Frequent Activating Mutations Of JAK-STAT Pathway Genes In Natural Killer Cell Lymphomas
Authors: Kucuk Can , Jiang Bei , Hu Xiaozhou , Gaulard Philippe , Zhang Wenyan , Xiao Wenming , Staudt Louis M. , Iqbal Javeed , Zhang Weiwei , Cornish Adam , Kavak Pinar , Alkan Can , Gong Qiang , Yang Qunpei , Sun Hong , d'Amore Francesco , Leppa Sirpa , Liu Weiping , McKeithan Timothy , Chan Wing C. 
Published: October 2019

Abstract

Background
Natural killer cell lymphomas (NKCLs) are rare diseases with poor prognosis. There are few studies that reported oncogenic mutations in this disease. Identifying mutations critical to the neoplastic transformation of NK cells is crucial for the development of targeted therapies.

Methods
We performed RNA sequencing (RNA-Seq) on NKCL cases (n=17), malignant NK cell lines (n=3) and resting or activated normal NK cells (n=3) to analyze the genome-wide mutation profile in NKCLs. All SNVs detected by RNA-Seq were validated by Sanger sequencing using the corresponding genomic DNA (gDNA). We expanded our analysis to include specific sequencing of the SH2 domains of STAT3 and STAT5B using 37 additional NKCL cases and 6 NK cell lines. Retroviral or lentiviral transduction was performed to express SOCS6 or STAT3 shRNA in NK cell lines, respectively. After transduction, cells were tracked by quantifying the % of GFP+ cells. Apoptosis was assessed by quantifying AnnexinV-PE stained cells. Western blot was performed on 6 NK cell lines using p-STAT3 (Tyr705) antibody. q-MSP and q-RT-PCR were used to detect SOCS6 promoter methylation and mRNA expression in NK cell lines, respectively.

Results
RNA-Seq showed frequent oncogenic mutations in JAK/STAT pathway members STAT3 (18%), STAT5B (6%), BRAF (6%) and MAP2K1 (6%). Targeted Sanger sequencing of 37 additional NKCL cases showed one patient with activating STAT3 and two patients with activating STAT5B mutations leading to 7.4% (4 of 54) and 6% (3 of 50) total mutation frequency, respectively. STAT3 and STAT5B mutations were located in the SH2 domain and three of four STAT3 mutations and all STAT5B mutations were previously observed in NK- or T-LGL leukemia cases. Oncogenic activities of two other JAK-STAT pathway genes, BRAF (G469A), MAP2K1 (K57N) have been reported in solid tumors and leukemias. The JAK/STAT pathway mutations were present in a total of 53% of the NKCL cases with RNA-Seq data available (n=17). Intriguingly, targeted sequencing revealed oncogenic STAT3 mutations in 50% of malignant NK cell lines (n=6), which were associated with p-STAT3 expression detected by western blot.STAT3 knock-down resulted in reduced growth in a NK cell line with STAT3 mutation. In agreement with Kimura et. al. Leuk Lymphoma 2013, we were not able to detect JAK3 mutations as reported previously. In an accompanying DNA methylation analysis, we observed epigenetic silencing of SOCS6, a negative regulator of JAK-STAT3 signaling, in NKCL samples. Reintroduction of SOCS6 showed negative selection pressure associated with increased apoptosis in limiting concentrations of IL2 in two SOCS6-null NK cell lines with activating STAT3 mutations suggesting a possible cooperation of oncogenic JAK-STAT pathway mutations and the epigenetic silencing of a negative regulator of this pathway.

Conclusions
We have identified a high incidence of activating mutations of STAT3, STAT5B and other oncogenic JAK-STAT pathway genes in NKCLs. SOCS6 was frequently methylated in NKCLs with corresponding low gene expression. There was evidence suggestive of cooperation of genetic and epigenetic mechanisms in the activation of the JAK-STAT pathway in NKCLs. This study suggests that JAK-STAT pathway inhibition may be a therapeutic option in NKCLs.

Disclosures:
No relevant conflicts of interest to declare.