Defective Hematopoiesis and Erythroid Differentiation Resulting From Telomerase Deficiency

Abstract 2410

Telomere shortening and anemia are common in bone marrow failure syndromes (BMF) such as dyskeratosis congenita (DKC), acquired aplastic anemia and myelodysplastic syndromes (MDS). Components of the telomerase complex including TERT, TERC and Dyskerin are mutated in multiple BMF syndromes, strongly suggesting a link between aberrant telomere maintenance and BMF. However, the specific defects in hematopoiesis that lead to BMF have not been completely defined. To better understand the effects of telomere shortening on hematopoiesis, we crossed Tert−/− mice for five successive generations to derive 5^th generation Tert−/− (G5 Tert−/−) mice with short telomeres. The G5 Tert−/− mice were smaller in size and their peripheral blood cell counts had significantly reduced numbers of red blood cells and hemoglobin content (p-value < 0.01). There was also a significant reduction in immature erythroid (CD71+; p-value < 0.03) and mature erythroid (CD71-Ter119+; p-value < 0.05) cells in the bone marrows of these mice as compared to the G0 Tert+/− controls, while myeloid (Mac-1, Gr1+) cell numbers were not decreased. Annexin V and PI staining showed increased apoptosis in immature erythroid cells from G5 Tert−/− mice. In order to determine if the reduction in erythrocytes was due to defective hematopoiesis, we studied hematopoietic stem cell (HSC), committed erythroid progenitor (MEP) and myeloid progenitor (GMP) cell numbers in these mice. We observed a marked decrease in MEP (Lin-cKit+Sca1-CD34-CD16/CD32lo; p-value < 0.02) and HSC (Lin-cKit+Sca1+CD34-CD150+; p-value < 0.03) populations in G5 Tert−/− mice marrow, while GMP (Lin-cKit+Sca1-CD34+CD16/CD34hi; p-value < 0.6) cells were not affected. In contrast, there was an increase in the number of immature erythroid cells (CD71+), hematopoietic progenitor cells (Lin-cKit+Sca1+) and HSC in the spleens of G5 Tert−/− mice, strongly suggesting extramedullary hematopoiesis. These data demonstrate that there is a defect in the regulation of hematopoiesis, starting at the level of HSC, in the bone marrow of G5 Tert−/− mice. MEPs from G5 Tert−/− mice showed increased DNA damage, as demonstrated by phospho-H2Ax staining by flow cytometry, although enhanced apoptosis was not present. In contrast, GMPs did not show any difference in phospho-H2Ax staining, suggesting that MEPs may be selectively sensitive to telomere shortening or that telomeres shorten more rapidly in these cells. Total bone marrow cells from G0 Tert+/− and G5 Tert−/− mice were plated in Methocult supporting erythroid colony formation; we observed that the G5 Tert−/− cells had significantly reduced numbers of CFU-E and BFU-E colonies (p-value < 0.01) compared to cells from the G0 mice, suggesting that the ability of erythroid progenitor cells to differentiate is compromised by dysfunctional telomeres. These data show for the first time a direct and distinctive link between the loss of telomerase function and erythropoiesis. These results, in conjunction with single-cell “mass cytometry” experiments to explore the molecular pathways that are selectively deregulated in the HSC and erythroid precursors of G5 Tert−/− mice, will elucidate the mechanisms underlying the cell-specific effects of telomere shortening on erythropoiesis and help to elucidate the relationship between shortened telomeres and the anemia of bone marrow failure syndromes.