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

The 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. <b>Abbreviations:</b> 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

通过线粒体自噬（mitophagy）实现的线粒体选择性降解，对于维持线粒体稳态至关重要，进而影响急性髓系白血病（AML）的病情维持与疾病进展。线粒体自噬由多种线粒体自噬受体协同调控，但其相互作用机制尚未完全阐明。本研究构建了靶向线粒体自噬受体的成对多重CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats）筛选体系，以解析其功能冗余性，并深入阐明调控AML中线粒体自噬的功能互作组。我们鉴定出视神经蛋白（optineurin, OPTN）是唯一的非冗余线粒体自噬受体，并阐明了其在AML中的独特功能。敲低（knockdown, KD）与过表达（overexpression, OE）实验证实，OPTN的表达水平是AML细胞增殖的限速因素。在MN1驱动的小鼠移植模型中，OPTN敲除（knockout, KO）可使小鼠中位总生存期延长7天（增幅达21%）。机制研究显示，OPTN缺失会广泛损伤线粒体呼吸功能与线粒体完整性，并伴随线粒体活性氧（reactive oxygen species, ROS）水平升高，这极有可能是导致细胞增殖缺陷的原因。本研究解析了AML中依赖泛素的线粒体自噬与受体介导的线粒体自噬所涉及的线粒体自噬受体互作网络，证实OPTN是白血病背景下研究线粒体自噬的非冗余工具，并提示OPTN抑制有望成为极具潜力的治疗策略。<b>缩写说明：</b> AML: 急性髓系白血病（acute myeloid leukemia）；CRISPR: 成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats）；CTRL: 对照组（control）；DFP: 去铁酮（deferiprone）；GI: 遗传互作（genetic interaction）；KD: 敲低（knockdown）；KO: 敲除（knockout）；ldMBM: 谱系剔除小鼠骨髓（lineage-depleted murine bone marrow）；LFC: 对数2倍变化（log2 fold change）；LIR: LC3互作区域（LC3-interacting region）；LSC: 白血病干细胞（leukemic stem cell）；MAGeCK: 全基因组CRISPR-Cas9敲除模型分析（Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout）；MDIVI-1: 线粒体分裂抑制剂1（mitochondrial division inhibitor 1）；MOI: 感染复数（multiplicity of infection）；MOM: 线粒体外膜（mitochondrial outer membrane）；NAC: N-乙酰-L-半胱氨酸（N-acetyl-L-cysteine）；OA: 寡霉素-抗霉素A（oligomycin-antimycin A）；OCR: 氧消耗速率（oxygen consumption rate）；OE: 过表达（overexpression）；OPTN: 视神经蛋白（optineurin）；PINK1: PTEN诱导推定激酶1（PTEN induced putative kinase 1）；ROS: 活性氧（reactive oxygen species）；SEM: 均值标准误（standard error of the mean）；TCGA: 癌症基因组图谱（The Cancer Genome Atlas）；TEM: 透射电子显微镜（transmission electron microscopy）；UBD: 泛素结合结构域（ubiquitin-binding domain）；WT: 野生型（wild type）