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

• Published in: Cancer research (Chicago, Ill.) Vol. 75; no. 18; pp. 3865 - 3878
• Main Authors: Vangamudi, Bhavatarini, Paul, Thomas A, Shah, Parantu K, Kost-Alimova, Maria, Nottebaum, Lisa, Shi, Xi, Zhan, Yanai, Leo, Elisabetta, Mahadeshwar, Harshad S, Protopopov, Alexei, Futreal, Andrew, Tieu, Trang N, Peoples, Mike, Heffernan, Timothy P, Marszalek, Joseph R, Toniatti, Carlo, Petrocchi, Alessia, Verhelle, Dominique, Owen, Dafydd R, Draetta, Giulio, Jones, Philip, Palmer, Wylie S, Sharma, Shikhar, Andersen, Jannik N
• Format: Journal Article
• Language: English
• Published: United States 15.09.2015
• Subjects: Azabicyclo Compounds - pharmacology; Binding, Competitive; Catalysis; Cell Line, Tumor; Chromatin - metabolism; Chromatin Assembly and Disassembly - drug effects; Chromosomal Proteins, Non-Histone - deficiency; Chromosomal Proteins, Non-Histone - genetics; DNA Helicases - antagonists & inhibitors; DNA Helicases - chemistry; DNA Helicases - deficiency; DNA, Complementary - genetics; Gene Knockout Techniques; Genetic Complementation Test; Humans; Lung Neoplasms - pathology; Microarray Analysis; Molecular Targeted Therapy; Neoplasm Proteins - antagonists & inhibitors; Neoplasms - drug therapy; Neoplasms - genetics; Nuclear Proteins - antagonists & inhibitors; Nuclear Proteins - chemistry; Nuclear Proteins - deficiency; Protein Structure, Tertiary; Pyridines - pharmacology; Rhabdoid Tumor - genetics; Rhabdoid Tumor - pathology; RNA Interference; RNA, Small Interfering - pharmacology; Sarcoma, Synovial - genetics; Sarcoma, Synovial - pathology; Transcription Factors - antagonists & inhibitors; Transcription Factors - chemistry; Transcription Factors - deficiency; Transcription Factors - genetics
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-14-3798

The SWI/SNF multisubunit 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 pharmacologic 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 pharmacologic 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. Furthermore, 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 pharmacologic studies exemplify a general strategy for multidomain 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.