Introduction 介绍

Brain tumors are the leading cause of cancer death in children under 201. Medulloblastoma (MB) is the most common primary pediatric brain malignancy, representing over 20% of newly diagnosed childhood central nervous system cancers2. Over the past decade, proteogenomic profiling of hundreds of MB tumors from several independent groups has classified MB tumors into at least four consensus molecular subgroups: Wingless (WNT) MB, Sonic Hedgehog (SHH) MB, group 3 (G3) MB and group 4 (G4) MB3,4. The prognosis for MB patients varies greatly depending on the molecular subgroup of the tumor, with the best survival rate being >95% in the WNT subgroup and the deadliest subgroup, G3 MB, harboring a survival rate of <60%5.
脑肿瘤是 20 岁以下儿童癌症死亡的主要原因1 。髓母细胞瘤 (MB) 是最常见的原发性儿童脑恶性肿瘤,占新诊断儿童中枢神经系统癌症的 20% 以上2 。在过去的十年中,对来自几个独立组的数百个 MB 肿瘤进行了蛋白质组学分析,将 MB 肿瘤分为至少四个一致的分​​子亚组:Wingless (WNT) MB、Sonic Hedgehog (SHH) MB、第 3 组 (G3) MB 和第 4 组(G4)MB 3 , 4 。 MB患者的预后根据肿瘤的分子亚型而有很大差异,WNT亚型的最佳生存率>95%,而最致命的G3 MB亚型的生存率<60% 5

The oncogene c-MYC (MYC hereafter) is commonly amplified and overexpressed in G3 MB but not in other subgroups6. High levels of MYC are associated with poor outcomes (~40% survival) and unresponsiveness to almost all current therapies6. Treatment options for G3 MB patients are limited, where the current standard of care involves surgery followed by radiation therapy and cytotoxic chemotherapy (i.e., cisplatin and vincristine with lomustine or cyclophosphamide)7. Moreover, patients who survive often suffer severe long-term health problems due to the treatments they received as a child8. While there are limited targeted therapies under clinical evaluation for MB brain tumors (NCT03434262; NCT04023669; and NCT01878617), further efforts are needed to expand treatment options and to identify effective agents with less toxic effects on the developing nervous system.
癌基因 c- MYC (以下简称 MYC)通常在 G3 MB 中扩增和过表达,但在其他亚组中则不然6 。高水平的 MYC 与不良预后(约 40% 的生存率)和对几乎所有现有疗法无反应有关6 。 G3 MB 患者的治疗选择有限,目前的护理标准包括手术后进行放射治疗和细胞毒性化疗(即顺铂和长春新碱联合洛莫司汀或环磷酰胺) 7 。此外,幸存下来的患者往往由于小时候接受的治疗而遭受严重的长期健康问题8 。虽然针对MB脑肿瘤的临床评估中的靶向治疗有限(NCT03434262;NCT04023669;和NCT01878617),但仍需要进一步努力扩大治疗选择,并找出对发育中的神经系统毒性较小的有效药物。

Although various cancer types, including many G3 MB tumors, exhibit aberrantly high MYC abundance, clinical targeting of MYC has remained elusive9,10,11,12. MYC is a key transcription factor that potentially regulates ~15% of the genome, controlling almost every cellular process13. Moreover, MYC regulates different targets depending on cell type. Still, the broad spectrum of MYC-controlled processes means that direct targeting of MYC can cause undesirable side effects and toxicity to normal cells that depend on MYC function14. Moreover, MYC is an intrinsically disordered protein that lacks defined targetable structures for small molecule inhibitors10. Hence, we posit efforts can be re-directed to exploit indirect means of targeting MYC within hyperactive MB cells. Unfortunately, the regulatory mechanisms that support MYC abundance remain unclear. Hence, a better understanding of the processes that help maintain high MYC levels in G3 MB cells is much needed to identify targets that could be exploited for therapeutic purposes.
尽管各种癌症类型,包括许多 G3 MB 肿瘤,表现出异常高的 MYC 丰度,但 MYC 的临床靶向仍然难以捉摸9 , 10 , 11 , 12 。 MYC 是一个关键的转录因子,可能调节约 15% 的基因组,控制几乎所有细胞过程13 。此外,MYC 根据细胞类型调节不同的靶点。尽管如此,MYC 控制过程的广谱性意味着直接靶向 MYC 可能会对依赖 MYC 功能的正常细胞造成不良副作用和毒性14 。此外,MYC 是一种本质上无序的蛋白质,缺乏小分子抑制剂的明确可靶向结构10 。因此,我们认为可以将努力转向利用间接方法来靶向过度活跃的 MB 细胞中的 MYC。不幸的是,支持 MYC 丰度的调控机制仍不清楚。因此,迫切需要更好地了解有助于维持 G3 MB 细胞中高 MYC 水平的过程,以确定可用于治疗目的的靶标。

One of the significant functions of MYC is to regulate cellular bioenergetics15. MYC controls multiple metabolic pathways, including glycolysis, glutaminolysis, fatty acid oxidation, and oxidative phosphorylation (OXPHOS)15,16. Cancer cells can leverage these metabolic pathways to maintain their increased bioenergetic and biosynthetic demands. Hence, MYC amplification is critical for supporting tumor metabolic reprogramming15. On the other hand, emerging evidence demonstrates that metabolism is far more essential in maintaining cellular expression profiles than previously appreciated17. Metabolism can directly influence gene and protein expression by modulating the supply of precursors for epigenetic and post-translational modifications17,18. Although targeting metabolism to block tumor energy production has been a sought-after therapeutic strategy for several years, manipulating metabolism to target oncogenic factors represents a unique approach. While the role of MYC in regulating metabolism has been extensively studied over three decades, it is unclear whether metabolism may reciprocally regulate MYC to support its enhanced abundance in cancer. Such reciprocal regulatory relationships have been demonstrated for other major transcription factors such as TP53, which has been shown to regulate the transcription of NAD+ synthesizing enzymes19. These NAD + synthesizing enzymes can in turn modulate TP53 stability by mediating the activity of NAD+-dependent deacetylases20. Yet, the role of metabolism for regulating MYC remains relatively unexplored.
MYC 的重要功能之一是调节细胞生物能量15 。 MYC 控制多种代谢途径,包括糖酵解、谷氨酰胺分解、脂肪酸氧化和氧化磷酸化 (OXPHOS) 15 , 16 。癌细胞可以利用这些代谢途径来维持其增加的生物能量和生物合成需求。因此, MYC扩增对于支持肿瘤代谢重编程至关重要15 。另一方面,新出现的证据表明,新陈代谢在维持细胞表达谱方面的重要性比之前认识的要重要得多17 。代谢可以通过调节表观遗传和翻译后修饰前体的供应来直接影响基因和蛋白质表达17 , 18 。尽管靶向代谢来阻止肿瘤能量产生多年来一直是一种广受欢迎的治疗策略,但操纵代谢来靶向致癌因子代表了一种独特的方法。虽然 MYC 在调节代谢中的作用已被广泛研究了三十年,但尚不清楚代谢是否可以相互调节 MYC 以支持其在癌症中丰度的增加。这种相互调节关系已在其他主要转录因子(例如 TP53)中得到证实,TP53 已被证明可以调节 NAD +合成酶的转录19 。这些 NAD + 合成酶可以通过介导 NAD +依赖性脱乙酰酶的活性来调节 TP53 的稳定性20 。 然而,代谢在调节 MYC 中的作用仍然相对未被探索。

Here, we investigate the concept of leveraging metabolism-targeting interventions as an indirect means of modulating MYC abundance and activity in G3 MB. We identify a targetable metabolic vulnerability in which G3 MB cells demonstrate exquisite sensitivity towards inhibitors of complex-I in the electron transport chain (ETC) but not to other metabolism-targeting agents, including glycolytic inhibitors. Moreover, we uncover a mechanism whereby targeting OXPHOS via inhibition of complex-I leads to inactivating acetylation of antioxidant enzyme SOD2, inducing rapid and specific oxidation of MYC followed by proteasomal degradation in a mitochondrial pyruvate carrier (MPC)-dependent manner. The therapeutic implications of these findings are demonstrated by the observations that OXPHOS inhibition decreases MYC levels and significantly suppresses the growth and self-renewal capacity of various well-characterized G3 MB cell lines, and other MYC-amplified cancer cells from multiple tumor types, including ovarian, colorectal and breast carcinomas. Ultimately, treatment with an orally bioavailable and blood-brain barrier (BBB) permeable complex-I inhibitor impairs the growth of intracerebellar orthotopic G3 MB tumor xenografts, induces differentiation, and significantly prolongs animal survival. These findings reveal a targetable role for OXPHOS metabolism that contributes to MYC abundance via the MPC-SOD2 axis in G3 MB.

Results

Complex I inhibition suppresses MYC abundance

MYC amplification is common in many aggressive tumors including, ovarian cancer, breast cancer, pancreatic ductal adenocarcinoma, colorectal carcinoma, and others (Fig. 1A)21. MYC is amplified in approximately 11% of G3 MB tumors5,22, making it one of the top 5 tumor types with the highest incidence of MYC amplification (Fig. 1A). MYC amplification universally corresponds with a worse prognosis across cancer types (Supplementary Fig. 1A). In G3 MB tumors specifically, high MYC levels correspond with an overall 10-year survival rate of <50%, compared to >70% survival in patients with MYCLOW tumors (Fig. 1B). Because of this, MYC is an attractive therapeutic target for cancer therapy. Despite the challenges associated with designing small-molecule inhibitors of MYC activity, progress has been made in developing agents that interfere with MYC interactions and block MYC transcriptional activity11. For example, 10058-F4 targets the interaction between MYC and its essential binding partner MAX23. However, these inhibitors have yet to be translated into clinical settings due to concerns associated with toxicity11. Indeed, we observed that although 10058-F4 effectively reduced the cell number of the well-established MYC-amplified G3 MB cell line HD-MB03 at various doses, it had a similar effect on normal human astrocytes, which constitute a major cellular component of the brain (Supplementary Fig.