2033 - REPLICATION STRESS MEDIATED CHECKPOINT ACTIVATION DURING PHYSIOLOGIC HSC EXPANSION IN THE FETAL LIVER LEADS TO HEMATOPOIETIC DEFICITS IN FANCONI ANEMIA

Abstract

Fanconi Anemia (FA) patients show depletion of hematopoietic stem cell (HSC) pools early in life, the onset and pathophysiology of which remain to be clarified. While HSC from FA mice generally show no unprovoked bone marrow failure, we and others observed spontaneous fetal HSC deficits in Fancc- and Fancd2- KO littermates. This affords a unique opportunity to explore the physiologic basis of hematopoietic failure in FA in utero. Here, we find that differences in HSC frequency between WT and KO specifically emerge due to a lack of physiologic expansion in the fetal liver (FL) between E12.5 and 14.5, and not as a result of apoptotic loss seen in adult FA HSCs. We undertook detailed cell cycle studies using a sequential EdU-BrdU pulse chase technique and showed a delay in G1/S phase transition and S-phase progression in Fancd2 FL cells. We then confirmed an underlying Replication Stress (RS) response, via alkaline comet assays, increased replication associated protein p-Rpa expression and activation p-Chk1. This cell cycle checkpoint activation further coincided with nuclear localization of p21, a known negative cell cycle regulator, but not p53 phosphorylation. TGF-b compromises adult FA HSC function, and is a critical developmental morphogen that regulates p21 activity. Here, pharmacological inhibition of TGF-b signaling in FA FL HSC provided functional rescue of myeloid progenitors as well as reversal of p21 translocation in FA. Intriguingly, expression of Chk1 under these circumstances was increased. Thus, TGF-b signaling acts as a rheostat for p21 activity and progenitor clonogenicity, albeit at the cost of increasing RS and Chk1 phosphorylation. Our data in show for the first time that FA HSC are vulnerable to RS during physiologic expansion in the FL, which acts as a principal constraint on HSC pool formation and origin of hematopoietic failure in FA.