Human pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities

Abstract

TSC2 inactivating mutations elicit mTORC1 hyperactivation and underlie neurological dysfunction and the development of neural and mesenchymal tumors in the monogenic disease tuberous sclerosis complex (TSC). We present a multi-lineage model of TSC2-deficiency employing CRISPR-Cas9 engineering in human pluripotent stem cells (hPSCs) and differentiation into neural and neural crest lineages, cell types predicted to drive TSC manifestations. Temporal RNA-sequencing reveals a massive proteostatic stress response underlying early neuroepithelial induction of TSC2-deficient cells, which is resolved upon neural crest cell (NCC) specification but persists in neural precursor cells (NPCs). This culminates in long-term endosomal and metabolic reprogramming as cells age, and sensitivity of TSC2 -/-NPCs, but not NCCs, to death via proteasome inhibition independent of mTORC1 activity. Thus, TSC2-deficiency induces lineage-specific stress adaptations which confer differential sensitivity to a commonly targeted pathway. These results exemplify the complexity of elucidating underlying biological mechanisms and therapeutic approaches for multisystem diseases, illustrating the power of utilizing hPSC disease models with tissue-specific relevance.

Organelle Cooperation in Stem Cell Fate: Lysosomes as Emerging Regulators of Cell Identity

“…AMPK −/− ESCs maintain pluripotency but fail to generate chimeric embryos and exhibit a preference for ectodermal compared to endodermal differentiation, due to hypophosphorylation of Tfeb and its reduced nuclear localization (Young et al, 2016). This study and others position coordinated regulation of AMPK and mTORC1 signaling to be an essential regulatory node in PSCs that determines an ectodermal or endodermal cell fate, with hyper-active mTORC1 signaling consistently leading to preferential ectodermal differentiation (Zhou et al, 2009;Young et al, 2016;Delaney et al, 2019;Jaiswal and Kimmel, 2019).…”

“…Our recent work further implicates lysosome activation as a critical requirement for NSC fate regulation, in the initial determination of neural lineage identity (Delaney et al, 2019; Figure 1). We developed a modeling system of the multi-system tumor disorder tuberous sclerosis by engineering inactivating TSC2 mutations in human PSCs, thereby inducing mTORC1 hyperactivation, and subsequently differentiating them in a directed manner into NSCs and the developmentally related neural crest (NC).…”

“…Of note, a recent study revealed that cells undergoing NSC specification undergo a reprogramming of protein chaperone networks, which are intimately connected with proteostatic mechanisms; this finding underscores the importance of proteostasis signaling integrity during early NSC development (Vonk et al, 2020). Furthermore, lysosome content changed dynamically throughout NSC induction in TSC2 −/− cells, and these patterns correlated with alterations in the expression of cell fate markers (Delaney et al, 2019).…”

“…Demonstrating that lysosomes and other organelles can affect cell identity determination in distinct ways, this mitochondrial remodeling was strikingly independent of autophagy, evidenced by findings that mitochondria-lysosome co-localization could not be observed and lysosom inhibition with bafilomycin or shRNA targeted knock-down of autophagy regulators (Atg5, Beclin or Vps34) did not affect mitochondrial mass (Wang et al, 2013). The requirement for mTORC1 inhibition in regulating pluripotency appears to be limited to the initial stage of cell identity determination, as we and others have demonstrated that the loss or reduction of TSC2, limiting the capacity to inhibit mTORC1, does not affect the maintenance of a pluripotent state in established human PSCs (Julian et al, 2017a;Blair et al, 2018;Delaney et al, 2019). Thus, mTORC1 inhibition and consequent autophagy activation appears to be critical specifically at a very early stage of cell fate transition (Figure 1).…”

“…The aberrant lysosome activation driven by TSC2-deficiency was transient during neural development but became reactivated with time as NSC cultures were aged, in a manner that paralleled altered expression of cell fate markers (Delaney et al, 2019). These findings suggest a close association of lysosome biogenesis with cell fate in NSC populations, both during their initial specification and long-term maintenance.…”