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    • The ensuing replication stress during rapid expansion elicit

    2018-10-24

    The ensuing replication stress during rapid expansion elicits a broad DDR, when Fancd2-deficient HSPC accumulate DNA damage and activate p38 MAPK in response to physiological replication demands encountered during development. The lack of a more robust γH2AX phosphorylation seemed initially surprising to us, but has been reported before (Burma et al., 2001; Ciccia and Elledge, 2010; Zha et al., 2008). However, a significant accumulation of DNA strand breaks, as well as RAD51 foci, along with the transcriptional elevation of repair wnt inhibitor in the ASL-sorted population clearly indicate the overall activation of DDR in Fancd2 HSPCs (Beerman et al., 2014; Mohrin et al., 2010; Zhang et al., 2016). Not surprisingly, the magnitude of DDR was comparatively more modest in Fancd2 FL HSPCs than that observed after typical interstrand crosslinker exposure or chronic injury models (Hu et al., 2013; Walter et al., 2015). This is entirely consistent with an operational model of diminished, but not eliminated in utero HSPC function, and experimentally supported by fetal deficits in Fancd2 that are not due to apoptosis but rather result from diminished proliferative capacity (Alvarez et al., 2015; Shen et al., 2013; Zhang et al., 2011). Our observations are further consistent with spontaneous developmental deficits that arise during rapid mitotic cycles in Fancc and Fancm germ cell proliferation (Luo et al., 2014; Nadler and Braun, 2000). Fetal HSPCs display unique molecular and functional properties before they transition postnatally to the definitive adult phenotype (Bowie et al., 2007). Although p53 is readily phosphorylated by irradiation in both WT and Fancd2 FL HSPCs and active during development in general (reviewed in Molchadsky et al., 2010), our studies reveal that fetal FA HSPC DDRs are independent of p53 activation. This finding diverges from a well-described phenotype in adult FA models (Ceccaldi et al., 2012; Garaycoechea and Patel, 2014; Zhang et al., 2013). A recent study shows that the p53 response is indeed linearly correlated with the number of DNA breaks (Loewer et al., 2013). Hence, it is conceivable FL HSPCs at 14.5 dpc may not have accumulated enough DNA damage to reach threshold p53 activity to induce apoptosis or cell-cycle arrest. The data consistently show that Fancd2 FL cells encounter genomic stress that activates DDR elements during replication, not seen in adult HSPCs (Fleming et al., 1993). Similar to a previous report on HSPC development (Nygren et al., 2006), we find no accumulation in G0, G1, or S-G2-M cell-cycle phase, suggesting that cell-cycle transit is evenly prolonged, whereby ATM activation is insufficient to activate checkpoints, leaving cell-cycle phase distribution unchanged, even as Fancd2 proliferation and repopulation decline. Others showed that constitutive activation of the p38 MAPK “stress kinase” results in HSPC pool depletion (Bakker and Passegue, 2013; Ito et al., 2006). Our studies herein not only corroborate activation of p38 MAPK in Fancd2 FL cells, but demonstrate rescue of the proliferative capacity after pharmacological p38 MAPK inhibition both in vitro and in vivo (Ito et al., 2004, 2006). Although our in vivo treatment with SB203580 in Fancd2 FL recipients precluded full evaluation of the long-term effect on HSCs, rescue strategies involving short-term p38 MAPK inhibition or using a more potent and less toxic compound, such as LY2228820 (Campbell et al., 2014), might provide a more definitive answer. Yet while several reports and our data imply that p38 MAPK could be a potential therapeutic target to ameliorate hematopoietic failure in FA patients, it is important to consider that this leaves HSPCs vulnerable to DNA-damage accumulation and genomic instability (Anur et al., 2012; Briot et al., 2008; Garbati et al., 2013; Pearl-Yafe et al., 2004; Saadatzadeh et al., 2009). Taken together, we demonstrate that Fancd2 mice exhibit a pervasive developmental HSPC defect that echoes the constitutional defects evident at birth in a subset of FA patients. Mechanistically, the data support a model whereby the physiological replication stress in rapidly cycling Fancd2 fetal HSPCs spontaneously elicits DNA damage and promotes the activation of p38 MAPK, resulting a diminished proliferative fitness, but without the characteristic postnatal p53-mediated apoptotic response.