SLC29A3 lysosomal transporter in hematopoietic homeostasis and disease

Mutations exclusively in SLC29A3, which encodes the lysosomal transporter termed equilibrative nucleoside transporter-3 (ENT3), cause an expanding spectrum of human genetic disorders, such as H syndrome, PHID syndrome, RDD syndrome, SHML syndrome, dysosteosclerosis etc. They share common mutations and overlapping clinical manifestations with anemia, erythroid hypoplasia and hepatosplenomegaly as characteristic signs. Our group has recently described an indispensable role of ENT3 in the maintenance of hematopoietic stem cell (HSC) homeostasis via the genetic deletion of ENT3 in mice. Intriguingly, ENT3- deleted mice manifest clinical signs closely resembling human SLC29A3 disorders. The objective of this application is to evaluate the molecular disease pathogeneses and treatments of SLC29A3 genetic disorders. Specifically, it is our hypothesis that interference with the lysosome-to-ER mobilization of bile acid (BA) chemical chaperones and ER stress signaling in HSCs underlies the pathogeneses of SLC29A3 dysfunctions and that endogenous or synthetic chaperones will serve as suitable treatment agents to overcome these disorders. This hypothesis is based on preliminary data showing that BA chemical chaperones are novel cargos of ENT3, showing ENT3 conferring the BA-dependent amelioration of ER stress signaling in HSCs, and showing improved function of ENT3-deleted HSCs after treatment with salubrinal, a chemical ER stress reducer. The rationale for this project is that the identification of the mechanisms of ER stress regulation by ENT3 in HSCs will determine the molecular disease pathogenesis of SLC29A3 disorders, which would provide therapeutic opportunities to treat the SLC29A3 genetic disorders. This central hypothesis will be tested by pursuing two specific aims. Specific Aim 1 will evaluate ENT3 transport and the subcellular disposition of BA chemical chaperones in HSCs. The working hypothesis is that ENT3 promotes the lysosome-to-ER mobilization of BA and that the loss of ENT3 will reduce the ER accumulation of BAs with increased sequestration in the lysosome. We will use new investigational models, ENT3 structure-function analysis, novel BA molecular probes, time lapse imaging, subcellular pharmacokinetics, and mass spectrometry studies to address this aim. Specific Aim 2 will evaluate aberrant ER stress signaling as the basis of HSC dysfunction in SLC29A3 disorders. Key questions connecting ENT3-regulated ER stress signaling and SLC29A3 disease pathologies will be evaluated in steady-state and stressed HSCs using HSC-specific, inducible and conditional ENT3 KO mice. Furthermore, endogenous and synthetic chaperones and pharmacological modifiers of ENT3 misfolding, trafficking, and degradation will be evaluated to identify suitable treatment agent(s) for overcoming SLC29A3 disorders. Completion of this project will illuminate the pathogenetic basis and treatments for SLC29A3 genetic disorders and will provide unique insight into the ENT3-regulated BA chaperone defense mechanisms in the physiological ER stress signaling in HSCs.