An old cancer drug's degrading new look Typically, cancer drugs that help only a small number of patients in clinical trials are not pursued. This might change in a future world of precision medicine, where biomarkers will match specific drugs to the patients most likely to respond. Han et al. identified the mechanism of action of a cancer drug called indisulam, a sulfonamide tested previously in patients with solid tumors. Indisulam and related sulfonamides killed cells by disrupting precursor mRNA splicing. The drugs targeted a specific RNA splicing factor for degradation by “gluing” it to the CUL4-DCAF15 ubiquitin ligase. Experiments with cancer cell lines suggest that future clinical trials of these drugs should focus on leukemias and lymphomas with high DCAF15 expression levels. Science, this issue p. eaal3755 Sulfonamide drugs kill cancer cells by targeting a specific RNA splicing factor for degradation. INTRODUCTION Indisulam 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. RATIONALE At 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. RESULTS Using 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, DCAF15 mRNA expression levels and DCAF15 gene copy number variation directly correlated with indisulam sensitivity. CONCLUSION Cancer 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, SPLAM derivatives potentially could be used to target DCAF15 to novel neosubstrates that, like RBM39, are otherwise undruggable. SPLAMs target the splicing factor RBM39 for proteasomal degradation. A class of clinically tested anticancer sulfonamides, including indisulam, tasisulam, and CQS, functions by promoting the interaction of the RBM39 splicing factor and the CUL4-DCAF15 E3 ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of RBM39. Cancer cell lines from hematopoietic and lymphoid lineages that show high expression levels of DCAF15 are more sensitive to the cytotoxic effects of SPLAMs, suggesting that DCAF15 is a potential biomarker to guide future clinical trials of SPLAMs. Indisulam is an aryl sulfonamide drug with selective anticancer activity. Its mechanism of action and the basis for its selectivity have so far been unknown. Here we show that indisulam promotes the recruitment of RBM39 (RNA binding motif protein 39) to the CUL4-DCAF15 E3 ubiquitin ligase, leading to RBM39 polyubiquitination and proteasomal degradation. Mutations in RBM39 that prevent its recruitment to CUL4-DCAF15 increase RBM39 stability and confer resistance to indisulam’s cytotoxicity. RBM39 associates with precursor messenger RNA (pre-mRNA) splicing factors, and inactivation of RBM39 by indisulam causes aberrant pre-mRNA splicing. Many cancer cell lines derived from hematopoietic and lymphoid lineages are sensitive to indisulam, and their sensitivity correlates with DCAF15 expression levels. Two other clinically tested sulfonamides, tasisulam and chloroquinoxaline sulfonamide, share the same mechanism of action as indisulam. We propose that DCAF15 expression may be a useful biomarker to guide clinical trials of this class of drugs, which we refer to as SPLAMs (splicing inhibitor sulfonamides).

中文翻译：

---

抗癌磺胺类药物通过募集到 DCAF15 诱导 RBM39 降解来靶向剪接

旧的抗癌药有辱人格的新面貌 通常情况下，在临床试验中只对少数患者有帮助的抗癌药是不会被追求的。在未来的精准医学世界中，这可能会发生变化，生物标志物将使特定药物与最有可能产生反应的患者相匹配。韩等人。确定了一种名为消地舒仑的抗癌药物的作用机制，这是一种以前在实体瘤患者中测试过的磺胺类药物。Indisulam 和相关磺胺类药物通过破坏前体 mRNA 剪接来杀死细胞。这些药物通过将特定的 RNA 剪接因子“粘合”到 CUL4-DCAF15 泛素连接酶来靶向降解。癌细胞系实验表明，这些药物未来的临床试验应侧重于具有高 DCAF15 表达水平的白血病和淋巴瘤。科学，这个问题 p。eaal3755 磺胺类药物通过靶向特定的 RNA 剪接因子进行降解来杀死癌细胞。介绍 Indisulam 是一种芳基磺酰胺药物，可抑制某些人类癌细胞系的增殖。其作用机制及其选择性背后的机制知之甚少。基于其在体外和小鼠体内的抗癌活性，已在晚期实体瘤患者中进行了广泛的试验。在接受 indisulam 单药治疗的患者中没有报告不可接受的毒性，但只有不到 10% 的患者表现出临床反应。基本原理 目前，没有办法预测哪些癌症患者最有可能从吲地磺胺治疗中受益。我们推断，更好地了解 indisulam 抗癌活性背后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。这反过来可能导致药物的更有效的临床使用。为了研究消炎痛的作用机制，我们确定了对其细胞毒性作用具有抗性的基因突变。结果 使用正向遗传策略，我们发现称为 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培养的癌细胞和具有肿瘤异种移植物的小鼠的 indisulam 毒性作用的抗性。在消炎痛存在下，RBM39 与 CUL4-DDB1-DDA1-DCAF15 E3 泛素连接酶复合物 (CUL4-DCAF15) 相关联，导致 RBM39 的多泛素化和蛋白酶体降解。相比之下，导致消炎痛抗性的 RBM39 突变与 CUL4-DCAF15 无关，因此既没有被多聚泛素修饰，也没有被蛋白酶体降解。在纯化重组蛋白的实验中，我们发现消炎痛与 RBM39 和 E3 泛素连接酶受体 DCAF15 形成三元复合物，单独对任一物种都没有可检测到的亲和力。RBM39 突变导致 indisulam 抗性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茚地磺胺具有相似的结构——他西磺胺和氯喹喔啉磺胺 (CQS)——与茚地磺胺具有相同的作用机制。RBM39 是一种核蛋白，参与前体 mRNA (pre-mRNA) 剪接。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。我们发现消炎痛对 RBM39 的降解导致数百个基因中的前 mRNA 剪接异常，包括内含子保留和外显子跳跃。在对 800 多种癌细胞系的消炎痛敏感性进行的大型调查中，我们发现源自造血和淋巴 (HL) 谱系的癌细胞比源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。结论 癌症基因组测序研究强调了前体 mRNA 剪接在肿瘤发生中的重要性。药物如吲地磺胺、他西磺胺、和 CQS——我们统称为 SPLAMs（剪接抑制剂磺胺）——提供了一种策略来靶向癌症中依赖 RBM39 的前 mRNA 剪接。许多早期的吲哚美辛临床试验都集中在实体瘤患者身上。我们的研究结果表明，地舒仑可能对表达相对高水平 DCAF15 的白血病和淋巴瘤患者最有效。SPLAMs 的活性类似于 IMiDs（免疫调节药物）。IMiD 是一种抗癌药物，可充当“分子胶”，将 E3 泛素连接酶受体 cereblon 和各种新底物结合在一起。以类似的方式，SPLAM 衍生物可能用于将 DCAF15 靶向新底物，如 RBM39，否则无法成药。SPLAM 靶向剪接因子 RBM39 以进行蛋白酶体降解。一类经过临床测试的抗癌磺胺药，包括吲地磺胺、他西磺胺和 CQS，通过促进 RBM39 剪接因子和 CUL4-DCAF15 E3 泛素连接酶的相互作用，导致 RBM39 的多泛素化和蛋白酶体降解。DCAF15 高表达水平的造血和淋巴系癌细胞系对 SPLAM 的细胞毒性作用更敏感，这表明 DCAF15 是指导 SPLAM 未来临床试验的潜在生物标志物。Indisulam 是一种具有选择性抗癌活性的芳基磺酰胺药物。迄今为止，其作用机制及其选择性的基础尚不清楚。在这里，我们表明 indisulam 促进 RBM39（RNA 结合基序蛋白 39）向 CUL4-DCAF15 E3 泛素连接酶的募集，导致 RBM39 多泛素化和蛋白酶体降解。RBM39 中阻止其募集到 CUL4-DCAF15 的突变会增加 RBM39 的稳定性并赋予对吲哚美林细胞毒性的抗性。RBM39 与前体信使 RNA (pre-mRNA) 剪接因子相关，因地磺胺对 RBM39 的失活会导致前体 mRNA 剪接异常。许多源自造血和淋巴系的癌细胞系对 indisulam 敏感，它们的敏感性与 DCAF15 表达水平相关。其他两种经过临床测试的磺胺类药物，tasisulam 和氯喹喔啉磺胺，与 indisulam 具有相同的作用机制。我们提出 DCAF15 表达可能是指导此类药物临床试验的有用生物标志物，我们将其称为 SPLAM（剪接抑制剂磺胺类药物）。RBM39 中阻止其募集到 CUL4-DCAF15 的突变会增加 RBM39 的稳定性并赋予对吲哚美林细胞毒性的抗性。RBM39 与前体信使 RNA (pre-mRNA) 剪接因子相关，因地磺胺对 RBM39 的失活会导致前体 mRNA 剪接异常。许多源自造血和淋巴谱系的癌细胞系对 indisulam 敏感，它们的敏感性与 DCAF15 表达水平相关。其他两种经过临床测试的磺胺类药物，tasisulam 和氯喹喔啉磺胺，与 indisulam 具有相同的作用机制。我们提出 DCAF15 表达可能是指导此类药物临床试验的有用生物标志物，我们将其称为 SPLAM（剪接抑制剂磺胺类药物）。RBM39 中阻止其募集到 CUL4-DCAF15 的突变会增加 RBM39 的稳定性并赋予对吲哚美林细胞毒性的抗性。RBM39 与前体信使 RNA (pre-mRNA) 剪接因子相关，因地磺胺对 RBM39 的失活会导致前体 mRNA 剪接异常。许多源自造血和淋巴系的癌细胞系对 indisulam 敏感，它们的敏感性与 DCAF15 表达水平相关。其他两种经过临床测试的磺胺类药物，tasisulam 和氯喹喔啉磺胺，与 indisulam 具有相同的作用机制。我们提出 DCAF15 表达可能是指导此类药物临床试验的有用生物标志物，我们将其称为 SPLAM（剪接抑制剂磺胺类药物）。