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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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A Menin-MLL small molecule inhibitor impairs prostate cancer growth in mice. (a) Heat map representation of the impact of 5μM MI-136 treatment on DHT induced genes in VCaP cells as assessed by microarray. (b) GSEA was performed using an AR target gene signature (supplementary table S2). (c) VCaP cells were treated with 5μM MI-503 and incubated at indicated temperatures. Cells were lysed and soluble proteins were detected by immunoblotting. Shown are representative blots (n=2). (d) VCaP, LNCaP or PNT2 cells were treated with either DMSO or MI-503 for 48 hours and the effects on protein levels of PSA and cleaved PARP were determined by immunoblotting Shown are representative blots (n=3). (e) The effect on AR target gene expression (TMPRSS2, FKBP5, and KLK3) was quantified by qPCR in VCaP cells pre-treated with either DMSO or MI-503 and subsequently stimulated with 10nM DHT for 6 hours. *, P<0.01; **, P<0.001, compared with untreated by one-way ANOVA. (n = 3, mean ± s.e.m) (f) Heat map representation of the impact of 5μM MI-503 on DHT induced genes in VCaP cells was assessed by microarray. (g) GSEA was performed using an AR target gene signature (supplementary table S2). (h) Heat map representation demonstrates the effects on gene expression after ASH2L KD and MI-503 treatment. (i) Impact of MI-503 on growth of LNCaP-AR xenografts in castrated mice. LNCaP-AR xenografts were implanted in castrated mice. Once the tumors reach 80–100mm3, mice were treated daily with vehicle (n=20) or 60mg/kg MI-503 (n=18) intraperitoneally. (j) Impact of MI-503 on growth of castrate resistant VCaP xenografts. Castrate resistant VCaP tumors were generated as described in online methods. Once tumors reach 80–100mm3, mice were treated with vehicle (n=20), MI-503 (75mg/kg, n=16), MDV-3100 (10mg/kg, n=20) or combination (n=20). MI-503 was given intraperitoneally and MDV-3100 was given by oral route. Tumors are measured using caliper measurements taken bi-weekly. *, P<0.05; **, P<0.005. Compared to vehicle by a Student’s t test.
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Figure 6: A Menin-MLL small molecule inhibitor impairs prostate cancer growth in mice. (a) Heat map representation of the impact of 5μM MI-136 treatment on DHT induced genes in VCaP cells as assessed by microarray. (b) GSEA was performed using an AR target gene signature (supplementary table S2). (c) VCaP cells were treated with 5μM MI-503 and incubated at indicated temperatures. Cells were lysed and soluble proteins were detected by immunoblotting. Shown are representative blots (n=2). (d) VCaP, LNCaP or PNT2 cells were treated with either DMSO or MI-503 for 48 hours and the effects on protein levels of PSA and cleaved PARP were determined by immunoblotting Shown are representative blots (n=3). (e) The effect on AR target gene expression (TMPRSS2, FKBP5, and KLK3) was quantified by qPCR in VCaP cells pre-treated with either DMSO or MI-503 and subsequently stimulated with 10nM DHT for 6 hours. *, P<0.01; **, P<0.001, compared with untreated by one-way ANOVA. (n = 3, mean ± s.e.m) (f) Heat map representation of the impact of 5μM MI-503 on DHT induced genes in VCaP cells was assessed by microarray. (g) GSEA was performed using an AR target gene signature (supplementary table S2). (h) Heat map representation demonstrates the effects on gene expression after ASH2L KD and MI-503 treatment. (i) Impact of MI-503 on growth of LNCaP-AR xenografts in castrated mice. LNCaP-AR xenografts were implanted in castrated mice. Once the tumors reach 80–100mm3, mice were treated daily with vehicle (n=20) or 60mg/kg MI-503 (n=18) intraperitoneally. (j) Impact of MI-503 on growth of castrate resistant VCaP xenografts. Castrate resistant VCaP tumors were generated as described in online methods. Once tumors reach 80–100mm3, mice were treated with vehicle (n=20), MI-503 (75mg/kg, n=16), MDV-3100 (10mg/kg, n=20) or combination (n=20). MI-503 was given intraperitoneally and MDV-3100 was given by oral route. Tumors are measured using caliper measurements taken bi-weekly. *, P<0.05; **, P<0.005. Compared to vehicle by a Student’s t test.

Mentions: Next we monitored the effect of MI-136 on the AR transcriptional program using qRT-PCR on VCaP cells treated with MI-136, MDV-3100, or MI-nc (non-active control). Treatment with MI-136 and MDV-3100 inhibited DHT-induced expression of AR target genes (Supplementary Fig. 9d). Inhibition of PSA protein expression was also observed in both VCaP and LNCaP cells (Supplementary Fig. 9e,f). To examine the effects of menin inhibition on global AR signaling, we performed microarray analysis on DHT stimulated VCaP cells pre-treated with MI-136. GSEA revealed that MI-136 treatment blocked the induction of AR-upregulated genes (Fig. 6a,b). Treatment with MI-136 also inhibited the expression of genes that were bound to ASH2L after AR stimulation (Supplementary Fig. 9g, Supplementary Table 2). We further showed that treatment with MI-136 induced apoptosis of VCaP cells as evidenced by PARP (cPARP) cleavage (Supplementary Fig. 9h).


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)

A Menin-MLL small molecule inhibitor impairs prostate cancer growth in mice. (a) Heat map representation of the impact of 5μM MI-136 treatment on DHT induced genes in VCaP cells as assessed by microarray. (b) GSEA was performed using an AR target gene signature (supplementary table S2). (c) VCaP cells were treated with 5μM MI-503 and incubated at indicated temperatures. Cells were lysed and soluble proteins were detected by immunoblotting. Shown are representative blots (n=2). (d) VCaP, LNCaP or PNT2 cells were treated with either DMSO or MI-503 for 48 hours and the effects on protein levels of PSA and cleaved PARP were determined by immunoblotting Shown are representative blots (n=3). (e) The effect on AR target gene expression (TMPRSS2, FKBP5, and KLK3) was quantified by qPCR in VCaP cells pre-treated with either DMSO or MI-503 and subsequently stimulated with 10nM DHT for 6 hours. *, P<0.01; **, P<0.001, compared with untreated by one-way ANOVA. (n = 3, mean ± s.e.m) (f) Heat map representation of the impact of 5μM MI-503 on DHT induced genes in VCaP cells was assessed by microarray. (g) GSEA was performed using an AR target gene signature (supplementary table S2). (h) Heat map representation demonstrates the effects on gene expression after ASH2L KD and MI-503 treatment. (i) Impact of MI-503 on growth of LNCaP-AR xenografts in castrated mice. LNCaP-AR xenografts were implanted in castrated mice. Once the tumors reach 80–100mm3, mice were treated daily with vehicle (n=20) or 60mg/kg MI-503 (n=18) intraperitoneally. (j) Impact of MI-503 on growth of castrate resistant VCaP xenografts. Castrate resistant VCaP tumors were generated as described in online methods. Once tumors reach 80–100mm3, mice were treated with vehicle (n=20), MI-503 (75mg/kg, n=16), MDV-3100 (10mg/kg, n=20) or combination (n=20). MI-503 was given intraperitoneally and MDV-3100 was given by oral route. Tumors are measured using caliper measurements taken bi-weekly. *, P<0.05; **, P<0.005. Compared to vehicle by a Student’s t test.
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Figure 6: A Menin-MLL small molecule inhibitor impairs prostate cancer growth in mice. (a) Heat map representation of the impact of 5μM MI-136 treatment on DHT induced genes in VCaP cells as assessed by microarray. (b) GSEA was performed using an AR target gene signature (supplementary table S2). (c) VCaP cells were treated with 5μM MI-503 and incubated at indicated temperatures. Cells were lysed and soluble proteins were detected by immunoblotting. Shown are representative blots (n=2). (d) VCaP, LNCaP or PNT2 cells were treated with either DMSO or MI-503 for 48 hours and the effects on protein levels of PSA and cleaved PARP were determined by immunoblotting Shown are representative blots (n=3). (e) The effect on AR target gene expression (TMPRSS2, FKBP5, and KLK3) was quantified by qPCR in VCaP cells pre-treated with either DMSO or MI-503 and subsequently stimulated with 10nM DHT for 6 hours. *, P<0.01; **, P<0.001, compared with untreated by one-way ANOVA. (n = 3, mean ± s.e.m) (f) Heat map representation of the impact of 5μM MI-503 on DHT induced genes in VCaP cells was assessed by microarray. (g) GSEA was performed using an AR target gene signature (supplementary table S2). (h) Heat map representation demonstrates the effects on gene expression after ASH2L KD and MI-503 treatment. (i) Impact of MI-503 on growth of LNCaP-AR xenografts in castrated mice. LNCaP-AR xenografts were implanted in castrated mice. Once the tumors reach 80–100mm3, mice were treated daily with vehicle (n=20) or 60mg/kg MI-503 (n=18) intraperitoneally. (j) Impact of MI-503 on growth of castrate resistant VCaP xenografts. Castrate resistant VCaP tumors were generated as described in online methods. Once tumors reach 80–100mm3, mice were treated with vehicle (n=20), MI-503 (75mg/kg, n=16), MDV-3100 (10mg/kg, n=20) or combination (n=20). MI-503 was given intraperitoneally and MDV-3100 was given by oral route. Tumors are measured using caliper measurements taken bi-weekly. *, P<0.05; **, P<0.005. Compared to vehicle by a Student’s t test.
Mentions: Next we monitored the effect of MI-136 on the AR transcriptional program using qRT-PCR on VCaP cells treated with MI-136, MDV-3100, or MI-nc (non-active control). Treatment with MI-136 and MDV-3100 inhibited DHT-induced expression of AR target genes (Supplementary Fig. 9d). Inhibition of PSA protein expression was also observed in both VCaP and LNCaP cells (Supplementary Fig. 9e,f). To examine the effects of menin inhibition on global AR signaling, we performed microarray analysis on DHT stimulated VCaP cells pre-treated with MI-136. GSEA revealed that MI-136 treatment blocked the induction of AR-upregulated genes (Fig. 6a,b). Treatment with MI-136 also inhibited the expression of genes that were bound to ASH2L after AR stimulation (Supplementary Fig. 9g, Supplementary Table 2). We further showed that treatment with MI-136 induced apoptosis of VCaP cells as evidenced by PARP (cPARP) cleavage (Supplementary Fig. 9h).

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