Intrinsic BET inhibitor resistance in SPOP-mutated prostate cancer is mediated by BET protein stabilization and AKT-mTORC1 activation

Abstract

Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anticancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is the most frequently mutated in primary prostate cancer. Here we demonstrate that wild-type SPOP binds to and induces ubiquitination and proteasomal degradation of BET proteins (BRD2, BRD3 and BRD4) by recognizing a degron motif common among them. In contrast, prostate cancer-associated SPOP mutants show impaired binding to BET proteins, resulting in decreased proteasomal degradation and accumulation of these proteins in prostate cancer cell lines and patient specimens and causing resistance to BET inhibitors. Transcriptome and BRD4 cistrome analyses reveal enhanced expression of the GTPase RAC1 and cholesterol-biosynthesis-associated genes together with activation of AKT-mTORC1 signaling as a consequence of BRD4 stabilization. Our data show that resistance to BET inhibitors in SPOP-mutant prostate cancer can be overcome by combination with AKT inhibitors and further support the evaluation of SPOP mutations as biomarkers to guide BET-inhibitor-oriented therapy in patients with prostate cancer.

Figure 1. SPOP interacts with and promotes BRD2/3/4 protein ubiquitination and degradation

a, Diagram showing portions of BRD2/3/4 proteins identified by yeast two-hybrid screen in a human fetal brain cDNA library using the full-length SPOP as bait. The region between two dashed red lines is the minimal interaction region shared by positive clones, and the bolded red vertical line represents the SBC motif. BD1, bromodomain 1; BD2, bromodomain 2; ET, extraterminal domain; CTM, C-terminal motif. b, Western blot of co-IP samples of IgG or anti-BRD2/3/4 antibodies from cell lysate of LNCaP cells treated with 20 μM MG132 for 8 h. c, Western blot of whole cell lysate (WCL) of 293T cells transfected with indicated plasmids and treated with or without 20 μM MG132 for 8 h. Actin was used as a loading control. d, Western blot of WCL of different cell lines transfected with indicated siRNAs. e, Western blot of the products of in vivo ubiquitination assay performed using cell lysate of 293T cells transfected with indicated plasmids and treated with 20 μM MG132 for 8 h. f, Western blot of the products of in vitro ubiquitination assay performed by incubating the reconstituted SPOP-CUL3-RBX1 E3 ligase complex with E1, E2, Ub, and His-BRD4-N (amino acids 1–500) at 30°C for 2 h.

Figure 2. The SBC motif in BRD2/3/4 is a SPOP-recognized degron

a, Amino acid (aa) sequencing alignment of a putative SBC motif in BRD2/3/4. MacroH2A and DEK, positive controls. Φ represents a nonpolar residue and π represents a polar residue. S, serine; T, threonine. b, Diagram showing the wild-type BRD2/3/4 and SBC motif-deleted mutants. c, Western blot of WCL and co-IP samples of anti-FLAG antibody from 293T cells transfected with indicated plasmids and treated with 20 μM MG132 for 8 h. d, Western blot of WCL of 293T cells transfected with indicated plasmids. e and f, Western blot of WCL of 293T cells transfected with indicated plasmids and treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points (e). At each time point, the intensity of BET protein was normalized to the intensity of actin and then to the value at 0 h (f). g, Western blot of the products of in vivo ubiquitination assay from 293T cells transfected with indicated plasmids and treated with 20 μM MG132 for 8 h.

Figure 3. Expression of BET proteins is elevated in SPOP mutant-expressing prostate cancer cells and patient specimens

a, Western blot of WCL and co-IP samples of anti-FLAG antibody from 293T cells transfected with indicated Myc- or FLAG-tagged plasmids and treated with 20 μM MG132 for 8 h. b, Western blot of the products of in vivo ubiquitination assay from 293T cells transfected with indicated Myc- or FLAG-tagged plasmids and treated with 20 μM MG132 for 8 h. c, Western blot of indicated proteins in WCL of C4-2 cells infected with lentivirus expressing empty vector (EV), wild-type (WT) or mutated SPOP. d and e, Representative images of BRD2/3/4 IHC in SPOP-WT and -mutated (MUT) prostate cancer tissues (d). The quantitative data of BRD2/3/4 staining are shown in (e). Statistical significance was determined by Wilcoxon rank sum test.

Figure 4. Mechanism of BET inhibitor resistance in SPOP-mutated prostate cancer cells

a, Western blot of indicated proteins including p-AKT (Ser473) and p-S6K (Thr389) in C4-2 cells infected with lentivirus expressing empty vector (EV) or SPOP-F133V mutant in combination with control shRNA (shC) or BRD2/3/4-specific shRNAs. Cells were treated with or without JQ1 (1 μM) for 24 h before harvested. Asterisk in red indicates exogenous SPOP-F133V mutant. b, C4-2 cells infected with lentivirus as in (a) were implanted subcutaneously in mice (n = 6/group). When tumors reached a size of approximately 100 mm³, xenografted mice were treated with vehicle or JQ1 (50 mg/kg) 5 days a week. Tumors were measured by caliper twice a week. Data are shown as means ± SD. Statistical significance was determined by two-tailed Student’s t-test for tumors at day 21 of drug treatment. c, Image of tumors isolated from each group of mice at day 21 of drug treatment as shown in (b). d, Heat map of RNA-seq data shows expression of a cluster of genes (n = 1,017) in C4-2 cells infected with lentivirus expressing EV or F133V and treated with or without JQ1 (1 μM) for 24 h. e, Heat map showing expression of 129 genes associated with JQ1 resistance was upregulated in SPOP-mutated (MUT) prostate tumors compared to SPOP-WT tumors in the TCGA cohort. f, Venn diagram shows that JQ1-resistant genes upregulated in SPOP-mutated prostate tumors significantly overlapped with the common BRD4 target genes of SPOP F133V and HA-BRD4 overexpressed (OE) in C4-2 cells (P = 9.407e-12, Permutation test). g, UCSC genome browser screen shots showing BRD4 ChIP-seq signal profiles in the RAC1 gene locus in C4-2 cells expressing EV, F133V or HA-BRD4 treated with DMSO or JQ1 (1 μM) for 24 h. H3K4me3 ChIP-seq was acquired from LNCaP cells as reported previously. h, C4-2 cells infected with lentivirus as in (a) were implanted subcutaneously in mice (n = 6/group). When tumors reached a size of approximately 100 mm³, xenografted mice were treated with vehicle, JQ1 (50 mg/kg) or GDC-0068 (100 mg/kg) individually or in combination 5 days a week. Tumors were measured by caliper twice a week. Data are shown as means ± SD. Statistical significance was determined by two-tailed Student’s t-test for tumors at day 21 of drug treatment. i, Image of tumors isolated from each group of mice at day 21 of drug treatment as shown in (h).