Abstract
Introduction: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency, skeletal defects, neutropenia, and an increased risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Biallelic mutations in SBDS account for ~90% of patients. SBDS interacts physically with EFL1 to release EIF6 from the pre-60S ribosomal subunit and promote the assembly of the mature 80S ribosome in the cytoplasm. A subset of SDS patients acquire del(20q), resulting in loss of EIF6. Additionally, somatic heterozygous EIF6 mutations have been found in SDS patients. SDS patients harboring del(20q) and EIF6 mutations are linked to a lower risk of developing MDS/AML when compared to other SDS patients. Together these factors point to a potential mechanism whereby a reduction in EIF6 levels may lead to a suppression in ribosome biogenesis defects.
Methods and Results: We have created sbds knockout (KO) zebrafish strains that phenocopy the human syndrome with stunted growth, neutropenia and atrophy in the pancreas, liver, and intestine. Unlike the Sbds-null mice which die in early embryogenesis, sbds-null fish survive to early juvenile stages. The sbds KO fish display an accumulation of Eif6 in liver and intestine. Interestingly, we also found an accumulation of EIF6 in SDS patient-derived lymphoblasts. EIF6 has been described as a key regulator of metabolism, specifically stimulating glycolytic and fatty acid synthesis. Lipid metabolomics of sbds KO fish show a decrease in free fatty acids and phosphatidylcholine. Expression of several genes critical in lipid metabolism (srebp1, fasn and pparg) is upregulated in sbds KO fish. To study the role of Eif6 in the pathophysiology of SDS, we created an eif6 KO zebrafish with an insertion of one base pair resulting in a premature stop codon. By immunoblotting we demonstrated that mutants did not display Eif6 by 6 days post fertilization (dpf), and heterozygous fish displayed less protein than wildtype siblings. This new strain showed early mortality (~10 dpf), neutropenia, tp53 pathway activation, with a pronounced upregulation of cdkn1a as early as 5 dpf. RNA-Seq data of eif6 KO fish at 5 dpf showed a total of 2225 differentially expressed genes (DEGs): 668 downregulated and 1557 upregulated. These DEGs are involved in two main gene expression pathways: ribosome biogenesis and oxidative phosphorylation. We also observed a dysregulation of lipid metabolism markers in the eif6 KO fish. Furthermore, we bred the sbds KO with the eif6 KO, we observed a partial rescue on survival only in sbds-/- with one copy of eif6(eif6+/-) but failed to rescue their neutropenia.
Conclusions: Our organismal models of sbds or eif6 deletion provide new insights into the pathophysiology of human SDS: 1) SBDS affects lipid metabolism possibly due to an accumulation of EIF6, 2) Loss of eif6 affects development/survival at an earlier stage than loss of sbds, and 3) Loss of either sbds or eif6 markedly upregulates cdkn1a, which is downstream of tp53. Interestingly, Eif6 partially rescues survival of sbds-null organisms, but only in the haploinsufficient state. EIF6 may offer a promising target for a novel therapeutic strategy in SDS.
Disclosures
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
