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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 directly interacts with menin. (a) Purified untagged full-length (FL) AR was incubated with purified menin, ASH2L, Max or Ring1b proteins. Anti-AR antibody or control IgG immunoprecipitates (IP) were subjected to immunoblot (IB) analysis using indicated antibodies. (b) Halo-tagged FL-AR or FL-Max was incubated with purified menin. AR was immunoprecipitated using anti-AR and immunoprecipitates were subjected to immunoblot analysis with anti-menin. Immunoblots were stripped and re-probed using anti-Halo. (c) Purified menin was incubated with Halo-tagged FL-AR and FL-Max. Menin was immunoprecipitated using anti-menin and binding to AR was analyzed by immunoblotting with anti-Halo. (d) Schematic representation of Halo-tagged AR truncation mutants used in this study. (e,f) Halo-tagged FL and truncation mutants of AR were incubated with purified menin. Menin was immunoprecipitated and binding to either FL or truncation mutants of AR was analyzed by immunoblot analysis using anti-Halo. The amounts of Halo-tagged proteins used in the pull-down experiments were estimated by anti-Halo immunoblot analysis. All panels show representative blots of three independent experiments.
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Figure 4: AR directly interacts with menin. (a) Purified untagged full-length (FL) AR was incubated with purified menin, ASH2L, Max or Ring1b proteins. Anti-AR antibody or control IgG immunoprecipitates (IP) were subjected to immunoblot (IB) analysis using indicated antibodies. (b) Halo-tagged FL-AR or FL-Max was incubated with purified menin. AR was immunoprecipitated using anti-AR and immunoprecipitates were subjected to immunoblot analysis with anti-menin. Immunoblots were stripped and re-probed using anti-Halo. (c) Purified menin was incubated with Halo-tagged FL-AR and FL-Max. Menin was immunoprecipitated using anti-menin and binding to AR was analyzed by immunoblotting with anti-Halo. (d) Schematic representation of Halo-tagged AR truncation mutants used in this study. (e,f) Halo-tagged FL and truncation mutants of AR were incubated with purified menin. Menin was immunoprecipitated and binding to either FL or truncation mutants of AR was analyzed by immunoblot analysis using anti-Halo. The amounts of Halo-tagged proteins used in the pull-down experiments were estimated by anti-Halo immunoblot analysis. All panels show representative blots of three independent experiments.

Mentions: Having demonstrated the recruitment of MLL complex proteins to AR bound chromatin regions; we sought to further characterize this interaction. We performed in vitro pull-down experiments and detected direct interaction between AR and menin (Fig. 4a). Further, we observed binding between purified untagged menin and Halo-AR (Fig. 4b). Immunoprecipitation of purified menin also pulled down Halo-AR (Fig. 4c). Next, to fine-map the AR-menin interaction, we generated deletion constructs for Halo-tagged AR (Fig. 4d), and found that menin interacts with the N-terminal domain of AR (Fig. 4e), specifically to amino acids 469–559 of the AR NTD (Fig. 4f). We examined the effect of AR stimulation on distribution of menin and saw no change (Supplementary Fig. 7a,b). Both AR and menin were mostly localized to the nucleus. Taken together, our experiments suggest a direct interaction between AR and menin.


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 directly interacts with menin. (a) Purified untagged full-length (FL) AR was incubated with purified menin, ASH2L, Max or Ring1b proteins. Anti-AR antibody or control IgG immunoprecipitates (IP) were subjected to immunoblot (IB) analysis using indicated antibodies. (b) Halo-tagged FL-AR or FL-Max was incubated with purified menin. AR was immunoprecipitated using anti-AR and immunoprecipitates were subjected to immunoblot analysis with anti-menin. Immunoblots were stripped and re-probed using anti-Halo. (c) Purified menin was incubated with Halo-tagged FL-AR and FL-Max. Menin was immunoprecipitated using anti-menin and binding to AR was analyzed by immunoblotting with anti-Halo. (d) Schematic representation of Halo-tagged AR truncation mutants used in this study. (e,f) Halo-tagged FL and truncation mutants of AR were incubated with purified menin. Menin was immunoprecipitated and binding to either FL or truncation mutants of AR was analyzed by immunoblot analysis using anti-Halo. The amounts of Halo-tagged proteins used in the pull-down experiments were estimated by anti-Halo immunoblot analysis. All panels show representative blots of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390530&req=5

Figure 4: AR directly interacts with menin. (a) Purified untagged full-length (FL) AR was incubated with purified menin, ASH2L, Max or Ring1b proteins. Anti-AR antibody or control IgG immunoprecipitates (IP) were subjected to immunoblot (IB) analysis using indicated antibodies. (b) Halo-tagged FL-AR or FL-Max was incubated with purified menin. AR was immunoprecipitated using anti-AR and immunoprecipitates were subjected to immunoblot analysis with anti-menin. Immunoblots were stripped and re-probed using anti-Halo. (c) Purified menin was incubated with Halo-tagged FL-AR and FL-Max. Menin was immunoprecipitated using anti-menin and binding to AR was analyzed by immunoblotting with anti-Halo. (d) Schematic representation of Halo-tagged AR truncation mutants used in this study. (e,f) Halo-tagged FL and truncation mutants of AR were incubated with purified menin. Menin was immunoprecipitated and binding to either FL or truncation mutants of AR was analyzed by immunoblot analysis using anti-Halo. The amounts of Halo-tagged proteins used in the pull-down experiments were estimated by anti-Halo immunoblot analysis. All panels show representative blots of three independent experiments.
Mentions: Having demonstrated the recruitment of MLL complex proteins to AR bound chromatin regions; we sought to further characterize this interaction. We performed in vitro pull-down experiments and detected direct interaction between AR and menin (Fig. 4a). Further, we observed binding between purified untagged menin and Halo-AR (Fig. 4b). Immunoprecipitation of purified menin also pulled down Halo-AR (Fig. 4c). Next, to fine-map the AR-menin interaction, we generated deletion constructs for Halo-tagged AR (Fig. 4d), and found that menin interacts with the N-terminal domain of AR (Fig. 4e), specifically to amino acids 469–559 of the AR NTD (Fig. 4f). We examined the effect of AR stimulation on distribution of menin and saw no change (Supplementary Fig. 7a,b). Both AR and menin were mostly localized to the nucleus. Taken together, our experiments suggest a direct interaction between AR and menin.

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