austrian Academy of Sciences in Vienna and his colleagues describe the development of a chemical genetic approach that has identified mechanisms that can lead to resistance to PI3K inhibitors.

As Professor Nijman explained to the cancernetwork, “Even for [targeted drugs] that have been designed to act specifically on cells carrying a certain cancer mutation, only a fraction of the patients respond. The reasons for this are often unclear. But obviously, if one could know beforehand who would respond and who would not, this could be a great advantage. Patients that would not benefit could be saved the side effects of treatment and given an alternative treatment. Or perhaps one could extend the duration of response by designing a combination therapy. So what is needed is being able to predict response to treatment. Our study had this aim and we found one such ‘mechanism of resistance.'"

The researchers developed a way to measure the cellular fitness of isogenic human cell lines in order to quantitatively assess functional drug-gene interactions. As the authors explain, “this method assists the systematic assessment of the impact of cancer aberrations on proliferation in response to a collection of drugs."

The actual screen used an isogenic breast cancer cell line to which a genetic alteration was introduced linked to a molecular "barcode." The barcode is a non-transcribed short sequence of DNA that can be easily identified and quantitated. After addition of the drug, measuring the abundance of the barcode DNA via PCR in the population of cells served as a proxy for the measure of cellular fitness/growth.

The researchers tested the interaction of 87 drugs with 70 gene mutations (either knock-down or overexpression) in the breast cancer cells, and this resulted in data from over 6000 drug-gene pairs. The genes were selected based on literature and database searches and included important genes linked to breast cancer such as HER2, BRCA1, BRCA2, c-MYC, NOTCH1, and PTEN. The cell line selected had a normal karyotype and is responsive to most signaling pathways present in normal breast epithelial cells. The study identified previously known interactions, confirming the utility of the approach. 

Importantly, the study also identified a new mechanism of resistance to a group of PI3K inhibitors that are currently being investigated in clinical trial. Many breast tumors harbor activating mutations in the PI3K pathway. The screen identified that the intracellular active domain of NOTCH1 confers resistance to the dual PI3K-mTOR (mammalian target of rapamycin) inhibitor BEZ-235. Further testing with other cancer cell lines suggested that NOTCH1 activation generally uncouples proliferation from the PI3K-mTOR pathway: “We have evidence that other cancer types may show the same mechanism of resistance” Nijman confirmed. NOTCH1 appears to override the need for the PI3-K-mTOR pathway through increased c-MYC transcription. The published work demonstrates that transcriptional activation of m-MYC via NOTCH1 is sufficient to confer the resistance to BEZ235. This result was validated by inhibiting c-MYC expression with RNAi in the NOTCH1 upregulated cells.

"In screening thousands of drug-gene interactions, one of our hits was the resistance caused by activation of the NOTCH pathway and downstream activation of c-MYC to PI3K/mTOR inhibitors. These drugs are of great interest as many are in clinical development for a variety of cancers. However, so far mechanisms of resistance have not been reported” explained professor Nijman.

NOTCH activation is known to occur in a subset of breast cancers and is associated with tumor progression and poor prognosis. MYC amplification is a relatively frequent event in breast cancer. BEZ235 is being developed by Novartis and is currently in phase 1 and 2 clinical trials for patients with solid tumors, including advanced breast cancers as both a monotherapy and in combination with chemotherapy and other targeted agents. 

In terms of next steps, Professor Nijman says that his laboratory is still “digesting the many hits that came out of the screen." The researchers are also continuing to develope the technology and performing more screens. They plan to continue to focus on data generation that will need to be validated by further benchwork and clinical trials. Nijman added, “What excites me is that using our approach, [which] was not guaranteed to work, we have made some very interesting and clinically relevant discoveries. I believe that this is only the beginning!”  

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