Abstract

Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure caused by mutations in SBDS, which encodes a conserved ribosome assembly factor. Despite simple genetic underpinnings, SDS is surprisingly complex. Patients suffer varying degrees of neutropenia, thrombocytopenia and anemia, and are predisposed to myelodysplasia and acute myeloid leukemia. The only curative treatment is stem cell transplant, but patients are unusually susceptible to toxic side effects. Mapping molecular pathways in affected lineages is a critical step towards developing safer, targeted therapies.

The affected cell type(s) and genetic networks in SDS have remained elusive, but bone marrow hypocellularity and the involvement of multiple lineages points to a defect in the CD34+ hematopoietic stem and progenitor cell (HSPC) population. To identify the molecular basis of SDS, we set out to transcriptionally profile HSPC from SDS patients. However, these cells are rare and heterogeneous, even in normal donors. To overcome this challenge, we developed a pipeline to define the transcriptional architecture of early hematopoiesis at single cell resolution.Key features include rapid conversion of fresh cells to stable cDNA libraries which preserves their natural biology and unbiased sampling to capture all aspects of HSPC heterogeneity. To date, we have sequenced full-length cDNA libraries from 92 normal donor and 176 SDS patient cells.

To deconvolute developmental heterogeneity among HSPC, we clustered cells based on the expression of empirically-determined lineage signature genes. Normal and SDS cells were similarly distributed along the developmental continuum, though there was an increased proportion of SDS cells in the stem and multipotent progenitor stage (HSC/MPP). This analysis produced the first single cell roadmap of human hematopoiesis, which illustrates that hematopoiesis is a continuous process rather than a series of discrete steps.

To identify genes that contribute to impaired hematopoiesis in SDS, we used the MAST statistical framework for single cell expression analysis. The top upregulated pathway in SDS was TNF-alpha signaling via NF-kB. Interestingly, when we mapped this pathway back to our single cell roadmap we found that it was activated to varying degrees in cells at the HSC/MPP stage, but not in more committed progenitors. This finding is intriguing given that TNF-alpha has suppressive effects on HSC growth and long-term engraftment in mice. Moreover, HSC from patients with Fanconi Anemia, a related bone marrow failure, are hypersensitive to TNF-alpha-mediated suppression.

Our study establishes the first link between an inflammatory pathway, TNF-alpha, and bone marrow failure in SDS. We are currently investigating the mechanistic basis for this link using patient-derived induced pluripotent stem cells. In the future, we will examine whether TNF-alpha inhibition is a viable therapy to counteract bone marrow failure in SDS.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.