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Targeting the MLL complex in castration-resistant prostate cancer.

Malik R, Khan AP, Asangani IA, Cieślik M, Prensner JR, Wang X, Iyer MK, Jiang X, Borkin D, Escara-Wilke J, Stender R, Wu YM, Niknafs YS, Jing X, Qiao Y, Palanisamy N, Kunju LP, Krishnamurthy PM, Yocum AK, Mellacheruvu D, Nesvizhskii AI, Cao X, Dhanasekaran SM, Feng FY, Grembecka J, Cierpicki T, Chinnaiyan AM - Nat. Med. (2015)

Bottom Line: Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling.AR directly interacts with the MLL complex via the menin-MLL subunit.Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice.

View Article: PubMed Central - PubMed

Affiliation: 1] Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan, USA. [2] Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration-resistant prostate cancer (CRPC). Although prior work has focused on targeting AR directly, co-activators of AR signaling, which may represent new therapeutic targets, are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin-MLL subunit. Menin expression is higher in CRPC than in both hormone-naive prostate cancer and benign prostate tissue, and high menin expression correlates with poor overall survival of individuals diagnosed with prostate cancer. Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a crucial co-activator of AR and a potential therapeutic target in advanced prostate cancer.

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AR and ASH2L are recruited to the same genomic loci upon androgen stimulation. (a) A heat map representation of AR and ASH2L binding to promoter regions, 2.5kb flanking transcriptional start sites (TSS, indicated by 0) in vehicle and R1881 stimulated VCaP cells as assessed by ChIP-Sequencing. Gene promoters are rank-ordered by the level of AR enrichment at the TSS. (b) Average TSS-aligned profiles of AR and ASH2L occupancy for all annotated genes before and after vehicle or R1881 stimulation. (c) The overlap between R1881-induced AR and ASH2L peaks. (d) Representative gene (NDRG1) co-occupied by AR and ASH2L before and after AR stimulation. (e) De novo motif detection with MEME program identified enrichment of two half-androgen response elements (ARE) among ASH2L binding sites. (MEME E value 2.1e-025).
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Figure 3: AR and ASH2L are recruited to the same genomic loci upon androgen stimulation. (a) A heat map representation of AR and ASH2L binding to promoter regions, 2.5kb flanking transcriptional start sites (TSS, indicated by 0) in vehicle and R1881 stimulated VCaP cells as assessed by ChIP-Sequencing. Gene promoters are rank-ordered by the level of AR enrichment at the TSS. (b) Average TSS-aligned profiles of AR and ASH2L occupancy for all annotated genes before and after vehicle or R1881 stimulation. (c) The overlap between R1881-induced AR and ASH2L peaks. (d) Representative gene (NDRG1) co-occupied by AR and ASH2L before and after AR stimulation. (e) De novo motif detection with MEME program identified enrichment of two half-androgen response elements (ARE) among ASH2L binding sites. (MEME E value 2.1e-025).

Mentions: Given the role of MLL complex proteins in AR transcriptional regulation, we hypothesized that the MLL complex may co-localize with AR on a genome-wide scale. To investigate this, we identified genome-wide ASH2L binding by chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) in VCaP cells upon synthetic androgen (R1881) or vehicle-treatment and compared the data with published AR ChIP-Seq data27. First, we noted an overlap between ASH2L binding sites and androgen-stimulated AR binding sites (Fig. 3a,b). Next we identified a total of 15,637 distinct genome-wide individual AR peaks (FDR < 0.05) out of which 12,243 peaks increased upon androgen stimulation (Fig. 3c). For ASH2L, we identified a total 30,114 peaks (FDR < 0.05) out of which 2,187 showed increased binding upon androgen stimulation (Supplementary Fig. 5a). Importantly, we noted a significant overlap of 1,410 target regions (64.4% of ASH2L-binding sites) where both ASH2L and AR are concomitantly recruited following androgen stimulation (Fig. 3c). Representative gene promoter with overlapping AR and ASH2L binding patterns is shown in Fig. 3d, and others are shown in Supplementary Fig. 5b–e. To investigate the presence of potential cis regulatory elements among ASH2L genomic binding regions, we performed de novo motif discovery using Multiple EM for Motif Elicitation (MEME)39, on the ASH2L ChIP-Seq data. We identified significant enrichment of two androgen responsive element (ARE) half sites in the ASH2L binding site, further supporting the overlap observed between ASH2L and AR binding in AR dependent cell lines (Fig. 3e). We next examined the expression profile of genes that were within 10kb of androgen-induced ASH2L peaks, and observed a significant decrease in their expression upon ASH2L knockdown (Supplementary Fig. 5f). Similar to ASH2L, we observed an enrichment of MLL and menin on AR target genes by ChIP-PCR (Supplementary Fig. 6a–c). Taken together these data suggest that upon androgen stimulation the MLL complex is co-recruited to direct AR targets and modulates their transcriptional activation.


Targeting the MLL complex in castration-resistant prostate cancer.

Malik R, Khan AP, Asangani IA, Cieślik M, Prensner JR, Wang X, Iyer MK, Jiang X, Borkin D, Escara-Wilke J, Stender R, Wu YM, Niknafs YS, Jing X, Qiao Y, Palanisamy N, Kunju LP, Krishnamurthy PM, Yocum AK, Mellacheruvu D, Nesvizhskii AI, Cao X, Dhanasekaran SM, Feng FY, Grembecka J, Cierpicki T, Chinnaiyan AM - Nat. Med. (2015)

AR and ASH2L are recruited to the same genomic loci upon androgen stimulation. (a) A heat map representation of AR and ASH2L binding to promoter regions, 2.5kb flanking transcriptional start sites (TSS, indicated by 0) in vehicle and R1881 stimulated VCaP cells as assessed by ChIP-Sequencing. Gene promoters are rank-ordered by the level of AR enrichment at the TSS. (b) Average TSS-aligned profiles of AR and ASH2L occupancy for all annotated genes before and after vehicle or R1881 stimulation. (c) The overlap between R1881-induced AR and ASH2L peaks. (d) Representative gene (NDRG1) co-occupied by AR and ASH2L before and after AR stimulation. (e) De novo motif detection with MEME program identified enrichment of two half-androgen response elements (ARE) among ASH2L binding sites. (MEME E value 2.1e-025).
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Related In: Results  -  Collection

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Figure 3: AR and ASH2L are recruited to the same genomic loci upon androgen stimulation. (a) A heat map representation of AR and ASH2L binding to promoter regions, 2.5kb flanking transcriptional start sites (TSS, indicated by 0) in vehicle and R1881 stimulated VCaP cells as assessed by ChIP-Sequencing. Gene promoters are rank-ordered by the level of AR enrichment at the TSS. (b) Average TSS-aligned profiles of AR and ASH2L occupancy for all annotated genes before and after vehicle or R1881 stimulation. (c) The overlap between R1881-induced AR and ASH2L peaks. (d) Representative gene (NDRG1) co-occupied by AR and ASH2L before and after AR stimulation. (e) De novo motif detection with MEME program identified enrichment of two half-androgen response elements (ARE) among ASH2L binding sites. (MEME E value 2.1e-025).
Mentions: Given the role of MLL complex proteins in AR transcriptional regulation, we hypothesized that the MLL complex may co-localize with AR on a genome-wide scale. To investigate this, we identified genome-wide ASH2L binding by chromatin immunoprecipitation coupled with high throughput sequencing (ChIP-Seq) in VCaP cells upon synthetic androgen (R1881) or vehicle-treatment and compared the data with published AR ChIP-Seq data27. First, we noted an overlap between ASH2L binding sites and androgen-stimulated AR binding sites (Fig. 3a,b). Next we identified a total of 15,637 distinct genome-wide individual AR peaks (FDR < 0.05) out of which 12,243 peaks increased upon androgen stimulation (Fig. 3c). For ASH2L, we identified a total 30,114 peaks (FDR < 0.05) out of which 2,187 showed increased binding upon androgen stimulation (Supplementary Fig. 5a). Importantly, we noted a significant overlap of 1,410 target regions (64.4% of ASH2L-binding sites) where both ASH2L and AR are concomitantly recruited following androgen stimulation (Fig. 3c). Representative gene promoter with overlapping AR and ASH2L binding patterns is shown in Fig. 3d, and others are shown in Supplementary Fig. 5b–e. To investigate the presence of potential cis regulatory elements among ASH2L genomic binding regions, we performed de novo motif discovery using Multiple EM for Motif Elicitation (MEME)39, on the ASH2L ChIP-Seq data. We identified significant enrichment of two androgen responsive element (ARE) half sites in the ASH2L binding site, further supporting the overlap observed between ASH2L and AR binding in AR dependent cell lines (Fig. 3e). We next examined the expression profile of genes that were within 10kb of androgen-induced ASH2L peaks, and observed a significant decrease in their expression upon ASH2L knockdown (Supplementary Fig. 5f). Similar to ASH2L, we observed an enrichment of MLL and menin on AR target genes by ChIP-PCR (Supplementary Fig. 6a–c). Taken together these data suggest that upon androgen stimulation the MLL complex is co-recruited to direct AR targets and modulates their transcriptional activation.

Bottom Line: Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling.AR directly interacts with the MLL complex via the menin-MLL subunit.Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice.

View Article: PubMed Central - PubMed

Affiliation: 1] Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan, USA. [2] Department of Pathology, University of Michigan, Ann Arbor, Michigan, USA.

ABSTRACT
Resistance to androgen deprivation therapies and increased androgen receptor (AR) activity are major drivers of castration-resistant prostate cancer (CRPC). Although prior work has focused on targeting AR directly, co-activators of AR signaling, which may represent new therapeutic targets, are relatively underexplored. Here we demonstrate that the mixed-lineage leukemia protein (MLL) complex, a well-known driver of MLL fusion-positive leukemia, acts as a co-activator of AR signaling. AR directly interacts with the MLL complex via the menin-MLL subunit. Menin expression is higher in CRPC than in both hormone-naive prostate cancer and benign prostate tissue, and high menin expression correlates with poor overall survival of individuals diagnosed with prostate cancer. Treatment with a small-molecule inhibitor of menin-MLL interaction blocks AR signaling and inhibits the growth of castration-resistant tumors in vivo in mice. Taken together, this work identifies the MLL complex as a crucial co-activator of AR and a potential therapeutic target in advanced prostate cancer.

Show MeSH
Related in: MedlinePlus