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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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Androgen receptor interacts with MLL complex proteins. (a) VCaP nuclear lysate fractions from Superose-6 gel filtration column were immunoblotted using indicated antibodies. Arrows indicate the approximate mass of the complexes eluted. Distribution of cytoplasmic or nuclear proteins demonstrates the efficiency of lysate fractionation (inset western blot). Shown are representative blots (n=3). (b–c) VCaP (b) and LNCaP (c) nuclear lysates were immunoprecipitated (IP) with AR, ASH2L and menin antibodies followed by immunoblotting (IB) using indicated antisera. Shown are representative blots from three independent experiments. (d) VCaP cells were fixed and immunostained using AR (green) and ASH2L (red) or menin (red) antibodies. Confocal microscopic images were analyzed by ImageJ software. Co-localization is represented by yellow puncta. Bar = 10μm. Inset bar = 2.5μm. Shown are representative images from three independent experiments.
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Figure 1: Androgen receptor interacts with MLL complex proteins. (a) VCaP nuclear lysate fractions from Superose-6 gel filtration column were immunoblotted using indicated antibodies. Arrows indicate the approximate mass of the complexes eluted. Distribution of cytoplasmic or nuclear proteins demonstrates the efficiency of lysate fractionation (inset western blot). Shown are representative blots (n=3). (b–c) VCaP (b) and LNCaP (c) nuclear lysates were immunoprecipitated (IP) with AR, ASH2L and menin antibodies followed by immunoblotting (IB) using indicated antisera. Shown are representative blots from three independent experiments. (d) VCaP cells were fixed and immunostained using AR (green) and ASH2L (red) or menin (red) antibodies. Confocal microscopic images were analyzed by ImageJ software. Co-localization is represented by yellow puncta. Bar = 10μm. Inset bar = 2.5μm. Shown are representative images from three independent experiments.

Mentions: Using co-immunoprecipitation (co-IP) assays in the AR-dependent prostate cancer cell line VCaP, we previously reported that AR interacts with proteins of the MLL complex26. To further study the nature of this interaction, we fractionated VCaP cell nuclear extracts by size-exclusion chromatography and measured the presence of AR and MLL complex proteins by immunoblot analysis. AR eluted in a fraction that contains high-molecular weight complexes, akin to the elution pattern of MLL complex components including MLL, MLL4, WDR5, ASH2L, and menin (Fig. 1a). Next we co-immunoprecipitated endogenous ASH2L, menin and AR from VCaP and another AR-dependent prostate cancer cell line, LNCaP, to confirm an association between AR and MLL complex proteins. Subsequent immunoprecipitation with AR, ASH2L and menin antibodies followed by immunoblot analysis for AR and MLL complex proteins demonstrated their association (Fig. 1b,c). To test the robustness of this interaction, we performed co-IP experiments in VCaP cells under stringent condition (350 mM NaCl), and we used a different AR antibody; in both instances, MLL complex proteins co-immunoprecipitated with AR (Supplementary Fig. 1a,b). Confocal immunofluorescence microscopy in VCaP cells also demonstrated that ASH2L and menin co-localize with AR in the nucleus (Fig. 1d). To corroborate this interaction in situ, we stained sections from benign, localized and metastatic human prostate cancer tissue with antibodies against menin, MLL and AR; AR and menin staining was predominantly nuclear in epithelial cells with some staining observed in the stroma (Supplementary Fig. 1c,d). Likewise, most of the MLL staining was primarily limited to epithelial cells; however, some smooth muscle cells also showed nuclear staining (Supplementary Fig. 1e). Collectively, these results show that AR physically associates with the MLL complex in prostate cancer cells and tissues.


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)

Androgen receptor interacts with MLL complex proteins. (a) VCaP nuclear lysate fractions from Superose-6 gel filtration column were immunoblotted using indicated antibodies. Arrows indicate the approximate mass of the complexes eluted. Distribution of cytoplasmic or nuclear proteins demonstrates the efficiency of lysate fractionation (inset western blot). Shown are representative blots (n=3). (b–c) VCaP (b) and LNCaP (c) nuclear lysates were immunoprecipitated (IP) with AR, ASH2L and menin antibodies followed by immunoblotting (IB) using indicated antisera. Shown are representative blots from three independent experiments. (d) VCaP cells were fixed and immunostained using AR (green) and ASH2L (red) or menin (red) antibodies. Confocal microscopic images were analyzed by ImageJ software. Co-localization is represented by yellow puncta. Bar = 10μm. Inset bar = 2.5μm. Shown are representative images from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390530&req=5

Figure 1: Androgen receptor interacts with MLL complex proteins. (a) VCaP nuclear lysate fractions from Superose-6 gel filtration column were immunoblotted using indicated antibodies. Arrows indicate the approximate mass of the complexes eluted. Distribution of cytoplasmic or nuclear proteins demonstrates the efficiency of lysate fractionation (inset western blot). Shown are representative blots (n=3). (b–c) VCaP (b) and LNCaP (c) nuclear lysates were immunoprecipitated (IP) with AR, ASH2L and menin antibodies followed by immunoblotting (IB) using indicated antisera. Shown are representative blots from three independent experiments. (d) VCaP cells were fixed and immunostained using AR (green) and ASH2L (red) or menin (red) antibodies. Confocal microscopic images were analyzed by ImageJ software. Co-localization is represented by yellow puncta. Bar = 10μm. Inset bar = 2.5μm. Shown are representative images from three independent experiments.
Mentions: Using co-immunoprecipitation (co-IP) assays in the AR-dependent prostate cancer cell line VCaP, we previously reported that AR interacts with proteins of the MLL complex26. To further study the nature of this interaction, we fractionated VCaP cell nuclear extracts by size-exclusion chromatography and measured the presence of AR and MLL complex proteins by immunoblot analysis. AR eluted in a fraction that contains high-molecular weight complexes, akin to the elution pattern of MLL complex components including MLL, MLL4, WDR5, ASH2L, and menin (Fig. 1a). Next we co-immunoprecipitated endogenous ASH2L, menin and AR from VCaP and another AR-dependent prostate cancer cell line, LNCaP, to confirm an association between AR and MLL complex proteins. Subsequent immunoprecipitation with AR, ASH2L and menin antibodies followed by immunoblot analysis for AR and MLL complex proteins demonstrated their association (Fig. 1b,c). To test the robustness of this interaction, we performed co-IP experiments in VCaP cells under stringent condition (350 mM NaCl), and we used a different AR antibody; in both instances, MLL complex proteins co-immunoprecipitated with AR (Supplementary Fig. 1a,b). Confocal immunofluorescence microscopy in VCaP cells also demonstrated that ASH2L and menin co-localize with AR in the nucleus (Fig. 1d). To corroborate this interaction in situ, we stained sections from benign, localized and metastatic human prostate cancer tissue with antibodies against menin, MLL and AR; AR and menin staining was predominantly nuclear in epithelial cells with some staining observed in the stroma (Supplementary Fig. 1c,d). Likewise, most of the MLL staining was primarily limited to epithelial cells; however, some smooth muscle cells also showed nuclear staining (Supplementary Fig. 1e). Collectively, these results show that AR physically associates with the MLL complex in prostate cancer cells and tissues.

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