A Mouse Model Of Rothmund-Thomson Syndrome Reveals An Essential Role For Recql4 In Maintenance Of Hematopoiesis

Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disorder that presents with congenital skeletal malformations, premature ageing and an increased incidence of malignant disease including osteosarcoma and hematologic malignancy. The majority of RTS patients have deleterious germ-line mutations in the RECQL4 helicase. RECQL4 is a member of a family of DNA helicases including Bloom (BLM) and Werner (WRN) syndrome helicases and is thought to play an important role in maintaining chromosome stability. Recql4 deficiency is associated with karyotypic abnormalities and increased rates of aneuploidy. To enable lineage-restricted deletion of Recql4 we generated a mouse Recql4fl/fl line where exon 9 and 10 are flanked by loxP sites. This region encodes the start of the ATP dependent helicase domain and corresponds to human exons 8 and 9, mutations of which are commonly associated with cancers in RTS patients. Germ-line deletion of Recql4 led to embryonic lethality before E10.5. To understand the role of Recql4 in adult hematopoiesis, Rosa26-CreERT2 Recql4+/+, Recql4fl/+ and Recql4fl/fl mice were fed a tamoxifen containing diet (which activates Cre) for up to 4 weeks from 7 weeks of age to somatically delete Recql4. The most striking phenotype observed in these mice was that all R26-CreERT2 Recql4fl/fl animals became anaemic and developed white extremities, which in nearly all cases necessitated euthanasia by 30 days after starting tamoxifen. No differences were observed between R26-CreERT2Recql4+/+ and R26-CreERT2Recql4fl/+. Hematopoietic analyses revealed a severe multi-lineage cytopenia including profound red cell aplasia in the peripheral blood, spleen and bone marrow of Recql4 deficient animals. Effects were observed across all lineages with myeloid, B-lymphoid and T-lymphoid cell numbers severely reduced with a phenotype consistent with acute bone marrow failure. Loss of Recql4 led to a block in B-lymphoid development in the BM at the PrePro-B to Pro-B cell stage. T-lymphoid development was severely interrupted and most populations in the thymus were impacted. Within the stem and progenitor fractions, phenotypic HSCs were preserved in the absence of Recql4 but we found that the erythroid progenitors were nearly completely lost and only CMP/GMP remained. Colony formation was reduced by ∼70% in the deleted bone marrow. Bone marrow transplantation was used to assess the functionality of the Recql4 deleted HSCs. Strikingly, and consistently observed using both R26-CreERT2 and an additional hScl-CreER model, there was a strong selection against deficient cells and a recovery of hematopoiesis by cells with incomplete deletion of Recql4. This result demonstrates an essential role for Recql4 in the maintenance of hematopoiesis. In vitro B and T cell cultures recapitulated the developmental defects observed in the R26-CreERT2 Recql4fl/fl demonstrating a cell intrinsic requirement for Recql4. Loss of Recql4 lead to an accumulation of cells in S-phase and increased levels of DNA damage as measured by gH2Ax staining. Interestingly, the failure in B cell, T cell development and LKS+ CFC formation could be rescued by overexpression of a either WT or a helicase-dead Recql4-K508A mutant. Collectively these data demonstrate that Recql4 is essential for the maintenance of hematopoiesis and acts to maintain the committed progenitor pool. Loss of Recql4 leads to bone marrow failure, demonstrating a unique requirement for Recql4 in the regulation of hematopoiesis. Disclosures: No relevant conflicts of interest to declare.