This blog post is a continuation of my last post so I will first review what was found in the first post. A 76-year-old man was diagnosed with stage IV BRAF V600K mutant melanoma. He was put on a BRAF mutant inhibitor (vemurafenib or PLX4032) and responded well to the chemotherapy until it was discovered that the drug was causing the patient to develop chronic melomoncytic leukemia. Analysis of the patient’s leukemia cells showed an upstream mutation in the NRAS oncoprotein. The doctors then hypothesized that vemurafenib was causing the hyper activation of ERK (Map Kinase) and stimulating the growth of preexisting NRAS-mutated chronic myelomonocytic leukemia cells. This is now widely known as the paradoxical activation of ERK.
Clarifying the two types of cancer:
The paper includes this table to help us understand where the mutations are in which cells:
It is pointing out that the BRAF V600K mutation is only in the melanoma cells and the four other cells that are blood cells are wild type for this mutation. It also shows that the monocytes, megakaryocyts, and erythroid cells express a NRAS G12R mutation that the melanoma cells are wild type for. Finally, they have the lymphoctyes which are wild type for both mutations and are used as a control in the following experiments. It is important to know where the different mutations are to understand that these two types of cancer are not related and are driven by different oncogenes. The blood cells do no have the BRAF mutation and therefore should not be affected by the BRAF inhibitor.
Understanding the Activation of the RAS pathway:
To further explore this paradoxical activation the researchers collected peripheral-blood mononuclear cells (PBMCs, any blood cell having a round nucleus) from the patient when he was receiving treatment with vemurafenib (shown with a + sign in the graph) and when he was not receiving treatment with vemurafenib (shown with a – sign in the graph). They wanted to test for the activation of the RAS pathway so they chose to take a closer look at ERK, which is downstream of RAS and is phosphorylated when active. When the pathway is on and the cells are stimulated to proliferate ERK is phosphorylated (shown as pERK). The samples of monocytes (with the NRAS mutation) and Lymphocytes (wild type) taken during treatment with vemurafenib and also not during treatment were stained for pERK and also tERK (total ERK). Total ERK refers to all of the ERK protein in the cell regardless of it being phosphoylated or not. The purpose of staining for tERK as well as pERK is to see the relative concentrations of protein in the cells so that we are evaluating each sample from the same baseline concentration of protein. Fluorescently labeled antibodies that were either specific to pERK or tERK were placed in each sample and then analyzed using flow cytometry. The ratio of the median fluorescence intensity (MFI) of staining for pERK was compared with the MFI for total ERK.
The graph shows that monocytes being treated with vemurafenib had an elevated pERK:tERK ratio as compared with the sample taken when the patient was not being treated with vemurafenib. A change in the pERK:tERK ratio was not seen in the NRAS wildtype lymphoctye samples with or without the presence of vemurafenib. The reason the ratios are smaller in the control group compared is probably due to the fact that the blood sample was taken after the patient had already received some treatment and therefore the monocytes had not come back down to basal protein levels yet. The paper did not specify when the blood sample was taken relative to how long the patient was off of the drug. My concern with this data is that the p-value is higher than one would like to be able to trust the findings. The graph depicts the mean of the experimental triplicates so the data is more trustworthy knowing the experiment was repeated 3 times. The T-bars represent the standard error of the mean.
A Possible Solution:
The final experiment they did in this paper was to further understand the paradoxical activation of the RAS pathway by introducing a MEK inhibitor (PD325901). MEK is directly upstream of ERK and directly downstream of RAF and since the MEK inhibitor is known to be effective in vitro it was hypothesized that it would be a solution to blocking the paradoxical activation of the RAS pathway that was causing the leukemia. They cultured the leukemic PBMCs and treated them with either PLX4023(vemurafenib), PD325901 (MEK inhibitor), or both at different concentrations for 4 hours. As in the last example the levels of pERK:tERK were measured using flow cytometry. The + and – signs indicate the presence of the MEK inhibitor (100nM).
As expected the cells treated with vemurafenib had increased pERK:tERK levels showing RAS pathway activation. Also as expected the cells treated with only the MEK inhibitor had lower pERK:tERK levels. Interestingly, the cells that had the MEK inhibitor added as well as the vemurafenib showed lower ratios of pERK:tERK levels meaning that the paradoxical activation was being shut off to some extent by the interruption of the MEK inhibitor. This data is represented by good p-values for statistical significance expect in the case of the cells treated with only the vemurafenib which is what we saw in the first experiment mentioned. This graph is a representation of the mean of the experimental quadruplicates giving it more validity. In conclusion this experiment has shown that ERK activation that is induced by the RAF inhibitor in this RAS-mutant leukemia can be suppressed by the addition of a MEK inhibitor.
Clinical Application:To bring the in vitro results back into the clinic the researchers concluded the paper by speculating that the addition of a MEK inhibitor to the patient’s chemotherapy would suppress the paradoxical activation of the RAS pathway by vemurafenib that developed into leukemia. However, at the time there was no FDA approved MEK inhibitor to be able to test this hypothesis. This is most likely because MEK inhibitors have been shown to be very toxic.
The Next Step: I am working on trying to find a paper that uses other chemotherapies, possibly a now approved MEK inhibitor, to treat other patients with paradoxically activated cancers.
Callahan, Margaret K., Raajit Rampal, James J. Harding, Virginia M. Klimek, Young
Rock Chung, Taha Merghoub, Jedd D. Wolchok, David B. Solit, Neal Rosen, Omar Abdel-Wahab, Ross L. Levine, and Paul B. Chapman. "Progression of RAS-Mutant Leukemia during RAF Inhibitor Treatment." New England Journal of Medicine 367.24 (2012): 2316-321. Print.