|Summary of the probe development effort to identify activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM) - BioAssay Summary
Name: Summary of the probe development effort to to identify activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM). ..more
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: David Reisman, University of Florida
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number 1R03DA028854-01
Grant Proposal PI: David Reisman
External Assay ID: BRM_ACT_SUMMARY
Name: Summary of the probe development effort to to identify activators of the function of SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 (SMARCA2, BRM).
The protein encoded by the BRM gene (SMARCA2, SNF2, SWI2) is a member of the SWI/SNF family of proteins and is highly similar to the brahma protein of Drosophila (1). Members of this family have helicase and ATPase activities and are thought to regulate transcription of certain genes by altering the chromatin structure around those genes (2). The encoded protein is part of the large ATP-dependent chromatin remodeling complex SNF/SWI, which is required for transcriptional activation of genes normally repressed by chromatin. BRM is an epigenetically suppressed anti-cancer gene, which is silenced in wide variety of solid tumors (2). Because BRM function is key for growth control, restoring its expression routinely inhibits cancer cell growth. For this reason, restoring BRM has potential as an anticancer therapeutic modality (3-4). Its expression prevents cancer development as seen in murine models system where BRM loss potentiates cancer development 10-fold (5). It is known from preliminary studies that histone deacetylase (HDAC) inhibitors are found to restore BRM expression in cancer cell lines, but not its function (2). However, BRM is involved in many pathways and required by numerous transcription factors which control development, differentiation, DNA repair, adhesion, and growth control (6). As such, the impact of inactivating BRM and/or restoring its expression goes well beyond growth control (7).
Although pan-histone deacetylase (HDAC) inhibitors are found to restore BRM expression, but not its function in cancer cell lines, specific inhibitors of either HDAC3 or HDAC9, as well as the transcription factor GATA3 and/or MEF2D, induce a functional BRM. These other constituents involved in BRM regulation may also be the molecular targets. However, since HDAC9 and GATA3 are highly overexpressed and given the limited scope of expression of HDAC9, these proteins would be preferred targets. The fact that each are highly over-expressed is akin to EGFR in lung cancer or HER2 in breast cancer. Candidate compounds that are positive on the primary screen will be re-screened using our counter-screen, where BRM has been suppressed using anti-BRM shRNA (MG2-KDM). We have found that even the most potent inducers of BRM, are blocked by at least 50% or more by this assay. Hence, false positives will yield readouts of luciferase activity that show >50% inductions, closely approximating the levels found in the primary BRM (MG213) screen and will not have inductions <50% as observed with essentially all BRM-specific inducers.
Compounds will be then be verified as positive hits by a series of assays beginning with a third confirmatory (dose response) screening. Following the dose response screening, hits will be screened by directly measuring BRM induction by qPCR since the BRM gene is controlled by transcription. Additionally, we will determine the relative specificity the hits identified by screening each for its potential to induce a number (~10) of BRM-dependent genes. Since each of these BRM-dependent genes are controlled by different signal transduction pathways, interference in one or more cellular pathways (due to lack of specificity for BRM) will be demonstrated by a lack or reduced induction of one or more of the BRM-dependent genes. Moreover, the level of induction observed for each BRM-dependent gene is an indirect marker for the potency of BRM induction.
A secondary goal of this project is to group these verified compounds based upon their relative site of action in the pathway of BRM induction. We will use a secondary assay to differentiate compounds affecting upstream and downstream sections in the regulatory pathway. A possible additional screen will then be optionally performed using cells harboring anti-HDAC2 shRNA (MGH2KD cells). Since the deacetylation of BRM is a requirement for function thus generating a luciferase signal in these reporter cells, only the compound(s) that affect higher-level regulatory genes (such as MAP kinase inhibition) will be identified with a positive result using this assay. For those compounds having an impact lower in the pathway, we will use them to determine if they inhibit BRN2, GATA3, HDAC3, MEF2D, or perhaps HDAC9, thereby assisting us in determining which motifs of the compound underlie the re-expression of BRM. After this series of screens, all hits will then undergo a series of secondary assays to determine how well they restore BRM expression and its function in BRM-deficient cell lines. The assay provider will explore these secondary MOA studies in additional collaboration beyond the scope of this CPDP.
1. Bourachot, B., M. Yaniv, and C. Muchardt, The activity of mammalian brm/SNF2alpha is dependent on a high-mobility-group protein I/Y-like DNA binding domain. Mol Cell Biol, 1999. 19(6): p. 3931-9.
2. Glaros, S., et al., The reversible epigenetic silencing of BRM: implications for clinical targeted therapy. Oncogene, 2007. 26(49): p. 7058-66.
3. Reisman, D., S. Glaros, and E.A. Thompson, The SWI/SNF complex and cancer. Oncogene, 2009. 28(14): p. 1653-68.
4. Reyes, J.C., et al., Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha). EMBO J, 1998. 17(23): p. 6979-91.
5. Liu, G., et al., Two novel BRM insertion promoter sequence variants are associated with loss of BRM expression and lung cancer risk. Oncogene, 2011. 30(29): p. 3295-304.
6. Coisy-Quivy, M., et al., Role for Brm in cell growth control. Cancer Res, 2006. 66(10): p. 5069-76.
7. Gramling, S., et al., Pharmacologic reversal of epigenetic silencing of the anticancer protein BRM: a novel targeted treatment strategy. Oncogene, 2011. 30(29): p. 3289-94.
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