Use of human iPSCs and kidney organoids to develop a cysteamine/mTOR inhibition combination therapy to treat cystinosis

Abstract

Cystinosis is a lysosomal storage disease caused by mutations in CTNS, encoding a cystine transporter, and in its severest form is characterized by cystine accumulation, renal proximal tubule dysfunction and kidney failure. Cystinosis is treated with the cystine-depleting drug cysteamine, however this only slows progression of the disease and there is an urgent need for better treatments. Here, we have generated and characterized the first human induced pluripotent stem cell (iPSC) and kidney organoid models of cystinosis. These models exhibit elevated cystine and cysteine levels, enlarged lysosomes and a block in basal autophagy flux.Cysteamine treatment ameliorates this phenotype except for the basal autophagy flux defect.We found that treatment with Everolimus, an inhibitor of the mTOR pathway, reduces the number of large lysosomes and activates autophagy but does not rescue the cystine/cysteine loading defect. However, dual treatment of cystinotic iPSCs or kidney organoids with cysteamine and Everolimus corrects all of the observed phenotypes indicating that a combination therapy has therapeutic potential to improve the treatment of cystinosis.

Cysteamine-bicalutamide combination treatment restores alpha-ketoglutarate and corrects proximal tubule phenotype in cystinosis

“…This in line with the study of Rega et al where stimulation of endogenous TFEB activity by genistein was shown to lower cystine levels and rescue the delayed endocytic cargo processing in cystinotic proximal tubule cells 16 . Furthermore, inhibition of mTOR signaling by everolimus was shown to activate autophagy, rescue the number of large lysosomes, and in combination with cysteamine, reverse the cystine/cysteine loading defect in patient-specific and CRISPR-edited cystinotic induced pluripotent stem cells and kidney organoids 48 .…”

Investigating the pathophysiology and potential therapeutic approaches for nephropathic cystinosis

“…Jamalpoor et al provided evidence that CTNS loss affects lysosomal function by hampering the delivery of newly synthesized lysosomal enzymes from Golgi to lysosomes and/or by inhibiting the maturation of these enzymes within (endo) lysosomal compartments. The latter hypothesis was further confirmed by Festa et al where they have shown the impairment of lysosome proteolysis is a result of defective lysosomal enzymes activation in in vitro and in vivo model of cystinosis [79]. mTOR inhibition has been found to resolve the impaired autophagic flux observed in cystinotic cells.…”

“…Similarly, several studies showed that the disruption in tight junction proteins triggers a cascade of events resulting in abnormal cell proliferation, reduced differentiation and repression of apical endocytic receptors, causing epithelial dysfunction in cystinosis [81][82][83]. Mito_TEMPO, a mitochondrial targeted antioxidant, restored the integrity, differentiation and transport function in cystinotic proximal tubular cells as well as in Ctns -/mice [79]. On the other hand, Janssens et al recently showed that blocking the uptake of disulfide-rich plasma proteins, through inhibiting megalin-mediated endocytosis, efficiently prevents accumulation of cystine and delays progression of kidney disease in Ctns -/mice [84].…”

“…Our current understanding of the pathogenesis of cystinosis is largely based on in vitro cell models. The pioneering studies which established intra-lysosomal accumulation of cystine as the hallmark of cystinosis were mainly performed on human cystinotic leukocytes [77], fibroblasts [78], or other lymph node cells [79]. Moreover, due to their relative availability, cystinotic skin fibroblasts are commonly used to study cystinosis pathology.…”

“…Being a major site of ROS production, mitochondria are more susceptible to damage [62] and together with the observed defective gamma glutamyl cycle and increased susceptibility of cystinotic cells to oxidative stress, this may explain the increase in mitophagy. Festa et al demonstrated that, oxidative stress resulting from abnormal mitophagy stimulates Gα12/Src-mediated phosphorylation of tight junction protein Zonula occludin-1 (ZO-1), and its subsequent misrouting to the endolysosome leading to disrupted tight junction integrity [79]. As a result, ZO-1-associated Y-box factor ZONAB is released, and further leads to epithelial dysfunction and dedifferentiation by promoting abnormal cell proliferation and repressing apical endocytic receptors such as megalin [80].…”