Recurrent mutations of the STAT6 DNA binding domain in primary mediastinal B-cell lymphoma.

Primary mediastinal B-cell lymphoma (PMBL) is a separate entity of aggressive B-cell lymphoma, characterized by a constitutive activation of janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, also observed in Hodgkin lymphoma. Although many cancers exhibit constitutive JAK-STAT pathway activation, mutations of STAT genes have not been reported in neoplasms. Here, we show that MedB-1 PMBL-derived and L1236 Hodgkin-derived cell lines and 20 of 55 (36%) PMBL cases harbor heterozygous missense mutations in STAT6 DNA binding domain, whereas no mutation was found in 25 diffuse large B-cell lymphoma samples. In 3 cases, somatic origin was indicated by the absence of the mutations in the nontumoral tissue. The pattern of STAT6 mutations was different from the classical features of somatic hypermutations. The mutant STAT6 proteins showed a decreased DNA binding ability in transfected HEK cells, but no decrease in expression of STAT6 canonical target genes was observed in PMBL cases with a mutated STAT6 gene. Although the oncogenic properties of STAT6 mutant proteins remain to be determined, their recurrent selection in PMBL strongly argues for their involvement in the pathogenesis of this aggressive B-cell lymphoma.

Recurrent somatic mutations of PTPN1 in primary mediastinal B cell lymphoma and Hodgkin lymphoma.

Classical Hodgkin lymphoma and primary mediastinal B cell lymphoma (PMBCL) are related lymphomas sharing pathological, molecular and clinical characteristics. Here we discovered by whole-genome and whole-transcriptome sequencing recurrent somatic coding-sequence mutations in the PTPN1 gene. Mutations were found in 6 of 30 (20%) Hodgkin lymphoma cases, in 6 of 9 (67%) Hodgkin lymphoma-derived cell lines, in 17 of 77 (22%) PMBCL cases and in 1 of 3 (33%) PMBCL-derived cell lines, consisting of nonsense, missense and frameshift mutations. We demonstrate that PTPN1 mutations lead to reduced phosphatase activity and increased phosphorylation of JAK-STAT pathway members. Moreover, silencing of PTPN1 by RNA interference in Hodgkin lymphoma cell line KM-H2 resulted in hyperphosphorylation and overexpression of downstream oncogenic targets. Our data establish PTPN1 mutations as new drivers in lymphomagenesis.

Human IL4I1 is a secreted L-phenylalanine oxidase expressed by mature dendritic cells that inhibits T-lymphocyte proliferation.

Interleukin-4-induced gene 1 (IL4I1) was first described as a B-cell IL4-inducible gene and is highly expressed in primary mediastinal B-cell lymphomas. We established stable HEK293 clones expressing human and mouse IL4I1 to examine their biochemical properties and function. Both proteins were secreted into the culture medium, and we observed the secretion of endogenous human IL4I1 (hIL4I1) protein in a mediastinal lymphoma B-cell line, MedB-1. We showed that IL4I1 has l-amino acid oxidase activity, optimal at physiological pH and primarily directed toward phenylalanine. Immunohistochemical analysis of secondary lymphoid organs showed staining of germinal center macrophages and inflammatory myeloid cells. In vitro, functional enzyme was highest in mature dendritic cells (DCs), suggesting a role in antigen-presenting cell/T-lymphocyte cross-talk. Indeed, hIL4I1 inhibited the proliferation of CD3-stimulated T lymphocytes with a similar effect on CD4(+) and CD8(+) T cells. In contrast, memory T cells were more strongly affected by hIL4I1 and its catabolite H(2)O(2) than naive T cells. hIL4I1 inhibitory effect was dependent on enzymatic activity and H(2)O(2) production and associated with a transient down-regulation of TCRzeta expression. Altogether these data suggest IL4I1 as a new immunomodulatory enzyme produced by DCs.

Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL), currently recognized as a diffuse large B-cell lymphoma (DLBCL) subtype, shows increased expression of interleukin 4 (IL-4)/IL-13 signaling effectors and targets, suggesting constitutive activation of these pathways. We therefore investigated the functional state of the signal transducer and activator of transcription 6 (STAT6), mediating IL-4/IL-13 transcriptional effects. Constitutive STAT6 phosphorylation and DNA-binding activity were detected in PMBL cell lines but not DLBCL cell lines. Moreover, immunohistochemical analysis revealed nuclear phosphorylated STAT6 (P-STAT6) in 8 of 11 PMBL, compared with 1 of 10 DLBCL primary tumors (P =.01). IL-4 and IL-13 transcripts were absent in PMBL cell lines and expressed at low levels in tumors, indicating that, contrary to classical Hodgkin lymphoma (cHL), STAT6 activation is not due to an autocrine IL-4/IL-13 secretion. We demonstrated an amplification of the JAK2 gene in 2 of 6 PMBL cases, and showed higher JAK2 mRNA levels in PMBL compared with DLBCL (P =.005). The Janus kinase 2 (JAK2) was constitutively phosphorylated in the PMBL MedB1 cell line. MedB1 treatment with JAK2 inhibitor AG490 partially decreased STAT6 phosphorylation, suggesting that JAK2 is partially involved in STAT6 activation in these cells. Our findings highlight phosphorylated STAT6 as a characteristic distinguishing PMBL from DLBCL, but a common feature to PMBL and cHL, supporting the hypothesis of common pathogenic events in these 2 lymphomas.