Aggressive NK-cell leukemia (ANKL) is a rare form of NK cell neoplasm that is more prevalent among people from Asia and Central and South America. Patients usually die within days to months, even after receiving prompt therapeutic management. Here we performed the first comprehensive study of ANKL by integrating whole genome, transcriptome and targeted sequencing, cytokine array as well as functional assays. Mutations in the JAK-STAT pathway were identified in 48% (14/29) of ANKL patients, while the extracellular STAT3 stimulator IL10 was elevated by an average of 56-fold (P < 0.0001) in the plasma of all patients examined. Additional frequently mutated genes included TP53 (34%), TET2 (28%), CREBBP (21%) and MLL2 (21%). Patient NK leukemia cells showed prominent activation of STAT3 phosphorylation, MYC expression and transcriptional activities in multiple metabolic pathways. Functionally, STAT3 activation and MYC expression were critical for the proliferation and survival of ANKL cells. STAT signaling regulated the MYC transcription program, and both STAT signaling and MYC transcription were required to maintain the activation of nucleotide synthesis and glycolysis. Collectively, the JAK-STAT pathway represents a major target for genomic alterations and IL10 stimulation in ANKL. This newly discovered JAK/STAT-MYC-biosynthesis axis may provide opportunities for the development of novel therapeutic strategies in treating this subtype of leukemia.
We thank all the faculties and staffs in the Clinical and Laboratory Unit of the Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology for their clinical and technical support; the Core Genomic Facility of Beijing Institute of Genomics, Chinese Academy of Sciences for the construction and sequencing of libraries; Drs Qing Li and Jiguang Wang for critical reading and valuable comments on the manuscript; Dr Kai Fu for providing the cell line YT. This study was supported by the National Natural Science Foundation of China (81570196 to JZ, 81425003 to QW, 81670152 to Liang H, 81600120 to NW, 81300410 to DW, 81500100 to YL and 81400122 to KZ), the National Key Basic Research Program of China (2014CB542001 to QW), the Key Program of the National Natural Science Foundation of China (81230052 to JZ), the Key Research Program of the Chinese Academy of Sciences (Precious Medicine Research in Chinese Population; KJZD-EW-L14-3 to QW), and the National High Technology Research and Development Program of China (863 program; 2012AA02A507 to JZ and 2014AA020532 to Liang H).
Strategies for the integrative analysis of ANKLs.
Isolation of granulocytes, leukemia NK cells and normal NK cells.
Frequency of somatic SNV of ANKL and 40 additional cancer types.
EBV reads number of leukemia NK cells and granulocytes from 8 ANKL patients.
KEGG pathway enrichment analysis of protein-altering somatic SNVs.
Functional enrichment analysis of genomic alterations with HotNet2.
STAT3 and STAT5B mutations identified in ANKL patients.
TET2 mutations identified in ANKL patients.
Semi-quantitative immunoreactivity histological scores of MYC staining in bone marrow biopsy of ANKL and control samples.
Functional enrichment map for MYC-signature genes that were upregulated in ANKLs compared to healthy donors.
The effect of IL10 or a STAT3 inhibitor (Stattic) on the apoptosis of ANKL cell lines.
The effect of IL10 and the STAT3 inhibitor (Stattic) on the mRNA expression of MYC in ANKL cell lines.
The rate of EdU incorporation in JQ1-treated ANKL cell lines.
Gene-set enrichment analysis (GSEA) of known MYC and STAT3 signatures.
Enrichment of metabolic pathways in primary ANKL leukemia cells and ANKL cell lines.
The effect of STAT3 Y640F mutant on the phosphorylation of STAT3 and mRNA expression of MYC target gene.
The effect of IL10 on the mRNA expression of MYC and MYC target genes in KHYG-1 cell line transfected with STAT3 Y640F mutant.
The effect of IL10 on STAT3 phosphorylation, MYC expressionand the proliferation of STAT3 Y640F-mutant ANKL cell line YT.
Quantitative analysis of the Epstein-Barr virus (EBV) load in ANKL patients and healthy donors with whole-transcriptome sequencing (WTS) data.
Expression of EBV-encoded small RNAs in primary ANKL leukemia cells.
A large gain across MYC and an inter-chromosomal translocation detected by GVC-CNV and GVC-SV in ANKL No.19.
Patient characteristics of ANKL.
SNVs identified in ANKL NK leukemia cells.
CNVs identified in ANKL NK leukemia cells.
SVs identified in ANKL NK leukemia cells.
AmpliSeq targeted sequencing results of ANKLs.
H-score of phosphorylated STAT3 and corresponding STAT mutation status of ANKL cases.
Differentially upregulated genes in ANKL leukemia cells in comparison with normal controls.
Differentially downregulated genes in ANKL leukemia cells in comparison with normal controls.
KEGG pathway enrichment analysis of upregulated genes in ANKL leukemia cells.
KEGG pathway enrichment analysis of downregulated genes in ANKL leukemia cells.
IPA metabolic pathways analysis of ANKL and tumors that have known metabolic features.
Sequencing depth information of 8 WGS ANKL patients.
Primers for quantitative RT-PCR
Materials and methods
(Supplementary information is linked to the online version of the paper on the Cell Research website.)