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Combating castration-resistant prostate cancer by co-targeting the epigenetic regulators EZH2 and HDAC [1]

['Amy E. Schade', 'Department Of Medicine', 'Division Of Genetics', 'Brigham', 'Women S Hospital', 'Boston', 'Massachusetts', 'United States Of America', 'Harvard Medical School', 'Ludwig Center At Harvard']

Date: 2023-05 

While screening and early detection have reduced mortality from prostate cancer, castration-resistant disease (CRPC) is still incurable. Here, we report that combined EZH2/HDAC inhibitors potently kill CRPCs and cause dramatic tumor regression in aggressive human and mouse CRPC models. Notably, EZH2 and HDAC both transmit transcriptional repressive signals: regulating histone H3 methylation and histone deacetylation, respectively. Accordingly, we show that suppression of both EZH2 and HDAC are required to derepress/induce a subset of EZH2 targets, by promoting the sequential demethylation and acetylation of histone H3. Moreover, we find that the induction of one of these targets, ATF3, which is a broad stress response gene, is critical for the therapeutic response. Importantly, in human tumors, low ATF3 levels are associated with decreased survival. Moreover, EZH2- and ATF3-mediated transcriptional programs inversely correlate and are most highly/lowly expressed in advanced disease. Together, these studies identify a promising therapeutic strategy for CRPC and suggest that these two major epigenetic regulators buffer prostate cancers from a lethal response to cellular stresses, thereby conferring a tractable therapeutic vulnerability.

Funding: This work was supported by the National Cancer Institute (R01 CA198097 to K.C.) and the Cancer Research UK Grand Challenge and the Mark Foundation For Cancer Research (C5470/A27144 to K.C. as a member of the SPECIFICANCER Team). A.E.S. was supported by an American Cancer Society – Cancer Research Racquet and Women’s Tennis Association - Postdoctoral Fellowship, PF-22-040- 01-ET. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The PRC2 complex confers a repressive transcriptional signal by methylating histone H3 at lysine 27 (H3K27me3). Accordingly, EZH2 inhibitors promote a loss of this repressive mark [ 12 ]. However, the loss of this repressive signal may not be sufficient to restore transcription at some PRC2-regulated sites, if H3K27 acetylation (H3K27ac), which is associated with transcriptional activation, is suppressed. Interestingly, histone deacetylases (HDACs) are also overexpressed in prostate cancer and physically interact with PRC2 [ 13 – 15 ]. Therefore, HDACs often work in concert with the PRC2, by removing histone acetylation marks so that methylation may occur. As such, we hypothesized that the combined suppression of both EZH2 and HDAC might be required to derepress an important subset of PRC2 target genes in CRPC, by promoting the demethylation and the subsequent acetylation of H3K27 at some promoters. Here, we show that this is indeed the case. More importantly, we have discovered that EZH2 and HDAC inhibitors kill prostate cancers, in part, by activating a broad stress response gene, which is normally repressed in advanced tumors.

Agents that inhibit EZH2 are being clinically evaluated in a variety of tumor types [ 8 ], and one EZH2 inhibitor has been approved for the treatment of epithelioid sarcomas [ 9 , 10 ]. However, in animal and cell culture models of prostate cancer, EZH2 inhibitors modestly reduce proliferation, but exhibit little efficacy as single agents [ 7 , 11 ]. In neuroendocrine-like CRPC models, EZH2 inhibition reverses the neuroendocrine phenotype, restores AR expression, and partially resensitizes cells to androgen deprivation therapy [ 7 ]. Nevertheless, while combined AR and EZH2 inhibitors exert a more potent cytostatic response in these models, tumors still do not regress. Therefore, we set out to identify an EZH2 inhibitor-based drug combination that was capable of killing CRPC and inducing frank tumor regression.

Relatively little is known about the signals that drive and maintain advanced CRPC. However, one gene implicated in various aspects of prostate cancer progression is EZH2 (enhancer of zeste homologue 2). EZH2 encodes the histone methyltransferase component of the Polycomb Repressive Complex 2 (PRC2), which regulates epigenetic gene silencing [ 4 ]. It is overexpressed and/or amplified in prostate cancer, and expression levels are highest in advanced, metastatic tumors [ 5 ]. Moreover, EZH2 plays a direct causal role in driving prostate tumor progression and metastasis in animal models [ 6 ]. EZH2 has also been implicated in maintaining a neuroendocrine-like state in a subset of CRPCs, characterized, in part, by the loss of AR expression and an acquisition of AR independence [ 7 ]. These distinct roles for EZH2 in promoting prostate cancer progression, metastasis, and AR independence raise the possibility that it could be a potential therapeutic target in advanced disease.

The development of prostate cancer is dependent on androgen receptor (AR) signaling [ 1 ]. However, while androgen deprivation therapy is used to treat advanced disease, tumors that are refractory to anti-androgen therapies or are completely androgen independent can emerge [ 2 , 3 ]. Consequently, prostate cancer remains the third-leading cause of cancer death in men [ 2 ]. Therefore, it may be critical to develop therapeutic strategies for castration-resistant prostate cancer (CRPC) that extend beyond targeting AR pathways.

Results

EZH2 and HDAC inhibitors trigger the regression of CRPC in vivo We next evaluated the efficacy of these agents in vivo. To eliminate any potential effects of AR signaling, we used PC3 xenografts, which do not express the AR. Animals with established tumors (100 to 200 mm3) were either pretreated with the EZH2 inhibitor or vehicle to permit loss of H3K27me3. After 7 days of the pretreatment phase, mice were further segregated into 4 treatment arms (vehicle, HDACi, EZH2i, or HDAC/EZH2i) and were exposed to drugs for a total of 18 days. Consistent with in vitro observations, EZH2 and HDAC inhibitors exerted minimal effects on their own, but when combined triggered potent tumor regression (Fig 1G and S1 Data). Analysis of H3K27me3 and H3K27ac in tumor tissue confirmed that both agents effectively suppressed their targets in vivo (S1D Fig). Growth curves of responsive tumors demonstrate that EZH2 inhibition had no effect on tumor growth alone during the pretreatment phase, but that tumors immediately began to regress once HDACi was included in the regimen, and continued to shrink over time (Fig 1H and S1 Data). It should be noted that PC3 xenografts are an extremely aggressive tumor model, and few, if any, agents have been shown to cause this type of response. Cell death was readily observed within 4 days in tumors treated with both agents, as demonstrated by extensive cleaved caspase 3 (CC3) staining, which was not detected in tumors treated with vehicle or each single agent alone (Fig 1I). Notably, EZH2 and HDAC inhibitors also triggered tumor regression in an immunocompetent mouse model of AR-independent prostate cancer, PT-09, indicating that this combination remains effective in the context of an intact immune system (Figs 1J and S1E and S1 and S2 Data) [18].

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002038

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