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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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MLL complex proteins are important for AR signaling and cell growth. (a) Microarray profiling of ASH2L knockdown (two independent siRNAs) and control non-target (NT) VCaP cells following 24 hr androgen (R1881) treatment. Heatmap displays the altered expression of the androgen-induced genes upon ASH2L knockdown. (b) Gene set enrichment analysis (GSEA) of microarray data shows enrichment of AR target gene signature. (c) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent ASH2L shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=2). (d) Expression of FKBP5 and TMPRSS2 AR-target genes was measured using qPCR in LNCaP cells stably expressing non-targeting (NT) shRNA or one of two independent menin shRNAs, after DHT stimulation for indicated time points. * P < 0.01, **P < 0.001, compared with vehicle by one-way ANOVA. (n = 3, mean ± s.e.m) (e) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent menin shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=3). (f,g) VCaP cells expressing NT shRNA or one of two independent ASH2L (f), MLL (g) or menin (h) shRNAs respectively were injected subcutaneously into both sides of mouse dorsal flank. Tumor volumes of xenografts were measured at 7 weeks post implantation. Each dot represents individual tumors. *, P<0.01; **, P<0.001, ***P<0.0001, compared with shNT by one-way ANOVA. All graphs are shown with mean ± s.e.m.
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Figure 2: MLL complex proteins are important for AR signaling and cell growth. (a) Microarray profiling of ASH2L knockdown (two independent siRNAs) and control non-target (NT) VCaP cells following 24 hr androgen (R1881) treatment. Heatmap displays the altered expression of the androgen-induced genes upon ASH2L knockdown. (b) Gene set enrichment analysis (GSEA) of microarray data shows enrichment of AR target gene signature. (c) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent ASH2L shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=2). (d) Expression of FKBP5 and TMPRSS2 AR-target genes was measured using qPCR in LNCaP cells stably expressing non-targeting (NT) shRNA or one of two independent menin shRNAs, after DHT stimulation for indicated time points. * P < 0.01, **P < 0.001, compared with vehicle by one-way ANOVA. (n = 3, mean ± s.e.m) (e) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent menin shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=3). (f,g) VCaP cells expressing NT shRNA or one of two independent ASH2L (f), MLL (g) or menin (h) shRNAs respectively were injected subcutaneously into both sides of mouse dorsal flank. Tumor volumes of xenografts were measured at 7 weeks post implantation. Each dot represents individual tumors. *, P<0.01; **, P<0.001, ***P<0.0001, compared with shNT by one-way ANOVA. All graphs are shown with mean ± s.e.m.

Mentions: Next we conducted knockdown experiments to study the role of MLL complex in AR-driven transcription. Compared to VCaP cells treated with two independent siRNAs against the MLL subunit ASH2L, cells treated with control siRNA showed higher induction of AR target gene expression after treatment with synthetic androgen (R1881), as revealed by microarray and gene set enrichment analysis (GSEA) with an AR gene signature (Fig. 2a,b, Supplementary Fig. 2a, Supplementary table S2). Similar effects of ASH2L knockdown on AR signaling (both at transcript and protein levels) were observed in LNCaP cells (Fig. 2c and Supplementary Fig. 2b–e). Next, we assessed the role of menin on AR signaling using qPCR and immunoblotting. Analogous to ASH2L, knockdown of menin resulted in a significant decrease in expression of DHT induced AR target genes (Fig. 2d,e). This was further confirmed by negative enrichment of the AR target gene signature in menin knockdown cells (Supplementary Fig. 2f,g).


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)

MLL complex proteins are important for AR signaling and cell growth. (a) Microarray profiling of ASH2L knockdown (two independent siRNAs) and control non-target (NT) VCaP cells following 24 hr androgen (R1881) treatment. Heatmap displays the altered expression of the androgen-induced genes upon ASH2L knockdown. (b) Gene set enrichment analysis (GSEA) of microarray data shows enrichment of AR target gene signature. (c) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent ASH2L shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=2). (d) Expression of FKBP5 and TMPRSS2 AR-target genes was measured using qPCR in LNCaP cells stably expressing non-targeting (NT) shRNA or one of two independent menin shRNAs, after DHT stimulation for indicated time points. * P < 0.01, **P < 0.001, compared with vehicle by one-way ANOVA. (n = 3, mean ± s.e.m) (e) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent menin shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=3). (f,g) VCaP cells expressing NT shRNA or one of two independent ASH2L (f), MLL (g) or menin (h) shRNAs respectively were injected subcutaneously into both sides of mouse dorsal flank. Tumor volumes of xenografts were measured at 7 weeks post implantation. Each dot represents individual tumors. *, P<0.01; **, P<0.001, ***P<0.0001, compared with shNT by one-way ANOVA. All graphs are shown with mean ± s.e.m.
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Related In: Results  -  Collection

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Figure 2: MLL complex proteins are important for AR signaling and cell growth. (a) Microarray profiling of ASH2L knockdown (two independent siRNAs) and control non-target (NT) VCaP cells following 24 hr androgen (R1881) treatment. Heatmap displays the altered expression of the androgen-induced genes upon ASH2L knockdown. (b) Gene set enrichment analysis (GSEA) of microarray data shows enrichment of AR target gene signature. (c) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent ASH2L shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=2). (d) Expression of FKBP5 and TMPRSS2 AR-target genes was measured using qPCR in LNCaP cells stably expressing non-targeting (NT) shRNA or one of two independent menin shRNAs, after DHT stimulation for indicated time points. * P < 0.01, **P < 0.001, compared with vehicle by one-way ANOVA. (n = 3, mean ± s.e.m) (e) Immunoblots for TMPRSS2 protein expression in VCaP cells stably expressing NT shRNA or one of two independent menin shRNAs after DHT stimulation for indicated time points. Shown are representative blots (n=3). (f,g) VCaP cells expressing NT shRNA or one of two independent ASH2L (f), MLL (g) or menin (h) shRNAs respectively were injected subcutaneously into both sides of mouse dorsal flank. Tumor volumes of xenografts were measured at 7 weeks post implantation. Each dot represents individual tumors. *, P<0.01; **, P<0.001, ***P<0.0001, compared with shNT by one-way ANOVA. All graphs are shown with mean ± s.e.m.
Mentions: Next we conducted knockdown experiments to study the role of MLL complex in AR-driven transcription. Compared to VCaP cells treated with two independent siRNAs against the MLL subunit ASH2L, cells treated with control siRNA showed higher induction of AR target gene expression after treatment with synthetic androgen (R1881), as revealed by microarray and gene set enrichment analysis (GSEA) with an AR gene signature (Fig. 2a,b, Supplementary Fig. 2a, Supplementary table S2). Similar effects of ASH2L knockdown on AR signaling (both at transcript and protein levels) were observed in LNCaP cells (Fig. 2c and Supplementary Fig. 2b–e). Next, we assessed the role of menin on AR signaling using qPCR and immunoblotting. Analogous to ASH2L, knockdown of menin resulted in a significant decrease in expression of DHT induced AR target genes (Fig. 2d,e). This was further confirmed by negative enrichment of the AR target gene signature in menin knockdown cells (Supplementary Fig. 2f,g).

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