Emmanuel de Billy

Putting the heat on cancer

Two studies highlight the role of the heat shock response in initiating and maintaining cancer.

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.

HSF1 Is Essential for Myeloma Cell Survival and A Promising Therapeutic Target

Purpose: Myeloma is a plasma cell malignancy characterized by the overproduction of immunoglobulin, and is therefore susceptible to therapies targeting protein homeostasis. We hypothesized that heat shock factor 1 (HSF1) was an attractive therapeutic target for myeloma due to its direct regulation of transcriptional programs implicated in both protein homeostasis and the oncogenic phenotype. Here, we interrogate HSF1 as a therapeutic target in myeloma using bioinformatic, genetic, and pharmacologic means. Experimental Design: To assess the clinical relevance of HSF1, we analyzed publicly available patient myeloma gene expression datasets. Validation of this novel target was conducted in in vitro experiments using shRNA or inhibitors of the HSF1 pathway in human myeloma cell lines and primary cells as well as in in vivo human myeloma xenograft models. Results: Expression of HSF1 and its target genes were associated with poorer myeloma patient survival. ShRNA-mediated knockdown or pharmacologic inhibition of the HSF1 pathway with a novel chemical probe, CCT251236, or with KRIBB11, led to caspase-mediated cell death that was associated with an increase in EIF2α phosphorylation, CHOP expression and a decrease in overall protein synthesis. Importantly, both CCT251236 and KRIBB11 induced cytotoxicity in human myeloma cell lines and patient-derived primary myeloma cells with a therapeutic window over normal cells. Pharmacologic inhibition induced tumor growth inhibition and was well-tolerated in a human myeloma xenograft murine model with evidence of pharmacodynamic biomarker modulation. Conclusions: Taken together, our studies demonstrate the dependence of myeloma cells on HSF1 for survival and support the clinical evaluation of pharmacologic inhibitors of the HSF1 pathway in myeloma. Clin Cancer Res; 24(10); 2395–407. ©2018 AACR. See related commentary by Parekh, p. 2237

Abstract LB-304: Discovery of chemical probe CCT251236: An orally bioavailable efficacious pirin ligand from an HSF1 phenotypic screen

Heat shock factor 1 (HSF1) was originally identified as a master regulator of the classical ‘cytoprotective’ heat shock response. However, a large body of evidence has now verified the importance of HSF1 to tumorigenesis and cancer progression. HSF1 is activated by various elements of the cancer state, reprogramming the transcriptome in a way that is overlapping with, but distinct from, the canonical heat-shock response. Also, there is a strong correlation between the expression of activated HSF1 in tumors and adverse clinical outcomes. This evidence indicates that the inhibition of HSF1-mediated transcription could be a viable strategy in cancer treatment. Inhibiting the HSF1 stress pathway represents an attempt at targeting non-oncogene addiction and proteotoxic stress, which has been proposed to be advantageous. However, HSF1 is a ligandless transcription factor and is unlikely to be amenable to standard drug discovery strategies and direct inhibition with small molecules. Therefore, we proposed that inhibitors of HSF1-mediated transcription, which antagonize the HSF1 pathway but without necessarily binding directly to HSF1, could be discovered and developed via a cell-based phenotypic screen. We carried out a high throughput Arrayscan assay of 200,000 compounds to measure the inhibition of HSF1-mediated HSP72 expression stimulated by pre-treatment with an HSP90 inhibitor. We identified a singleton hit with a bisamide core, CCT245232. This compound showed potent growth inhibition in a range of human cancer cell lines but had poor physicochemical properties leading to an unacceptable pharmacokinetic profile. Improvement of the physicochemical properties of CCT245232 whilst maintaining potency versus our cell-based assays led to the orally bioavailable tool compound CCT251236. This compound shows potent growth inhibition (GI50 values in low nanomolar range) of human ovarian cancer cell lines in vitro and good efficacy against human ovarian cancer xenografts in nude mice in vivo. We applied chemo-proteomic strategies to identify the molecular target using a probe based on CCT251236 and discovered pirin as a high affinity molecular target. Binding of CCT251236 to recombinant pirin was confirmed in biophysical assays. CCT251236 recapitulates the reported anti-migratory phenotype for a pirin ligand although binding to pirin alone does not explain the cellular phenotype observed with our chemical tool. We are currently using CCT251236 as a chemical probe while further optimizing its properties to identify a clinical candidate. Citation Format: Matthew D. Cheeseman, Nicola E. Chessum, Carl S. Rye, Elisa A. Pasqua, Michael J. Tucker, Birgit Wilding, Lindsay E. Evans, Susan Lepri, Meirion Richards, Swee Y. Sharp, Salyha Ali, Martin Rowlands, Lisa O9Fee, Asadh Miah, Angela Hayes, Alan T. Henley, Marissa Powers, Robert te Poele, Emmanuel De Billy, Loredana Pellegrino, Florence Raynaud, Rosemary Burke, Robert L. van Montfort, Suzanne A. Eccles, Keith Jones, Paul Workman. Discovery of chemical probe CCT251236: An orally bioavailable efficacious pirin ligand from an HSF1 phenotypic screen [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 LB-304. doi:10.1158/1538-7445.AM2017-LB-304

Correction: Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9. See DOI: 10.1039/c6md00159a

Correction for ‘Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9’ by Carl S. Rye et al., Med. Chem. Commun., 2016, 7, 1580–1586.

Abstract 2998: Molecular and cellular effects of individual and combinatorial silencing of HSP90 paralogs

HSP90 is an ATP-dependent molecular chaperone critical for the folding, stability and function of over 350 client proteins, including many that are oncogenic. Pharmacologic inhibition of the essential ATPase domain inhibits HSP90 function and produces a well-documented molecular and cellular response comprising depletion of client proteins, induction of heat shock proteins and reduction of tumor cell proliferation. There are four paralogs within the HSP90 family, HSP90α, HSP90β, GRP94 and TRAP1, and it is not currently clear what precise role each one plays in the molecular and therapeutic response to HSP90 inhibitors. Here we use an siRNA approach to selectively reduce the expression of each HSP90 paralog, alone or in combination, in an attempt to dissect the individual roles of each chaperone in the molecular and cellular response associated with pharmacologic HSP90 inhibition. In the human cancer models used we found that by simultaneously silencing HSP90α and HSP90β we could phenocopy aspects of pharmacologic HSP90 inhibition including reduction of cell proliferation, induction of HSP72 and other HSF-1 target genes and depletion of oncogenic client proteins. Through analysing representative HSP90 client proteins we observed interesting patterns of paralog-dependence that were related to their overall sensitivity to pharmacologic HSP90 inhibition. Our results reveal the importance of HSP90 paralogs in particular aspects of the molecular and cellular response to pharmacologic HSP90 inhibition. Citation Format: Marissa V. Powers, Robert H. Te Poele, Ravindhi L. Murphy, Emmanuel de Billy, Paul A. Clarke, Paul Workman. Molecular and cellular effects of individual and combinatorial silencing of HSP90 paralogs. [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 2998.

Abstract B166: A RNA helicase siRNA screen to identify potential therapeutic targets in castration-resistant prostate cancer

Castration-resistant prostate cancers (CRPC) are resistant to androgen-deprivation therapies commonly used to treat carcinoma of the prostate, resulting in death from this disease. CRPC can remain AR-driven through upregulation of the expression of wild-type, mutated or alternatively spliced constitutively active AR. Targeting both full-length AR and AR splice variants may overcome endocrine resistance. Since RNA helicases play crucial roles in various aspects of RNA metabolism including transcription, pre-mRNA splicing, translation, RNA export and RNA decay, we evaluated potentially druggable RNA helicases implicated in the regulation of AR and AR-V7 expression. We performed an siRNA screen targeting RNA helicases in which the readout was the expression of AR and AR-V7 mRNA as determined by quantitative RT-PCR. Out of the 75 human RNA helicases silenced, we identified 11 that induced a 50% or greater decrease in both AR and AR-V7 mRNA expression. These included 3 RNA helicases that have no known described function, 2 that regulate ribosomal RNA transcription, 3 required in nuclear export of spliced and unspliced mRNA, 2 in translation initiation and the DEAD-box helicase EIF4A3 involved in nonsense-mediated decay. Among these, only EIF4A3 reduced cell numbers by 40% in addition to decreasing AR and AR-V7 expression. Silencing EIF4A3 resulted in a 50-80% inhibition of cell proliferation and between 2- to 11-fold induction of apoptosis in a panel of 6 prostate cancer cell lines. Inhibiting EIF4A3 in CRPC may block the expression of important oncogenes including AR and counteract the emergence of splice variants involved in resistance mechanisms. Citation Format: Hannah Wang, Emmanuel de Billy, Johann de Bono, Paul Workman. A RNA helicase siRNA screen to identify potential therapeutic targets in castration-resistant prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B166.

Abstract 1775: Identification of small molecule inhibitors of HSF1 stress pathway activation in cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Heat Shock Factor 1 (HSF1) is a key transcription factor involved in proteostasis and response to stress, as well as being implicated in many diseases including cancer. Up-regulation of its activity by environmental stress or oncogenesis leads to transcriptional induction of genes involved in diverse cellular processes supporting the cancer state, including proteostasis, proliferation, survival and metastasis. Inhibition of HSF1 is therefore potentially beneficial for cancer treatment, but HSF1 is not technically druggable. We developed a high-throughput cell-based reporter gene assay to screen a library of small-molecule kinase inhibitors and identified 3 compound series as validated inhibitors of HSF1 activation by the HSP90 inhibitor 17-AAG. Further characterization of imidazo[1,2-b]pyridazine compounds showed inhibition of HSF1 target gene expression and the ability to mimic other features of the HSF1 knockdown phenotype. Using these tool compounds and by siRNA knockdown of HSF1 expression we demonstrate that HSF1 inhibition leads to cell cycle arrest and enhances the antiproliferative effect of 17-AAG. In addition, we show that HSP90 inhibition induces HSF1 phosphorylation at two serine residues, Ser326 and Ser320, highlighting the importance of Ser320 phosphorylation in HSP72 up-regulation by 17-AAG-induced HSF1 activation, and show that this activation is inhibited by our tool compounds. Our findings support the optimization and development of small-molecule inhibitors of the HSF1 pathway for cancer treatment. Note: This abstract was not presented at the meeting. Citation Format: Emmanuel de Billy, Nicola Chessum, Robert Te Poele, Jennifer Smith, Lorenzo Zani, Swee Sharp, Mark Stubbs, Wynne Aherne, Keith Jones, Paul Workman. Identification of small molecule inhibitors of HSF1 stress pathway activation in cancer cells. [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 1775. doi:10.1158/1538-7445.AM2014-1775