Telomere Dysfunction-Induced DNA Damage Drives Hematopoietic Stem Cell Fate

Abstract

Accumulating evidence supports the view that DNA damage checkpoints activated by telomere erosion can drive hematopoietic stem cell (HSC) decline, thereby compromising HSC self-renewal, repopulating capacity, and differentiation. However, the precise mechanisms underlying telomere dysfunction-related HSC defects are still largely unknown. In this study, we employed the inducible telomerase deficient mice TERTER/ER to molecularly define the adverse effects of wide-spread endogenous telomere dysfunction-induced DNA damage signaling on stem cell function in vivo.The HSC compartment of 3-month-old telomere dysfunctional mice (G4/G5 TERTER/ER) showed an increased expansion in the steady-state absolute number of long-term HSCs (LT-HSC) and short-term HSCs with a concomitant decrease of multipotent progenitor cells. Accordingly, telomere dysfunctional LT-HSC showed a significant decrease of the quiescence state (p=0.018) associated with an increase of cells in the G1/G2-M phase of the cell cycle (p=0.038), although the preferential accumulation of phospho-H2AX foci (p=7x10-4). Furthermore, peripheral blood analysis revealed that the total CD45.2-derived reconstitution was significantly compromised in mice competitively transplanted with G4/G5 TERTER/ER LT-HSC, which shows that they have a finite potential for self-renewal under regenerative stress. Overall, these findings suggest the existence of a telomere dysfunction-induced differentiation checkpoint, which occurs at the level of LT-HSC and is responsible for their premature exhaustion. Correspondingly, aged telomere-dysfunctional mice (n=20) showed a significant decrease in the absolute number of LT-HSC in comparison to aged mice with intact telomeres (n=10) (p=0.04). On the contrary, leukemic transformation which occurred in about 5% of G4/G5 TERTER/ER mice both in homeostatic conditions and in the setting of competitive transplantation induced a significant expansion of the HSC pool, suggesting the existence of secondary events able to overcome the decline of telomere dysfunction-induced HSC self-renewal capability.One way in which cells can balance renewal with differentiation is through the control of asymmetric and symmetric division. During asymmetric division, one daughter cell remains a stem cell, while the other becomes a committed progenitor cell. In contrast, during symmetric divisions, a stem cell divides to become two HSCs (symmetric self-renewal) or two committed cells (symmetric commitment). Asymmetric cell division involves the polarized distribution of determinants, such as Numb, within the mother cell and their unequal inheritance by each daughter cell; in contrast, symmetric division allows both daughter cells to adopt equivalent fates.To determine if telomere dysfunction-induced DNA damage was directly responsible for HSC exhaustion by altering the mechanism of HSC self-renewal versus differentiation cell fate decisions, we evaluated Numb inheritance and expression in sorted telomere dysfunctional LT-HSC (n=310 LT-HSC isolated from 12 mice) in comparison to LT-HSC with intact telomeres (n=273 LT-HSC, isolated from 7 mice) induced to proliferate in culture. Specifically, we found that the frequency of symmetric self-renewal divisions was approximately 1.5-fold lower in telomere dysfunctional LT-HSC compared with those with intact telomeres (p=0.02), with a concomitant 2-fold increase in the frequency of symmetric commitment (p=0.006). Thus, telomere dysfunction-induced DNA damage is associated with a cell-intrinsic skewing toward symmetric commitment, which leads to compromised self-renewal capability. In contrast, and consistent with our in vivo data, LT-HSC isolated from G4/G5 TERTER/ER mice in leukemic transformation preferentially underwent symmetric self-renewal divisions. Next, we performed unbiased RNA sequencing on sorted G4/G5 TERTER/ER LT-HSC induced to proliferate in vitro, which underwent to preferential symmetric commitment or symmetric self-renewal divisions. Results of these analyses will provide insights into the mechanistic basis of how telomere dysfunction-induced DNA damage drives aberrant commitment of HSC, which results in their exhaustion, whereas leukemic transformation leads to deregulated and enhanced self-renewal, which results in their expansion and suppression of normal hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.