Hematopoietic Defects In ACD/TPP1-Deficient Mice Reveal An Essential Role for the Shelterin Complex In Blood-Forming Stem Cell Homeostasis

Abstract 882

Bone marrow failure syndromes can be caused by loss-of-function mutations in telomerase components. Besides the enzymatic activity of telomerase, telomere maintenance requires the protective function of the shelterin protein complex. This complex consists of six proteins that bind to the telomere and prevent an inappropriate DNA damage response from being elicited by exposed telomeric DNA. Within this complex, TPP1, encoded by the gene Acd, plays a central role in complex organization as it links elements that bind the double-stranded portion of the telomere to those that bind the single-stranded overhang. We report an essential function for the shelterin complex and specifically for TPP1 in the maintenance of hematopoietic stem cells (HSCs). Utilizing adrenocortical dysplasia (acd), a spontaneous autosomal recessive mouse mutation causing profoundly hypomorphic Acd expression, we identified phenotypic and functional abnormalities in fetal hematopoietic progenitors. acd mutant Fetal liver HSCs were larger, more granular, and expressed higher levels of the cell surface protein Sca-1, as compared to wild-type counterparts. BrdU incorporation analysis showed an accumulation of Acd-deficient progenitors in the G2M phase of the cell cycle, consistent with G2M arrest. Absolute quantification of fetal liver HSCs revealed a significant reduction in stem cell numbers in acd mutants as compared to control littermates. p53 deficiency rescued the HSC depletion phenotype, but induced a paradoxical exacerbation of the increased progenitor cell size and Sca-1 expression. These findings suggest that p53-dependent and p53-independent elements contribute to the acd HSC phenotype. In competitive repopulation assays, acd HSCs failed to provide tri-lineage hematopoietic reconstitution in lethally irradiated recipients, indicating a profound functional defect. To further study the effects of Acd deficiency in adult HSCs, we generated an Acd allele in which exons 3–8 are flanked by loxP sites, allowing conditional inactivation following Mx1-Cre induction via poly(I:C) administration. Acd inactivation resulted in rapid HSC depletion within 2 weeks after initiation of poly(I:C) injections. To assess whether Acd exerted exclusively cell-autonomous effects in HSCs, we transplanted Acd^f/–Mx1-Cre⁺ HSCs into wild-type recipients. Poly(I:C)-mediated Acd inactivation in these bone marrow chimeric mice resulted in rapid HSC loss and failure of tri-lineage hematopoiesis in animals reconstituted with Acd-deficient bone marrow. These data indicate an exquisite cell-autonomous requirement for Acd/TPP1 in adult mouse HSCs. In view of the extremely rapid HSC loss after Acd inactivation, these observations cannot be explained by telomere shortening. Together, our findings represent the first report of an essential role for Acd/TPP1 and the shelterin complex in HSC maintenance and hematopoietic homeostasis, independently of telomerase function.