Review:
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.
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.
