Down-Regulated Expression Of Protection Of Telomeres 1 (POT1) Gene In Bone Marrow Hematopoietic Progenitor Cell Compartment Has Prognostic Value In Myelodysplastic Syndromes (MDS)

Abstract

Genomic instability is a hallmark for MDS and AML and is also important for the evolution of MDS to AML. One major cause of genomic instability is telomere dysfunction. Abnormal telomere shortening have been observed in MDS/AML and a spectrum of bone marrow failure syndromes such as dyskeratosis congenita and aplastic anemia. Studies in telomerase deficient mice also indicate that the activation of cell intrinsic checkpoints in response to telomerase dysfunction limits the repopulating capability of hematopoietic progenitor cells after serial bone marrow transplantation, eventually leading to bone marrow failure condition. POT1 is a telomere maintenance gene that encodes a telomere protection protein of the shelterin complex, and is the first member of this this structure found to be mutated in human cancer. Most recently, somatic mutations of POT1 implicated in loss of biological function have been identified in human chronic lymphocytic leukemia (CLL) (Nature Genetics 2013), indicating that POT1 dysfunction is involved in pathogenesis of hematological neoplasms. To study the role of POT1 in MDS, we first sequenced all coding exons of POT1 known to have genomic mutations in CLL. This sequencing analysis was performed via PCR-Sanger method in bone marrow mononuclear cells (BM-MNNC) of a cohort of thirty patients with MDS (15 with RAEB/RAEBT, 11 with RA/RARS/RCMD/MDS-U, 2 with CMML, and 2 with 5q- syndromes). No genetic mutation of POT1 gene was detected in this cohort. We then studied expression levels of POT1 in CD34+ bone marrow hematopoietic progenitor cells. In a cohort of sixty-five patients with MDS (partially overlapping with the patients sequenced for POT1 gene), we performed Q-RTPCR to compare POT1 RNA expression levels of patients with control CD34+ cells of healthy individuals (N=8). Results indicate that, although the overall POT1 RNA level of this patient cohort is not significantly different from controls, the subset of patients (N=13) with cytogentic deletions of chromosome 7 or 7q have a significant reduction of POT1 RNA expression (40% of controls, p<0.00001). Ten out of thirteen (77%) patients of this subset presented over a 50% down-regulation of POT1 expression. POT1 gene is located on chromosome 7q. Therefore this result suggests that haploinsufficiency is a key molecular mechanisms underlying reduced POT1 expression in hematopoietic progenitor cells of MDS. We further analyzed clinical implications of POT1 down-regulation by setting the cutoff RNA expression level of POT1 at 50% of controls. Compared to other patients (N=51), patients with POT1 down-regulation (N=14) have significant higher levels of bone marrow blasts (12.6% v.s. 6%, p=0.002), lower platelet counts (63.6 v.s. 121.2 X109/L, p=0.007), and shorter overall survivals (12.3 v.s. 24 month, p=0.02). All patients in this subset had higher risk diseases based on IPSS scores (five IPSS-high and nine INT-2), compared to 29% of high/ INT-2 in the subset of patients without POT1 down-regulation. A majority of patients in this subset (10 out of 14 cases, 71%) are diagnosed as RAEB/RAEB-T, which is significantly (p=0.03) from the 35% of RAEB/RAEB-T in the group without POT1 down-regulation. In patients with POT1 down-regulation, we identified the expression of EZH2, another 7q gene implicated in the pathogenesis of MDS, was lower than other patients (74% decrease, p=0.001). There was also a tendency of increased expression of IL-8 (3.5-fold increase, p=0.08), a cytokine recently found to be overexpressed in MDS. Finally, we evaluated possible impact of POT1 down-regulation on telomere integrity of patients. Telomere lengths were measured in patients of the cohort with available BM-MNNCs genomic DNA (N=22) through an established Q-PCR based approach. Patients with lower POT1 expression (N=8) presented shorter relative telomere lengths than patients without POT1 down-regulation (38% decrease, p=0.04). Taken together, results of this study indicate that down-regulation of POT1 gene expression, rather than its genomic mutation, plays a role in the pathogenesis and prognosis of MDS, particularly in higher-risk disease and perhaps by affecting telomere integrity. Characterization of POT1 expression in more patients and investigation of biological molecular mechanisms underlying POT1 down-regulation in MDS should be performed in future studies. Disclosures:No relevant conflicts of interest to declare.