Deletion of Sbds from Hematopoietic Progenitors Causes Neutropenia in a Mouse Model of Shwachman-Diamond Syndrome By Specifically Blocking Myeloid Lineage Progression at Late Differentiation Stages

• Published in: Blood 2014-12, Vol.124 (21), p.355-355
• Main Authors: Zambetti, Noemi A., Van Strien, Paulina M.H., Valkhof, Marijke G., Adisty, Maria N., Bindels, Eric M.J., Hoogenboezem, Remco M., Sanders, Mathijs A., Rommens, Johanna M., Touw, Ivo P., Raaijmakers, Marc H.G.P.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V124.21.355.355

Shwachman-Diamond Syndrome (SDS) is a congenital bone marrow failure disorder characterized by neutropenia and predisposition to leukemia. SDS is associated with loss-of-function mutations in the SBDSgene, involved in ribosome biogenesis, but the cellular and molecular events driving neutropenia in SDS remain poorly defined, largely due to a lack of mammalian disease models recapitulating the hematopoietic features of SDS. To achieve deletion of Sbds in early hematopoietic/myeloid progenitors, we generated Cebpacre/+Sbdsf/f R26 EYFP/+ mice (Sbds Δ/Δ), which were non-viable. Analysis of E14.5 embryos demonstrated global conservation of the hematopoietic hierarchy in Sbds Δ/Δ embryos compared to Cebpacre/+ R26 EYFP/+ controls (Sbds +/+). Functional relevance of Sbds deletion in Cebpa+progenitors was tested by transplanting fetal liver cells from E14.5 Sbds Δ/Δ or Sbds +/+ embryos into lethally irradiated B6.SJL mice. Deficiency of Sbds in different EYFP+ hematopoietic subsets was confirmed by qPCR (log2 fold change: LKS -2.25, CMP -9.42, GMP -9.1 and neutrophils -7.29). Mice transplanted with Sbds Δ/Δ cells developed profound neutropenia (log2FC: -2.56; p=0.002, n=7), which was stable during the time of analysis (16 weeks). FACS and morphological studies of bone marrow EYFP+ cells demonstrated increased frequencies of early progenitor populations, recapitulating the left-shifted hematopoiesis observed in human SDS patients (Dror et al., Ann N Y Acad Sci 2011), with a pronounced accumulation of cKitint Gr1low EYFP+ myelocytes-metamyelocytes (MC-MMs) (frequency of EYFP+ cells: Sbds +/+ 15.3±1.7 %, Sbds Δ/Δ 39.4±1.1 %; p= 8.7x10-8) and a marked decrease in Gr1+ Mac1+ cells (Sbds +/+: 54.5±7.5 %, Sbds Δ/Δ: 22.3±2.8 %; p= 1.6x10-4), indicating that neutropenia was caused by disrupted lineage progression from MC-MMs to neutrophils. In line with this, whole transcriptome analysis of prospectively isolated EYFP+ MC-MMs (RNA-seq, n=4) revealed enrichment for hematopoietic stem and progenitor cell signatures in Sbds Δ/Δ recipients, while myeloid signatures were enriched in Sbds +/+ mice (GSEA). Transcript analysis further showed reduced expression of granule components produced at the MC-MM stage, such as Ltf, Mmp8, Mmp9. As expected, reduced expression of Sbds (FDR=6.6x10-9, log2FC=-2.67) and dysregulation of ribosome proteins (RP) production were observed, with increased expression of over 60 RP genes in SbdsΔ/Δ MC-MMs. To investigate the molecular events underlying