Following results of a phosphoproteomic screen, University of Montreal researchers have tracked down a protein kinase, ribosomal S6 kinase, or RSK, that plays a central role in melanoma's resistance to chemotherapy through the Ras/MAPK pathway. According to the group, the results suggest RSK could be a promising therapeutic target in the disease.
The researchers described the finding in a study published in Oncogene late last month. According to the group, RSK phosphorylates a kinase called checkpoint kinase 1, or Chk1, at an important inhibitory site. This affects Chk1 activity in response to DNA-damaging agents, a mechanism the group hypothesized could be at the root of melanoma's resistance to chemotherapies.
In addition, the team showed that RSK inhibitors were able to greatly increase cells' sensitivity in vitro to DNA-damaging chemotherapy treatment.
The study's senior author, Philippe Roux, told ProteoMonitor that the idea that the Ras/MAPK pathway might be involved in chemoresistance isn't new.
"There are previous reports that have shown that the whole pathway – when inhibited more upstream – was involved in chemoresistance," he said. "We basically identified the mechanism by which this occurs. By knowing that, we can target it more downstream and more specifically."
In the study, Roux and his colleagues showed that two kinases in the RSK family, RSK1 and RSK2, are the predominant kinases that phosphorylate Chk1at the inhibitory site ser280 in response to mitogens and growth factors.
The group wrote that RSK inhibits Chk1 activation in response to DNA damage, and promotes G2 DNA damage checkpoint recovery.
Roux said his team became interested in the potential chemoresistant effect of RSK after his lab's phosphoproteomic screening experiments identified Chk1 as one of RSKs targets. Chk1 is involved with cell response to DNA damage, acting to arrest cell activity to allow DNA repair, he said.
"We thought it was interesting to follow up because it is a protein that is conserved through evolution, will respond to DNA damage in cells, and help the cells react to such an insult," Roux said. "That's also when we became more aware of the fact that melanomas are known for being difficult to treat, and the main reason for that is that tumors are resistant to the most classic chemotherapeutics, which induce DNA damage."
Based on this link, the group went on to examine the phosphorylation of Chk1 by RKS, to see whether it caused activation or inactivation of the kinase.
"We found it was a mechanism to inhibit Chk1, and so reduce the [cell cycle arrest] response to DNA damage," Roux said.
Then the group tested cells to see if inhibiting RSK would increase Chk1 activity and affect cells' response to DNA damage, which it did.
"If we inhibited RSK with pharmacological inhibitors, we found the cells were arresting longer in response to the same insult," Roux said.
"Finally we thought, 'Lets see what happens in terms of the response of different melanoma cells to DNA-damaging agents by looking at cell survival and proliferation,' to see if they will respond to lower [chemotherapy] doses if we inhibit the pathway," he said.
Roux said the group tested melanoma cell lines, first identifying a chemotherapy dose — using common melanoma treatments — that would not induce a response on its own, and then adding RSK inhibitors at different concentrations.
"We found that it was a pretty good way to increase the sensitivity of those cells," Roux said.
The group didn't do a detailed dose-response analysis, he said, but the researchers did show that adding a high dose of an RSK inhibitor called SL0101 to normally ineffective chemotherapy doses yielded a combination treatment that could completely inhibit cell proliferation.
Roux said that the handful of RSK inhibitors that have been identified thus far are not precise enough in their activity to be used in mouse studies, which is the group's next goal. "Although they efficiently inhibit RSK, some of them have secondary targets so they are not ideal at the moment," he said.
According to Roux, the team is planning to use a high-throughput screening platform to try to identify novel RSK inhibitors that would be more amenable to use in mice.
In the meantime, Roux and his colleagues have conceived an alternative plan to test the potential of RSK inhibition in mouse models of melanoma by creating a melanoma cell line that carries an RNAi construct, which the group can turn on to eliminate RSK.
"This will allow us to inject the melanoma cells in the animal, wait for tumors to grow, [and] then, at a specific moment, induce our construct to completely get rid of RSK," Roux said. "Then we'll be able to see the impact of that and the impact if we combine it with chemotherapeutics."