Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, ...implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome.
Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly ...defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.
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•Mesenchymal deletion of Sbds in mice recapitulates bone defects in SDS•Mesenchymal niche cells induce genotoxic stress in HSPCs in this model•p53-S100A8/9-TLR4 signaling, activated in SDS and MDS, drives these phenotypes•Mesenchymal S100A8/9 predicts leukemic evolution and disease outcome in human MDS
Cell-extrinsic factors driving malignant transformation remain understudied. In a mouse model of pre-leukemia, Zambetti and colleagues establish a concept of mesenchymal niche-induced genotoxic stress in hematopoietic stem cells through p53-S100A8/9-TLR4 signaling, with relevance to human leukemia. The findings provide conceptual and mechanistic insights into the link between inflammation and cancer.
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 impaired lineage progression, we focused on p53 activation, a common mechanism of tissue failure in ribosomopathies. P53 protein was significantly and selectively accumulated in EYFP+ MC-MMs of Sbds Δ/Δ recipients as measured by FACS (Mean Fluorescence Intensity FC: 1.8; p=0.01), with increased expression of p53 targets such as Cdkn1a (p21), Bbc3 (PUMA) and Bax. This was accompanied by increased apoptosis (annexin V FACS analysis) in both MC-MMs and mature neutrophils (FC: 1.36; p=0.02 and FC=2.44; p=1.6x10-4, respectively). However, pharmacological inhibition of the p53 pathway by pifithrin-α (2 months, i.p.) failed to rescue neutropenia, suggesting that alternative mechanisms, such as disrupted expression of transcription factors, may contribute to neutropenia in SDS. Transcript analysis of Sbds-deficient MC-MMs revealed downregulation of Rara (Retinoic acid receptor α) and its target Cdkn1b (p27), implicated in cell cycle arrest and terminal granulopoiesis (Walkley et al., Blood 2004). Congruent with this finding, Ki67 staining showed that end-stage granulocytes from Sbds Δ/Δ recipients fail to exit cell cycle.
In conclusion, we established a mouse model of neutropenia caused by Sbds deficiency in the hematopoietic system, providing experimental support for a direct causative link between Sbds deficiency and neutropenia in mammals. The data reveal a previously unanticipated, selective dependency of late myeloid cells on this ubiquitous protein, while its deficiency spares the function of rapid cycling hematopoietic progenitors. Mechanistically, disrupted expression transcription factors governing terminal myeloid differentiation may be implicated. We anticipate the mouse model to be a valuable tool in further dissecting the molecular pathogenesis of SDS and enabling preclinical studies for disease modulation.
No relevant conflicts of interest to declare.
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