Telomere Dysfunction-Induced DNA Damage Drives Hematopoietic Stem Cell Fate
Authors: Marchesini Matteo , Ogoti Yamini , Ganan-Gomez Irene , Wei Yue , Bueso-Ramos Carlos E. , Garcia-Manero Guillermo , DePinho Ronald A. , Colla Simona 
Published: October 2019

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.

Disclosures
No relevant conflicts of interest to declare.