Constitutive Activation of Ras and Mitogen-Activated Protein Kinase (MAPK/ERK) in LGL Leukemia Unrelated to Activating Mutations in N and KRas Genes.
Authors: Painter Jeffrey S. , Costellanos Angela , Bai Fanqi , Wei Sheng , Loughran Thomas P. , Epling-Burnette P.K. 
Published: October 2019

Abstract
Background: Patients with Large Granular Lymphocyte (LGL) leukemia have defects in the CD95/CD95L (Fas-Ligand)-apoptotic pathway and a clinical syndrome with similarities to autoimmune lymphoproliferative diseases (ALPS). Patients with ALPS have autoimmunity and lymphocyte accumulation of CD3+CD4−CD8− aβ T-cells; whereas, the expanded population of lymphocytes in LGL leukemia are characterized by a CD3+CD4−CD8+ T-cell or a CD3−CD56+CD16+ NK-cell phenotype. Patients with ALPS usually possess mutations in genes involved in the CD95/CD95L apoptosis pathway but it was recently shown that a germline activating mutation in NRas exon 1 (Gly13Asp) resulted in clinical pathologic features of ALPS. In our previous studies, we found leukemic NK-LGLs to have evidence of constitutively activated extracellular-regulated kinase (ERK) and Ras that contributed to survival. Ablation of this signaling pathway with a dominant-negative form of Ras (N17Ras) and pharmacologic inhibition with the farnesyltransferase inhibitor FTI2153 resulted in ERK inhibition and enhanced apoptosis. Therefore, we hypothesize that expansion of leukemic LGL may be secondary to an acquired or germline mutation in the Ras gene. In this study, we performed allele-specific amplification of the NRas and KRas genes to determine the incidence of activating mutations in codons 12, 13, and 61.
Methods: PBMCs were collected from 5 healthy controls and twelve patients with T-LGL and one patient with NK-LGL leukemia (n=13). Apoptosis assessed using annexin/7-AAD flow cytometry was performed in response to inhibition of Ras and MEK using FTI2153 and PD98059, respectively. Western blot analysis was performed for phosphorylated-activate MAPK (ERK1/2), total MAPK, Bcl-2, XIAP, and CD95-FasR in five patients. Allele-specific PCR was performed using primers that recognized mutated Ras bases at their 3′ end and multiplex reactions were performed that simultaneously detected the wild type sequence, as previously described (Bezieau et al., Hum Mutat18:221, 2001). A total of 40 unique mutations were analyzed and primer sensitivity and specificity was determined in cell lines with confirmed mutations in each of these exons.
Results: Apoptosis was induced in a time and dose-dependent fashion using both MEK and Ras inhibitors. Constitutively activated MAPK/ERK and higher amounts of CD95-FasR was detected in LGL patients compared to healthy controls; whereas, Bcl-2 showed variable expression in LGL leukemia. XIAP was expressed in patients but was not different than controls. After treatment with the MEK inhibitor (PD98059), activated MAPK was reduced along with Bcl-2 when expressed. We detected the appropriate mutations as expected in all tumor cell lines but there were no mutations detected in LGL leukemia cases including NRas exon1 (Gly13Asp), which was mutated in ALPS. Wild-type primers were capable of detecting wild-type Ras in each reaction validating the amplification procedure.
Conclusions: These results show that LGL leukemia has dysregulated apoptosis in association with an activated Ras/MAPK signaling pathway unrelated to mutations in the NRas or KRas genes examined. Ras-inactivating drugs are currently being investigated in LGL leukemia.