During my internship last summer I came across this 2012 article from the New England Journal of Medicine titled “Progression of RAS-Mutant Leukemia during RAF Inhibitor Treatment.” I found it particularly interesting because it took what I was working on in vitro with mouse cells and put it in a clinical setting with actual patients. I thought this article would be perfect for the blog for this class because its main focus is on the RAS and RAF oncoproteins that we have been talking about.
Background: We have already covered the RAS signaling pathway in class so I wont go into too much detail but would like to add that there are 3 isoforms (different forms of the same protein) of RAS (KRAS, HRAS, and NRAS) and of RAF (ARAF, BRAF, and CRAF). BRAF V600E (V600E just means that there is a change from a Valine to a Glutamic Acid at the 600 position) is particularly important because it is mutated in about 50% of patients with Metastatic Melanoma, a cancer in melanocytes which are the cells that produce the melanin pigment that is responsible for the color of our skin. The treatment of this disease by Vemurafenib was not only a break through in the treatment of Metestatic Melenoma but in all cancers. The reason for this is because Vemurafenib is a drug that is targeted to inhibit the mutant form of BRAF. This is a big step forward in chemotherapy because the drug does not inhibit wild-type BRAF and therefore healthy cells are not inhibited from performing their normal functions and the patients will not suffer from as many side effects. Here is a way for you to visualize the RAS pathway and how Vemurafenib works: (RAS is directly upstream of RAF but is not shown and the thicker arrows indicate heightened signaling) PLX 4032 is the name for vemurafenib used in research.
The Problem: This paper presents a 76 year old male patient with stage IV BRAF mutant melanoma. He began treatment with 960 mg of Vemurafenib twice a day and after 11 days the patient reported an improvement in his breathing but a new feeling of fatigue. It was noted that his white cell, monocyte, and neutophil counts had increased significantly since the start of treatment, which is an indication of chronic myelomoncytic leukemia (cancer of the white blood cells). This diagnosis was verified by clinical and hematopathological (pathology of the cells in the bone marrow) findings. At the same time it was found that one of his subcutaneous (just below the skin surface) tumors had resolved. The patient was instructed to stop taking the vemurafenib and two weeks later his white cell and monocyte counts had decreased. Following this the doctor put the patient back on a smaller (720 mg) dose of vemurafenib but with in days the patient’s white cell count had began to rise. The graph below shows the progression of the patients treatment over 300 days during which time they put the patient on and off the vemurafenib and tracked the white cell and absolute monocyte counts.
The patient was initially treated will Ipilimumab (another melenoma chemotherapy) but was not responsive and it also did not have an affect on his white cell and monocyte counts. From there on it is clear to see that as the Vemurafenib was added (shaded areas) and taken away the white cell and moncoyte counts went up and down in the same pattern. This led the doctors to believe that it was something about the BRAF mutant inhibitor that was causing the development of the chronic myelomonocytic leukemia. The patient was also diagnosed with splenomegaly (enlargement of the spleen), which is consistent with progressive chromic myelomonocytic leukemia. This means that the BRAF mutant inhibitor is reacting in some way within the cell to turn on growth pathways.
Problems with this Data: The most obvious issue with determining how valid these results are is that our sample size is only one patient. The paper does mention at the beginning that this drug has been seen to induce cutaneous neoplasias (skin growths) such as ketatocanthomas (skin tumors unlikely to become metastatic) and squamous-cell carcinomas (cancer of the squamous cells in the main part of the epidermis). So induced abnormal growth has been documented after treatment for melanoma with this drug but the paper gives not statistics to support the findings such as in what percentage of patients this occurs and whether or not this is a statistically significant finding. Since this is the first known case of development of leukemia during treatment with vemurafenib it is impossible to that it is definitely the BRAF inhibitor that is causing these results. The data shown does look to be convincing of the fact that it is the vemurafenib causing the rise in the white cell and monocyte counts however it lacks any statistical analysis to back up what we see.
What is going on: The next step was for the doctors to analyze the patient’s leukemia cells for mutations in NRAS and KRAS which are known to be associated with chronic myelomoncytic leukemia. This analysis revealed an NRAS G12R (change from a Glycine to an Arginine at position 12) mutation in the genomic DNA of the myeloid lineage (blood precursor cells in the bone marrow). 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 and the biochemical mechanisms behind it is being studied for further understanding.
The Next Step: In my next blog I will discuss the next part of the paper which goes into the details of how they tested their paradoxical activation hypothesis in vitro.
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.