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代謝重新布線對于 AML 細(xì)胞存活以克服 ATG3 缺失導(dǎo)致的自噬抑制至關(guān)重要

發(fā)表日期：2022-03-03

Metabolic Rewiring Is Essential for AML Cell Survival to Overcome Autophagy Inhibition by Loss of ATG3

代謝重新布線對于 AML 細(xì)胞存活以克服 ATG3 缺失導(dǎo)致的自噬抑制至關(guān)重要

The importance of autophagy in leukemia progression and survival has been studied previously. However, little is known about the development of resistance mechanisms to autophagy inhibition in leukemia. Here, we present data on the mechanisms by which leukemia cells maintain their cell survival after inhibition of autophagy by the loss of ATG3. After the loss of ATG3, leukemia cells upregulated their energy metabolism by increasing glycolysis and mitochondrial metabolism, in particular oxidative phosphorylation, which resulted in higher ATP levels. Moreover, inhibition of mitochondrial function strongly impaired cell survival in ATG3 deficiency, thus demonstrating the importance of ATG3 in the regulation of metabolism and survival of leukemic cells. Therefore, our data provide a rationale for combining autophagy inhibitors with inhibitors targeting mitochondrial metabolism for the development of leukemia therapy to overcome the potential obstacle of emerging resistance to autophagy inhibition.

先前已經(jīng)研究了自噬在白血病進展和存活中的重要性。然而，對于白血病中自噬抑制的抗性機制的發(fā)展知之甚少。在這里，我們提供了白血病細(xì)胞在 ATG3 缺失抑制自噬后維持細(xì)胞存活的機制的數(shù)據(jù)。在失去 ATG3 后，白血病細(xì)胞通過增加糖酵解和線粒體代謝，特別是氧化磷酸化，上調(diào)其能量代謝，從而導(dǎo)致更高的 ATP 水平。此外，線粒體功能的抑制會嚴(yán)重?fù)p害 ATG3 缺乏癥的細(xì)胞存活，從而證明 ATG3 在調(diào)節(jié)白血病細(xì)胞代謝和存活中的重要性。

細(xì)胞工廠

Acute myeloid leukemia (AML) develops from malignant clonal expansion of undifferentiated myeloid progenitors, causing bone marrow failure. Despite advances in the treatment of AML, prognosis remains poor for most patients. Consequently, there is an unmet medical need to find novel therapeutic approaches targeting AML onset and progression.

Several studies have underlined the pivotal role of autophagy in the development and progression of AML [1,2]. Autophagy is a well-known regulator of cellular metabolism contributing to homeostasis and cell survival [3]. The self-degradative property of cytosolic macromolecules is central to autophagy thereby serving as a nutrient source for building blocks during limited energy supply [4]. Proliferating cancer cells are exposed to permanent nutrient deprivation and maintain high energy levels by rewiring metabolic pathways [5,6]. In particular, AML cells are highly dependent on mitochondrial metabolism, as we and others have demonstrated the fundamental role of mitophagy in maintaining mitochondrial integrity in AML [7,8,9]. Therefore, numerous studies have presented the promising approach of targeting autophagy together with different chemotherapies for cancer therapy [10]. However, inherent and acquired resistance to autophagy inhibition has been reported in clinical trials, resulting in further tumor progression after initial response [11]. Importantly, autophagy-related proteins (ATG) including ATG5 and ATG7, which are well known critical autophagy genes, have been reported to promote cell proliferation in leukemia [12,13]. Furthermore, the core autophagy gene ATG3, which directly participates in autophagosome formation by conjugating phosphatidyl-ethanolamine with LC3 [14,15,16], was shown to be essential for leukemogenesis [17].

Despite the vast number of studies exploring autophagy, the mechanisms involved in overcoming autophagy inhibition are poorly understood. In this study, we identified ATG3 as a key player for survival of leukemia cells and present conclusive evidence supporting that upon loss of ATG3 AML cells rewire their central carbon metabolism to evade survival disruption upon autophagy inhibition. Firstly, we performed a CRISPR/Cas9 screen using an autophagy library to identify genes essential for AML proliferation. We found ATG3 to be important for AML cell proliferation and essential for autophagy. By analyzing the effect of ATG3 deficiency in AML cell lines, we found increased levels of mitochondrial reactive oxygen species and upregulated oxidative phosphorylation. By utilizing uniformly labeled 13C-glucose nuclear magnetic resonance (NMR) analysis, we observed increased glycolysis and enhanced activity of mitochondrial metabolism upon ATG3 loss. Furthermore, ATG3 deficiency sensitized AML cells to inhibition of mitochondrial respiration. These results suggest a combination of inhibitors targeting autophagy and mitochondrial metabolism as promising approach to treat AML.

細(xì)胞搖瓶

急性髓性白血病 (AML) 由未分化的髓系祖細(xì)胞的惡性克隆擴增發(fā)展而來，導(dǎo)致骨髓衰竭。盡管 AML 的治療取得了進展，但大多數(shù)患者的預(yù)后仍然很差。因此，尋找針對 AML 發(fā)病和進展的新治療方法的醫(yī)療需求尚未得到滿足。

一些研究強調(diào)了自噬在 AML 的發(fā)展和進展中的關(guān)鍵作用 [ 1 , 2 ]。自噬是眾所周知的細(xì)胞代謝調(diào)節(jié)劑，有助于體內(nèi)平衡和細(xì)胞存活 [ 3 ]。細(xì)胞溶質(zhì)大分子的自降解特性是自噬的核心，因此在有限的能量供應(yīng)期間可作為構(gòu)建模塊的營養(yǎng)來源 [ 4 ]。增殖的癌細(xì)胞暴露在永久的營養(yǎng)缺乏中，并通過重新連接代謝途徑來維持高能量水平 [ 5 , 6]。特別是，AML 細(xì)胞高度依賴線粒體代謝，因為我們和其他人已經(jīng)證明了線粒體自噬在維持 AML 中線粒體完整性方面的基本作用 [ 7 , 8 , 9 ]。因此，許多研究提出了一種有前景的靶向自噬的方法以及用于癌癥治療的不同化學(xué)療法[ 10 ]。然而，在臨床試驗中報告了對自噬抑制的固有和獲得性抗性，導(dǎo)致初始反應(yīng)后腫瘤進一步進展 [ 11 ]。重要的是，據(jù)報道，自噬相關(guān)蛋白 (ATG) 包括 ATG5 和 ATG7，它們是眾所周知的關(guān)鍵自噬基因，可促進白血病細(xì)胞增殖。12、13 ]。_ 此外，通過將磷脂酰乙醇胺與 LC3 結(jié)合直接參與自噬體形成的核心自噬基因 ATG3 [ 14 , 15 , 16 ]，被證明對白血病發(fā)生至關(guān)重要 [ 17 ]。

盡管有大量研究探索自噬，但對克服自噬抑制的機制知之甚少。在這項研究中，我們將 ATG3 確定為白血病細(xì)胞存活的關(guān)鍵因素，并提供確鑿的證據(jù)支持在 ATG3 喪失后，AML 細(xì)胞重新連接其中心碳代謝，以逃避自噬抑制后的存活中斷。首先，我們使用自噬文庫進行了 CRISPR/Cas9 篩選，以鑒定 AML 增殖所必需的基因。我們發(fā)現(xiàn) ATG3 對 AML 細(xì)胞增殖很重要，對自噬至關(guān)重要。通過分析 ATG3 缺乏對 AML 細(xì)胞系的影響，我們發(fā)現(xiàn)線粒體活性氧物質(zhì)水平升高和氧化磷酸化上調(diào)。通過使用統(tǒng)一標(biāo)記的13C-葡萄糖核磁共振 (NMR) 分析，我們觀察到 ATG3 損失后糖酵解增加和線粒體代謝活性增強。此外，ATG3 缺乏使 AML 細(xì)胞對線粒體呼吸的抑制敏感。這些結(jié)果表明，靶向自噬和線粒體代謝的抑制劑組合是治療 AML 的有希望的方法。

細(xì)胞培養(yǎng)瓶

In our study, we identified ATG3 as a core autophagy gene essential for AML cell survival by performing a CRISPR/Cas9 proliferation screen targeting 192 autophagy-related genes. By genetically depleting ATG3 in human leukemia cell lines, we demonstrate that AML cells rewire their energy metabolism to sustain cell survival when autophagy is impaired. NMR analysis revealed upregulation of glycolysis and OXPHOS upon ATG3 deficiency, which was accompanied by increased mitochondrial ROS and ATP production. Importantly, while ATG3-deficient cells are resistant to inhibition of glycolysis, we show that inhibition of OXPHOS severely reduces AML cell survival, identifying their dependence on mitochondrial metabolism as arising vulnerability upon autophagy inhibition (Figure 6).Taken together, our findings indicate the importance of ATG3 in the regulation of metabolism and survival of AML cells, as inhibition of mitochondrial oxidative phosphorylation severely impaired cell survival upon loss of ATG3. Moreover, targeting both autophagy and mitochondrial oxidative phosphorylation might be a potential therapeutic strategy for AML.

在我們的研究中，我們通過針對 192 個自噬相關(guān)基因進行 CRISPR/Cas9 增殖篩選，將 ATG3 鑒定為對 AML 細(xì)胞存活至關(guān)重要的核心自噬基因。通過基因消耗人類白血病細(xì)胞系中的 ATG3，我們證明 AML 細(xì)胞重新連接其能量代謝以在自噬受損時維持細(xì)胞存活。NMR分析顯示ATG3缺乏時糖酵解和OXPHOS的上調(diào)，伴隨著線粒體ROS和ATP產(chǎn)生的增加。重要的是，雖然 ATG3 缺陷細(xì)胞對糖酵解的抑制具有抗性，但我們表明 OXPHOS 的抑制會嚴(yán)重降低 AML 細(xì)胞的存活率，從而確定它們對線粒體代謝的依賴性是自噬抑制后出現(xiàn)的脆弱性（圖 6）。總之，我們的研究結(jié)果表明 ATG3 在調(diào)節(jié) AML 細(xì)胞的代謝和存活中的重要性，因為抑制線粒體氧化磷酸化會在 ATG3 缺失后嚴(yán)重?fù)p害細(xì)胞存活。此外，靶向自噬和線粒體氧化磷酸化可能是 AML 的潛在治療策略。