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ABSTRACTThe selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors 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.Abbreviations:AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type

ABSTRACTMacroautophagy/autophagy is a cellular process in which cytosolic contents are degraded by lysosome in response to various stress conditions. Apart from its role in the maintenance of cellular homeostasis, autophagy also involves in regulation of cell cycle progression under nutrient-deprivation conditions. However, whether and how autophagy is regulated by the cell cycle especially during mitosis remains largely undefined. Here we show that WIPI2/ATG18B (WD repeat domain, phosphoinositide interacting 2), an autophagy-related (ATG) protein that plays a critical role in autophagosome biogenesis, is a direct substrate of CUL4-RING ubiquitin ligases (CRL4s). Upon mitosis induction, CRL4s are activated via neddylation, and recruit WIPI2 via DDB1 (damage specific DNA binding protein 1), leading to polyubiquitination and proteasomal degradation of WIPI2 and suppression of autophagy. The WIPI2 protein level and autophagy during mitosis could be rescued by knockdown of CRL4sor treatment with MLN4924/Pevonedistat, a selective inhibitor of CRLs, via suppression of NAE1 (NEDD8 activating enzyme E1 subunit 1). Moreover, restoration of WIPI2 rescues autophagy during mitosis and leads to mitotic slippage and cell senescence. Our study thus discovers a novel function of CRL4s in autophagy by targeting WIPI2 for polyubiquitination and proteasomal degradation during mitosis.Abbreviations: ACTB, actin beta; ATG, autophagy-related; AMPK, AMP-activated protein kinase; AURKB/ARK2, aurora kinase B; BafA1, bafilomycin A1; CCNB1, cyclin B1; CDK1, cyclin dependent kinase 1; CHX, cycloheximide; CQ, chloroquine; CRL4s, CUL4-RING ubiquitin ligases; DDB1, damage specific DNA binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; GST, glutathione S-transferase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; STK11/LKB1,serine/threonine kinase 11; MTORC1/MTOR complex 1, mechanistic target of rapamycin kinase complex 1; NAE1, NEDD8 activating enzyme E1 subunit 1; NOC, nocodazole; RING, really interesting new gene; RBX1, ring-box 1; SA-GLB1/β-gal, senescence-associated galactosidase beta 1; TSC2, TSC complex subunit 2; TUBA, tubulin alpha; WIPI2, WD repeat domain, phosphoinositide interacting 2