More than 60% of breast cancers harbor genomic changes in the PI3K-AKT-mTOR, or phosphatidylinositol-3-kinase/AKT/mechanistic target of rapamycin, pathway, but attempts to develop drugs that target components in the pathway have been largely unsuccessful. Potentially hundreds of clinical trials have failed, but scientists haven’t really understood why. Researchers headed by a team at the University of California, San Francisco (UCSF), have now used a new unbiased chemoproteomics technique to identify an unrelated protein, Aurora kinase A (AURKA), which effectively acts to protect cancer cells from PI3K pathway-targeted drugs.

Their in vitro and in vivo studies showed that treating cancer using the FDA-approved PI3K pathway component inhibitor everolimus in combination with a Phase II-stage experimental AURKA inhibitor stopped the growth of estrogen receptor-positive tumors and also killed cancer cells.

“There have been more than 200 clinical trials with experimental drugs that target the PI3K pathway, and probably more than $1 billion invested, with only one approved drug treatment so far,” says senior researcher Sourav Bandyopadhyay, Ph.D., UCSF assistant professor of bioengineering and therapeutic sciences and a member of the UCSF Helen Diller Family Comprehensive Cancer Center. “Every pharma company in the cancer space has tried to target the PI3 kinase pathway, with little success. Now we may know why. “This work has immediate clinical implications, and we hope this study spurs interest in combining these two classes of embattled cancer drugs.” 

Reporting their results today in Nature Chemical Biology, the team concludes,”Our findings reveal that the combination of Aurora kinase inhibitors and PI3K-pathway inhibitors is synergistic and could be a promising clinical strategy to enhance the treatment response in breast cancer.” The team is currently working toward a Phase I clinical trial to evaluate drug combinations in patients. Their published paper is entitled “Kinome Rewiring Reveals AURKA Limits PI3K-Pathway Inhibitor Efficacy in Breast Cancer.

Mutations and aberrant signaling in the PI3K-AKT-mTOR pathway is a “prominent feature” of breast and many other types of cancer, but drugs targeting the pathway have demonstrated only “modest responses” across clinical trials, the authors write. “A prominent feature of this pathway is drug-induced signaling adaptation and feedback mechanisms resulting in suboptimal drug responses.”

However, what scientists don't yet know is how these changes are orchestrated. “The failure of PI3 kinase drugs has been a huge mystery,” Dr. Bandyopadhyay states. To try and understand the underlying mechanisms that might impact on PI3K inhibitor drug resistance, the researchers employed a new chemoproteomics approach, MIBs/MS, which uses multiplexed inhibitor beads (MIBs) to capture and purify active kinases, with identification using quantitative mass spectrometry (MS), to generate a dynamic kinome signaling map and essentially identify kinase-related signaling changes associated with PI3K drug efficacy. The team focused on 151 kinases that are known to be expressed in cancer cells and generated data on how drugs that target the PI3K pathway affect kinase activity in breast cancer cell lines. “Comparison of kinome activity profiles between drug-sensitive and -resistant cells allowed us to generate a kinome-response signature associated with drug sensitivity,” they explain.

The results indicated that resistance to drugs that target PI3K components, including AKT and mTOR, was associated with a failure to inhibit AURKA, a protein that is unrelated to the PI3K pathway and which is involved in regulating centrosome alignment, mitotic spindle formation, and chromosome segregation during mitosis, and is known to regulate apoptosis. Initial in vitro tests showed that inhibiting the PI3K pathway led to reductions in AURKA protein levels in drug-sensitive laboratory-grown breast cancer cells, but not in drug-resistant cell lines. “The presence of Aurora kinase A causes resistance to every drug targeting the PI3K-pathway that we tested,” Bandyopadhyay states.

The team next showed that combining PI3K pathway inhibition with the experimental AURKA inhibitor MLN8237 resulted in an increase in cancer cell apoptosis, across 12 cell lines and independent of dose. The results were encouraging given that trials using aurora kinase inhibitors on their own against cancers other than breast have previously shown limited success. “Tested as monotherapy, Aurora kinase inhibitors have reached Phase III clinical trials for lymphoma with manageable toxicities but limited efficacy,” the authors state.  

Interestingly, prior in vitro tests using single drug treatments to target the PI3K pathway had been able to halt cancer growth, but had not eliminated cancer cells outright. MLN8273 (alisertib) is a Phase II–stage drug designed to specifically target and block AURKA activity. In contrast with the limited efficacy of PI3K inhibitor monotherapy, addition of the Aurura A kinase inhibitor prompted breast cancer cells to self-destruct. “AURKA mediates cellular survival in the context of PI3K-pathway inhibition, and because the drug combinations are synergistic in inducing apoptosis in breast cancer cells, we propose that it may be a promising companion target to enhance the efficacy of PI3K-pathway inhibitors,” the researchers write.

They next evaluated combining a low, nontoxic dose of MLN8237 with everolimus (RAD001)—the only mTOR inhibitor approved for treating breast cancer—in mice carrying tumors derived from human breast cancer cell lines. They found that while monotherapy with either drug partially impaired tumor growth, the combination therapy resulted in far greater levels of tumor growth inhibition than either drug alone. “Furthermore, all animals receiving the combination therapy (9/9) showed marked tumor regression, whereas no regressions were observed with monotherapy.”

Further investigations suggested that combination therapy effectively suppressed mTORC1 signaling via AKT. “Aurora kinases contribute to resistance to PI3K-pathway inhibitors through the maintenance of AKT and residual mTORC1 activity,” they state. “Hence, targeting this survival mechanism results in a more durable and complete repression of the PI3K pathway.”

The observation that combination therapy led to cell death spurred a set of studies designed to determine how Aurora kinase mediates cell survival in response to PI3K pathway suppression. As the researchers note, Aurora kinases and mTOR both regulate components of cells' native apoptosis pathway, and they hypothesized that dual therapy deregulates the balance of pro- and antiapoptotic cellular proteins, which might trigger cell death. They found that treating breast cancer-bearing mice using combination therapy caused changes in the levels of a protein known as BAX, which promotes apoptosis, and BCL2, which prevents apoptosis by inhibiting BAX activity, “leading to an increase in the ratio of BAX/BCL2.” A similar effect was demonstrated when MLN8237 and other PI3K inhibitors were combined against multiple breast cancer cell lines in vitro. “Taken together, we propose a model whereby Aurora kinase inhibitors potentiate the activity of PI3K-pathway inhibitors by enabling a durable and complete suppression of AKT/mTOR signaling and drive cell death by altering the balance of pro- and anti-apoptotic factors,” they comment.

“These data clearly warrant clinical testing and we are working to put together a Phase I clinical trial to test such drug combinations in patients,” Dr. Bandyopadhyay notes. “We believe the reason these drugs have not been more successful in the clinic is because of proteins that, when they are present in tumor cells, cause innate resistance to these drugs. We now have methods and technology we can use to identify these proteins.” 

The authors maintain that their results demonstrate that the expression of AURKA limits the efficacy of PI3K pathway–targeted therapy, which represents “a new vulnerability that can be used to enhance therapeutic responses to this class of drugs.…As single-agent responses to both PI3K- pathway and Aurora kinase inhibitors have been modest, these findings may unlock the full potential of these agents in realizing a clinical benefit.”
 








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