The UBE2J2/UBE2K-MARCH5 ubiquitination machinery regulates apoptosis in response to venetoclax in acute myeloid leukemia.
The UBE2J2/UBE2K-MARCH5 ubiquitination machinery regulates apoptosis in response to venetoclax in acute myeloid leukemia.
复制标题
UBE2J2/UBE2K-MARCH5 泛素化机制在急性髓系白血病中调节细胞凋亡以响应 Venetoclax。
DOI：
10.1038/s41375-024-02178-x
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发表时间：
2024
期刊：
影响因子：
11.4
通讯作者：
Stegmaier,Kimberly
中科院分区：
文献类型：
--
作者：
Lin,Shan;Schneider,Constanze;Su,AngelaH;Alexe,Gabriela;Root,DavidE;Stegmaier,Kimberly
细胞凋亡的逃避对于许多癌症类型（包括急性髓性白血病（AML））的生长、存活和化学抗性是至关重要的;因此，细胞凋亡的再激活可以被开发为治疗方法。细胞凋亡诱导主要由抗细胞凋亡和促细胞凋亡BCL 2家族蛋白之间的平衡控制，这决定了效应物BAX和巴克的寡聚化，从而决定了线粒体膜的透化[1]。维奈托克是一种拮抗抗凋亡蛋白BCL 2的选择性抑制剂，已成为AML的一种有前途的治疗方法。尽管与低甲基化药物联合治疗的缓解率较高，但一些患者表现出前期耐药，大多数患者最终会复发[2-4]。因此，确定维奈托克联合治疗的协同作用靶点对于提高该药物的临床应用具有重要意义。几个潜在的目标已经被发现。例如，抑制BCLXL或MCL 1（两种抗凋亡BCL 2家族成员）可引发与维奈托克的协同抗肿瘤活性[2]。我们还报道，抑制RING型泛素E3连接酶MARCH 5可诱导BAX/BAK依赖性细胞凋亡，并导致AML细胞中的维奈托克致敏[5]。为了系统地鉴定可以调节venetoclax效应的其他关键基因，我们在人AML细胞系MV 4 -11中进行了基因组规模的CRISPR-Cas9筛选。MV 4 -11细胞用Ammonia基因组规模的sgRNA文库转导，其用四种sgRNA靶向每个基因。将转导的细胞分为两组，并与溶媒对照DMSO或10 nM维奈托克一起培养16天，此时收集细胞沉淀用于测序分析（补充图1a）。这种浓度的维奈托克导致细胞活力降低30%，使我们能够确定基因靶点，这些基因靶点的丢失可以赋予耐药性或诱导致敏。鉴定了与DMSO对照相比，维奈托克处理的细胞中显著富集或耗尽的sgRNA。超几何方法用于评估靶向相同基因的sgRNA组的p值和log 2倍数变化（LFC）。与先前的报道一致，凋亡效应物BAX或促凋亡基因NOXA（PMAIP 1）的丧失引起维奈托克抗性，而靶向抗凋亡基因BCLXL（BCL 2L 1）、BCL 2L 2和BCL 2A 1的sgRNA在维奈托克处理的细胞中显著更多地耗尽（图1a）[6-8]。MCL 1和MARCH 5在该分析中没有得分，因为这两个靶标的缺失强烈抑制MV 4 -11细胞的生长，导致处理组和对照组中它们的sgRNA的强烈消耗。我们接下来关注E2泛素缀合酶UBE 2 J2和UBE 2K，这两种高度排名的venetoclax致敏剂尚未得到很好的表征（图1a）。我们验证了UBE 2 J2或UBE 2 K的消耗增加了MV 4 -11和另外两种AML细胞系对维奈托克的敏感性（图1 B）。成功的基因敲除通过UBE 2K的免疫印迹和UBE 2 J2的基因组位点测序来证实（补充图1b，c）。org/），其包括在超过1000种癌细胞系中的基因组规模CRISPR-Cas9筛选，揭示了在所有癌细胞系中对UBE 2 J2或UBE 2K的依赖性与对MARCH 5的依赖性显著相关;并且在AML细胞系中观察到类似的趋势（图Ic）。由于E2酶与泛素E3连接酶协调执行泛素化过程，这一观察表明UBE 2 J2...
Evasion of apoptosis is crucial for the growth, survival and chemoresistance of many cancer types, including acute myeloid leukemia (AML); thus, the reactivation of apoptosis can be exploited as a therapeutic approach. Apoptosis induction is mainly controlled by the balance between anti-apoptotic and pro-apoptotic BCL2 family proteins, which determines the oligomerization of effectors BAX and BAK and thus the permeabilization of mitochondrial membranes [1]. Venetoclax, a selective inhibitor antagonizing the anti-apoptotic protein BCL2, has emerged as a promising therapy in AML. Despite high response rates in combination with hypomethylating agents, some patients display upfront resistance, and most patients will ultimately relapse [2-4]. Therefore, identification of synergistic targets for combination therapies with venetoclax is important for improving the clinical application of this drug. Several potential targets have been revealed. For instance, inhibiting BCLXL or MCL1, two anti-apoptotic BCL2 family members, can trigger synergistic anti-tumor activity with venetoclax [2]. We also reported that inhibition of the RING-type ubiquitin E3 ligase MARCH5 can induce BAX/BAK-dependent apoptosis and lead to venetoclax sensitization in AML cells [5]. To systematically identify other key genes that can modulate the venetoclax effect, we performed a genome-scale CRISPR-Cas9 screen in the human AML cell line MV4-11. MV4-11 cells were transduced with the Avana genome-scale sgRNA library, which targets each gene with four sgRNAs. Transduced cells were divided into two groups and cultured with the vehicle control DMSO or 10 nM venetoclax for 16 days at which point cell pellets were collected for sequencing analysis (Supplementary Fig. 1a). This concentration of venetoclax resulted in a 30% decrease in cell viability, allowing us to identify gene targets whose loss can either confer resistance or induce sensitization. Significantly enriched or depleted sgRNAs in venetoclax-treated cells compared to DMSO controls were identified. The hypergeometric method was used to assess p-values and the log2 fold-change (LFC) of the set of sgRNAs targeting the same gene. Consistent with previous reports, loss of the apoptosis effector BAX or the pro-apoptotic gene NOXA (PMAIP1) caused venetoclax resistance, while sgRNAs targeting the anti-apoptotic genes BCLXL (BCL2L1), BCL2L2 and BCL2A1 were significantly more depleted in venetoclax-treated cells (Fig. 1 a)[6-8]. MCL1 and MARCH5 did not score in this analysis because deletion of these two targets strongly inhibits the growth of MV4-11 cells, causing a strong depletion of their sgRNAs in both treatment and control groups. We next focused on the E2 ubiquitin-conjugating enzymes UBE2J2 and UBE2K, two highly ranked venetoclax sensitizers that have not been well characterized (Fig. 1 a). We validated that depletion of either UBE2J2 or UBE2K increased sensitivity to venetoclax in MV4-11 and two additional AML cell lines (Fig. 1 b). Successful gene knockout was confirmed by immunoblot for UBE2K and genomic loci sequencing for UBE2J2 (Supplementary Fig. 1b, c).Intriguingly, exploiting the Broad InstituteLs Cancer Dependency Map (DepMap) dataset (https://depmap. org/), which includes genome-scale CRISPR-Cas9 screens in over 1000 cancer cell lines, revealed that dependency on UBE2J2 or UBE2K significantly correlated with a dependency on MARCH5 across all cancer cell lines; and similar trends were observed within AML cell lines (Fig. 1 c). Since E2 enzymes coordinate with ubiquitin E3 ligases to execute ubiquitination processes, this observation suggests that UBE2J2 …