Abstract
Telomeres are important in maintaining the integrity of the chromosomes. A series of shelterin proteins (TRF1, TRF2, RAP1, POT1a and b, TIPP1 and TIN2), are involved in the maintenance of telomeres. In chronic lymphocytic leukemia (CLL), the telomeres are shorter than in normal B cells, with very short telomeres being associated with poor survival. In the present study, we demonstrate that the leukemia cell telomere length shortens over time were CLL are followed over year, indicating that telomere shortening in these cells is an ongoing process. When CLL cell metaphases were examined by Q-FISH, telomere length shortening was found to affect all chromosomes equally. To determine whether these telomere changes were related to alterations in the shelterin complex, protein levels of the six shelterins were determined in CLL cells and normal pooled B cells. In general, apart from TIN2, the shelterin protein levels were increased in CLL cells and TRF2 levels were higher in IgVH unmutated cells than in mutated cells. TIN2 protein levels were dramatically reduced in CLL cells as compared to normal B cells and an alternative isoform of TIN2 was observed, both in IgVH mutated and unmuted cases. TIN2 targeted RT-PCR followed by sequencing revealed a deletion of the entire exon 2, totaling 105 bp. This abnormality appeared in monoclonal B-cell lymphocytosis (MBL) indicating that this was an early event. In addition, a subgroup of patients showed an increase in the spliced form of TIN2, when followed over time, suggesting that this might provide the cell with a survival advantage. A protein translated from the spliced variant RNA is expected to be deleted for 35 amino acids at the N-terminus, which is the binding site for TRF2. As TIN2 maintains telomere structure by forming a complex of TRF1 and TRF2 with the telomere, this should not occur with the splice variant. Indicating a pathological role of spliced TIN2 in CLL, TRF2 was localized in both the nuclei and cytoplasm in CLL cells compared to localization only to the nucleus in normal B cells. To confirm the function of the spliced TIN2, we have overexpressed the both full length and spliced clones of TIN2 in HEK293 cells and are presently determining the localization of TRF2 and the ability to form a TRF2/TIN2 complex. Thus, these studies demonstrate a unique splice variant of TIN2 in CLL, which potentially may influence the formation of the shelterin complex and contribute to telomere shortening in this disease.
Citation Format: Ganchimeg Ishdorj, Sara EF Kost, Yunli Zang, Spencer B. Gibson, James B. Johnston. Role of shelterin proteins for telomere shortening in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2850.
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