Supporting data for "Genome-wide CRISPR/Cas9 Knockout Library Screening Identified PTPMT1 for Cardiolipin Synthesis as a Crucial Metabolic Regulator for Hypoxic Survival in Hepatocellular Carcinoma"

Hypoxia, low oxygen (O₂) tension, due to deficiency of blood supply and dysregulated cancer cell proliferation, is a universal feature for all solid tumours, including hepatocellular carcinoma (HCC). Hypoxia induces metabolic rewiring that culminates in refractory and aggressive phenotypes in cancer. Novel targetable vulnerabilities are urgently warranted to combat the continuous dismal clinical outcomes of hypoxic HCC. We employed genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockout library screening to unbiasedly identify candidates that confer hypoxic survival advantages to HCC cells. HIF-1α and HIF-1β, which dimerize to form HIF-1 complex, the well-studied master regulator for oxygen sensing and hypoxic adaptation, ranked as the first and second most important candidates in our screening. The prioritization of these positive control genes highlights the validity and reliability of our screening. In contrast to the conventional view that mitochondria were insignificant hypoxic condition, we, from the screening, identified protein tyrosine phosphatase mitochondrial 1 (PTPMT1) as the third most significant gene for the survival of HCC cells under hypoxia, right after the HIF-1 complex. PTPMT1 is a pivotal enzyme for the synthesis of cardiolipin (CL), which is exclusively found in the mitochondrial with the greatest abundance at the mitochondrial inner membrane (MIM). CL physically interacts with and stabilizes the assembly of electron transport chain (ETC) complexes located at MIM to optimize electron transfer and oxidative phosphorylation (OXPHOS) to produce ATP. We envisioned that the loss of PTPMT1 prevented CL maturation, leading to the disruption of ETC. Electron leakage further resulted in excessive reactive oxygen species (ROS) accumulation and extensive cell death in hypoxic HCC cells where O₂, the final electron accepter, is depleted. PTPMT1 knockout substantially disrupted mitochondrial cristae structure, reduced mitochondrial capacity and activity, increased cellular ROS level and induced apoptotic cell death of hypoxic HCC cells. Lipidomics and targeted mass spectrometry (MS) analyses confirmed the impairment of CL synthesis pathway and decrease of CL in PTPMT1 KO HCC cells. <i>In vivo</i>, loss of PTPMT1 greatly reduced HCC growth and tumor aggressiveness in orthotopic implantation model and liver-specific Ptpmt1 knockout model. Intriguingly, HCC cells treated with a specific PTPMT1 inhibitor, alexidine dihydrochloride (AD), recapitulated the phenotypes of PTPMT1 knockout cells. AD greatly sensitized HCC cells under hypoxia by inducing extensive cell death. Importantly, we showed that AD consistently induced oxidative stress in different cancer types <i>in vitro</i> and effectively suppressed development of ovarian cancer and colorectal cancer <i>in vivo</i>. Clinically, PTPMT1 was frequently upregulated in HCC patients and its overexpression was associated with poor survival and prognosis. Taken together, we unprecedented leveraged CRISPR/Cas9 library screening to identify PTPMT1, an important metabolic regulator for hypoxic survival by maintaining mitochondrial integrity in cancer cells. PTPMT1 inhibitor, AD, also represents a novel therapeutic regimen to combat not only HCC, but also other hypoxic solid tumors.

缺氧（hypoxia）指血氧分压降低，多由血液供应不足与癌细胞增殖失调引发，是包括肝细胞癌（hepatocellular carcinoma, HCC）在内的所有实体瘤的普遍特征。缺氧会诱导代谢重编程，最终使癌症产生难治性与侵袭性表型。面对缺氧型肝细胞癌持续不佳的临床预后，亟需开发新型可靶向的治疗脆弱靶点。我们采用全基因组成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats, CRISPR）/Cas9敲除文库筛选，以无偏方式筛选可赋予肝癌细胞缺氧存活优势的候选基因。HIF-1α与HIF-1β可二聚化形成HIF-1复合物，这是研究较为充分的氧感知与缺氧适应核心调控因子，在本次筛选中位列最重要候选基因的前两位。这两类阳性对照基因的优先筛选结果，证明了本次筛选的有效性与可靠性。与传统认为线粒体在缺氧条件下作用无关紧要的观点相悖，我们通过本次筛选鉴定出蛋白酪氨酸磷酸酶线粒体1（protein tyrosine phosphatase mitochondrial 1, PTPMT1），其为缺氧环境下肝癌细胞存活的第三大关键基因，仅次于HIF-1复合物。PTPMT1是心磷脂（cardiolipin, CL）合成的关键酶，心磷脂仅特异性分布于线粒体中，在线粒体内膜（mitochondrial inner membrane, MIM）丰度最高。心磷脂可物理结合并稳定线粒体内膜上的电子传递链（electron transport chain, ETC）复合物组装，以优化电子传递效率与氧化磷酸化（oxidative phosphorylation, OXPHOS）过程，进而合成ATP。我们推测，PTPMT1缺失会阻断心磷脂的成熟过程，导致电子传递链结构与功能受损。电子渗漏会进一步引发活性氧（reactive oxygen species, ROS）过度积累，并在作为最终电子受体的氧气匮乏的缺氧肝癌细胞中引发广泛的细胞死亡。PTPMT1敲除会显著破坏线粒体嵴结构，降低线粒体容量与活性，提高细胞内活性氧水平，并诱导缺氧肝癌细胞发生凋亡性死亡。脂质组学与靶向质谱（mass spectrometry, MS）分析证实，PTPMT1敲除的肝癌细胞中心磷脂合成通路受损，且心磷脂含量显著降低。体内（in vivo）实验结果显示，PTPMT1缺失会大幅降低原位移植模型与肝特异性Ptpmt1敲除模型中的肝癌生长速率与肿瘤侵袭性。有趣的是，使用特异性PTPMT1抑制剂双盐酸阿来啶（alexidine dihydrochloride, AD）处理的肝癌细胞，重现了PTPMT1敲除细胞的表型。AD可通过诱导广泛细胞死亡，显著增敏缺氧环境下的肝癌细胞。重要的是，我们发现AD在体外（in vitro）可持续诱导不同癌症类型的氧化应激，并在体内有效抑制卵巢癌与结直肠癌的发展。临床数据分析显示，肝细胞癌患者组织中PTPMT1常出现上调，其过表达与不良生存预后显著相关。综上，我们首次利用CRISPR/Cas9文库筛选鉴定出PTPMT1，它是通过维持癌细胞线粒体完整性来调控缺氧存活的重要代谢调控因子。PTPMT1抑制剂AD不仅可作为治疗肝细胞癌的新型治疗方案，也可用于对抗其他缺氧型实体瘤。