Deciphering the mitophagy receptor network identifies a crucial role for optineurin in acute myeloid leukemia

Background: The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, for disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Aims: We intended to systematically investigate the molecular mechanisms of mitophagy in AML and identify critical non-redundant components of the mitophagy receptor network required for leukemia maintenance and progression. Methods: We established a multiplexed CRISPR/Cas9 screen targeting mitophagy receptors and their pairwise combinations to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. Follow-up in vitro studies utilizing clonogenic assays, flow cytometry, fluorescence microscopy, transmission electron microscopy, immunoblotting, metabolic profiling, genetic manipulations with shRNA & CRISPR/Cas9 techniques as well as in vivo studies on syngeneic murine AML models with genetic or pharmacologic impairment of mitophagy were performed. Results: CRISPR/Cas9 experiments with a multiplexed gRNA library targeting all possible combinations of mitophagy receptors in AML cells constitutively expressing a mitophagy receptor (mt-mKeima) mapped the functional interactome of mitophagy receptors in AML. This pinpointed OPTN as a critical and independent mitophagy receptor and we subsequently characterized its unique role in both murine and human AML. Knockdown and overexpression experiments as well as rescue experiments reconstituting autophagy-deficient mutants or wild-type OPTN demonstrated that OPTN expression is rate-limiting for mitophagy and AML cell proliferation. Loss of OPTN delayed leukemia development in a MN1-driven murine transplantation model. Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS upon loss of OPTN. Exogeneous addition of the ROS scavenger N-acetylcystein rescued the impaired proliferation of OPTN-deficient cells. Summary/Conclusion: Our results decipher the mitophagy receptor network in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy in AML. Keywords: AML, Acute myeloid leukemia