The SMARCA2/4 ATPase Domain Surpasses the Bromodomain as a Drug Target in SWI/SNF-Mutant Cancers: Insights from cDNA Rescue and PFI-3 Inhibitor Studies.
The SMARCA2/4 ATPase Domain Surpasses the Bromodomain as a Drug Target in SWI/SNF-Mutant Cancers: Insights from cDNA Rescue and PFI-3 Inhibitor Studies.
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DOI：
10.1158/0008-5472.can-14-3798
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发表时间：
2015-09-15
期刊：
影响因子：
11.2
通讯作者：
Andersen JN
中科院分区：
文献类型：
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作者：
Vangamudi B;Paul TA;Shah PK;Kost-Alimova M;Nottebaum L;Shi X;Zhan Y;Leo E;Mahadeshwar HS;Protopopov A;Futreal A;Tieu TN;Peoples M;Heffernan TP;Marszalek JR;Toniatti C;Petrocchi A;Verhelle D;Owen DR;Draetta G;Jones P;Palmer WS;Sharma S;Andersen JN
SWI/SNF多亚基复合物通过两个相互排斥的催化亚基SMARCA 2和SMARCA 4的活性调节染色质结构，这两个亚基都含有溴结构域和ATP酶结构域。使用RNAi，已经在SWI/SNF突变肿瘤（包括SMARCA 4缺陷型肺癌）中鉴定出癌症特异性脆弱性，然而，保守的可药物化蛋白结构域对这种抗癌表型的贡献是未知的。在这里，我们使用生物信息学，遗传学和药理学工具功能性地解构癌细胞的SMARCA 2/4依赖性。我们评估了一种选择性SMARCA 2/4布罗莫结构域抑制剂（PFI-3），并在染色质结合和细胞功能测定中表征了其活性，这些测定侧重于具有改变的SWI/SNF复合物的细胞（例如，肺、滑膜肉瘤、白血病和横纹肌样肿瘤）。我们证明，PFI-3是一种有效的，细胞可渗透的探针，能够取代异位表达，GFP标记的SMARCA 2-布罗莫结构域从染色质，但相反的目标敲除，抑制剂未能显示抗增殖表型。从机制上讲，通过原位细胞提取、染色质免疫沉淀和靶基因表达研究确定，溴结构域抑制未能从染色质中置换内源性全长SMARCA 2，从而协调了药理学功效的缺乏。此外，使用可诱导的RNAi和cDNA互补（布罗莫结构域和ATP酶死亡构建体），我们明确地鉴定了SMARCA 2的ATP酶结构域而不是布罗莫结构域作为具有抑制定义的转录程序的催化活性的相关治疗靶标。总之，我们的互补遗传和药理学研究为多结构域蛋白质药物靶标验证提供了一种通用策略，并且在SMARCA 2/4的情况下，突出了对更具挑战性的解旋酶/ATP酶结构域进行药物化以实现合成致死性治疗的潜力。
The SWI/SNF multi-subunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF mutant tumors, including SMARCA4-deficient lung cancer, however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic and pharmacological tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g. Lung, Synovial Sarcoma, Leukemia, and Rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacological efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation and target gene expression studies. Further, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacological studies exemplify a general strategy for multi-domain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy.