Cancer cells that develop resistance to one or more drugs will simultaneously develop a new weakness that could feasibly make them susceptible to other anticancer treatments. The trick is knowing what that new vulnerability is and how to exploit it. Scientists headed by a team at the Oncode Institute and Netherlands Cancer Institute have now identified a weakness that emerges in BRAF-mutant melanoma cells as they develop resistance to BRAF inhibitor therapy. The drug-resistant cells produce more reactive oxygen species (ROS), and this makes them susceptible to another approved anticancer drug, vorinostat, which is normally used to treat a rare form of lymphoma.

The team, headed by René Bernards, Ph.D., devised a sequential treatment strategy that they tested in vitro and in melanoma-bearing mice, and subsequently started a clinical trial to evaluate the use of vorinostat in suitable melanoma patients who have developed resistance to BRAF inhibitor therapy.  “It is unique that a clinical trial has already been part of a fundamental scientific publication,” comments Dr. Bernards. “This is how we, increasingly, want to do it.”

The researchers report their work in Cell, in a paper entitled “An Acquired Vulnerability of Drug-Resistant Melanoma with Therapeutic Potential.”

About half of melanoma skin cancers carry mutations in the BRAF oncogene that lead to activation of the mitogen-activated protein kinase (MAPK) pathway, which signals to the cells to keep dividing. Although drugs that block mutated BRAF oncoprotein do help to hold back tumor growth and progression, most patients will ultimately relapse with resistant disease, the authors explain. Treatment with inhibitors of both BRAF and downstream MEK kinases can help to improve response, but resistance is still “mostly inevitable.”

Resistance to MAPK pathway inhibitors in melanoma is commonly caused by reactivation of signaling through the MAPK pathway, even when inhibitor therapy is being administered. Multiple mechanisms that underpin this reactivation have been reported, the team adds. Interestingly, withdrawing drug therapy in patients that have developed resistance doesn’t immediately lead to rapid disease progression, the researchers note. Rather, there is a pause in tumor growth, which is known as the “drug holiday effect.” The phenomenon can partly be explained by drug withdrawal-related hyperactivation of MAPK pathway signaling, which leads to a cellular state that the researchers describe as having hallmarks of oncogene-induced senescence. However, this lull in tumor growth doesn’t last. Signaling hyperactivation is subsequently dampened, which reinitiates tumor growth, and also results in the tumor regaining sensitivity to BRAF inhibitor therapy.  

The Netherlands Cancer Institute researchers reasoned that the drug withdrawal–related temporary halt in growth of BRAF inhibitor–resistant melanomas may be linked with the drug-resistant cells acquiring a new vulnerability that wasn’t present in the drug-sensitive parent cells. “That drug resistance of cancer cells comes at a fitness cost that in turn can cause sensitivity to other drugs was identified over 50 years ago and is referred to as ‘collateral sensitivity,’” the authors state. “'Drug resistance seems inevitable because tumors are constantly adapting,” Dr.  Bernards adds. “For over 40 years, we have been devising ways to prevent drug resistance in cancer. Now I think: Let's just accept that this is the way it is, and go and see if we can find the new vulnerability associated with resistance. Then we can exploit this sensitivity clinically and keep the cancer under control for a longer time.”

The researchers wanted to look for any weaknesses in drug-resistant BRAF-mutant melanomas, so they first cultured human BRAF-mutant melanoma cells in the presence of BRAF inhibitor, until the cells developed resistance. Interestingly, the different cell lines exhibited four resistance mutations that were commonly found in human patients that develop resistance to BRAF and/or MEK kinases.

When the team then analyzed the cells, they found that all four of the resistant cell lines exhibited at least twofold higher than normal levels of ROS. This hinted at the possibility of using another drug to increase the levels of ROS even further in these cells, which could have catastrophic effects on cell survival.

“Then we thought: Suppose we can give those hyperactive resistant tumor cells the last push toward cell death, by allowing them to produce even more free radicals,” Dr. Bernards notes. “We hypothesized that this increase in ROS levels may represent an acquired vulnerability in the sense that a further increase in ROS levels could become detrimental to the drug-resistant cells,” the authors write.

The experiments demonstrated that the drug-resistant cell lines were, as projected, susceptible to the ROS inducer paraquat. The team then searched for approved drugs that might have the equivalent effect of pushing up ROS levels in the drug-resistant cells. They selected vorinostat, a histone deacetylase inhibitor (HDACi) that was approved in 2006 for treating cutaneous T-cell lymphoma. The drug has a safe pharmacological profile in the clinic, and HDAC inhibitors are known to induce ROS, the researchers explain.

Initial in vitro tests confirmed that while the drug induced elevated ROS levels in both nonresistant parent BRAF-mutant melanoma cells and the drug-resistant cells, tumor cell growth was inhibited to a much larger extent in the resistant cells, “most likely explained by the much higher ROS levels induced in MAPKi-resistant melanoma cells as compared to the ROS levels induced by vorinostat in parental cells,” the authors suggest. Further analyses indicated that HDAC inhibitors such as vorinostat induce ROS by suppressing a gene called SLC7A11.

Interestingly, increasing ROS levels also boosted MAPK signaling in melanoma cell lines, indicating that if given at the wrong time, vorinostat treatment could actually counteract the effects of MAPK inhibitors in cells that are still sensitive to MAPK inhibition. The antagonistic effects of HDAC inhibitors and MAPK inhibitors on melanoma tumor cells suggest that in a therapeutic setting it would be important to administer MAPK and HDAC inhibitors sequentially, rather than at the same time, the scientists point out.  This was a surprising finding, they acknowledge. “The finding that BRAF and HDAC inhibitors must be used sequentially was unexpected as a recent publication demonstrated that combination of BRAF and HDAC inhibitors upfront can prevent emergence of resistant melanoma cells in a short-term assay.”

“It is not a combination drug,” emphasizes Bernards, who is no stranger to the concept of devising drug combinations. “It is very important that you first stop the signaling pathway inhibitors because they suppress the free radicals and thus eliminate the effects of vorinostat.”

With this in mind, the team first tested the sequential treatment strategy in a mouse model. BRAF-mutant melanoma-bearing mice were treated using an MAPK inhibitor until their tumors developed resistance. When these drug-resistant animals were then treated with vorinostat, their tumors shrank.

The researchers then started a human study as an early evaluation of the effects of sequential MAPK inhibitor–HDAC inhibitor therapy. The trial enrolled eligible patients with advanced BRAF V600E-mutated melanoma patients who had progressed on dabrafenib plus trametinib therapy. After a period of MAPK inhibitor washout, the participants were started on therapy using vorinostat, which the researchers synthesized in their own pharmacy. While detailed findings from the study haven’t yet been published, and the trial is continuing, data from three patients indicated that the treatment repressed SLC7A11 expression in the tumors and eradicated the cells that had become resistant to therapy with the BRAF and MEK inhibitors.

The team is now planning a clinical follow-up study that will be carried out under the umbrella of the Oncode Institute. “In our clinical proof-of-concept study, we gave the patients BRAF inhibitors for one year, until the cancer had become resistant,” Dr. Bernards says. “We then exterminated the resistant cells in one month with vorinostat. Now that we know that this principle works, we want to go a step further. We are going to check the patients' blood every month for mutations in the tumor DNA. As soon as we see a trace of resistance, we will briefly treat with vorinostat to nip the resistance in the bud. Then, we again transfer to the BRAF inhibitors, until we see resistance emerge again. With such a pulse-treatment, we think we can keep the cancer under control longer.”

Bernards will also soon initiate another major study that will look at how to exploit senescence in other cancers, for which he has been awarded a European Research Council grant of €2.5 million ($3 million).

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