Role of BRAF Mutations in Hematopoietic Malignancies and the Induction of Cytokine-Independence-Synergy with PI3K/Akt Pathway.
Authors: Libra Massimo , Mazzarino Maria C. , De Re Valli , Steelman Linda S. , Abrams Steven L. , Bertrand Fred E. , Shelton John G. , Molton Sarah , McMahon Martin , McCubrey James A. 
Published: April 2020

Abstract
Mutations in the Ras pathway have been detected in approximately 50% of hematopoietic malignancies. These mutations usually occur at KRAS, NRAS or upstream FLT3. Recent results from an Asian based leukemia study indicated that the BRAF gene is mutated in 4% of AMLs and 3% of NHL. We sought to determine whether the BRAF, KRAS, PI3K genes were mutated in various hematopoietic malignancies of European ancestry as differences in mutation frequencies for other genes has been reported among various ethnic groups. As controls for BRAF mutations, the structure of the BRAF gene was examined in melanoma biopsies from European patients with various stages of melanoma. 61% of the melanoma patients had the BRAF V600E mutation. In contrast, essentially no mutations were detected in the European based NHL and AML patients. The presence of CMV infection in NHL did not change the genetic status of BRAF. To determine the effects of the BRAF V600E mutation on the cytokine-dependency of hematopoietic cells, cDNAs encoding BRAF wild type (WT) and BRAF V600E mutation were ligated to the hormone binding domain of a mutated estrogen receptor (ER*) which responds to 4 hydroxyl tamoxifen (4HT). These modified cDNAs were inserted into retroviral vectors. BRAF V600E induced the morphological transformation of NIH-3T3 cells in a 4HT-dependent fashion. Prolonged expression of BRAF V600E induced anoikis, results consistent with our previous studies indicating that BRAF overexpression can induce cell cycle arrest and apoptosis. Three cytokine dependent hematopoietic cell lines (human TF-1, murine FL5.12 and FDC-P1) were infected with WT and V600E BRAF encoding retroviruses. Expression of WT or V600E BRAF did not efficiently abrogate cytokine dependence. To determine if expression of the PI3K/Akt pathway could synergize with BRAF to induce cytokine independence, WT and V600E BRAF transduced cells were infected with WT and activated PI3K (p110 catalytic subunit), active and inactive Akt and an empty retroviral vector as a control. Conditional (Akt:ER*Myr+) and constitutive (Akt−Myr+) and PI3K but not inactive (Akt:ER*Myr−) synergized at least 250-fold with activated V600E but not WT BRAF to abrogate cytokine-dependence. Conditional activation of BRAF V600E induced downstream MEK and ERK, which was associated with cell cycle progression and prevention of both caspase 3 activation and apoptosis. Prevention of apoptosis in the BRAF V600E and Akt transformed hematopoietic cells was highly sensitive to 3 different MEK inhibitors and apoptosis was synergistically enhanced when the cells were treated with MEK and the mTOR inhibitor rapamycin. Our results indicate that the BRAF gene is infrequently mutated in hematopoietic malignancies and that the BRAF V600E mutant does not efficiently abrogate the cytokine dependence of 3 hematopoietic cell lines. However activated V600E but not WT BRAF synergized with the activated PI3K/Akt pathway to induce cytokine independence which was MEK1 dependent. Our data suggest that the BRAF mutation may not be detected frequently in hematopoietic malignancies because it is relatively inefficient by itself in conferring a growth advantage to hematopoietic cells (e.g., cytokine-independence) which is often associated with the induction of leukemia.