P300-Mediated Acetylation of Histone Demethylase JMJD1A Prevents Its Degradation by Ubiquitin Ligase STUB1 and Enhances Its Activity in Prostate Cancer

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Author Manuscript Published OnlineFirst on June 10, 2020; DOI: 10.1158/0008-5472.CAN-20-0233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

p300-mediated acetylation of histone demethylase JMJD1A prevents its degradation by ubiquitin ligase STUB1 and enhances its activity in prostate cancer

Songhui Xu1,2,*, Lingling Fan1,2,*, Hee-Young Jeon1,2, Fengbo Zhang1,2,3, Xiaolu Cui1,2,4, McKayla B. Mickle1,2, Guihong Peng1,2, Arif Hussain2,5, Ladan Fazli6, Martin E. Gleave6, Xuesen Dong6, Jianfei Qi1,2,#

1Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD, USA. 2Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD, USA. 3Department of Urology, Beijing Friendship Hospital, Capital Medical University, Beijing, 100050, China. 4Department of Urology, First Hospital of China Medical University, Shenyang, 110001, China. 5Baltimore VA Medical Center, Baltimore, MD, USA. 6Vancouver Prostate Centre, University of British Columbia, Vancouver, BC, Canada.

* These authors contributed equally to the study.

Running Title: Regulation of JMJD1A protein stability in prostate cancer

# Correspondence – Jianfei Qi, Marlene and Stewart Greenebaum Comprehensive Cancer Center, Department of Biochemistry and Molecular Biology, University of Maryland, 655 W Baltimore St, Baltimore, MD, 21201 USA. Office Phone: 410-706-2192. Email: [email protected]

The authors declare no conflicts of interest.

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 10, 2020; DOI: 10.1158/0008-5472.CAN-20-0233 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

The androgen receptor (AR) pathway plays a central role in the development of castration- resistant prostate cancer (CRPC). The histone demethylase JMJD1A has been shown to regulate activities of AR and c-Myc transcription factors and promote prostate cancer progression. Here we report that JMJD1A protein stability is controlled by the ubiquitin ligase STUB1. High levels of JMJD1A were strongly correlated with low STUB1 levels in human CRPC specimens. STUB1 inhibited AR activity, AR-V7 levels, and prostate cancer cell growth partly through degradation of JMJD1A. Furthermore, the acetyltransferase p300 acetylated JMJD1A at lysine (K) 421, a modification that recruits the BET family member BRD4 to block JMJD1A degradation and promote JMJD1A recruitment to AR targets. Increased levels of both total and K421-acetylated JMJD1A were observed in prostate cancer cells as they developed resistance to the AR antagonist enzalutamide. Treatment of prostate cancer cells with either p300 or BET inhibitors destabilized JMJD1A and enzalutamide-resistant prostate cancer cells were more sensitive than parental cells to these inhibitors. Together, our findings identify a critical role for acetylation of JMJD1A in regulating JMJD1A stability and AR activity in CRPC. These newly identified mechanisms controlling JMJD1A protein stability provide potential druggable targets to encourage the development of additional therapies for advanced prostate cancer.

Statement of Significance Identification of mechanisms regulating JMJD1A protein stability reveals new strategies to destabilize JMJD1A and concomitantly inhibit AR activities as potential prostate cancer therapy.

Introduction

Androgen deprivation therapy (ADT) is the primary therapy for metastatic prostate cancer (PCa). However, most PCa tumors become resistant to ADT and progress to a lethal stage called castration-resistant prostate cancer (CRPC). AR plays a central role in driving CRPC progression. AR transcriptional activity in CRPC can be restored via multiple mechanisms, among them, AR overexpression/mutation/splicing, overexpression of AR co-factors, and intratumoral androgen biosynthesis (1). The second-generation AR-pathway inhibitors (such as enzalutamide or abiraterone) and chemotherapy are used as CRPC therapies, but they extend a patient's life by only a few months (2,3). Thus it is critical to identify new therapeutic targets against CRPC. Methylation of histone 3 lysine-9 (H3K9) is a repressive epigenetic modification. The JMJD1A histone demethylase removes mono- and di-methyl groups from H3K9 (specifically, from H3K9me1 or H3K9me2), enabling transcriptional activation in processes such as spermatogenesis, metabolism, sex determination and stem cell self-renewal (4-7). JMJD1A is upregulated and plays a tumor-promoting role in a variety of malignancies (8-15). We previously reported that JMJD1A promotes proliferation and survival of PCa cells through AR and c-Myc pathways (16-18). JMJD1A regulates AR activity as an AR co-activator or by enhancing the alternative splicing of AR-V7, a hormone-independent truncated form of AR (16,17,19,20). JMJD1A also elevates c-Myc levels by enhancing transcription of c-Myc mRNA or attenuating c-Myc protein degradation (16). These findings indicate that JMJD1A is a potential target for PCa therapy. The ubiquitin ligase STUB1 promotes ubiquitination and degradation of several substrates (among them oncogenes and tumor suppressors, such as EGFR, c-Myc, AR-V7, PTEN and p53) context-dependently (21-25). Here, we identified STUB1 as the first known E3 ligase to target JMJD1A. JMJD1A degradation by STUB1 was antagonized by JMJD1A acetylation and subsequent recruitment of BRD4, a member of the Bromodomain and Extra-Terminal Domain (BET) family. The bromodomain (BRD) itself recognizes acetylated lysines on histones or other proteins (26), and the BET family members are promising targets for PCa therapy (27-29).

In this study, we identified mechanisms underlying regulation of JMJD1A stability and AR activity in CRPC. Our study suggests that targeting JMJD1A stability may be a novel treatment strategy for advanced PCa.

Materials and Methods

Antibodies. Antibodies were purchased from the following companies: JMJD1A (12835-1-AP), Proteintech (Rosemont, IL). STUB1 (A301-572A) and BRD4 (A301-985A), Bethyl Laboratories (Montgomery, TX). JMJD1A (sc-376608), AR (sc-816), p300 (sc-585, sc-48343), ubiquitin (sc- 6085, sc-8017), HA (sc-7392, sc-805), GST (sc-138) and c-Myc (sc-40, sc-789), Santa Cruz Biotechnology (Dallas, TX). AR-V7 (31-1109-00), RevMAb Biosciences (South San Francisco, CA). Acetyl-lysine (#9441), GST (#2622) and c-Myc (#9402), Cell Signaling (Danvers, MA). Ki-67 (ab8191), Abcam (Cambridge, United Kingdom). AR (06-680), H3K9me2 (07-441) and acetyl- Histone H3 (06-599), EMD Millipore (Burlington, MA). STUB1 (S1073), Flag (F7425, F3165) and actin (A5441), Sigma-Aldrich (St. Louis, MO). Acetyl-K421 JMJD1A antibody was generated using a commercial service provided by Abclonal (Woburn, MA).

Cell Lines. Rv1 cells (also called CWR22Rv1 cells) were provided by Dr. James Jacobberger (Case Western Reserve University, Cleveland, Ohio). C4-2 cells were provided by Dr. Leland Chung (Cedars-Sinai Medical Center, Los Angeles, CA). Enzalutamide-resistant C4-2 cells were generated by serial growth and passaging of parental C4-2 cells in sequentially increasing concentrations of enzalutamide (2 to 20 M) for over 6 months. PC3, LNCaP or primary human prostate epithelia cells (HPrECs) were purchased from and authenticated by American Type Culture Collection (ATCC). Other cell lines were authenticated by short tandem repeats (STR) analysis (Genomics Core Facility, University of Maryland Baltimore, MD). All cells were maintained in RPMI 1640 media supplemented with 10% FBS and antibiotics. Cells were periodically checked for Mycoplasma by PCR analysis, and cells of <20 passages were used for experiments.

Animal studies. Athymic nude mice (NU/NU) were purchased from the Jackson Laboratory (Bar Harbor, Maine) and housed in the animal facility at University of Maryland School of Medicine. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC #

0613011) and conducted following the institute’s animal policies in accordance with NIH guidelines.

Prostate cancer samples. A total of 219 PCa specimens were obtained from the Vancouver Prostate Tissue Bank at University of British Columbia (Clinical Research Ethics Board number: H09-01628). Among them were 140 primary PCa and 79 CRPC specimens. Primary PCa specimens were obtained from radical prostatectomy and CRPC specimens from transurethral resections of the prostate. H&E slides were reviewed and desired areas marked. Tissue microarrays (TMAs) were manually constructed (Beecher Instruments, MD, USA) by punching duplicate 1 mm cores from each sample.

Statistical analysis. The in vitro experiments were done in biological triplicate each time and independently repeated at least three times. Data are presented as the mean ± s.d. Student’s t- test (two-tailed) was used to compare differences between two groups of datasets with similar variance. Analysis of variance (ANOVA) test was used to compare differences among more than two groups of datasets. For immunohistochemistry staining of JMJD1A or STUB1, groups of PCa tissues displaying a given staining score (low, moderate or high) were compared, and the Mann- Whitney U test was used for the statistical analysis. For all statistical analyses, differences were labeled as * (p<0.05), ** (p<0.01), or *** (p<0.001). p values <0.05 were considered statistically significant. Additional Experimental Procedures can be found in supplemental information.

Results

JMJD1A protein levels increase in CRPC specimens After validating the specificity of JMJD1A antibody (Figure S1A, S1B), we performed immunohistochemistry staining for JMJD1A on a human PCa tissue microarray (TMA) consisting of 219 specimens of primary PCa and CRPC. JMJD1A staining revealed strong nuclear with weak cytoplasmic localization (Figure 1A, S1B). Based on staining intensity, we defined JMJD1A levels as low, moderate or high. Relative to the primary PCa, the percentage of CRPC specimens exhibiting JMJD1A-high staining increased over 2.7-fold (Figure 1A). Despite JMJD1A protein upregulation in CRPC, GEO datasets of PCa tissues showed no increase of JMJD1A mRNA levels

in metastatic PCa or CRPC relative to primary PCa (Figure S1C, S1D), suggesting that elevated JMJD1A protein levels seen in the CRPC specimens may be due to post-transcriptional mechanism(s). JMJD1A interacts with the ubiquitin ligase STUB1 To identify factors that potentially regulate JMJD1A protein levels, we conducted mass spectrometry analysis for proteins co-precipitating with JMJD1A in PCa cells. That analysis identified three candidate ubiquitin ligases including HUWE1, DDB1 (an adaptor protein in the Cul4 ubiquitin ligase complex) and STUB1 (Table S1). To determine which ubiquitin ligase may regulate JMJD1A, we knocked down HUWE1, DDB1 or STUB1 in Rv1 cells (Figure S1E). STUB1 knockdown increased the protein levels of JMJD1A (Figure S1F) and mRNA levels of AR targets (Figure S1G), whereas knockdown of HUWE1 or DDB1 had no such effects, indicating STUB1 as a promising candidate. We confirmed that endogenous STUB1 and JMJD1A co-precipitated in Rv1 cells (Figure 1B). To map interacting domains of both, we performed co- immunoprecipitation (co-IP) experiments using truncated mutants of JMJD1A and STUB1. Myc- STUB1 co-precipitated with the N-terminal half of Flag-JMJD1A, but not the C-terminal half (Figure 1C). STUB1 consists of an N-terminal TPR domain that interacts with chaperones, a central region, and a C-terminal U-box domain that recruits the E2 ubiquitin-conjugating enzyme (Figure S1H). Flag-JMJD1A co-precipitated with mutant STUB1 lacking the TPR domain, but not a mutant lacking the U-box domain (Figure 1D). Finally, to determine whether STUB1 and JMJD1A directly interact, we mixed purified GST-STUB1 with Flag-JMJD1A and tested binding in vitro. GST-STUB1, but not GST protein, co-precipitated with Flag-JMJD1A (Figure 1E, S1I), indicative of direct interaction. Together, these results demonstrate that JMJD1A N- terminal half interacts with the STUB1 U-box domain. STUB1 induces ubiquitination and degradation of JMJD1A To determine whether STUB1 induces the degradation of JMJD1A, we overexpressed both Myc- STUB1 and Flag-JMJD1A in 293T cells, and found that overexpression of Myc-STUB1 reduced Flag-JMJD1A protein levels (Figure 1F, lane 2 vs. lane 1). To determine which STUB1 domain is required for JMJD1A degradation, we conducted similar analysis using STUB1-K30A (a TPR domain mutant that cannot interact with chaperones) or STUB1-H260Q (a U-box domain

mutant lacking ubiquitin ligase activity (30). Overexpression of STUB1-K30A reduced Flag- JMJD1A protein levels (Figure 1F, lane 3 vs. lane 1), whereas overexpression of STUB1-H260Q had no effect on Flag-JMJD1A levels (Figure 1F, lane 4 vs. lane 1). Thus, STUB1-induced JMJD1A degradation requires its ubiquitin ligase activity but not its chaperone binding activity. To test whether STUB1 ubiquitinates JMJD1A, we co-expressed Flag-JMJD1A, Myc- STUB1 and HA-ubiquitin in 293T cells, IP'd Flag-JMJD1A with anti-Flag M2 beads and performed western blotting with HA antibodies to detect ubiquitination. Overexpression of STUB1 induced poly-ubiquitination of JMJD1A (Figure 1G, lane 3), whereas much less of ubiquitinated JMJD1A was seen in the absence of STUB1 overexpression (Figure 1G, lane 2). To test whether STUB1 directly ubiquitinates JMJD1A, we performed an in vitro ubiquitination assay using purified Flag- JMJD1A, GST-STUB1 and E1/E2/ubiquitin. Following precipitation of Flag-JMJD1A and western blotting with ubiquitin antibodies, we found that GST-STUB1 directly ubiquitinated Flag-JMJD1A in vitro (Figure 1H, lane 2). To determine whether STUB1 alters JMJD1A stability, we performed cycloheximide chase experiments to determine JMJD1A half-life in Rv1 cells. JMJD1A half-life in control and STUB1-knockdown cells was ~4 and 7 hours, respectively (Figure 1I), indicating that STUB1 knockdown prolongs JMJD1A half-life. Knockdown of STUB1 in various PCa cells increased JMJD1A protein levels (Figure 1J, S1J), but had no effect on JMJD1A mRNA levels (Figure S1K). These results indicate overall that STUB1 induces the ubiquitination-mediated degradation of JMJD1A in PCa cells. STUB1 and JMJD1A staining is inversely correlated in CRPC specimens To determine the in vivo relevance of our findings, we performed immunohistochemistry staining of STUB1 on the same PCa TMA used for JMJD1A staining, after validating the specificity of STUB1 antibody (Figure S1L, S1M). STUB1 staining revealed strong cytoplasmic with weak nuclear localization (Figure 1K, S1M). Based on staining intensity, we defined STUB1 levels as low, moderate or high. The percentage of specimens exhibiting STUB1-low staining was 2.4-fold greater in CRPC compared to primary PCa (Figure 1K). We conclude that low levels of STUB1 are correlated with high levels of JMJD1A in CRPC specimens (Figure 1K, Figure 1A). GEO datasets of PCa tissues showed no decrease of STUB1 mRNA levels in metastatic PCa or

CRPC relative to primary PCa (Figure S1N, S1O), suggesting a post-transcriptional mechanism(s) in the regulation of STUB1 protein in CRPC. STUB1 regulates AR activities and AR-V7 levels partly via JMJD1A STUB1 has been reported to induce AR degradation when overexpressed or under stress conditions (31,32). However, STUB1 knockdown in Rv1 or C4-2 cells had little effect on AR protein levels (Figure 1J), indicating that AR is not a STUB1 substrate in PCa cells under physiological conditions. Nonetheless, STUB1 knockdown in Rv1 (Figure 2A) or C4-2 (Figure S2A) cells did increase mRNA levels of AR targets such as PSA and KLK2. As JMJD1A interacts with AR and functions as an AR coactivator (19,20), we asked whether STUB1 regulated binding of JMJD1A or AR to these AR targets by knocking down STUB1 in Rv1 cells and performing chromatin immunoprecipitation (ChIP) with antibodies for JMJD1A, AR or the JMJD1A substrate H3K9me2. Relative to controls, STUB1 knockdown increased enrichment of JMJD1A or AR and decreased enrichment of H3K9me2 marks at the ARE region of PSA (Figure 2B) and KLK2 (Figure S2B) genes, indicating that STUB1 knockdown increases recruitment of AR to targets, possibly by increasing JMJD1A levels and JMJD1A-dependent H3K9 demethylation. Consistent with a reported role for JMJD1A in alternative splicing of AR-V7 (17), STUB1 knockdown in Rv1 cells increased AR-V7 at both mRNA and protein levels (Figure 1J, 2A). Interestingly, STUB1 was recently reported to induce ubiquitination and degradation of AR-V7 (25). As STUB1 destabilizes JMJD1A (Figure 1) and JMJD1A promotes the splicing of AR-V7 mRNA (17), we determined to test whether STUB1 also affects the AR-V7 splicing. We transfected the AR-null PC3 cells (control or STUB1 knockdown) with an AR mini-gene reporter and used the qRT-PCR to check the spliced products of AR full-length (AR-FL) or AR-V7 as previously described (17). Compared with control cells, STUB1-knockdown cells exhibited an increase in the splicing of AR-V7, but not that of AR-FL (Figure 2C). Thus, STUB1 may also inhibit the AR-V7 splicing likely through JMJD1A, in addition to its reported role in destabilizing AR-V7 protein. To determine whether effects of STUB1 on AR activities and AR-V7 levels depend on JMJD1A, we knocked down STUB1 in Rv1 cells, which increased JMJD1A levels, and then partially knocked down JMJD1A in those cells to levels seen in control cells (Figure 2D). STUB1-

knockdown Rv1 cells showed increased mRNA levels of AR targets, an effect attenuated upon partial JMJD1A knockdown (Figure 2E). Elevated levels of AR-V7 mRNA and protein observed in STUB1-knockdown Rv1 cells were also attenuated upon partial JMJD1A knockdown (Figure 2D, 2E). Similarly, knockdown of STUB1 in C4-2 cells increased mRNA levels of AR targets, an effect attenuated upon partial JMJD1A knockdown (Figure S2C). These results indicate that STUB1 reduces AR activities and AR-V7 levels partly via JMJD1A. STUB1 plays a tumor-suppressive role by degrading JMJD1A STUB1 can play a tumor-promoting or -suppressing role depending on context. To determine its function in prostate cancer, we knocked down STUB1 in PCa cells and measured cell proliferation by colony formation or soft agar assays. Relative to controls, STUB1 knockdown in AR-positive Rv1 (Figure 3A, 3B) or C4-2 (Figure S3A, S3B) cells increased cell proliferation in the presence or absence of androgen, with this effect being more apparent under androgen deprived conditions. STUB1 knockdown also increased proliferation of AR-negative PC3 cells independent of androgens (Figure S3C, S3D). To evaluate these effects in vivo, we subcutaneously injected control or STUB1-knockdown Rv1 cells into nude mice and monitored xenograft tumor growth. Compared with control Rv1 cells, STUB1-knockdown cells showed an increase in the xenograft tumor growth (Figure 3C-3E), concomitant with increased staining of proliferation marker Ki-67 in tumor sections (Figure 3F). To determine whether STUB1 inhibits prostate cancer cell growth via its effect on JMJD1A, we analyzed growth of control, STUB1-knockdown, and STUB1/JMJD1A-double knockdown PCa cells. Increased colony formation seen in STUB1-knockdown Rv1 (Figure 3G), C4-2 (Figure S3E) or PC3 (Figure S3F) cells was attenuated upon partial JMJD1A knockdown. Next, we analyzed xenograft tumor growth by control, STUB1-knockdown or STUB1/JMJD1A- double knockdown Rv1 cells in the castrated nude mice. STUB1 knockdown in xenografts enhanced tumor growth (Figure 3H-3J), elevated levels of JMJD1A, AR-V7, PSA and c-Myc (Figure S3G), and increased staining of proliferation marker Ki-67 (Figure 3K). However, these phenotypes of STUB1-knockdown xenografts were attenuated upon partial JMJD1A knockdown (Figure 3H-3K, S3G). Together, these results indicate that STUB1 inhibits PCa cell growth in part through JMJD1A degradation.

p300 acetylates JMJD1A at K421 to increase JMJD1A stability Removal of repressive H3K9 methylation in chromatin by JMJD1A was reportedly accompanied by increased histone acetylation (33-35), suggesting that JMJD1A cooperates with histone acetyltransferase(s) to activate transcription. Indeed, our ChIP analysis showed that JMJD1A knockdown in Rv1 cells increased enrichment of H3K9me2, and decreased enrichment of p300 and acetyl-H3 at the PSA gene ARE (Figure 4A). By contrast, STUB1 knockdown in Rv1 cells decreased H3K9me2 enrichment, and increased enrichment of JMJD1A, p300 and acetyl-H3 at the PSA gene ARE (Figure 4B). These results suggest that JMJD1A may recruit p300 to mediate H3K9 demethylation and histone acetylation on AR targets. Consistent with this possibility, we found that Myc-JMJD1A co-precipitated with Flag- p300, but not with the other acetyltransferases (NCOA1, NCOA2 or NCOA3) known to regulate AR activities (Figure S4A), indicating the specificity of JMJD1A/p300 interaction. JMJD1A co- precipitated with p300 in Rv1 or C4-2 cells (Figure 4C), indicating endogenous interaction of the two proteins. To determine how they interact, we overexpressed truncated p300 or JMJD1A mutants for co-IP experiments in 293T cells. Myc-JMJD1A co-precipitated with the N-terminal p300 fragment containing the Taz1 domain, but not with the other four p300 fragments (Figure 4D, S4B), indicating interaction of JMJD1A with that p300 domain. On the other hand, Flag-p300 co-precipitated the N-terminal half, but not the C-terminal half, of Myc-JMJD1A (Figure 4E). Overexpression of wild type p300, but not its catalytically-inactive mutant (Y1467F), increased protein levels of JMJD1A ectopically expressed in 293T cells (Figure 4F), whereas co-expression of Myc-p300 and Flag-JMJD1A had no effect on mRNA levels of Flag-JMJD1A (Figure S4C). In contrast, p300 knockdown in PCa cells reduced JMJD1A protein levels (Figure 4G), effects attenuated by treating Rv1 cells with the proteasome inhibitor MG132 (Figure 4H). Knockdown of p300 in PCa cells had no effect on JMJD1A mRNA levels (Figure S4D). Given the JMJD1A/p300 interaction, we asked whether p300 acetylates JMJD1A. To do so, we co-expressed Flag-JMJD1A with WT or mutant forms of p300 in 293T cells, and performed IP with anti-Flag antibodies followed by western blotting with acetyl-lysine antibodies. Overexpression of WT p300, but not its catalytically-inactive mutant, induced acetylation of Flag-JMJD1A (Figure 4I). Moreover, p300 knockdown significantly reduced

acetylation of endogenous JMJD1A IP'd from Rv1 cells (Figure 4J). To identify JMJD1A lysine residues modified by p300, we co-expressed Flag-JMJD1A and Myc-p300 in 293T cells, and IP'd Flag-JMJD1A for trypsin digestion and mass spectrometry analysis. That analysis identified 6 candidate acetyl-lysine residues on the tryptic JMJD1A peptides (Table S2). We mutated each to arginine and found that one, the K421R mutation, abolished the p300-induced JMJD1A acetylation, whereas mutation of the other 5 sites had little effect (Figure 4K). We next transfected 293T cells with equal amounts of plasmids encoding either WT or K421R mutant JMJD1A, and observed reduced protein levels of JMJD1A-K421R relative to the WT form, an effect blocked when cells were treated with MG132 (Figure 4L). Furthermore, p300 overexpression in 293T cells increased levels of co-expressed WT JMJD1A but not the JMJD1A-K421R mutant (Figure 4M), supporting the notion that p300 stabilizes JMJD1A via K421 acetylation. We next asked whether JMJD1A acetylation altered its ubiquitination by STUB1. To do so, we co-expressed Flag-JMJD1A (WT or K421R) with Myc-STUB1 and HA-ubiquitin in 293T cells. The JMJD1A-K421R mutant exhibited higher levels of poly-ubiquitination than did WT JMJD1A (Figure 4N, lane 4 vs. lane 2). Consistently, quantities of co-precipitated STUB1 with JMJD1A-K421R were greater than quantities of co-precipitated STUB1 with WT JMJD1A (Figure 4O). These results indicate overall that p300 induces JMJD1A acetylation at K421 and this increases JMJD1A stability by interfering with its interaction with STUB1 and preventing its subsequent ubiquitination. BRD4 regulates stability of JMJD1A Acetylated lysines found on histones or transcription regulators are known to recruit bromodomain (BRD) proteins for the regulation of gene expression (26), and the BET family BRD proteins are key coactivators of AR (27). Therefore, we asked whether activities associated with JMJD1A acetylation required BET family members. JQ1 blocks interaction of acetylated lysines with a bromodomain and is a widely used BET inhibitor (36). JQ1 treatment of Rv1 cells reduced JMJD1A protein levels, an outcome attenuated by co-treatment with the proteasome inhibitor MG132 (Figure 5A). JQ1 treatment had no effect on JMJD1A mRNA levels (Figure S5A).

To determine which BET family members function in stabilization of JMJD1A protein, we knocked down BRD2, 3 or 4 in Rv1 cells (Figure S5B-S5D). BRD4 knockdown reduced JMJD1A protein levels, whereas knockdown of BRD2 or BRD3 had little effect (Figure 5B). Knockdown of BRD2, 3 or 4 had no effect on JMJD1A mRNA levels (Figure S5B-S5D). These results suggest that BRD4 may regulate the stability of JMJD1A protein. To determine whether BRD4 protein interacts with acetylated JMJD1A, we co-expressed Flag-JMJD1A and Myc-p300 in 293T cells to induce JMJD1A acetylation, treated cells with or without JQ1, and then performed Flag IP. p300 overexpression enhanced acetylation of Flag- JMJD1A (Figure 5C, IP-2 or IP-3 vs. IP-1), and BRD4 co-IP'd with acetylated but not non- acetylated Flag-JMJD1A (Figure 5C, IP-2 vs. IP-1). Pre-treatment with JQ1 blocked co- precipitation of BRD4 with acetylated JMJD1A (Figure 5C, IP-3 vs IP-2). Next, to determine whether JMJD1A-BRD4 interaction interferes with JMJD1A interaction with STUB1, we used Flag-JMJD1A IP’d from 293T cells (IP-1, IP-2 or IP-3 as described in Figure 5C) for an in vitro pull- down assay with GST-STUB1. Relative to non-acetylated JMJD1A, decreased amounts of GST- STUB1 were pulled down by acetylated JMJD1A isolated from cells not treated with JQ1 (Figure 5D, IP-2 vs. IP-1). However, in contrast, similar amounts of GST-STUB1 were pulled down by non-acetylated or acetylated JMJD1A isolated from JQ1-treated cells (Figure 5D, IP-3 vs. IP-1), suggesting that JMJD1A-BRD4 interaction inhibits JMJD1A-STUB1 interaction. Overall, these results show that BRD4 binds to acetylated JMJD1A and promotes JMJD1A stability by inhibiting the STUB1-JMJD1A interaction. BRD4 promotes recruitment of acetylated JMJD1A to AR targets Because acetylated JMJD1A interacts with BRD4, a key coactivator of AR, we asked whether BRD4 affects the recruitment of JMJD1A to AR targets. We knocked down JMJD1A in Rv1 cells and then transduced the cells with lentiviral constructs encoding WT JMJD1A or the JMJD1A- K421R mutant, both of which harbor silent mutations within the shRNA targeting site allowing escape from shRNA silencing. We adjusted this protocol to enforce comparable expression of WT and K421R proteins (Figure 5E), with a goal of replacing endogenous JMJD1A in the knockdown cells with similar levels of ectopic JMJD1A (WT or K421R). We designate these lines JMJD1AWT and JMJD1AK421R, respectively. JMJD1AWT and JMJD1AK421R Rv1 cells exhibited similar

levels of AR and AR-V7 (Figure 5E). However, compared with JMJD1AWT cells, JMJD1AK421R cells showed the decreased expression of AR targets (Figure 5F) and reduced enrichment of JMJD1A at the PSA gene ARE (Figure 5G). In contrast, similar enrichment of BRD4 at the PSA gene ARE was detected between JMJD1AWT and JMJD1AK421R cells (Figure 5G). These results suggest that BRD4 enhances the recruitment of K421-acetylated JMJD1A to AR targets. Further, we immunoprecipitated AR from JMJD1AWT or JMJD1AK421R cells, and tested the co-precipitation for JMJD1A and BRD4. Despite the similar co-precipitation of BRD4 with AR between the two lines, we found the reduced co-precipitation of JMJD1A with AR in JMJD1AK421R relative to JMJD1AWT cells (Figure 5H), suggesting that BRD4 enhances the recruitment of K421-acetylated JMJD1A to AR. Finally, we analyzed the cell proliferation and found the decreased colony formation by JMJD1AK421R relative to JMJD1AWT cells (Figure 5I, 5J). Together, analysis of cells expressing comparable levels of JMJD1A WT and K421R mutant reveals that JMJD1A acetylation can enhance JMJD1A recruitment to AR via BRD4 and promote PCa cell growth, an additional function beyond the JMJD1A stabilization. Enzalutamide-resistant C4-2 cells show relatively high levels of total and K421-acetylated JMJD1A Enzalutamide resistance is a roadblock to CRPC therapy. We generated enzalutamide-resistant C4-2 cells by growing parental C4-2 cells with gradually increasing concentrations of enzalutamide (2 to 20 M) for over 6 months, a widely used approach (25,37,38). We confirmed that enzalutamide treatment indeed had much less effect on proliferation of resistant than parental C4-2 cells in both the MTT (Figure 6A) and colony formation (Figure 6B) assays. In the enzalutamide-resistant C4-2 line, we detected increased levels of JMJD1A protein (Figure 6C), but not mRNA (Figure S6A), relative to parental cells. This line also showed a mild increase in p300 protein levels, but there were little changes in levels of STUB1, AR or c-Myc (Figure 6C). AR-V7 was not detectable in either parental or resistant cells (Figure 6C). Then, we asked whether increased JMJD1A protein seen in enzalutamide-resistant cells is due to p300- induced acetylation at K421. To this purpose, we generated a peptide antibody specific to acetyl-K421 JMJD1A using a commercial service (ABclonal Science). That antibody detected a single band of the same molecular weight as JMJD1A, with increased signal after treating cells

with TSA (an HDAC inhibitor) and lost signal upon JMJD1A knockdown (Figure S6B). The antibody also recognized precipitated WT JMJD1A but not the JMJD1A-K421R mutant after TSA treatment (Figure S6C). Both results validate antibody specificity to acetyl-K421 JMJD1A. To assess JMJD1A acetyl-K421 levels, we IP'd total JMJD1A from parental or enzalutamide-resistant C4-2 cells, and then analyzed similar amounts of JMJD1A immunoprecipitates. JMJD1A IP’d from enzalutamide-resistant cells showed higher levels of JMJD1A acetyl-K421 than did parental C4-2 cells (Figure 6D). Thus, the levels of both total and K421-acetylated JMJD1A were increased in the enzalutamide-resistant C4-2 cells (Figure 6C, 6D). Enzalutamide-resistant and parental cells exhibited similar levels of AR or BRD4 (Figure 6D). However, JMJD1A IP’d from enzalutamide-resistant cells showed the increased co-precipitation of AR and BRD4 relative to parental cells (Figure 6D). Thus, in the enzalutamide-resistant cells, increases of acetylated JMJD1A may elevate JMJD1A protein levels and also enhance the JMJD1A/AR interaction via BRD4. To determine whether increased JMJD1A levels underlie the enzalutamide resistance, we partially knocked down JMJD1A in enzalutamide-resistant C4-2 cells to comparable levels seen in parental cells (Figure 6E). The partial JMJD1A knockdown reduced levels of AR targets such as PSA and c-Myc (Figure 6E), and re-sensitized the resistant cells to enzalutamide in the colony formation assay (Figure 6F). These results suggest that increased levels of JMJD1A enhanced AR activity for enzalutamide resistance. To directly test whether increased levels of JMJD1A can cause enzalutamide resistance, we overexpressed JMJD1A in the enzalutamide- sensitive LNCaP cells (Figure 6G). Enzalutamide abolished the colony formation by control cells, but had less effect on the colony formation by JMJD1A-overexpressing LNCaP cells (Figure 6H). These results demonstrate that elevation of JMJD1A levels can confer enzalutamide resistance. P300 or BET inhibitors induce JMJD1A degradation Our above findings suggest that targeting either JMJD1A, STUB1, p300 or BRD4 will inhibit JMJD1A function and antagonize PCa cell growth. Although specific JMJD1A inhibitors or STUB1 activators are currently not available, selective inhibitors of p300 (39-41) and BET (36,42) have been developed. Thus, we asked whether the selective p300 inhibitors C646 or the BET inhibitor JQ1 could be used to inhibit JMJD1A. Treatment of Rv1 cells with C646 (Figure 7A) or

JQ1 (Figure 7B) decreased endogenous JMJD1A protein levels dose-dependently, effects accompanied by reduced levels of JMJD1A downstream targets such as AR-V7 and c-Myc. MG132 treatment of Rv1 cells attenuated the downregulation of JMJD1A proteins by C646 (Figure 7C) or JQ1 (Figure 5A). Treatment of Rv1 cells with C646 (Figure 7D) or JQ1 (Figure S5A) had no effect on JMJD1A mRNA levels. These results suggest that both C646 and JQ1 can destabilize JMJD1A protein. C646 or JQ1 treatment significantly reduced growth of control Rv1 cells but had less effect on growth of Rv1 cells overexpressing ectopic JMJD1A (Figure 7E, 7F), suggesting that high JMJD1A levels may contribute to the resistance of PCa cells to these inhibitors. To test the efficacy of C646 or JQ1 on PCa cell growth, we treated cells with various doses of C646 or JQ1 and performed an MTT assay to determine the IC50. The IC50 values of C646 or JQ1 on Rv1 and C4-2 cells was ~ 10-fold lower than that seen in assays of normal prostate epithelial cells (HPrECs) or AR-negative PC3 cells (Figure 7G), indicating that p300 or BET inhibitors are effective on AR-positive CRPC lines. The IC50 values of C646 or JQ1 for enzalutamide-resistant C4-2 cells were several-fold lower than those for the parental C4-2 cells (Figure 7G), indicating the increased sensitivity of enzalutamide-resistant cells to these inhibitors. Finally, we tested effects of combining C646 or JQ1 with enzalutamide on the growth of PCa cells. Either C646 or JQ1 inhibited colony formation by Rv1 (Figure 7H, 7I) or enzalutamide-resistant C4-2 (Figure 7J, 7K) cells. Combination of either C646 or JQ1 with enzalutamide was more effective in inhibiting colony formation (Figure 7H to 7K). These results overall indicate that p300 or BET inhibitors can inhibit the JMJD1A function associated with K421 acetylation to inhibit PCa cell growth (Figure 7L).

Discussion

Second-generation AR-pathway inhibitors such as enzalutamide or abiraterone are currently the major therapeutic agents for CRPC patients. However, these inhibitors target the AR ligand- binding domain (LBD) and thus cannot inhibit the AR splice variants such as AR-V7, which lacks an LBD. Increased levels of AR-V7 reportedly represent a major mechanism underlying resistance of CRPC to these inhibitors (43-46). Importantly, we and others have reported that

JMJD1A serves as an AR co-activator and promotes splicing of AR-V7 (16,17,19,20,47). Thus, targeting JMJD1A may inhibit both full-length AR and AR-V7, and serve as a promising approach to treat CRPC. Unfortunately, selective inhibitors of JMJD1A histone demethylase activity are not yet available. In this study, we identified mechanisms underlying JMJD1A modifications (ubiquitination and acetylation) and how they regulate JMJD1A stability. This work may provide a means to destabilize JMJD1A as potential CRPC therapy. The rationale of targeting JMJD1A stability is supported by our observations that JMJD1A protein levels are upregulated in CRPC tissues and JMJD1A stability increases as prostate cancer cells develop enzalutamide resistance. Elevated JMJD1A protein levels were associated with lower STUB1 levels in CRPC tissues and with increased acetylation at JMJD1A K421 in enzalutamide-resistant CRPC cells. We found that JMJD1A acetylation at K421 can block the ubiquitination and degradation of JMJD1A by STUB1. However, K421 is unlikely to be the actual site targeted by STUB1 because mutation of K421 does not inhibit, but in fact increases JMJD1A ubiquitination by STUB1. Mechanistically, we found that acetylation of JMJD1A at K421 leads to the recruitment of BRD4 to JMJD1A, and this stabilizes the latter by preventing its interaction with and ubiquitination by STUB1. In addition, we found that BRD4 can enhance the recruitment of acetylated JMJD1A to AR targets. Thus, the acetylation-mediated JMJD1A/BRD4 interaction enhances both JMJD1A stability and JMJD1A recruitment to AR targets. The mechanisms of JMJD1A stability regulation provide a means to induce JMJD1A degradation with either p300 inhibitors or BET inhibitors. P300 inhibitors reduce JMJD1A acetylation and subsequent BRD4 binding, whereas BET inhibitors have no effect on JMJD1A acetylation but block BRD4 binding to JMJD1A acetyl-K421. Treatment with either type of inhibitors impairs JMJD1A-BRD4 interaction and thus increases STUB1-induced ubiquitination and degradation of JMJD1A to inhibit prostate cancer cell growth. Importantly, we found that enzalutamide-resistant C4-2 cells, which exhibit elevated JMJD1A acetylation and JMDJ1A protein levels, were more sensitive to p300 or BET inhibitors than parental C4-2 cells. Future work is needed to determine whether elevated JMJD1A acetylation or JMJD1A protein levels can serve as a marker to select CRPC patients as candidates for treatment with either p300 or BET inhibitors.

Treatment with p300 or BET inhibitors will impact many proteins in addition to JMJD1A. We found that ectopic expression of JMJD1A rendered prostate cancer cells partially resistant to growth inhibition by p300 or BET inhibitors. Thus, JMJD1A acetylation appears to be an important target of these reagents in inhibition of prostate cancer cell growth. To more specifically repress JMJD1A acetylation, one could also identify inhibitors that block JMJD1A/p300 interaction. Given that not only lysine acetylation but also nearby amino acids determine BRD binding specificity (48), one could also develop inhibitors to block BRD4 interaction with acetylated JMJD1A. Or, one might also identify reagents that promote JMJD1A/STUB1 interaction in order to enhance JMJD1A degradation. Given that JMJD1A plays catalytic and catalysis-independent roles in gene regulation (16,49), development of reagents that target its stability could have added advantages over inhibitors designed to specifically block JMJD1A catalysis.

Acknowledgments

This study was supported by NCI grant R01CA207118 and a V Scholar award (to J. Qi). Part of A. Hussain’s time was supported by a Merit Review Award (I01 BX000545), Medical Research Service, Department of Veterans Affairs.

Author contributions Investigation & Data Analysis: SX, LF (Fan), HYJ, FZ, XC, MBM, GP, LF (Fazli); Resources: AH, MEG, XD; Editing: AH; Conceptualization, Supervision, Manuscript Writing, & Funding Acquisition: JQ.

References

1. Watson PA, Arora VK, Sawyers CL. Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. Nat Rev Cancer 2015;15:701-11 2. Fizazi K, Scher HI, Molina A, Logothetis CJ, Chi KN, Jones RJ, et al. Abiraterone acetate for treatment of metastatic castration-resistant prostate cancer: final overall survival analysis of the COU-AA-301 randomised, double-blind, placebo-controlled phase 3 study. Lancet Oncol 2012;13:983-92 3. Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med 2012;367:1187-97 4. Kuroki S, Matoba S, Akiyoshi M, Matsumura Y, Miyachi H, Mise N, et al. Epigenetic regulation of mouse sex determination by the histone demethylase Jmjd1a. Science 2013;341:1106-9 5. Loh YH, Zhang W, Chen X, George J, Ng HH. Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. Genes Dev 2007;21:2545-57 6. Okada Y, Scott G, Ray MK, Mishina Y, Zhang Y. Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis. Nature 2007;450:119-23 7. Tateishi K, Okada Y, Kallin EM, Zhang Y. Role of Jhdm2a in regulating metabolic gene expression and obesity resistance. Nature 2009;458:757-61 8. Krieg AJ, Rankin EB, Chan D, Razorenova O, Fernandez S, Giaccia AJ. Regulation of the histone demethylase JMJD1A by hypoxia-inducible factor 1 alpha enhances hypoxic gene expression and tumor growth. Mol Cell Biol 2010;30:344-53 9. Tee AE, Ling D, Nelson C, Atmadibrata B, Dinger ME, Xu N, et al. The histone demethylase JMJD1A induces cell migration and invasion by up-regulating the expression of the long noncoding RNA MALAT1. Oncotarget 2014;5:1793-804 10. Zhao QY, Lei PJ, Zhang X, Zheng JY, Wang HY, Zhao J, et al. Global histone modification profiling reveals the epigenomic dynamics during malignant transformation in a four-stage breast cancer model. Clin Epigenetics 2016;8:34 11. Liu J, Zhu M, Xia X, Huang Y, Zhang Q, Wang X. Jumonji domain-containing protein 1A promotes cell growth and progression via transactivation of c-Myc expression and predicts a poor prognosis in cervical cancer. Oncotarget 2016;7:85151-62 12. Wan W, Peng K, Li M, Qin L, Tong Z, Yan J, et al. Histone demethylase JMJD1A promotes urinary bladder cancer progression by enhancing glycolysis through coactivation of hypoxia inducible factor 1alpha. Oncogene 2017;36:3868-77 13. Parrish JK, Sechler M, Winn RA, Jedlicka P. The histone demethylase KDM3A is a microRNA-22- regulated tumor promoter in Ewing Sarcoma. Oncogene 2015;34:257-62 14. Peng K, Su G, Ji J, Yang X, Miao M, Mo P, et al. Histone demethylase JMJD1A promotes colorectal cancer growth and metastasis by enhancing Wnt/beta-catenin signaling. J Biol Chem 2018;293:10606-19 15. Wang Z, Yang X, Liu C, Li X, Zhang B, Wang B, et al. Acetylation of PHF5A Modulates Stress Responses and Colorectal Carcinogenesis through Alternative Splicing-Mediated Upregulation of KDM3A. Mol Cell 2019 16. Fan L, Peng G, Sahgal N, Fazli L, Gleave M, Zhang Y, et al. Regulation of c-Myc expression by the histone demethylase JMJD1A is essential for prostate cancer cell growth and survival. Oncogene 2016;35:2441-52 17. Fan L, Zhang F, Xu S, Cui X, Hussain A, Fazli L, et al. Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells. Proc Natl Acad Sci U S A 2018;115:E4584-E93

18. Fan L, Xu S, Zhang F, Cui X, Fazli L, Gleave M, et al. Histone demethylase JMJD1A promotes expression of DNA repair factors and radio-resistance of prostate cancer cells. Cell Death Dis 2020;11:214 19. Wilson S, Fan L, Sahgal N, Qi J, Filipp FV. The histone demethylase KDM3A regulates the transcriptional program of the androgen receptor in prostate cancer cells. Oncotarget 2017;8:30328-43 20. Yamane K, Toumazou C, Tsukada Y, Erdjument-Bromage H, Tempst P, Wong J, et al. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell 2006;125:483-95 21. Wang T, Yang J, Xu J, Li J, Cao Z, Zhou L, et al. CHIP is a novel tumor suppressor in pancreatic cancer through targeting EGFR. Oncotarget 2014;5:1969-86 22. Paul I, Ahmed SF, Bhowmik A, Deb S, Ghosh MK. The ubiquitin ligase CHIP regulates c-Myc stability and transcriptional activity. Oncogene 2013;32:1284-95 23. Ahmed SF, Deb S, Paul I, Chatterjee A, Mandal T, Chatterjee U, et al. The chaperone-assisted E3 ligase C terminus of Hsc70-interacting protein (CHIP) targets PTEN for proteasomal degradation. J Biol Chem 2012;287:15996-6006 24. Muller P, Hrstka R, Coomber D, Lane DP, Vojtesek B. Chaperone-dependent stabilization and degradation of p53 mutants. Oncogene 2008;27:3371-83 25. Liu C, Lou W, Yang JC, Liu L, Armstrong CM, Lombard AP, et al. Proteostasis by STUB1/HSP70 complex controls sensitivity to androgen receptor targeted therapy in advanced prostate cancer. Nat Commun 2018;9:4700 26. Fujisawa T, Filippakopoulos P. Functions of bromodomain-containing proteins and their roles in homeostasis and cancer. Nat Rev Mol Cell Biol 2017;18:246-62 27. Asangani IA, Dommeti VL, Wang X, Malik R, Cieslik M, Yang R, et al. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 2014;510:278-82 28. Fernandez-Salas E, Wang S, Chinnaiyan AM. Role of BET proteins in castration-resistant prostate cancer. Drug Discov Today Technol 2016;19:29-38 29. Zhang P, Wang D, Zhao Y, Ren S, Gao K, Ye Z, et al. Intrinsic BET inhibitor resistance in SPOP- mutated prostate cancer is mediated by BET protein stabilization and AKT-mTORC1 activation. Nat Med 2017;23:1055-62 30. Xu W, Marcu M, Yuan X, Mimnaugh E, Patterson C, Neckers L. Chaperone-dependent E3 ubiquitin ligase CHIP mediates a degradative pathway for c-ErbB2/Neu. Proc Natl Acad Sci U S A 2002;99:12847-52 31. Sarkar S, Brautigan DL, Parsons SJ, Larner JM. Androgen receptor degradation by the E3 ligase CHIP modulates mitotic arrest in prostate cancer cells. Oncogene 2014;33:26-33 32. Adachi H, Waza M, Tokui K, Katsuno M, Minamiyama M, Tanaka F, et al. CHIP overexpression reduces mutant androgen receptor protein and ameliorates phenotypes of the spinal and bulbar muscular atrophy transgenic mouse model. J Neurosci 2007;27:5115-26 33. Liu Z, Zhou S, Liao L, Chen X, Meistrich M, Xu J. Jmjd1a demethylase-regulated histone modification is essential for cAMP-response element modulator-regulated gene expression and spermatogenesis. J Biol Chem 2010;285:2758-70 34. Lin H, Zhu X, Chen G, Song L, Gao L, Khand AA, et al. KDM3A-mediated demethylation of histone H3 lysine 9 facilitates the chromatin binding of Neurog2 during neurogenesis. Development 2017;144:3674-85 35. Wang HY, Long QY, Tang SB, Xiao Q, Gao C, Zhao QY, et al. Histone demethylase KDM3A is required for enhancer activation of hippo target genes in colorectal cancer. Nucleic Acids Res 2019;47:2349-64

36. Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, et al. Selective inhibition of BET bromodomains. Nature 2010;468:1067-73 37. Liu C, Lou W, Zhu Y, Yang JC, Nadiminty N, Gaikwad NW, et al. Intracrine Androgens and AKR1C3 Activation Confer Resistance to Enzalutamide in Prostate Cancer. Cancer Res 2015;75:1413-22 38. Hoefer J, Akbor M, Handle F, Ofer P, Puhr M, Parson W, et al. Critical role of androgen receptor level in prostate cancer cell resistance to new generation antiandrogen enzalutamide. Oncotarget 2016;7:59781-94 39. Bowers EM, Yan G, Mukherjee C, Orry A, Wang L, Holbert MA, et al. Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor. Chem Biol 2010;17:471-82 40. Lasko LM, Jakob CG, Edalji RP, Qiu W, Montgomery D, Digiammarino EL, et al. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature 2017;550:128-32 41. Jin L, Garcia J, Chan E, de la Cruz C, Segal E, Merchant M, et al. Therapeutic Targeting of the CBP/p300 Bromodomain Blocks the Growth of Castration-Resistant Prostate Cancer. Cancer Res 2017;77:5564-75 42. Perez-Salvia M, Esteller M. Bromodomain inhibitors and cancer therapy: From structures to applications. Epigenetics 2017;12:323-39 43. Antonarakis ES, Lu C, Wang H, Luber B, Nakazawa M, Roeser JC, et al. AR-V7 and resistance to enzalutamide and abiraterone in prostate cancer. N Engl J Med 2014;371:1028-38 44. Zhu Y, Sharp A, Anderson CM, Silberstein JL, Taylor M, Lu C, et al. Novel Junction-specific and Quantifiable In Situ Detection of AR-V7 and its Clinical Correlates in Metastatic Castration- resistant Prostate Cancer. Eur Urol 2018;73:727-35 45. Seitz AK, Thoene S, Bietenbeck A, Nawroth R, Tauber R, Thalgott M, et al. AR-V7 in Peripheral Whole Blood of Patients with Castration-resistant Prostate Cancer: Association with Treatment- specific Outcome Under Abiraterone and Enzalutamide. Eur Urol 2017;72:828-34 46. Sharp A, Coleman I, Yuan W, Sprenger C, Dolling D, Rodrigues DN, et al. Androgen receptor splice variant-7 expression emerges with castration resistance in prostate cancer. J Clin Invest 2019;129:192-208 47. Lee HY, Yang EG, Park H. Hypoxia enhances the expression of prostate-specific antigen by modifying the quantity and catalytic activity of Jumonji C domain-containing histone demethylases. Carcinogenesis 2013;34:2706-15 48. Filippakopoulos P, Picaud S, Mangos M, Keates T, Lambert JP, Barsyte-Lovejoy D, et al. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 2012;149:214-31 49. Schneider P, Bayo-Fina JM, Singh R, Kumar Dhanyamraju P, Holz P, Baier A, et al. Identification of a novel actin-dependent signal transducing module allows for the targeted degradation of GLI1. Nat Commun 2015;6:8023

Figure Legends

Figure 1 A. Increased JMJD1A staining in CRPC specimens. (left) Example JMJD1A staining in primary PCa or CRPC. JMJD1A, brown; nuclei, blue. (right) Percentage of primary PCa or CRPC groups displaying a given staining score (low, moderate or high). Mann-Whitney U test was used for statistical analysis (***, p<0.001). B. Co-precipitation between endogenous JMJD1A and STUB1 in Rv1 cells. Left panel: JMJD1A co-precipitates with STUB1. Right panel: STUB1 co-precipitates with JMJD1A. C. STUB1 interaction with the N-terminal half (N), but not the C-terminal half (C), of JMJD1A. D. JMJD1A interaction with the STUB1 U-box domain. E. Direct interaction between recombinant JMJD1A and GST-STUB1. F. JMJD1A degradation requires STUB1 ubiquitin ligase activity, but not chaperone-binding activity. G. STUB1 overexpression induced JMJD1A ubiquitination. H. In vitro ubiquitination of JMJD1A by STUB1. I. STUB1 knockdown increased JMJD1A half-life. Rv1 cells (pLKO.1 or STUB1 knockdown) were treated with cycloheximide (CHX, 50 g/ml), and lysates were collected at indicated times afterwards and analyzed by western blotting with indicated antibodies. J. STUB1 knockdown in prostate cancer cells increased JMJD1A levels and levels of JMJD1A-dependent proteins such as AR-V7 and c- Myc. K. Downregulation of STUB1 protein in CRPC specimens. IHC staining of STUB1 was performed and analyzed on the same PCa TMA as described in A.

Figure 2 A. STUB1 knockdown in Rv1 cells increased mRNA levels of AR target genes and AR-V7. Rv1 cells were transduced with pLKO.1 control or STUB1 shRNA for 24 hours. Cells were then maintained in growth media containing 5% charcoal stripped FBS (CS-FBS) with or without supplementation of 1 nM R1881 (a synthetic androgen) for 24 hours. RNAs were collected and analyzed by qRT- PCR. B. STUB1 knockdown in Rv1 cells increased enrichment of JMJD1A and AR and reduced enrichment of H3K9me2 at the androgen responsive element (ARE) region of the PSA gene. Rv1 cells (control or STUB1 knockdown) were analyzed by ChIP assays using control, JMJD1A, AR or H3K9me2 antibodies. Precipitated chromatins were analyzed by qPCR using primers for ARE region of the PSA gene. Data were calculated as the percentage of input. C. STUB1 knockdown

increased the reporter activity of AR-V7 splicing. PC3 cells (pLKO.1 or shSTUB1) were transfected with an AR mini-gene reporter. After 72 hours, RNAs were analyzed by qRT-PCR using primers for the splicing junctions, which reflect the splicing of AR-V7 and AR-FL, respectively. D. Partial JMJD1A knockdown in the STUB1-knockdown Rv1 cells reduced the protein levels of AR-V7 and c-Myc. Transduction of JMJD1A shRNA was optimized to reduce JMJD1A levels in STUB1-knockdown cells to levels of control cells. E. Partial JMJD1A knockdown in STUB1-knockdown Rv1 cells decreased mRNA levels of AR target genes and AR-V7.

Figure 3 A. (left) STUB1 knockdown increased colony formation by Rv1 cells in cell-culture plates. Rv1 cells (pLKO.1 control or STUB1 knockdown) were seeded at low density and maintained for 3 weeks in media containing 5% CS-FBS without or with 1 nM R1881. Cells were fixed and stained with crystal violet. (right) Quantitation of colonies counted in 10 high-power fields. B. (left) STUB1 knockdown increased colony formation by Rv1 cells in soft agar. Rv1 cells (pLKO.1 control or STUB1 knockdown) were grown in sofa agar for 3 weeks in conditions described in A. (right) Quantification of colony number in 10 high-power fields. C, D, and E. STUB1 knockdown increased xenograft tumor growth. 5x105 Rv1 cells (pLKO.1 or STUB1 knockdown) were subcutaneously injected into the backs of athymic nude mice (n=6). Xenograft tumor size was measured every 4 days (C). Xenograft tumors were collected 4 weeks after injection (D), and average tumor weight determined (E). F. (left) Increased Ki-67 staining in STUB1-knockdown xenograft tumors of Rv1 cells. Ki-67, brown; nuclei, blue. (right) Percentage of Ki-67-positive cells, as determined in 5 high-power fields. G. Partial JMJD1A knockdown in STUB1-knockdown Rv1 cells reduced the colony formation in cell-culture plates. H, I, and J. Partial JMJD1A knockdown in STUB1-knockdown Rv1 cells decreased xenograft tumor growth in the castrated mice. Rv1 cells (pLKO.1, shSTUB1, shSTUB1/shJMJD1A) were subcutaneously injected into the backs of nude mice (n=5). One week later, the mice were castrated, and xenograft tumor size was measured every 4 days (H). Xenograft tumors were collected 4 weeks after injection (I), and average tumor weight determined (J). K. JMJD1A knockdown decreased Ki-67 staining in STUB1-knockdown Rv1 xenografts from the castrated mice.

Figure 4 A. JMJD1A Knockdown decreased enrichment of p300 and acetyl-H3 at the PSA gene ARE. B. STUB1 knockdown increased enrichment of JMJD1A, p300 and acetyl-H3 at the PSA gene ARE. C. Co-IP of endogenous JMJD1A with p300 in Rv1 or C4-2 cells. D. Mapping of the p300 domain interacting with JMJD1A. Myc-JMJD1A was co-expressed with Flag-tagged p300 fragments (see diagram, Figure S4B) in 293T cells. Flag IP was performed and analyzed by western blotting with Flag or myc antibodies. E. The N-terminal half of JMJD1A interacted with p300. F. p300 overexpression increased JMJD1A protein levels. G. p300 knockdown in prostate cancer cells decreased JMJD1A protein levels. H. MG132 treatment increased JMJD1A protein levels in p300-knockdown Rv1 cells. I. Overexpression of p300 induced JMJD1A acetylation. J. Reduced acetylation of JMJD1A upon p300 knockdown in Rv1 cells. K. p300 overexpression induced JMJD1A acetylation at K421. Myc-p300 was co-expressed with Flag-tagged JMJD1A (WT or indicated mutants) in 293T cells. Flag-JMJD1A was precipitated, and analyzed by western blotting with acetyl-lysine antibodies. Of note: the loading of Flag-IP’d samples was normalized to have similar levels of Flag-JMJD1A across the lanes. L. MG132 treatment increased the protein levels of JMJD1A-K421R mutant. M. p300 overexpression did not increase levels of JMJD1A-K421R mutant protein. N. STUB1 induced higher ubiquitination of JMJD1A-K421R mutant than WT JMJD1A. O. Increased co-precipitation of STUB1 with the JMJD1A-K421R mutant relative to WT JMJD1A.

Figure 5 A. Treatment with the BET inhibitor JQ1 reduced JMJD1A stability. Rv1 cells were treated with JQ1 (0.5 M) for 24 hours, then with MG132 (10 M) for 6 hours and analyzed by western blotting. B. BRD4 knockdown in Rv1 cells reduced JMJD1A protein levels. C. Isolation of acetylated JMJD1A complexes from cells treated with or without JQ1. D. Reduced pull-down of GST-STUB1 with the acetylated Flag-JMJD1A complex. E. Expression of JMJD1A-WT or JMJD1A- K421R in the JMJD1A-knockdown Rv1 cells to replace the endogenous JMJD1A. F. Reduced mRNA levels of AR targets in JMJD1AK421R relative to JMJD1AWT Rv1 cells. G. Reduced

enrichment of JMJD1A at the PSA gene ARE in JMJD1AK421R relative to JMJD1AWT Rv1 cells. H. Reduced co-precipitation of JMJD1A with AR in JMJD1AK421R relative to JMJD1AWT Rv1 cells. I and J. Reduced colony formation by JMJD1AK421R relative to JMJD1AWT Rv1 cells grown in cell- culture plates (I) or soft agar (J).

Figure 6 A. Survival of enzalutamide-resistant or parental C4-2 cells after treatment with increasing concentrations of enzalutamide for 48 hours as determined by MTT assays. B. Colony formation by enzalutamide-resistant and parental C4-2 cells in cell-culture plates in the presence of enzalutamide. C. Increased levels of JMJD1A protein in enzalutamide-resistant C4- 2 cells. D. Increased levels of acetyl-K421 JMJD1A and increased co-precipitation of AR/BRD4 with JMJD1A in enzalutamide-resistant C4-2 cells. JMJD1A was IP’d from parental and enzalutamide-resistant C4-2 cells, and similar amounts of JMJD1A precipitates were analyzed by western blotting. E. Partial JMJD1A knockdown in enzalutamide-resistant C4-2 cells reduced levels of PSA and c-Myc. Enzalutamide-resistant C4-2 cells were transduced with an optimized amount of JMJD1A shRNA to reduce JMJD1A levels to those seen in parental C4-2 cells. F. Partial JMJD1A knockdown in enzalutamide-resistant C4-2 cells reduced their colony formation in the presence of enzalutamide. G. Ectopic overexpression of JMJD1A in LNCaP cells by lentiviral transduction. H. Ectopic overexpression of JMJD1A in LNCaP cells confers enzalutamide resistance. Indicated cells were analyzed by colony formation assays in the presence of enzalutamide.

Figure 7 A. C646 treatment of Rv1 cells reduced JMJD1A protein levels. Cells were treated with indicated concentrations of C646 for 24 hours and analyzed by western blotting for indicated proteins. B. JQ1 treatment of Rv1 cells reduced JMJD1A protein levels. Procedure is as described in A. C. MG132 treatment attenuated C646-induced downregulation of JMJD1A protein in Rv1 cells. D. C646 treatment had no effect on JMJD1A mRNA levels. E and F. Ectopic overexpression of JMJD1A made Rv1 cells resistant to C646 (E) or JQ1 (F) treatment. Rv1 cells were transduced with a lentiviral plasmid encoding JMJD1A for 48 hours. Control or JMJD1A-overexpressing cells

were analyzed by colony formation assays in the presence of C646 or JQ1. G. IC50 values of C646 or JQ1 for the indicated cells. Cells were treated with various concentrations of C646 or JQ1 for 48 hours, and the percentage of surviving cells was determined by an MTT assay. IC50 values were calculated using GraphPad Prism software. H and I. Additive effect of C646/enzalutamide combination (H) or JQ1/enzalutamide combination (I) in inhibiting colony formation by Rv1 cells. J and K. Additive effect of C646/enzalutamide combination (J) or JQ1/enzalutamide combination (K) in inhibiting colony formation by enzalutamide-resistant C4- 2 cells. L. Summarizing diagram. Acetylation of JMJD1A at K421 mediates the BRD4-JMJD1A interaction, which inhibits STUB1-mediated JMJD1A degradation and also promotes recruitment of JMJD1A to AR targets. This leads to increased activity of AR and enhanced splicing of AR-V7, to drive CRPC progression and enzalutamide resistance. P300 inhibitors, which reduce JMJD1A acetylation, or BET inhibitors, which directly block JMJD1A-BRD4 interactions, can destabilize JMJD1A to reduce both AR activities and AR-V7 levels, thereby inhibiting CRPC progression and enzalutamide resistance.

p300-mediated acetylation of histone demethylase JMJD1A prevents its degradation by ubiquitin ligase STUB1 and enhances its activity in prostate cancer

Songhui Xu, Lingling Fan, Hee-Young Jeon, et al.

Cancer Res Published OnlineFirst June 10, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-20-0233

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