Marius S. Pop

Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer

Mutant protein in tumors hits the DEK Cancer genome sequencing projects have uncovered a multitude of mutations in human tumors. Understanding whether and how these mutations contribute to tumor development and progression could ultimately lead to new therapies. Theurillat et al. studied the protein product of a gene that is recurrently mutated in prostate cancer. Normally this protein helps attach a biochemical tag to cellular proteins that marks them for degradation. The new work shows that the tumor-associated mutant protein loses this tagging ability, which results in the stabilization of a handful of cellular proteins that would otherwise be degraded. One of the most intriguing of these proteins was DEK, which helps prostate cancer cells invade into surrounding tissue. Science, this issue p. 85 Mutations in human prostate tumors impair degradation of a protein that helps cancer cells invade into surrounding tissue. Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer–associated mutations of SPOP, an E3 ubiquitin ligase substrate-binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, which implicated DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.

A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein

Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewing sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed “undruggable” by conventional methods. Here, we used small-molecule microarray screens to identify and characterize drug-like compounds that modulate the biologic function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacologic ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins. Mol Cancer Ther; 13(6); 1492–502. ©2014 AACR.

Advances in discovering small molecules to probe protein function in a systems context.

High throughput screening (HTS) has historically been used for drug discovery almost exclusively by the pharmaceutical industry. Due to a significant decrease in costs associated with establishing a high throughput facility and an exponential interest in discovering probes of development and disease associated biomolecules, HTS core facilities have become an integral part of most academic and non-profit research institutions over the past decade. This major shift has led to the development of new HTS methodologies extending beyond the capabilities and target classes used in classical drug discovery approaches such as traditional enzymatic activity-based screens. In this brief review we describe some of the most interesting developments in HTS technologies and methods for chemical probe discovery.

Probing Small‐Molecule Microarrays with Tagged Proteins in Cell Lysates

The technique of small‐molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins. This type of interaction is easily detected by the presence of a fluorescence signal produced by labeled antibodies that specifically recognize a unique sequence (tag) present on the target protein. The fluorescent signal intensity values are determined based on signal‐to‐noise ratios (SNRs). SMM screening is a high‐throughput, unbiased method that can rapidly identify novel direct ligands for various protein targets. This binding‐based assay format is generally applicable to most proteins, but it is especially useful for protein targets that do not possess an enzymatic activity. SMMs enable screening a protein in a purified form or in the context of a cellular lysate, likely providing a more physiologically relevant screening environment.

Inhibition of Zinc-Dependent Histone Deacetylases with a Chemically Triggered Electrophile

Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound-enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization-mass spectrometry.

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

Abstract 882: TMPRSS2-ERG drives global mistargeting of mammalian SWI/SNF (BAF) complexes in prostate cancer

Prostate cancer remains one of the leading causes of cancer-related death in men. Chromosomal rearrangements resulting in the fusion of the androgen regulated gene TMPRSS2 and the ETS-family transcription factor ERG occur in over 50% of all prostate cancer cases. Recent studies enabled by genome-wide methodologies have implicated altered epigenomic landscapes and changes in DNA accessibility as major contributors to ERG-driven oncogenesis, however the precise mechanism underlying the ERG transcriptional signature has to date remained unclear. Here we performed the first endogenous purification and SILAC-mass spectrometric analysis of ERG in TMPRSS2-ERG prostate cancer cells. Remarkably, we demonstrate that ERG directly interacts with the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which was recently shown to be mutated in >20% of human malignancies. ERG co-localizes with BAF complexes genome-wide, resulting in specific ERG-dependent BAF complex targeting to sites enriched in known ERG, FOXA1, and HOXB13 motifs; additionally, loss of ERG in TMPRSS2-ERG driven cell lines results in dramatic retargeting of BAF complexes away from ERG-dependent sites, to sites enriched in known AR and CTCF motifs. Importantly, ERG-driven BAF complex retargeting contributes to activation of TMPRSS2-ERG prostate cancer gene expression signatures. We map the ERG-BAF interaction to a specific region within the ERG amino acid sequence and find that this region is required to bind BAF complexes. These studies reveal a novel, unexpected mechanism of action of ERG-driven oncogenesis and offers new strategies for therapeutic intervention. Citation Format: Gabriel J. Sandoval, John L. Pulice, David Y. Takeda, Monica A. Schenone, Marius Pop, Gaylor Boulay, Miguel N. Rivera, Lucienne Ronco, William C. Hahn, Cigall Kadoch. TMPRSS2-ERG drives global mistargeting of mammalian SWI/SNF (BAF) complexes in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 882.

Abstract 2519: A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Members of the ETS transcription factor family have been implicated in several cancers, where they are often dysregulated by genomic derangement. ETS variant 1 (ETV1) is an ETS factor gene that undergoes chromosomal translocation in prostate cancers and Ewings sarcomas, amplification in melanomas, and lineage dysregulation in gastrointestinal stromal tumors. Pharmacologic perturbation of ETV1 would be appealing in these cancers; however, oncogenic transcription factors are often deemed “undruggable” by conventional methods. Here, we used small-molecule microarray (SMM) screens to identify and characterize drug-like compounds that modulate the biological function of ETV1. We identified the 1,3,5-triazine small molecule BRD32048 as a top candidate ETV1 perturbagen. BRD32048 binds ETV1 directly, modulating both ETV1-mediated transcriptional activity and invasion of ETV1-driven cancer cells. Moreover, BRD32048 inhibits p300-dependent acetylation of ETV1, thereby promoting its degradation. These results point to a new avenue for pharmacological ETV1 inhibition and may inform a general means to discover small molecule perturbagens of transcription factor oncoproteins. Note: This abstract was not presented at the meeting. Citation Format: Marius Pop, Nicolas Stransky, Colin Garvie, Jean-Philippe Theurillat, Timothy Lewis, Cheng Zhong, Elizabeth Culyba, Fallon Lin, Douglas Daniels, Raymond Pagliarini, Lucienne Ronco, Angela Koehler, Levi Garraway. A small molecule that binds and inhibits the ETV1 transcription factor oncoprotein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2519. doi:10.1158/1538-7445.AM2014-2519

Abstract 3693: Small molecule microarray screens for inhibitors of oncogenic ETS factors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Members of the ETS transcription factors family are involved in many cancers and are often targeted by translocation events that result in oncogenic gene fusions or dysregulated ETS factor expression. For example, the vast majority of prostate cancers harbor translocations involving the ETS factors ERG, ETV1 and ETV4 (most commonly, these are fused to the TMPRSS2 androgen-regulated gene or other house keeping genes). Overexpression of ETS factor gene fusions can induce or sustain malignant characteristics of cancer cells such as survival or invasiveness. However transcription factors have often been deemed “undruggable” by conventional approaches. The focus of this project is to employ novel screening approaches to identify and characterize small molecules that are able to bind and modulate the biological function of oncogenic ETS factors. Toward this end, we have performed Small Molecule Microarray (SMM) screens that involve a total of 80,000 distinct small molecules spanning several categories, including diversity-oriented synthesis (DOS) products, natural products, and other biologically active chemotypes. The SMM screens identified six compound that selectively bind the ETS factor ETV1 with a stringent Zscore. Two of these compounds have shown inhibitory activity in an ETV1-specific promoter-induced luciferase system. Surface Plasmon Resonance analysis has confirmed that each of the two lead compounds binds ETV1 directly, with one compound showing submicromolar binding kinetics. This compound in particular was able to reduce the expression of several ETV1 downstream targets such as MMP1 and MMP7. These efforts suggest that we have identified novel small molecule “perturbagens” of ETV1, an oncogenic ETS transcription factor. More generally, our results suggest that SMM-based screens may offer a robust approach to identify novel compounds that bind and perturb the function of cancer-associated proteins deemed poorly “druggable” by conventional methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3693.