Extracellular Vesicle Trafficking Regulates Hematopoietic Stem and Progenitor Cell Fitness

Among regulatory pathways critical to hematopoietic stem and progenitor cell (HSPC) function, sphingolipid metabolism has recently been recognized for its role in safeguarding essential HSPC properties of self-renewal and lineage fate. In particular, de novo ceramide (Cer) synthesis appears to regulate a coordinated stress response that results in enhanced self-renewal of human HSPCs. However, the fundamental molecular mechanisms underlying these regenerative stress responses have yet to be uncovered and broadly validated. Moreover, alternative synthetic pathways regulating Cer and sphingomyelin (SM) levels may serve additional regulatory roles in HSPCs.

Here we target neutral sphingomyelinase-2 (nSMase2/SMPD3)-dependent SM hydrolysis through pharmacologic (GW4869) and genetic (Cas9-mediated) interruption, and similarly confirm an adaptive ATF4-mediated integrated stress response (ISR). Functionally, transient inhibition led to durable improvements in HSPC reconstitution after primary (>8 fold at steady-state, p<0.001) and secondary (>20 fold at steady-state, p<0.001) transplantation. Surprisingly, lipidomic profiling of GW4869-treated HSPCs demonstrated neither global Cer depletion nor SM accumulation, suggesting an alternative mechanism underlying the observed gains in HSPC fitness.

Sphingolipid metabolism also regulates the biogenesis of select subclasses of secreted extracellular vesicles (EVs). To segregate metabolic regulation of HSPCs by sphingolipids from their role in EV formation and secretion, we began to systematically modulate and compare several core EV biogenesis pathways by Cas9/ribonucleoprotein-mediated knockout (KO) of SMPD3, CD63, and TSG101 in HSPC ex vivo expansion cultures. Similar to pharmacologic nSMase2 inhibition, loss of all 3 genes suppressed EV secretion, whereas only depletion of SMPD3 or ESCRT protein TSG101, but not the tetraspanin protein CD63, led to ISR activation in HSPCs (p<0.05). As a functional corollary, clonogenicity and long-term transplantation outcomes were enhanced by KO of SMPD3 (1.5-fold; p<0.01) and TSG101 (2-fold; p<0.001), but reduced after CD63-KO (0.6-fold; p<0.05). One function of EVs is the export of protein and nucleic acid cargo from the cell. We reasoned the observed differences in stress response and HSPC fitness may reflect uniquely altered secretomes. Quantitative proteomic analysis of the vesicle secretome across KO cells revealed impaired secretion of mitochondrial (n=160, FDR=8.3E-22) and oxidative stress-related (n=82, FDR=2.8E-12) proteins from SMPD3- and TSG101- KO, but not CD63-KO HSPCs, highlighting the heterogeneity and specificity of EV cargo trafficking. We validated this observation by pharmacologic blockade of SMPD3 with GW4869, which similarly led to transient decreases in mitochondrial DNA (0.36-fold, p<0.01) and protein (0.6-fold, p<0.05) export. Finally, we confirmed that impaired mitochondrial cargo secretion into EVs (mtEV) after SMPD3 or TSG101, but not CD63, depletion led to transient accumulation of mitochondrial-sourced reactive oxygen species (p<0.01) that specifically triggered the observed ISR activation and downstream mTOR inhibition.

Our study reveals a novel mechanism by which secretory cargo trafficking into EVs guards HSPC homeostasis and fitness. We further demonstrate that transient modulation of this pathway, wherein EV cargo retention activates a molecularly-defined stress response pathway, can durably improve long-term transplantation outcomes. Sphingolipid biology inextricably links cellular metabolism and EV biogenesis, making it likely that prior descriptions of Cer-dependent adaptive stress response involve modulation of EV secretion. Finally, our work underscores the high degree of specificity that governs EV secretion in regulating intrinsic cell function. In aggregate, we show that differential EV biogenesis pathways regulate HSPC fate and fitness, with important translational implications in safeguarding stemness and regenerative capacity during ex vivo expansion and therapeutic transplantation.