Canonical autophagy remains inactive in induced pluripotent stem cells and neuronal progenitor cells following DNA damage induced by BPDE or etoposide

ABSTRACT (Macro-)Autophagy is a key cellular stress response mediating the recycling of long-lived or damaged proteins and organelles. In stem cells, autophagy is essential for the decision between quiescence, self-renewal and differentiation. We observed that induced pluripotent stem cells (iPSCs) and thereof derived neural progenitor cells (NPCs) have a functional autophagy machinery, as shown by starvation-induced autophagic flux and ULK1 activation. Using the human iPSC line iPS11 and thereof derived NPCs (niPS11), we investigated whether genotoxic stress induced by benzo[a]pyrene diolepoxide (BPDE) or etoposide can similarly activate autophagy, as previously reported for cancer cell lines. While both BPDE and etoposide induced the DNA damage markers phospho-p53 Ser15 and γH2AX and slightly altered the expression of DNA repair proteins such as XPC, they did not trigger autophagic flux in either iPSCs or NPCs. After genotoxin treatment, ULK1 activation was only observed in NPCs, but this was not sufficient to trigger a significant downstream autophagic response. Mass spectrometry revealed minimal proteomic changes in iPSCs and moderate changes in NPCs, mainly involving mitotic regulators. These results suggest that genotoxic agents do not strongly affect canonical autophagy in pluripotent stem cells or their neural derivatives despite an otherwise responsive autophagic system. Competing Interest Statement The authors have declared no competing interest. Abbreviations - AMPK - AMP-activated protein kinase - ATG - autophagy-related - AURKA - aurora kinase A - BafA1 - bafilomycin A1 - BECN1 - beclin 1 - DDR - DNA damage response - FIP200 - focal adhesion kinase family interacting protein of 200 kDa - IC50 - half maximal inhibitory concentration - iPSC - induced pluripotent stem cells - (MAP1)LC3 - (microtubule-associated proteins 1A/1B) light chain 3 - mTOR - mechanistic target of rapamycin - NPC - neural progenitor cell - NUCKS1 - nuclear ubiquitous casein kinase and cyclin-dependent kinase substrate 1 - PLK1 - polo like kinase 1 - RB1CC1 - retinoblastoma 1 inducible coiled-coil 1 - SQSTM1 - sequestosome 1 - V-ATPase - vacuolar-type H+-ATPase