NODAL/TGFβ signalling mediates the self-sustained stemness induced by PIK3CA^H1047R homozygosity in pluripotent stem cells

Activating PIK3CA mutations are known 'drivers' of human cancer and developmental overgrowth syndromes. We recently demonstrated that the 'hotspot' PIK3CA^H1047R variant exerts unexpected allele dose-dependent effects on stemness in human pluripotent stem cells (hPSCs). In this study, we combine high-depth transcriptomics, total proteomics and reverse-phase protein arrays to reveal potentially disease-related alterations in heterozygous cells, and to assess the contribution of activated TGFβ signalling to the stemness phenotype of homozygous PIK3CA^H1047R cells. We demonstrate signalling rewiring as a function of oncogenic PI3K signalling strength, and provide experimental evidence that self-sustained stemness is causally related to enhanced autocrine NODAL/TGFβ signalling. A significant transcriptomic signature of TGFβ pathway activation in heterozygous PIK3CA^H1047R was observed but was modest and was not associated with the stemness phenotype seen in homozygous mutants. Notably, the stemness gene expression in homozygous PIK3CA^H1047R hPSCs was reversed by pharmacological inhibition of NODAL/TGFβ signalling, but not by pharmacological PI3Kα pathway inhibition. Altogether, this provides the first in-depth analysis of PI3K signalling in hPSCs and directly links strong PI3K activation to developmental NODAL/TGFβ signalling. This work illustrates the importance of allele dosage and expression when artificial systems are used to model human genetic disease caused by activating PIK3CA mutations.