Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15
Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15
复制标题
DOI：
10.1126/science.aal3755
复制
发表时间：
2017-04-28
期刊：
影响因子：
56.9
通讯作者：
Nijhawan, Deepak
中科院分区：
文献类型：
--
作者：
Han, Ting;Goralski, Maria;Nijhawan, Deepak
Indisulam是一种芳基磺酰胺类药物，可抑制某些人类癌细胞系的增殖。其作用机制和选择性机制知之甚少。基于其在体外和小鼠中的抗癌活性，indisulam已在晚期实体瘤患者中进行了广泛的测试。在接受indisulam单药治疗的患者中没有报道不可接受的毒性，但不到10%的患者显示出临床反应。我们认为，更好地理解indisulam抗癌活性的分子机制可能会揭示为什么只有一部分肿瘤对它有反应，这反过来可能会导致该药物更有效的临床应用。为了研究indisulam的作用机制，我们确定了赋予其细胞毒性作用抗性的基因突变。通过正向遗传策略，我们发现，在培养的癌细胞和肿瘤异种移植小鼠中，称为RBM39（RNA结合基序蛋白39）的核蛋白中的几个单一氨基酸取代赋予了对indisulam毒性作用的抗性。在indisulam存在下，RBM39与CUL4-DDB1-DDA1-DCAF15 E3泛素连接酶复合物（CUL4-DCAF15）缔合，导致RBM39的多聚泛素化和蛋白酶体降解。在RBM39中引起indisulam抗性的突变，相比之下，不与CUL4-DCAF15相关，因此既不被多聚泛素修饰也不被蛋白酶体降解。在纯化的重组蛋白的实验中，我们发现indisulam与RBM39和E3泛素连接酶受体DCAF15形成三元复合物，单独对两种物种都没有可检测的亲和力。引起茚磺草胺抗性的RBM39突变阻碍了这种复合物的形成。有趣的是，我们发现另外两种临床测试的磺胺类药物与indisulam结构相似-他西磺草胺和氯喹喔啉磺酰胺（CQS）-与indisulam具有相同的作用机制。RBM39是参与前体mRNA（pre-mRNA）剪接的核蛋白。RBM39的生化分离揭示了与许多剪接因子和RNA结合蛋白的关联。我们发现，通过indisulam降解RBM39导致异常的前体mRNA剪接，包括内含子保留和外显子跳跃，在数百个genes.In一个大的调查indisulam敏感性超过800癌细胞系，我们发现，来自造血和淋巴（HL）谱系的癌细胞比来自其他谱系的癌细胞更敏感。在HL癌细胞系中，DCAF15 mRNA表达水平和DCAF15基因拷贝数变异与indisulam敏感性直接相关。药物如indisulam，tasisulam和CQS-我们统称为SPLAM（剪接抑制剂磺胺类药物）-提供了一种靶向癌症中RBM39依赖性前mRNA剪接的策略。许多早期的indisulam临床试验都集中在实体瘤患者身上。我们的研究结果表明，indisulam可能是最有效的白血病和淋巴瘤患者表达相对较高水平的DCAF15。SPLAM的活性类似于IMiDs（免疫调节药物）。IMiD是一种抗癌药物，充当“分子胶水”，将E3遍在蛋白连接酶受体cereblon和多种新底物结合在一起。以类似的方式…
INTRODUCTIONIndisulam is an aryl sulfonamide drug that inhibits the proliferation of certain human cancer cell lines. Its mechanism of action and the mechanism underlying its selectivity are poorly understood. On the basis of its anticancer activity in vitro and in mice, indisulam has been extensively tested in patients with advanced-stage solid tumors. No unacceptable toxicities were reported in patients receiving indisulam monotherapy, but fewer than 10% of patients showed a clinical response.RATIONALEAt present, there is no way to predict which cancer patients are most likely to benefit from indisulam treatment. We reasoned that a better understanding of the molecular mechanism underlying indisulam’s anticancer activity might reveal why only a subset of tumors respond to it. This in turn might lead to more effective clinical use of the drug. To study indisulam’s mechanism of action, we identified genetic mutations that confer resistance to its cytotoxic effect.RESULTSUsing a forward genetic strategy, we discovered that several single amino acid substitutions in a nuclear protein called RBM39 (RNA binding motif protein 39) conferred resistance to the toxic effects of indisulam in cultured cancer cells and in mice with tumor xenografts. In the presence of indisulam, RBM39 associated with the CUL4-DDB1-DDA1-DCAF15 E3 ubiquitin ligase complex (CUL4-DCAF15), leading to polyubiquitination and proteasomal degradation of RBM39. Mutations in RBM39 that cause indisulam resistance, in contrast, did not associate with CUL4-DCAF15 and were thus neither modified with polyubiquitin nor degraded by the proteasome.In experiments with purified recombinant proteins, we found that indisulam formed a ternary complex with RBM39 and the E3 ubiquitin ligase receptor DCAF15, with no detectable affinity for either species alone. RBM39 mutations that cause indisulam resistance impeded the formation of this complex. Interestingly, we found that two other clinically tested sulfonamides with structural similarity to indisulam—tasisulam and chloroquinoxaline sulfonamide (CQS)—share the same mechanism of action as indisulam. RBM39 is a nuclear protein that is involved in precursor mRNA (pre-mRNA) splicing. Biochemical isolation of RBM39 revealed an association with numerous splicing factors and RNA binding proteins. We found that degradation of RBM39 by indisulam led to aberrant pre-mRNA splicing, including intron retention and exon skipping, in hundreds of genes.In a large survey of indisulam sensitivity across more than 800 cancer cell lines, we found that cancer cells derived from the hematopoietic and lymphoid (HL) lineages were more sensitive than cancer cells derived from other lineages. In HL cancer cell lines,DCAF15mRNA expression levels andDCAF15gene copy number variation directly correlated with indisulam sensitivity.CONCLUSIONCancer genome–sequencing studies have highlighted the importance of pre-mRNA splicing in tumorigenesis. Drugs such as indisulam, tasisulam, and CQS—which we collectively refer to as SPLAMs (splicing inhibitor sulfonamides)—provide a strategy to target RBM39-dependent pre-mRNA splicing in cancer. Many of the earlier clinical trials of indisulam focused on patients with solid tumors. Our findings suggest that indisulam may be most effective in patients with leukemias and lymphomas that express relatively high levels of DCAF15.The activity of SPLAMs resembles that of IMiDs (immunomodulatory drugs). IMiDs are anticancer drugs that act as a “molecular glue,” bringing together the E3 ubiquitin ligase receptor cereblon and a variety of neosubstrates. In an analogous manner …