PB1871 ONCOGENIC MIRNAS CONTROL PROLIFERATION AND APOPTOSIS IN TCRαβ+ CD8+ LARGE GRANULAR LYMPHOCYTE (LGL) LEUKEMIA CELLS
Authors: Assmann J.L. , Erkeland S.J. , Leon L.G. , Langerak A.W. 
Published: June 2019

Background:

TCRαβ+ CD8+ large granular lymphocyte (LGL) leukemia is a rare heterogeneous hematological disorder that has a chronic disease course and mostly affects the elderly. LGL leukemia is estimated to account for 2‐5% of all chronic lymphoproliferative disorders and most commonly TCRαβ+ CD8+ T cells are affected. Although the precise disease etiology is largely unknown and disease course is heterogeneous, it is generally accepted that LGL cells arise through chronic antigenic stimulation and inflammation and thrive as a consequence of dysregulation of proliferation and apoptosis pathways. One level of dysregulation of these pathways could involve miRNAs. miRNAs are a class of endogenous noncoding RNAs (19‐25 nt) that are involved in regulation of gene expression. miRNAs primarily act at the post‐transcriptional level and are involved in various cellular processes such as proliferation and apoptosis. Moreover, dysregulated expression of miRNAs that hold tumor suppressor or oncogenic capacity has been associated with the pathogenesis of human cancers and leukemias. Here, we explored the miRNAome of TCRαβ+ CD8+ LGL leukemic cells by performing small noncoding RNA sequencing of sorted TCRαβ+ CD8+ LGL cells (n = 7) and sorted age‐matched healthy control TCRαβ+ CD8+ TEMRA effector T cells (n = 6). Furthermore, we evaluated whether differentially expressed miRNAs could potentially drive the dysregulation of proliferation and apoptosis pathways in LGL leukemia.

Aims:

To determine the miRNAome of TCRαβ+ CD8+ LGL cells and to elucidate whether the aberrant expression of miRNAs drives the dysregulation of proliferation and apoptosis pathways in LGL cells.

Methods:

Purified LGL leukemia cells and healthy control cell subsets were sorted with FACS Aria III. Cells were sorted directly in Trizol reagent and RNA was isolated according to manufacturer's protocol. Libraries were prepared with cleantag small RNA library prep kit and SMART‐seq v4 ultra low input RNA kit for sequencing according to manufacturer's protocol.

Results:

Using next generation sequencing, we determined differential expression of a subset of miRNAs between LGL leukemia cells and healthy control T cell subsets. Principle component analysis of miRNA expression profiles clearly showed LGL leukemia cells clustering away from healthy control T cells, with more than 100 miRNAs significantly upregulated or downregulated between patient cells and control cells. In silico pathway analysis of top differentially expressed miRNAs indicated that these miRNAs could be involved in cell cycle regulation through repression of cyclin dependent kinases CDKs, the NF‐κB pathway and potentially drive apoptosis resistance through inhibition of the p53 and BCL2 pathways. Currently we are exploiting the RNAome by RNA sequencing to validate the involvement of these pathways in LGL leukemia. By integrating both the miRNA and RNA datasets the association of top candidates in these pathways will be further investigated.

Summary/Conclusion:

Collectively our data implicate miRNAs as potential drivers in the maintenance of established LGL leukemic cells. Likewise, in silico prediction tools imply that long term survival of these cells is a result of miRNAs that enhance proliferation and inhibit apoptosis.