IDH2^R172 Mutations Define a Unique Subgroup of Patients in Angioimmunoblastic T-Cell Lymphoma

Background: Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL) with distinct pathological features and poor prognosis. Currently used chemotherapy is mostly unsuccessful with a 3-year overall survival of less than 30%. We and others have identified frequent mutations affecting IDH2 at arginine-172 (R172),TET2, DNMT3A and RHOA in AITL. The biochemical and functional consequences of IDH2^R172 mutations in T cells have not been demonstrated. In this study, we performed targeted re-sequencing of epigenetic regulators, including IDH2, TET2 and DNMT3A in molecularly defined PTCL cases and analyzed the biochemical changes associated with IDH2^R172 mutations as well as alterations in gene expression profiling (GEP), DNA methylation and histone modification that may improve our understanding of the pathogenetic mechanisms in AITL.

Methods: We performed targeted re-sequencing of epigenetic regulators IDH2, TET2 and DNMT3A in AITL (n = 39) and PTCL subtypes (n = 53) with corresponding GEP. Due to lack of appropriate cell lines derived from AITL, we chose to use Jurkat T cells, a T-ALL cell line frequently used for T-cell functional studies and normal CD4+T cell to study the biochemical consequences of IDH2^R172 mutations in T-cells. Liquid chromatography- tandem mass spectrometry was utilized for the detection of intracellular level of the 2-hydroxyglutarate and levels of 5-methycytosine and 5-hydroxymethycytosine in genomic DNA. Alterations of histone lysine tri-methylation were assessed by immunohistochemistry in AITL specimens and western blotting in vitro.

Results: TET2 mutations appear to be the founder mutations in AITL with 82.1% (32/39) mutated cases and present as the major clone in the majority of mutant cases (75%; 24/32). TET2 mutations were also observed at a lower frequency in PTCL-NOS molecular subgroups (TBX21 (46%; 10/18); GATA3 (41%; 5/12)) and ALK negative-ALCL (33.3%, 4/12). The mutations in DNMT3A were observed at similar frequency in AITL (38.5%, 15/39) and PTCL-NOS subgroups [TBX21 (33%; 6/18); GATA3 (25%; 3/12)). However, IDH2^R172 mutations were found predominantly in AITL (33.3%, 13/39), but rarely in PTCL-NOS subgroups (6.7%, 1/15 in PTCL-NOS). Remarkably, IDH2^R172 mutant cases formed a unique cluster in unsupervised hierarchical clustering, and IDH2^R172mutation defined a unique subset within AITL with a distinct gene expression signature. We observed that ectopic expression of IDH2^R172K in the Jurkat T cell line led to a markedly increased level of intracellular 2-HG and up-regulation of the repressive histone methylation mark H3K27me3. Furthermore, a significant increase in 5-methylcytosine and corresponding decrease in 5-hydroxymethycytosine in genomic DNA was also observed in IDH2^R172K transduced Jurkat and primary CD4+ T cells. Consistent with these findings, significant increase in aglobal DNA hypermethylation in proximal promoter regions and a global increase of the repressive histone mark H3K27me3 was observed in AITL harboring IDH2^R172 mutants. Integrative analysis of GEP and promoter methylation identified several recurrently hypermethylated genes including negative regulators of the NF-kB pathway.

Conclusion: IDH2^R172 mutations define a unique subgroup of patients in angioimmunoblastic T-cell lymphoma with a distinct gene expression profile. IDH2^R172 mutations are associated with global promoter hypermethylation in genomic DNA and trimethylation of H3K27 in AITL specimens. The current findings suggest that abnormal methylation associated with IDH2^R172 mutations contribute to lymphomagenesis in AITL.