Erythroleukemia Cells Acquire An Alternative Mitophagy Capability

Objective:To explore autophagic mechanism for leukemic advantage in erythroleukemia cells and to answer why leukemia cells are superior to normal hematopoietic cells with differentiation potential in buffering cellular stresses.Methods:(1) Construct Atg7-/- K562 cell line with the CRISPR/Cas9 system;(2) Check the expansion rate, apoptosis ratio and differentiation potential of Atg7-/- K562 using cell counts, Anexxin-Ⅴ/PI staining and CFU, respectively;(3) Check the status of autophagy under transmission electron microscope and mitochondrial mass, ROS levels with flow cytometry;(4) Detect the mitochondrial mass and the expression of TOMM20 in Atg7-/- K562 with flow cytometry and western blotting;(5) Check the expression of key alternative autophagy proteins by immunoblotting;(6) Detect the mitochondrial mass under brefeldin A(BFA) or knocking down RAB9A with flow cytometry;(7) Check the expression of key DNA damage repair proteins under autophagy inhibition post irradiation by immunoblotting.Results:(1) Successfully generation of Atg7-/- K562 cell line with the CRISPR/Cas9 system;(2) Atg7 deletion led to G2/M arrest and reduces differentiation potential, without alternation of mitochondral mass and reactive oxygen species(ROS) level;(3) Autophagosomes can still be observed in Atg7-/- K562 cells;(4) CCCP-induced mitophagy occurred independent of Atg7;(5) CCCP-induced mitophagy can be blocked by BFA or RAB9 A knocking down;(6) Apoptosis and ROS level were elevated when both canonical autophagy and alternative autophagy were blocked;(7) Alternative autophagy functioned in DNA damage repair.Conclusion:The erythroleukemia cells procure an Atg7-independent alternative mitophagy capabilityeven when their canonical autophagy is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage insult.