JAK-STAT and G-protein-coupled receptor signaling pathways are frequently altered in epitheliotropic intestinal T-cell lymphoma

Abstract

Epitheliotropic intestinal T-cell lymphoma (EITL, also known as type II enteropathy-associated T-cell lymphoma) is an aggressive intestinal disease with poor prognosis and its molecular alterations have not been comprehensively characterized. We aimed to identify actionable easy-to-screen alterations that would allow better diagnostics and/or treatment of this deadly disease. By performing whole-exome sequencing of four EITL tumor-normal pairs, followed by amplicon deep sequencing of 42 tumor samples, frequent alterations of the JAK-STAT and G-protein-coupled receptor (GPCR) signaling pathways were discovered in a large portion of samples. Specifically, STAT5B was mutated in a remarkable 63% of cases, JAK3 in 35% and GNAI2 in 24%, with the majority occurring at known activating hotspots in key functional domains. Moreover, STAT5B locus carried copy-neutral loss of heterozygosity resulting in the duplication of the mutant copy, suggesting the importance of mutant STAT5B dosage for the development of EITL. Dysregulation of the JAK-STAT and GPCR pathways was also supported by gene expression profiling and further verified in patient tumor samples. In vitro overexpression of GNAI2 mutants led to the upregulation of pERK1/2, a member of MEK-ERK pathway. Notably, inhibitors of both JAK-STAT and MEK-ERK pathways effectively reduced viability of patient-derived primary EITL cells, indicating potential therapeutic strategies for this neoplasm with no effective treatment currently available.

Figure 1

Graphical overview of the study, and mutation and subclonality profile of EITL tumors. (a) Types of analyses performed and the number of samples used for each experiment. Snap frozen cases are listed by their respective IDs. (b) Venn diagram illustrating the recurrent mutations in the discovery cohort. (c) The subclonal architecture of EITL sample 013T derived from SciClone analysis. The upper plot demonstrates kernel density estimation and the lower one variant allele frequencies plotted against read depth (⩾20 × coverage) in copy-neutral loss of heterozygosity-free regions. CNA, copy number analysis; FFPE, formalin-fixed paraffin-embedded; GEP, gene expression profiling.

Figure 2

Frequently mutated genes in EITL identified by WES and amplicon deep sequencing. (a) Mutation frequencies of STAT5B, JAK3 and GNAI2 correlating to CD8 dimer and TCR type. Distribution of mutations in (b) STAT5B, (c) JAK3 and (d) GNAI2 genes. The frequency of each alteration is denoted within parentheses after its label. Filled circles indicate known mutation hotspots and empty circles mutations with unknown functional consequence. *A total of four nonsynonymous missense mutations identified resulting in the change of two amino acids. G, GTP-binding; ND, not done; SH2, Src Homology 2; TA, transcriptional activation domain; TCR, T-cell receptor.

Figure 3

Acquired uniparental disomy in STAT5B locus. (a) Allele-specific copy number analysis of tumor (ASCAT) close-ups of two STAT5B mutant samples highlighting chromosome 17q. The upper panels demonstrate the raw (red) and segmented (green) B-allele frequencies (BAFs) of SNPs. The lower panels present allele-specific copy number profiles—red lines indicate the higher and green the lower copy number chromosomal haplotypes. The green line is at zero, which indicates loss of heterozygosity. (b) Sanger sequencing electropherograms demonstrating the presence of STAT5B somatic mutation in the same samples. (c) Representative images of pSTAT5 immunohistochemical staining in STAT5B wild-type and mutant cases. Magnification × 400. VAF, variant allele frequency from WES.

Figure 4

Effective targeted therapy by JAK, STAT and MEK inhibitors in primary EITL cells. (a) Stable cell lines were created by overexpressing GNAI2 wild-type and mutants in HuT78 and Jurkat cells. The expression of relevant proteins was assessed by western blot. (b) Representative images of pERK1/2 immunohistochemical staining in GNAI2 wild-type and mutant tumors. Magnification × 400. (c) Representative ex vivo cell viability assay in primary EITL cells treated with PD0325901 and Stattic for 72 h, and Tofacitinib, Trametinib and Gefitinib for 96 h. All results are normalized to the control (dimethyl sulfoxide (DMSO)) and presented as mean±s.d. The experiment was repeated at least two times with each inhibitor. (d) Immunoblots of indicated proteins in primary EITL cells treated with PD0325901, Tofacitinib and Trametinib for two and Stattic for 24 h at indicated concentrations.