Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome affecting multiple organ systems. However, the hematological defects in SDS remain poorly characterized. We used single cell RNA sequencing to identify increased TGF? signaling specifically in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), but not lineage-committed progenitors. We also identified significantly reduced expression of lncRNAs that normally down-regulate TGF? signaling. Our data implicate lncRNAs in SDS bone marrow failure. To define how lncRNAs function in normal myelopoiesis and how their dysregulation is pathogenic in SDS, we will use a recently developed yeast three hybrid (Y3H) assay to systematically define lncRNA-protein interactions for 100 lncRNAs identified in normal and disease hematopoiesis. Y3H is capable of detecting low- abundance and low-affinity interactions lncRNA-protein interactions. Overlaying the lncRNA-protein interacting onto existing PPI networks will define lncRNA-regulated pathways of normal myelopoiesis and their dysregulation in SDS. Because we will screen of overlapping lncRNA fragments of 100 individual lncRNAs, these studies will enable systematic lncRNA structure-function analyses. The information obtained through this structure-function analysis will be critical for establishing sequence and/or structural parameters which may enable prediction of lncRNA-protein binding activity, thereby advancing our understanding of the biology of lncRNAs, even those not included in this initial screen. We will then validate roles for lncRNAs in hematopoiesis and altered TGF? activation. Specifically, we will use CD34+ cells depleted of SBDS and increase (by ectopic expression) or decrease (by knockdown) lncRNAs and their interacting proteins and test these effects in self renewal, myeloid lineage commitment, proliferation, cell death, oxidative stress, inflammation and TGF? activation. To further test roles for specific lncRNAs in TGF? signaling, we will also test the ability of TGF? inhibitors to rescue normal hematopoietic function. We will identify TGF?-responsive genes in affected subpopulations that could be targeted for novel SDS therapies, and test their function in SDS hematopoiesis. Thus, these studies will (1) identify lncRNAs, proteins, and lncRNA- protein interactions directing normal and SDS pathogenic myeloid function; (2) develop a platform for systemically studying lncRNAs; and (3) provide pre-clinical evidence that could support a clinical trial targeting lncRNA-directed pathways for future SDS therapy.

Public Health Relevance

These studies will implicate long non-coding RNAs (lncRNAs) and their interacting proteins function in normal hematopoiesis and bone marrow failure; these studies may also provide pre-clinical evidence for targeting specific lncRNA-protein interactions for Shwachman Diamond Syndrome therapy.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZHL1)
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Bai, C Brian
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Dana-Farber Cancer Institute
United States
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