Search

// Miriam S. Butler 1, *, # , Mani Roshan-Moniri 1, *, # , Michael Hsing 1, *, # , Desmond Lau 2, *, # , Ari Kim 1 , Paul Yen 1 , Marta Mroczek 1 , Mannan Nouri 1 , Scott Lien 1 , Peter Axerio-Cilies 1 , Kush Dalal 1 , Clement Yau 1 , Fariba Ghaidi 1 , Yubin Guo 1 , Takeshi Yamazaki 1 , Sam Lawn 1 , Martin E. Gleave 1 , Cheryl Y. Gregory-Evans 3 , Lawrence P. McIntosh 2, * , Michael E. Cox 1, * , Paul S. Rennie 1, * and Artem Cherkasov 1, * 1 Vancouver Prostate Centre and the Department of Urologic Sciences, University of British Columbia, Vancouver, BC V6H 3Z6, Canada 2 Department of Biochemistry and Molecular Biology, Department of Chemistry, Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada 3 Department of Ophthalmology and Visual Sciences, Eye Care Centre, University of British Columbia, Vancouver, BC V5Z 3N9, Canada * These authors contributed equally to this work # Co-first authors Correspondence to: Michael E. Cox, email: mcox@prostatecentre.com Artem Cherkasov, email: acherkasov@prostatecentre.com Keywords: prostate cancer, ERG, rational drug design, small molecule inhibitor, TMPRSS2-ERG Received: July 29, 2016 Accepted: April 04, 2017 Published: April 15, 2017 ABSTRACT Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG-ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer.

Autoinhibition enables spatial and temporal regulation of cellular processes by coupling protein activity to surrounding conditions, often via protein partnerships or signaling pathways. We report the molecular basis of DNA-binding autoinhibition of ETS transcription factors ETV1, ETV4 and ETV5, which are often overexpressed in prostate cancer. Inhibitory elements that cooperate to repress DNA binding were identified in regions N- and C-terminal of the ETS domain. Crystal structures of these three factors revealed an α-helix in the C-terminal inhibitory domain that packs against the ETS domain and perturbs the conformation of its DNA-recognition helix. Nuclear magnetic resonance spectroscopy demonstrated that the N-terminal inhibitory domain (NID) is intrinsically disordered, yet utilizes transient intramolecular interactions with the DNA-recognition helix of the ETS domain to mediate autoinhibition. Acetylation of selected lysines within the NID activates DNA binding. This investigation revealed a distinctive mechanism for DNA-binding autoinhibition in the ETV1/4/5 subfamily involving a network of intramolecular interactions not present in other ETS factors. These distinguishing inhibitory elements provide a platform through which cellular triggers, such as protein–protein interactions or post-translational modifications, may specifically regulate the function of these oncogenic proteins.

The recruitment of transcriptional cofactors by sequence-specific transcription factors challenges the basis of high affinity and selective interactions. Extending previous studies that the N-terminal activation domain (AD) of ETV5 interacts with Mediator subunit 25 (MED25), we establish that similar, aromatic-rich motifs located both in the AD and in the DNA-binding domain (DBD) of the related ETS factor ETV4 interact with MED25. These ETV4 regions bind MED25 independently, display distinct kinetics, and combine to contribute to a high-affinity interaction of full-length ETV4 with MED25. Within the ETS family, high-affinity interactions with MED25 are specific for the ETV1/4/5 subfamily as other ETS factors display weaker or no detectable binding. The AD binds to a single site on MED25 and the DBD interacts with three MED25 sites, allowing for simultaneous binding of both domains in full-length ETV4. MED25 also stimulates the in vitro DNA binding activity of ETV4 by relieving autoinhibition. ETV1/4/5 factors are often overexpressed in prostate cancer and genome-wide studies in a prostate cancer cell line indicate that ETV4 and MED25 occupy enhancers that are enriched for ETS-binding sequences and are both functionally important for the transcription of genes regulated by these enhancers. AP1-binding sequences were observed in MED25-occupied regions and JUN/FOS also contact MED25; FOS strongly binds to the same MED25 site as ETV4 AD and JUN interacts with the other two MED25 sites. In summary, we describe features of the multivalent ETV4- and AP1-MED25 interactions, thereby implicating these factors in the recruitment of MED25 to transcriptional control elements.

The PAAD Death Domain is a 6-helix bundle domain that contains a disordered region in helix-3. This domain can fold by a two-state mechanism, has low stability and can undergo different partially folded conformations. To identify specific amino acids responsible for these structural features we focus our study on the PAAD domain of IFI16, a protein sensor that recognizes viral nucleic acids and triggers the (Interleukin-β) inflammatory response in response to infection. IFI16-PAAD was randomly mutated using GFP directed evolution and the resulting mutants with increased secondary structure and stability were identified by circular dichroism and tested for nucleic acid binding. Several IFI16-PAAD-GFP fusion mutants that contained I46N/S substitutions translated into improved secondary structure and stability and a direct correlation was observed between secondary structure/stability enhancement and their ability to destabilize dsDNA. In conclusion, GFP directed evolution can be used to obtain more structured and stable mutants and to probe conformational changes. These data suggest that changes in conformation of the PAAD domain of IFI16 occurs when the viral nucleic acid bind to IFI16 and must be a contributing factor to transduce the inflammatory response during viral infection.

Pointed-P2, the Drosophila ortholog of human ETS1 and ETS2, is a transcription factor involved in Ras/MAP kinase-regulated gene expression. In addition to a DNA-binding ETS domain, Pointed-P2 contains a PNT (or SAM) domain that serves as a docking module to enhance phosphorylation of an adjacent phosphoacceptor threonine by the ERK2 MAP kinase Rolled. Using NMR chemical shift, ¹⁵N relaxation, and amide hydrogen exchange measurements, we demonstrate that the Pointed-P2 PNT domain contains a dynamic N-terminal helix H0 appended to a core conserved five-helix bundle diagnostic of the SAM domain fold. Neither the secondary structure nor dynamics of the PNT domain is perturbed significantly upon in vitro ERK2 phosphorylation of three threonine residues in a disordered sequence immediately preceding this domain. These data thus confirm that the Drosophila Pointed-P2 PNT domain and phosphoacceptors are highly similar to those of the well-characterized human ETS1 transcription factor. NMR-monitored titrations also revealed that the phosphoacceptors and helix H0, as well as region of the core helical bundle identified previously by mutational analyses as a kinase docking site, are selectively perturbed upon ERK2 binding by Pointed-P2. Based on a homology model derived from the ETS1 PNT domain, helix H0 is predicted to partially occlude the docking interface. Therefore, this dynamic helix must be displaced to allow both docking of the kinase, as well as binding of Mae, a Drosophila protein that negatively regulates Pointed-P2 by competing with the kinase for its docking site.

Genomic alterations involving translocations of the ETS-related gene ERG occur in approximately half of prostate cancer cases. These alterations result in aberrant, androgen-regulated production of ERG protein variants that directly contribute to disease development and progression. This study describes the discovery and characterization of a new class of small molecule ERG antagonists identified through rational in silico methods. These antagonists are designed to sterically block DNA binding by the ETS domain of ERG and thereby disrupt transcriptional activity. We confirmed the direct binding of a lead compound, VPC-18005, with the ERG-ETS domain using biophysical approaches. We then demonstrated VPC-18005 reduced migration and invasion rates of ERG expressing prostate cancer cells, and reduced metastasis in a zebrafish xenograft model. These results demonstrate proof-of-principal that small molecule targeting of the ERG ETS domain can suppress transcriptional activity and reverse transformed characteristics of prostate cancers aberrantly expressing ERG. Clinical advancement of the developed small molecule inhibitors may provide new therapeutic agents for use as alternatives to, or in combination with, current therapies for men with ERG-expressing metastatic castration-resistant prostate cancer. Citation Format: Miriam S. Butler, Mani Roshan-Moniri, Michael Hsing, Desmond Lau, Ari Kim, Paul Yen, Marta Mroczek, Mannan Nouri, Scott Lien, Peter Axerio-Cilies, Kush Dalal, Clement Yau, Fariba Ghaidi, Yubin Guo, Takeshi Yamazaki, Sam Lawn, Martin Gleave, Cheryl Y. Gregory-Evans, Lawrence P. McIntosh, Paul S. Rennie, Artem Cherkasov, Michael E. Cox. Discovery and characterization of small molecules targeting the DNA-binding ETS domain of ERG in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 130. doi:10.1158/1538-7445.AM2017-130

Ras/MAPK signaling is often aberrantly activated in human cancers. The downstream effectors are transcription factors, including those encoded by the ETS gene family. Using cell-based assays and biophysical measurements, we have determined the mechanism by which Ras/MAPK signaling affects the function of Ets1 via phosphorylation of Thr38 and Ser41. These ERK2 phosphoacceptors lie within the unstructured N-terminal region of Ets1, immediately adjacent to the PNT domain. NMR spectroscopic analyses demonstrated that the PNT domain is a four-helix bundle (H2–H5), resembling the SAM domain, appended with two additional helices (H0–H1). Phosphorylation shifted a conformational equilibrium, displacing the dynamic helix H0 from the core bundle. The affinity of Ets1 for the TAZ1 (or CH1) domain of the coactivator CBP was enhanced 34-fold by phosphorylation, and this binding was sensitive to ionic strength. NMR-monitored titration experiments mapped the interaction surfaces of the TAZ1 domain and Ets1, the latter encompassing both the phosphoacceptors and PNT domain. Charge complementarity of these surfaces indicate that electrostatic forces act in concert with a conformational equilibrium to mediate phosphorylation effects. We conclude that the dynamic helical elements of Ets1, appended to a conserved structural core, constitute a phospho-switch that directs Ras/MAPK signaling to downstream changes in gene expression. This detailed structural and mechanistic information will guide strategies for targeting ETS proteins in human disease.

InterFeron-gamma Inducible protein 16 (IFI16) belongs to the interferon inducible HIN200 protein family that contains transcriptional regulators linked to cell cycle regulation and differentiation. All family members contain at most two domains of 200 amino acids, called HIN200, each containing two Oligonucleotide/Oligosaccharide Binding (OB) folds. IFI16 is involved in transcriptional repression and is a component of the DNA repair multi-protein complex known as BASC, which forms after UV-induced DNA damage. In this study, we used fold recognition and biophysical approaches as a tool to infer and validate functions to the HIN200 domain. Since the best template to model IFI16-HIN200 is Replication Protein A (RPA) in complex with single-stranded nucleic acids, we tested six RPA nucleic acid-binding characteristics for IFI16-HIN200. Our results indicate that IFI16-HIN200 is an RPA-like, OB-fold, nucleic acid-binding protein that binds to ssDNA with higher affinity than to dsDNA, recognizes ssDNA in the same orientation as RPA, oligomerizes upon ssDNA binding, wraps and stretches ssDNA, but does not destabilize dsDNA. We finally propose a framework model explaining how the HIN200 domain could prevent ssDNA from re-annealing.

Prostate cancer (PCa) is one of the leading causes of cancer-related death in men worldwide. The common treatment option for recurring and advanced PCa focuses on inhibiting the androgen receptor (AR). Unfortunately, despite an initial response to this treatment, drug resistance occurs, and the cancer relapses to an incurable, castration-resistant form; thus, there is a pressing need for new therapeutics. Previous research has shown that in up to 50% of all prostate cancer cases, the cause of the disease may be attributed to a common genomic rearrangement, the fusion between TMPRSS2 and ERG (ETS-related gene). ERG is a transcription factor mainly involved in hematopoiesis regulation during embryonic development, and it is not normally expressed in prostate cells in adults. However, its fusion with the TMPRSS2 promoter puts ERG under the regulation of AR, and as a consequence, ERG is one of the most commonly overexpressed genes in PCa. ERG overexpression in prostate epithelium has been shown to induce transformation and promote epithelial-mesenchymal transition (EMT) that gives cancer cells enhanced migratory and invasive characteristics. Currently, there is no approved therapeutic targeting ERG or any other member of the ETS family. While targeting transcription factors has been challenging, our integrated research team is specialized in targeting protein-DNA interaction sites. Using our established computer-aided drug discovery pipeline, we have identified several small molecules that can bind to and inhibit ERG. A total of 133 candidate compounds, pre-selected from the in silico screening of millions of chemical structures, were tested using a luciferase-based transcriptional reporter assay across two cell lines: the TMPRSS2-ERG fusion positive VCaP cell line, and the human prostate epithelial cell line (PNT1B) engineered to constitutively express ERG. In addition to transcriptional inhibition, the most potent compounds inhibited migration of PNT1B-ERG cells as demonstrated by a Real-Time Cell Analyzer. Significantly, both compounds shifted the binding spectra in protein NMR assays, indicating their direct interactions with residues located in the DNA binding domain of the ERG protein. We anticipate that results from this project will lead to the development of new drugs that can be used alternatively or synergistically with current anti-AR therapy to benefit patients with the most deadly forms of prostate cancer. (Supported by a grant from Prostate Cancer Canada) Citation Format: Mani Roshan-Moniri, Michael Hsing, Miriam S. Butler, Desmond Lau, Peter Axerio-Cilies, Paul Yen, Ari Kim, Scott Lien, Marta Mroczek, Dennis Ma, Huifang Li, Yubin Guo, Fuqiang Ban, Fariba Ghaidi, Eric LeBlanc, Lawrence McIntosh, Michael Cox, Artem Cherkasov, Paul S. Rennie. Therapeutic targeting of ETS factor ERG for the treatment of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1652. doi:10.1158/1538-7445.AM2015-1652

Despite the development of several successful targeted therapies, drug resistance and metastasis remain a significant challenge in the treatment of both breast (BCa) and prostate (PCa) cancers. For these reasons, there is an immediate need to identify a novel class of therapeutics targeting alternative factors, such as those that promote the metastatic capacity of tumour cells. ETS translocation variant 4 (ETV4) is a member of the ETS transcription factor family and is a significant mediator of tumorigenesis through its activation of several downstream pathways that are associated with migration and invasion. ETV4 is overexpressed in breast tumours and is associated with distant metastasis and poor prognosis particularly in triple negative BCa, which still lacks an approved targeted therapy. Similarly in PCa, overexpression of ETV4 is associated with the deregulation of the PI3K and Ras signalling pathways that are commonly implicated in metastatic disease. Like other ETS factors such as ERG and ETV1, fusion of the ETV4 gene can be found in a subtype of PCa cases and is associated with the disease progression. Thus, ETV4 is an important therapeutic candidate with potential applications in both advanced BCa and PCa. Drug development against ETV4 is made even more significant due to the lack of any approved therapy that directly targets it or any other members of the ETS family. Using a combination of in silico screening and in vitro assays, we have identified several small molecules with strong binding affinities and selectivity toward the DNA binding domain of ETV4. The prostate cell line, PC3, and the triple negative breast cancer cell line, MDA-MB-231, were used for in vitro studies as they endogenously express moderate to high levels of ETV4. Over 100 candidate compounds, which were selected from virtual screening against millions of small-molecular structures, were tested across the two cell lines using a luciferase-based transcriptional reporter assay. From this assay approximately 30 compounds were identified with significant inhibition on the transcriptional activity of ETV4 without a cytotoxic effect. The most potent of these compounds was shown by protein nuclear magnetic resonance (NMR) to directly interact with specific residues within the DNA binding domain. These compounds were also able to inhibit the migratory capacity of cancer cells. This data provides evidence for the direct targeting of ETV4 by small molecules, and future work will aim to further characterize their mechanism of action and effects on downstream targets in an effort to create a novel therapeutic strategy to treat metastatic breast and prostate cancers. (Supported by grants from the Canadian Cancer Society Research Initiative and Prostate Cancer Canada) Citation Format: Miriam S. Butler, Michael Hsing, Mani Roshan Moniri, Desmond Lau, Paul Yen, Ari Kim, Scott Lien, Marta Mroczek, Fariba Ghaidi, Eric LeBlanc, Lawrence McIntosh, Michael Cox, Artem Cherkasov, Paul S. Rennie. Targeting ETS factor ETV4 as a novel therapeutic for the management of breast and prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1648. doi:10.1158/1538-7445.AM2015-1648

Summary DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K D ∼60 μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.