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Supplementary Table 1. Human qRT-PCR primers Supplementary Table 2. Human ChIP-qPCR primers Figure S1. IDH2, IDH3A, IDH3B, and IDH3G are ubiquitously expressed across the human cancer spectrum. Figure S2. Relative mRNA expression of IDH genes in LAPC4 cells following 24h of treatment with 10 nM R1881 (right panel). KLK3 expression is shown as a positive control (left panel). Figure S3. Analysis of AR recruitment to IDH2 (A), IDH3A (B), IDH3B (C), and IDH3G (D) genes following 16h treatment with 10nM R1881. Figure S4. Achilles project results for cancer cell line sensitivity to shRNA-mediated knockdown of IDH1. Figure S5. IDH1 knockdown in PC3 and DU145 AR-negative PCa cells.

Mutations of the isocitrate dehydrogenase genes IDH1 and IDH2, key enzymes involved in citrate metabolism, are important oncogenic events in several cancer types, including in 1%–3% of all prostate cancer cases. However, if IDH1 and other IDH isoforms are associated with prostate cancer progression, as well as the regulatory factors controlling their expression and activity, remain mostly unknown. Using publicly available datasets, we showed that prostate cancer harbors the highest IDH1 expression across the human cancer spectrum and that IDH1 expression is altered during prostate cancer progression. We showed that the androgen receptor (AR), a key oncogene in prostate cancer, controls multiple IDH isoforms in both in vitro and in vivo models, predominantly positively regulating IDH1. Chromatin immunoprecipitation experiments confirmed the recruitment of AR at several regulatory regions of IDH1 and enzymatic assays demonstrated that AR significantly induces IDH activity. Genetic blockade of IDH1 significantly impaired prostate cancer cell proliferation, consistent with IDH1 having a key function in these cancer cells. Importantly, knockdown of IDH1 blocked the AR-mediated induction in IDH activity, indicating that AR promotes a mitochondrial to cytoplasmic reprogramming of IDH activity. Overall, our study demonstrates that IDH1 expression is associated with prostate cancer progression, that AR signaling integrates one of the first transcriptional mechanisms shown to regulate IDH1, and that AR reprograms prostate cancer cell metabolism by selectively inducing extra-mitochondrial IDH activity.Implications:The discovery that AR reprograms IDH activity highlights a novel metabolic reprogramming necessary for prostate cancer growth and suggests targeting IDH activity as a new therapeutic approach for prostate cancer treatment.

Cellular homeostasis requires the orderly expression of thousands of transcripts. Gene expression is regulated by numerous proteins that recognize post-translational modifications—in particular, the acetylation of lysine residues (Kac) on histones. In addition to affecting the general condensation state of the chromatin, acetylated histones act as anchor points for bromodomain (BRD)-containing adapter proteins. BRDs are the primary Kac reader domains in humans, and proteins containing them act as chromatin scaffolds that organize large networks of interactions to regulate transcription. To characterize BRD-dependent interaction networks, we established cell lines in which histone acetylation is dependent on acetate supplementation. To do this, we used genome editing to knock out ATP citrate lyase (ACLY), the enzyme responsible for converting citrate to oxaloacetate and acetyl-CoA in the cytoplasm and nucleus. In our cellular model, removing acetate from the culture medium resulted in the rapid catabolism of acetylated histones to restore the nucleocytoplasmic acetyl-CoA pool. Here we report the use of our new model in functional proteomics studies to characterize BRD-dependent interaction networks on the chromatin.

The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa), this phenotype is reprogrammed, making it an interesting therapeutic target. However, how the truncated prostate TCA cycle works is still not completely understood.We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis.First, mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate, thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis, several key nutrients were shown to allow the completion of the prostate TCA cycle, revealing a much more complex metabolic profile than originally anticipated. Indeed, along with the known pathway of aspartate replenishing oxaloacetate, glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids, aspartate entered the TCA cycle at the malate entry point, upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway, but also by at least three additional pathways: glutaminolysis, reductive carboxylation, and aspartate-malate conversion.Our results add a significant new dimension to the prostate citrate-secretory phenotype, with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa.

Sex-steroid hormones have been investigated for decades for their oncogenic properties in hormone-dependent cancers. The increasing body of knowledge on the biological actions of androgens in prostate cancer has led to the development of several targeted therapies that still represent the standard of care for cancer patients to this day. In the prostate, androgens promote cellular differentiation and proper tissue development. These hormones also promote the aberrant proliferation and survival of prostate cancer cells. Over the past few years, sequencing technologies for functional genomic analyses have rapidly expanded, revealing novel functions of sex-steroid hormone receptors other than their classic roles. In this article, we will focus on transcriptomic- and genomic-based evidence that demonstrates the importance of the androgen receptor signaling in the regulation of prostate cancer cell metabolism. This is significant because the reprogramming of cell metabolism is a hallmark of cancer. In fact, it is clear now that the androgen receptor contributes to the reprogramming of specific cellular metabolic pathways that promote tumor growth and disease progression, including aerobic glycolysis, mitochondrial respiration, fatty acid s-oxidation, and de novo lipid synthesis. Overall, beyond regulating development, differentiation, and proliferation, the androgen receptor is also a master regulator of cellular energy metabolism.

Prostate cancer cells (PCa) are dependent on the androgen receptor (AR) for their aberrant proliferation and survival. We have recently discovered that AR induces a reprogramming of PCa cell metabolism by controlling the cytoplasmic wild-type enzyme isocitrate dehydrogenase 1 (IDH1), which results in an enhanced proliferation of tumour cells. However, the specific metabolic functions of IDH1 in PCa or how to use the reliance of tumour cells on this enzyme as a therapeutic avenue, is elusive. In in vitro human PCa models, we showed that IDH1 protein levels and activity are increased in an AR-dependent manner. Using pharmacological and genetic tools, we showed that IDH1 is a major contributor to the replenishment of NADPH levels in PCa. This cofactor plays a key role in the synthesis of biomaterials required for cellular division, and our results indicate that IDH1 contribute to 30-40% of total cellular NADPH levels. In that context, blockade of IDH1 was shown to alter the mTOR signaling, a central regulator of cellular anabolism, which is linked to decreased cellular proliferation rates. FDA-approved pharmacological inhibitors of mutant IDH1 significantly inhibited IDH activity and proliferation in PCa cells, suggesting that such inhibitors could be used to treat PCa patients even in absence of IDH1 mutation. Globally, our results demonstrate that IDH1 is a key player in proliferative anabolic pathways in PCa. Importantly, they also support the hypothesis that inhibition of IDH1 using already-approved molecules represents one viable therapeutic solution. Citation Format: Kevin Gonthier, Cindy Weidmann, Lilianne Frégeau-Proulx, Étienne Audet-Walsh. Targeting the isocitrate dehydrogenase 1 (IDH1) metabolic enzyme in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3731.

Mutations of the isocitrate dehydrogenase genes IDH1 and IDH2, key enzymes involved in citrate metabolism, are important oncogenic events in several cancer types, including in 1%–3% of all prostate cancer cases. However, if IDH1 and other IDH isoforms are associated with prostate cancer progression, as well as the regulatory factors controlling their expression and activity, remain mostly unknown. Using publicly available datasets, we showed that prostate cancer harbors the highest IDH1 expression across the human cancer spectrum and that IDH1 expression is altered during prostate cancer progression. We showed that the androgen receptor (AR), a key oncogene in prostate cancer, controls multiple IDH isoforms in both in vitro and in vivo models, predominantly positively regulating IDH1. Chromatin immunoprecipitation experiments confirmed the recruitment of AR at several regulatory regions of IDH1 and enzymatic assays demonstrated that AR significantly induces IDH activity. Genetic blockade of IDH1 significantly impaired prostate cancer cell proliferation, consistent with IDH1 having a key function in these cancer cells. Importantly, knockdown of IDH1 blocked the AR-mediated induction in IDH activity, indicating that AR promotes a mitochondrial to cytoplasmic reprogramming of IDH activity. Overall, our study demonstrates that IDH1 expression is associated with prostate cancer progression, that AR signaling integrates one of the first transcriptional mechanisms shown to regulate IDH1, and that AR reprograms prostate cancer cell metabolism by selectively inducing extra-mitochondrial IDH activity. Implications: The discovery that AR reprograms IDH activity highlights a novel metabolic reprogramming necessary for prostate cancer growth and suggests targeting IDH activity as a new therapeutic approach for prostate cancer treatment.

A primary function of the prostate is to synthesize and secrete high levels of citrate by way of a unique metabolic profile regulated by the androgen receptor (AR). Prostate cancer (PCa) is an androgen-dependent disease that is characterized by early reprogramming of citrate metabolism. Accordingly, mutations of the isocitrate dehydrogenase genes IDH1 and IDH2, which are key enzymes involved in the regulation of cellular citrate levels, have been demonstrated as important oncogenic events in several cancer types, including in about 3% of all PCa cases. However, if IDH1 and other IDH isoforms are associated with PCa progression as well as the regulatory factors controlling their expression remain mostly unknown. Across the human cancer spectrum, PCa appears to be the cancer type with the highest expression of IDH1, with levels even higher than common cancers associated with IDH1 mutations such as gliomas. Using publicly available datasets and quantitative PCR, we showed that IDH1 is the predominant IDH isoform expressed in PCa cells. In PCa, the androgen receptor was found to regulate several IDH isoforms in both in vitro and in vivo models of PCa, predominantly positively regulating IDH1. Chromatin immunoprecipitation experiments confirmed the recruitment of AR at several regulatory regions of IDH1 and IDH2. IDH1 and other IDH isoforms were shown to be significantly altered during PCa progression, which is consistent with a reprogramming of citrate metabolism in PCa. In addition, modulation of IDH expression significantly altered PCa cell proliferation and metabolism. Overall, our study indicates that IDH gene regulation is associated with PCa progression and that AR plays a significant role in the regulation of IDH genes. Citation Format: Kevin Gonthier, Raghavendra TK Poluri, Cindy Weidmann, Etienne Audet-Walsh. Association of IDH genes with prostate cancer progression and their regulation by the androgen receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1853.