Genome-Wide microRNA Expression Profiling in Molecular Subgroups of Peripheral T-Cell Lymphoma Identified Role of Mir-126 in T-Cell Lymphomagenesis

Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas and approximately 30% of PTCLs are designated as not-otherwise specified (PTCL-NOS). Gene expression profiling (GEP) identified molecular classifiers for PTCL entities and identified 2 novel biological subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21), associated with T-cell differentiation subsets. To further investigate molecular oncogenesis, we performed microRNA expression profiling (miR-EP) in several molecular subtypes of PTCL including angioimmunoblastic T-cell lymphoma (AITL), PTCL-GATA3 and PTCL-TBX21 using formalin fixed paraffin embedded tissues. We also performed miR-EP of normal T-cell subsets polarized to represent different differentiation stages (T_FH, T_H1 and T_H2). We performed miR-EP on 102 PTCL cases using either quantitative real time PCR (ABI, Biosystem) or ultra-sensitive direct miRNA counting (nCounter, NanoString). Corresponding GEP (mRNA) were available for 67 PTCL cases. Normal T-cells were polarized in-vitro with different cytokine milieu and examined by flow cytometry.

We observed distinct miRNA profiles, with miRNA being uniquely expressed in T_FH polarized cells (miR-26a-5p, miR-17-5p, miR-30d-5p, miR-22-3p, miR-222-3p, miR-142-3p, let-7i-5p and miR-29b-3p). In contrast, the T_H1 lineage was enriched for expression of miR-155-5p, miR-146a-5p, miR-1246, miR-93-5p, miR-16-5p, miR-21-5p, miR-363-3p, miR-1260a, miR-186-5p, miR-148a-3p and miR-579-3p, whereas T_H2 polarized cells expressed miR-181a-5p, let-7a-5p, miR-191-5p, miR-15b-5p, let-7d-5p, let-7b-5p, miR-140-5p, miR-98-5p, miR-423-5p and miR-630. Several of these miRNA expressed in the T-cells subsets showed corresponding expression in their respective PTCL entity such as miR-142-3p, let7i-5p, miR-21-5p and miR-29b-3p with AITL, miR-146-5p, miR-155-5p and miR-16-5p in PTCL-TBX21 and miR-181a-5p, miR-630 and let7a-5p in PTCL-GATA3. We also performed the MiRNA Enrichment Analysis and Annotation (miEAA) for miRNA signatures and observed an enrichment of miRNA regulating epigenetic modifications in T_FH cells (p=0.028), whereas T_H1 showed an enrichment of miRNA regulating IFN-g signaling (p=0.0024), and miRNA signatures in T_H2 showed negative regulation of TGF-b signaling (p=0.023).

Supervised analysis (p=0.05) of the miRNA profiles identified significant association of miR-126, miR-145, and let-7c-5p with AITL, when compared to other PTCLs. Similarly, miR-92a, miR-25, miR-636, miR-210, miR-222 and miR-491-5p significantly associated with PTCL-GATA3 and miRNA 126-3p, 145-5p, miR-26a-5p and miR-34a-5p associated with PTCL-TBX21. The miEAA for tumor miRNA signatures revealed enrichment of miRNAs regulating histone methylation (h3 k4 methylation) and chemokine receptor signaling in AITL, whereas miRNA regulating T-cell receptor were enriched in PTCL-TBX21 and TP53 signaling pathway in PTCL-GATA3. We validated the expression of miR-126 in AITL by qRT-PCR and also observed its increased expression in IL21 stimulated CD4+ T-cells. Ectopic expression of miR-126 resulted in a ~3 fold increased expression in T-cell lines and led to reduced proliferation and increased apoptosis with expression of T-cell exhaustion makers PD1 and TIM3. Computational algorithmic programs identified relevant biological targets of miR-126, including p85/PIK3R2, S1PR2 and DNMT3A that were further validated in-vitro. We observed an inverse correlation of miR-126 expression with S1PR2 expression (r=-0.64). S1PR2 is a crucial G protein-coupled receptor regulating B and T-cell migration in the germinal center (GC) reaction. Migration assays demonstrated significant decreases in T to B-cell migration, when B-cells (Raji) were co-cultured with Jurkat cells with ectopic expression of miR-126. With the GC reaction holding an important role in AITL, we investigated the biological significance of miRNA-126 in the context of the AITL microenvironment. High expression of miRNA-126 significantly associated with inferior survival in AITL (p=0.008) and significant differences in tumor microenvironment signatures.

We identified distinct miRNA signatures for AITL and molecular subgroups of PTCL-NOS. Furthermore, elevated expression of miR-126 may contribute to the dysregulation and the homing of T_FH cells in GC reaction through S1PR2 and warrants further mechanistic investigation.