Impact of Dkc1 Deficiency on BM Niche Function during Hematopoietic Stem Cell Transplantation in a Conditional Murine Model of X-Linked Dyskeratosis Congenita
Authors: Zha Ji , Congdon Rheanna Grace , Kunselman Lori , Bigham Olivia , Olson Timothy S 
Published: December 2023

Dyskeratosis congenita (DC) is a telomere biology disorder associated with bone marrow failure (BMF) and the triad of dystrophic nails, skin rashes, and leukoplakia. X-linked DC (XDC) is caused by mutations in DKC1, which encodes dyskerin, a nucleolar protein associated with rRNA processing and telomerase complex formation. Hematopoietic stem cell transplantation (HSCT) is currently the only definitive curative treatment for BMF in XDC patients, though many patients exhibit only partial restoration of hematopoietic function. Our previous studies demonstrated that molecular and cellular defects in BM niches contribute to poor donor HSC engraftment in murine models of other BMF syndromes, including Shwachman-Diamond Syndrome (SDS), a disease whose pathogenesis intersects with the role of dyskerin in ribosome biogenesis. These studies led us to hypothesize that BM niche dysfunction contribute to poor graft function in DC patients post-HSCT. Defining specific deficits would enable development of novel targeting strategies to improve HSCT outcomes for patients with XDC. Here, we report that in a novel murine model of XDC driven by conditional Dkc1 deletion, Dkc1 deficiency within mesenchymal BM niche cells causes significant impairment of healthy donor HSC engraftment after HSCT. We have identified defects in multiple pathways within Dkc1-deficient BM niche in response to myeloablative conditioning that may drive this reduction in HSC supportive capacity.
To generate a murine model with conditional Dkc1 deletion in BM niche, we crossed Dkc1l/lmice with Mx1 Cre+mice, inducing Cre expression in hematopoietic cells and Mx1-inducible niche cells by polyinosinic-polycytidylic acid administration. At baseline, the resulting Mx1 CreDkc1 Exc mice exhibit mild hematopoietic abnormalities versus Dkc1 l/l controls, including variable thrombocytopenia, increased blood monocytes, and reduced BM lymphoid progenitors. To assess capacity of BM niches from Mx1 CreDkc1 Exc mice to efficiently engraft donor HSC, we transplanted Mx1 CreDkc1 Exc mice and Dkc1 l/l controls with healthy GFP + wild-type donor BM and assessed efficiency of donor HSC engraftment at 2 weeks following HSCT using competitive secondary HSCT. The Mx1 CreDkc1 Exc recipients exhibited significantly impaired efficiency of donor HSC engraftment compared with controls, as indicated by a 25-30% reduction of long-term GFP + cell reconstitution in mature blood lineages in secondary recipients (Figure 1).
We next sought to identify molecular and cellular defects in BM niche that contribute to the engraftment deficits in Mx1 CreDkc1 Exc mice. Flow cytometry analysis of BM niche cells 48 hours after total body irradiation (TBI) showed Mx1 CreDkc1 Exc mice exhibited a 20% reduction of CD11b+F4/80+ osteomacs compared to Dkc1 l/l controls. In contrast to our published findings in a model of SDS, the frequencies of osteoprogenitors, mesenchymal stem cell, endothelial cells and megakaryocytes in BM niche were similar between irradiated Dkc1-deficient and control mice. We then performed multiplex ELISA and RNA-seq analyses on isolated BM niche cells from Mx1 CreDkc1 Exc versus control BM at 48 hours post-TBI. Irradiated Mx1 CreDkc1 Exc BM niche demonstrated lower expression of multiple chemokines, including CXCL1, CXCL12, CCL1, CCL11 and CCL12, known to be critical for recruiting HSC and many leukocyte subsets. Dkc1 deficiency in post-TBI BM niche cells also led to lower expression of genes that regulate production of IFN-γ and IL-1, resulting in reduced expression level of both cytokines that may drive decreased osteomacs seen in Dkc1-deficient BM niches. Moreover, Gene Ontology analysis indicates that after TBI, Dkc1 deficiency within BM niches resulted in downregulation of gene expression involved in cell-cell adhesion and extracellular matrix, both of which are critical for HSC enlodgement and engraftment after HSCT.
Taken together, our results demonstrated that Dkc1 deficiency in BM niche cells that are not replaced by HSCT impairs the ability of these niches to engraft donor HSC efficiently. Disruptions of molecular and cellular pathways implicated in the niche dysfunction of this XDC model are distinct from our previous findings with SDS model. Ongoing and future studies are needed to define which of these pathways have the highest potential to serve as translational targets to improve HSCT success rate to treat BMF in patients with XDC.