Human Telomerase Reverse Transcriptase (hTERT) Deficiency in Myelodysplastic Syndrome (MDS) Demonstrates Mechanistic Linkage to Aplastic Anemia Pathophysiology

Lili Yang; Adam W Mailloux; Dana E Rollison; Jong Park; Jeffrey S. Painter; Rami S. Komrokji; Jaroslaw P Maciejewski; Ronald Paquette; Thomas P. Loughran; Sheng Wei; Alan F. List; Pearlie K Epling-Burnette

doi:10.1182/blood.V118.21.791.791

Abstract Abstract 791 Background: Myelodysplastic syndromes (MDS) are characterized by dysregulated myelopoiesis and peripheral cytopenias with enormous disease heterogeneity owing to diverse molecular pathobiology. The early manifestations of MDS, however, are relatively well conserved and include increased apoptosis coupled to excessive proliferation of myeloid progenitors. In addition to myeloid abnormalities, repertoire contraction and memory expansion is demonstrable in T cells. The notion that apoptosis of hematopoetic cells may be triggered through an immune–mediated mechanism arose from similarities with aplastic anemia (AA). Our recent data showed that MDS responsive to immunosuppressive therapy has accelerated naïve T cell turnover (ie, high proliferative index plus excessive cell death) which led us to hypothesize the presence of an inherent T cell abnormality impairing homeostatic regulation. AA can be caused by somatic mutations within telomere repair components. T-cells are one of a few somatic cells that retain telomerase function to control naïve T-cell survival, replication potential, and antigenic diversity. To this end, we examined telomere function and replicative burst capacity of MDS T cells as a possible mechanism for immune dysregulation. Methods: Primary specimens from MDS (n=37), AA (n=8), and controls (n=42) were investigated. Peripheral blood mononuclear cells were isolated from patient blood or buffy coats by Ficoll-Hypaque gradient centrifugation. Purified CD3+ T cells were isolated using negative selection and then stimulated with anti-CD3/anti-CD28 T cell activator beads (Dynabead®) for 3 days. Telomere length was assessed by quantitative PCR (q-PCR) and telomerase enzymatic function measured by Telomere Repeat Amplification Protocol (TRAP) assays. Results: Mean telomere length in purified T cells was significantly shorter among MDS patients compared to controls after adjusting for age and sex (p<0.0001). To assess telomerase repair function in MDS T-cells, we performed TRAP assays with purified T cells after stimulation and found that inducible telomerase activity is severely suppressed in MDS compare to controls. In comparison to controls, the inducible telomerase activity fell below the 95% confidence internal in all cases (MDS median 18.70, 95% CI, 15.93–20.54 vs control median 45.0, 95% CI, 45.79 – 64.5, p<0.0001) and the amount of telomerase activity was unrelated to risk stratification by the International Prognostic Scoring System (IPSS), World Health Organization (WHO) classification, and age indicating that it is a frequent abnormality in the disease. Analysis of telomerase function and telomere length in T cells from patients with AA showed a similar deficiency in telomerase repair function. The mechanism responsible for telomerase insufficiency in MDS was mediated by defective induction of telomerase reverse transcriptase (hTERT) transcription; the key enzyme involved in telomere maintenance. Next, to determine the functional consequences of the disturbance in telomere repair in MDS, the ability of T cells to enter S-phase and to undergo an antigen-induced proliferative burst were examined. TCR signaling was shown to be preserved, evidenced by induction of an early activation antigen CD69. Although some cells were capable of entering S-phase, the replicative burst potential was severely impaired in T cells form all patients. Telomere repair is exclusively present in naïve T cells and progressively declines after memory transition. TCR triggered telomerase activity was measured in sorted naïve (CD45RA+, CD45RO-) and memory (CD45RO+, CD45RA-) T cells. The telomere length in naïve cells was shorter in MDS patients compared to controls (p=0.018) and the telomerase activity was suppressed in naïve MDS T cells (p=0.0207) indicating that telomere dysfunction underlies the altered homeostasis of naïve T cells in MDS, a feature mechanistically akin to AA and other telomere repair disorders. Conclusion: Results of this study indicate that there is loss of telomere maintenance in naïve T cells due to a defect in hTERT transcription is associated with impaired replicative potential. This abnormality in naïve T cell homeostasis represents an inherent defect that contributes to a memory cell growth advantage and repertoire contraction associated with autoimmunity in AA and MDS. Disclosures: No relevant conflicts of interest to declare.

Human Telomerase Reverse Transcriptase (hTERT) Deficiency in Myelodysplastic Syndrome (MDS) Demonstrates Mechanistic Linkage to Aplastic Anemia Pathophysiology

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