A zebrafish model of parn deficiency identifies altered lipid metabolism as a novel therapeutic target for restoring hematopoiesis in dyskeratosis congenita Free

Dyskeratosis congenita is a type of inherited bone marrow failure syndrome (IBMFS) that is characterized by multilineage cytopenia and the classic triad of nail dystrophy, oral leukoplakia and reticular hyperpigmentation. Definitive treatment requires allogenic hematopoietic stem cell transplantation (HSCT). However, DC patients often develop organ dysfunction and secondary malignancies with HSCT. On the other hand, DC patients who do not undergo HSCT have a 13% cumulative risk of developing myelodysplastic syndrome (MDS). Whilst alternative therapies such as androgens improve hematopoietic function, they cause significant side effects including liver adenomas, splenic peliosis and increased risk of cardiovascular disease. There is hence an unmet need for developing targeted therapies informed by the underlying DC-biology. Germline mutations in the poly(A)-specific ribonuclease (PARN) gene are associated with a severe variant of DC called Hoyeraal-Hreidarsson syndrome, where bone marrow failure results in early mortality in the first decade of life.

We generated a novel zebrafish model of Parn deficiency by introducing germline deletions using CRISPR/Cas9 mutagenesis. Mutant fish exhibited reduced differentiation of multiple hematopoietic lineages during both embryonic and adult hematopoiesis, effectively phenocopying the multilineage cytopenia seen in DC patients. Additionally, parn^-/- whole kidney marrows (WKMs, human bone marrow equivalent) showed an abnormal expansion of myeloid progenitors. To determine the underlying molecular mechanisms supporting this expansion, we performed bulk RNA sequencing of WKMs. Transcriptional analysis identified upregulated expression of stem cell-promoting genes (Wnt and Hox). In addition, several pathways pertaining to lipid metabolism were dysregulated; linoleic acid metabolism and fatty acid biosynthesis were downregulated, whereas cholesterol biosynthesis and arachidonic acid metabolism were upregulated. Both rate-limiting enzymes of the cholesterol biosynthesis pathway, hmgcra and sqlea,were elevated. Downstream, these changes led to the aberrant activation of the MAPK/Erk pathway in both the liver and WKM of mutant fish.

We further characterized the lipid composition of WKMs using untargeted lipidomics by liquid chromatography-mass spectrometry. Mutant WKMs exhibited increased levels of saturated fatty acids such as eicosanoic acid, octadecanoic acid and nonadecanoic acid as well as several fatty acid esters of hydroxy fatty acids. Lipid reaction analysis showed a preferential upregulation of phosphatidylethanolamine (PE) production. On the other hand, several ether-linked PEs were downregulated. We also assessed the capacity for lipid transport using BODIPY-C₁₂, a long-chain fatty acid analogue tagged with the BODIPY fluorophore. Larvae were injected with BODIPY-C₁₂ in the yolk sac and fluorescence in the vasculature was assessed 48 hours post-injection. Mutant larvae exhibited reduced fluorescence suggesting poor lipid transport. Given the elevated levels of cholesterol synthesis genes and fatty acids in the mutants, we asked if treatment with the cholesterol synthesis inhibitors fatostatin or terbinafine hydrochloride would improve hematopoiesis. Larvae were treated with 10 nM fatostatin and 100 μM terbinafine hydrochloride from 24 hpf to 4 days post-fertilization. Excitingly, cholesterol inhibition with either drug significantly improved erythrocyte levels in the mutants.

In sum, leveraging novel mechanistic insights and preclinical testing in the zebrafish, we present the modulation of lipid metabolism as a promising therapy to restore hematopoiesis and prevent malignancy in DC.