SPOP-mutated Prostate Cancer Research Articles

The impact of SPOP gene alterations in men with metastatic prostate cancer: Results from the Prostate Cancer Precision Medicine Multi-Institutional Collaborative Effort (PROMISE) consortium.

225 Background: Inactivating mutations in the SPOP gene, encoding speckled-type poxvirus and zinc-finger protein, are common among men with localized and metastatic prostate cancer, occurring at a frequency of 6-15%. Previous studies have suggested the presence of an inactivating mutation in SPOP results in increased sensitivity to androgen deprivation therapy (ADT). In this multi-institutional clinical-genomic database, we evaluated the impact of SPOP alterations on survival outcomes in men with metastatic prostate cancer. Methods: Retrospective data from the PROMISE Consortium were utilized for this analysis. Eligible patients had metastatic prostate cancer and had undergone standard of care next-generation sequencing (NGS). Patients with inactivating mutations in SPOP were defined as SPOP mutated, where. as those lacking such alterations were defined as SPOP wild-type. The primary endpoint was overall survival defined as the time from diagnosis of metastatic prostate cancer to death from any cause, censored at the date of last follow-up. Secondary endpoints included time from metastatic disease to castration resistance and time from castration resistance to death. Results: Of the 2097 patients with available NGS testing, 5.5% (n=115) had SPOP alterations. The median age at diagnosis was 63 years, 427 were Black, and 83 were Hispanic. At last assessment, 66% had bone metastases, 7% lung metastases, and 5% liver metastases. The most frequent co-occurring alterations in the SPOP-mutated group were: TP53 (30.4%), APC (25.2%), and AR (21.7%). 1832 patients were included in the survival analysis [n=96 (5.2%) with and n=1736 (94.8%) without SPOP alterations]. Median overall survival was numerically longer, though not statistically significant, in the SPOP-mutated compared SPOP-wild-type group (75.9 versus 59.5 months, p=0.12). In patients with metastatic disease, median time to castration resistance was 14.6 months in the SPOP-mutated group versus 12.2 months in the SPOP-wild-type group (p=0.30). Median time from metastatic castration resistant to death was 45.0 months in the SPOP-mutated group and 40.2 months in the SPOP-wild type group. Conclusions: Our hypothesis generating data support that SPOP-mutated prostate cancer likely represents a unique molecular subtype of prostate cancer that may confer prolonged survival. Future studies of novel androgen-receptor targeting treatments should be tested in this molecular population.

Opposing Roles of SPOP Mutations in Human Prostate and Endometrial Cancers.

Recurrent gene mutations in speckle-type POZ protein (SPOP), the substrate-binding component of E3 ubiquitin ligase, are associated with tumor progression in prostate and endometrial cancers. Here, we characterized SPOP mutations in these cancers and explored their association with molecular and immune signatures and patient outcomes. There were 7,398 prostate cancer and 19,188 endometrial cancer samples analyzed for clinical and molecular profiles at Caris Life Sciences. Overall survival (OS) was analyzed using Kaplan-Meier survival curves. Statistical significance was determined using chi-square and Mann-Whitney U tests, with P values adjusted for multiple comparisons. SPOP mutations were identified in 9.2% of prostate and 4.3% of endometrial cancers. Mutations clustered in the SPOP meprin and TRAF-C homology domain, with no significant overlap between cancer types. SPOP mutation was associated with differential comutation profiles and opposing tumor immune microenvironment signatures for each cancer, with greater immune infiltration in SPOP-mutated endometrial cancer. SPOP-mutated prostate and endometrial cancers displayed altered epigenetic gene expression, including opposite regulation of BRD2 transcripts. In SPOP-mutant prostate cancer, higher expression of androgen receptor-regulated transcripts and improved OS after treatment with hormonal agents were observed. In endometrial cancer, hormone receptor expression was significantly lower in SPOP-mutated tumors and differences in OS were highly dependent on the particular hotspot mutation and histologic subtype. These data indicate that SPOP mutations drive opposing molecular and immune landscapes in prostate and endometrial cancers-suggesting a loss-of-function mechanism in prostate cancer and gain-of-function mechanism in endometrial cancer-and provide a rationale for tailored therapeutic approaches.

Abstract 937: Opposing roles of SPOP mutations in human prostate and endometrial cancers

Abstract Recurrent mutations in the gene encoding the Speckle Type POZ Protein, SPOP, the substrate-binding components of E3-ubiquitin ligase, have been identified as common alterations in prostate cancer (PCa) and endometrial cancer (EC). SPOP mutations are associated with PCa and EC growth and progression. Here, we characterized SPOP mutations in PCa and EC, and their association with molecular and immune signatures. 7,398 PCa and 19,188 EC samples were analyzed by next-generation sequencing (592, NextSeq; WES, NovaSeq), (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). Real world overall survival (rwOS) was extracted from insurance claims and depicted using Kaplan-Meier survival curves for molecularly defined cohorts from tissue collection to last contact. Statistical significance was determined using chi-square and Mann-Whitney U tests, with p-values adjusted for multiple comparisons (q<0.05). SPOP mutations were identified in 601 (8.1%) and 655 (3.4%) samples of PCa and EC, respectively. Mutations clustered in the SPOP MATH domain with hotspots at residues F133L (21.4%), F102C (11%), F133V (10.4%), F102V (7%) in PCa, and residues M117V (21.8%), R121Q (10.6 %), E47K (9%), E50K (8.5%) in EC. Importantly, no overlap of hotspot residues was noted in PCa and EC. SPOP-mutated PCa had higher frequency of APC (21.4% vs 5.8%) and BCOR (1.95% vs 0.2%) lesions, but SPOP-mutated EC had higher frequency of U2AF1 (4.28% vs 1.0%), POLE (5.24% vs 2.0%) and CASP8 (4.17% vs 1.79%) lesions (all q <0.05) compared to non-mutant SPOP. Analysis of epigenetic markers showed lower BRD2 (245.4 vs 312 TPM) transcripts in SPOP-mutated PCa, but higher BRD2 (227.0 vs 200.6) (all q <0.05) in SPOP-mutated EC, compared to non-mutant SPOP, the former diverging from earlier published data in PCa. Analysis of inferred immune cell infiltrates showed that in SPOP-mutated PCa, M1-MΦ (36.8% vs 48.1%), CD8 T cell (20.2% vs 30.4%) and DC (53.3% vs 79.4%) are decreased and M2-MΦ (100% vs 99.9%) are increased; while in SPOP-mutated EC, M1-MΦ (87.2% vs 83.3 %), CD8 T cells (56.8% vs 49.7%) and DC (94.9% vs 93.3%) are increased and M2-MΦ (98.8% vs 98.9%) are decreased (all q <0.05). In PCa, higher AR expression was found in SPOP-mutated vs non-mutated tumors (98.5% vs 97.3%, p=0.09), with improved OS outcomes after hormone targeting agents leuprolide (2125 vs 1803 days; HR: 0.71, 95% CI (0.51-0.99), p=0.04) and bicalutamide (2538 vs 1854 days; HR: 0.57, 95% CI (0.41-0.79), p<0.001). In EC, ER and PR expression was significantly lower in SPOP-mutated vs non-mutated tumors 50.8% vs 65.4% and 35.4 vs 49.5% (all q <0.05), respectively, although no difference in OS was noted in the overall EC population. These data indicate that SPOP-mutations drive different molecular and immune microenvironments in PCa and EC, with apparently opposing roles in these malignancies, and provide a rationale for tailored therapeutic approaches in SPOP-mutated PCa and EC. Citation Format: Ludimila Cavalcante, Sachin K. Desmukh, Benedito A. Carneiro, Kartik Angara, Tyler E. Mattox, Sharon Wu, Joanne Xiu, Philip Walker, Matthew Oberley, Chadi Nabhan, Emmanuel S. Antonarakis. Opposing roles of SPOP mutations in human prostate and endometrial cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 937.

Role of adenomatous polyposis coli (APC) gene in SPOP-mutant prostate cancer and clinical outcomes.

202 Background: Speckle-type POZ protein (SPOP) mutations are a frequent initiating event in prostate cancer (PC) and are associated with increased androgen receptor transcriptional output. While SPOP-Mutant (MT) PCs may be more sensitive to hormonal therapy, they are still common amongst castration-resistant PCs (CRPCs) and frequently lethal. It remains unknown which SPOPMT PCs will respond well to hormonal therapy, and which have the potential to develop early castration resistance and cause patient death. Methods: With IRB approval, we retrospectively analyzed NGS data obtained via Tempus|xT tissue assay (DNA sequencing of 648 genes in tumor and matched normal samples at 500x depth) and/or Tempus|xF liquid biopsy assay (ctDNA sequencing of 105 genes in peripheral blood samples at 5,000x depth) for germline and/or somatic mutations in 234 consecutive patients with high-risk localized, locally advanced or metastatic PC followed at Ben Taub Hospital (BTH), a public hospital serving a racially/ethnically diverse patient population (>50% of PC patients are African American). For confirmation, we examined de-identified NGS data from a nationwide cohort of 8812 PC patients sequenced with xT and/or xF by Tempus Labs (Chicago, IL), as well as publicly available data from four additional PC cohorts (PMIDs: 26544944, 34667026, 31061129 and 26855148). Results: We found SPOP mutations in 31/234 BTH PC patients (13.2%). Eleven of these 31 patients (35.5%) also exhibited loss-of-function (LOF) mutations or deletions in the tumor suppressor gene Adenomatous polyposis coli (APC). APC LOF events were found only in 15/203 (7.4%) of the SPOP-wild type (WT) PCs, and the enrichment of SPOPMT PCs for APC LOF events was highly significant (Chi square with Yates correction: P<0.00001). The co-mutation pattern between these two genes was observed even when the analysis was limited specifically to the tissue NGS (133 patients) or the liquid biopsy NGS subsets of the dataset, respectively. This enrichment was confirmed in the nationwide TEMPUS cohort and in the four publicly available clinical PC datasets (APC LOF found in 23.4, 27.0, 21.5, 32.0 and 30.8% of SPOPMT PCs, respectively; and only in 5.8, 2.4, 6.0, 6.7 and 1.0% of SPOP-Wild Type (WT) PCs, respectively (P<0.001 in each cohort). In the BTH PC cohort, SPOPMT PCs harboring APC LOF mutations were more likely to present with metastatic disease (M1) at diagnosis and to develop castration-resistance within 1 year of first-line hormonal therapy than SPOPMT PCs with WT APC. Conclusions: In SPOPMT PCs, inactivation of APC is a common event and associated with more aggressive disease (higher metastatic potential and faster progression to castration resistance). This observation may help identify SPOPMT PC patients who are at higher risk for early castration resistance and could benefit from more intensive treatment.

SPOP-mutant prostate cancer: Translating fundamental biology into patient care.

Comprehensive cancer genome studies have revealed genetically-defined subtypes of prostate cancer with distinct truncal driver mutations. Because prostate cancer has been largely seen as a rather uniform disease, the clinical significance of this discovery remained largely obscure. However, recent findings imply distinct biological features and therapeutic vulnerabilities linked to specific truncal mutations. Here we review our current understanding of prostate cancers harboring recurrent point mutations in the ubiquitin ligase adaptor protein SPOP and discuss opportunities for future clinical translation. More specifically, activation of the androgen receptor (AR) signaling emerges as the key oncogenic pathway. SPOP-mutant prostate cancer patients respond to AR inhibition in various clinical settings. Molecular insights on how mutant SPOP promotes tumorigenesis may open more specific therapeutic avenues which, in combination with conventional AR-targeting agents, could improve the outcome of patients with SPOP-mutant prostate cancer.

Clinical and genomic features of SPOP-mutant prostate cancer.

Inactivating missense mutations in the SPOP gene, encoding speckle-type poxvirus and zinc-finger protein, are one of the most common genetic alterations in prostate cancer. We retrospectively identified 72 consecutive prostate cancer patients with somatic SPOP mutations, through next-generation sequencing analysis, who were treated at the Johns Hopkins Hospital. We evaluated clinical and genomic characteristics of this SPOP-mutant subset. SPOP alterations were clustered in the MATH domain, with hotspot mutations involving the F133 and F102 residues. The most frequent concurrent genetic alterations were in APC (16/72 [22%]), PTEN (13/72 [18%]), and TP53 (11/72 [15%]). SPOP-mutant cancers appeared to be mutually exclusive with tumors harboring the TMPRSS2-ERG fusion, and were significantly enriched for Wnt pathway (APC, CTNNB1) mutations and de-enriched for TP53/PTEN/RB1 alterations. Patients with mtSPOP had durable responses to androgen deprivation therapy (ADT) with a median time-to-castration-resistance of 42.0 (95% confidence interval [CI], 25.7-60.8) months. However, time-to-castration-resistance was significantly shorter in SPOP-mutant patients with concurrent TP53 mutations (hazard ratio [HR] 4.53; p = 0.002), HRD pathway (ATM, BRCA1/2, and CHEK2) mutations (HR 3.19; p = 0.003), and PI3K pathway (PTEN, PIK3CA, and AKT1) alterations (HR 2.69; p = 0.004). In the castration-resistant prostate cancer setting, median progression-free survival was 8.9 (95% CI, 6.7-NR) months on abiraterone and 7.3 (95% CI, 3.2-NR) months on enzalutamide. There were no responses to PARP inhibitor treatment. SPOP-mutant prostate cancers represent a unique subset with absent ERG fusions and frequent Wnt pathway alterations, with potentially greater dependency on androgen signaling and enhanced responsiveness to ADT. Outcomes are best for SPOP-altered patients without other concurrent mutations.

Mutated SPOP E3 Ligase Promotes 17βHSD4 Protein Degradation to Drive Androgenesis and Prostate Cancer Progression.

Molecular mechanisms underlying intratumoral androgenesis and aberrant androgen receptor (AR) activation in prostate cancer remain poorly understood. Here we demonstrate that ectopic expression of the E3 ubiquitin ligase adaptor speckle-type poxvirus and zinc finger domain protein (SPOP) stabilizes 17βHSD4. SPOP bound a functional substrate-binding consensus (SBC) motif 315RATST319 in 17βHSD4 and promoted nondegradable K27- and K29-linked polyubiquitination of 17βHSD4. The effect of SPOP was antagonized by serum- and glucocorticoid kinase-3 (SGK3)-mediated phosphorylation of serine 318 (S318) in the SBC and S318 phosphorylation-dependent binding of SKP2 E3 ligase and subsequent K48-linked polyubiquitination and proteasomal degradation of 17βHSD4. Prostate cancer-associated SPOP mutations impaired the SPOP-17βHSD4 interaction, caused 17βHSD4 protein destruction in prostate cancer cells in culture and patient specimens, and increased testosterone production and prostate cancer cell growth in vitro and in mouse models. Thus, we have identified SPOP and SKP2 as two essential E3 ubiquitin ligases that exert opposite effects on 17βHSD4 protein degradation and intratumoral androgenesis in prostate cancer cells. We further demonstrate that SPOP mutations or SKP2 overexpression contribute to prostate cancer progression by decreasing 17βHSD4 expression and increasing intratumoral androgen synthesis. SIGNIFICANCE: This study reveals a novel mechanism of aberrant AR activation in SPOP-mutated prostate cancer and uncovers putative biomarkers for effective treatment by AR-targeted therapies.

Loss of Speckle-Type POZ Protein Promotes Prostate Cancer Cell Migration and Invasion Through Upregulation of MCP-1.

BackgroundThe goal of this study is to verify that the loss of speckle-type POZ protein (SPOP) promotes the migration and invasion of prostate cancer cells, and that this process is brought about by an increase in MCP-1.Material/MethodsSPOP knockout C4-2 cells (C4-2 SPOP−/−) were verified by western blotting. Transwell and wound-healing assays were applied to verify different migration and invasion abilities between the C4-2 SPOP−/− and control cells. We used an antibody array to find different soluble chemokine factors in the C4-2 SPOP−/− cells. ELISA and qRT-PCR were applied for confirmation. To test MCP-1 function in conditioned medium, a transwell assay was applied with or without anti-MCP-1 antibody.ResultsThe western blot showed that SPOP was knocked out in sgSPOP-1 and sgSPOP-2 (different clones of C4-2 SPOP−/−). The transwell and wound-healing assays indicated that, compared with control cells, sgSPOP-1 and sgSPOP-2 had stronger migration and invasion abilities. The antibody array found that the expression of MCP-1 was upregulated in sgSPOP-1 and sgSPOP-2 conditioned medium. This result was verified by ELISA and qRT-PCR. In the prostate cancer cells, migration and invasion activity was greatly increased in C4-2 SPOP−/− conditioned medium, while this activity was decreased after anti-MCP-1 antibody neutralization.ConclusionsOur findings suggest that the loss of SPOP in C4-2 cells promotes increased cell migration and invasion abilities. This may be realized by upregulating the expression of MCP-1. The inhibition of MCP-1 expression may be an effective treatment for SPOP-mutant prostate cancer.

Abstract 1753: Targeting cancer with selective cbp/p300 bromodomain inhibitors

Abstract Background: The Bromodomain (BRD) and Extra-Terminal domain (BET) family of proteins are key regulators of epigenetic control. Although pan BET Inhibitors show good clinical activity, progressive disease was seen after several months of treatment in clinical responders, likely due to secondary resistance mechanisms. The plausible resistance mechanisms are increased expression of TCF7L2, c-Myc, Survivin and PIM1. Cyclic AMP response element binding protein (CREB)-binding protein (CBP) and E1A interacting protein of 300 kDa (EP300 or p300) are two closely related histone acetyl transferases with oncogenic roles in a variety of cancers. They are known to be co-activators of several key transcription factors that contribute to tumor progression including HIF1a, BRCA-1, p53, c-Myc and androgen receptor (AR). Studies have indicated that p300 is also upregulated in SPOP-mutated prostate cancer. Thus, targeting CBP/p300 represents an attractive approach for developing novel therapies. Methods and Results: Multiple potent and selective CBP/p300 bromodomain inhibitors that are structurally unrelated to known inhibitors were identified by iterative medicinal chemistry and SAR based approaches. The compounds were optimized towards attaining good physicochemical properties and DMPK profile. The anti-proliferative activity of the lead compounds was studied across multiple tumor types in a 3-day assay. The lead compounds potently inhibited viability of a wide range of hematological and solid tumor cell lines including prostate cancer cell lines VCaP and 22Rv1. In H929 cell line the lead compounds showed dose-dependent inhibition of cMYC and increase in cPARP. In a single dose PK-PD study in MV4-11 xenograft model, the compounds showed modulation of cMYC and Survivin. Conclusions: In summary, our studies demonstrate that selective CBP/p300 bromodomain inhibitors are potent in models of hematologic malignancies and solid tumors in-vitro. Profiling of efficacy in xenograft models, and further toxicological evaluation are in progress. Citation Format: Mahaboobi Jaleel, Ramesh S. Senaiar, Chandrasekhar Abbineni, Girish A. Renukappa, Subhendu Mukherjee, Sivapriya Marappan, DS Samiulla, AB Aravind, Naveen R. Kumar, Venkata Siva N. Reddy, Asha Babu, Akhila P. Srinivas, Prasad Yadlapalli, Suraj Tgore, Raghavendra NR, Chandranath D. Naik, Sanjeev Giri, Thomas Antony, Kavitha Nellore, Shekar Chelur, Girish Daginakatte, laura Ravanti, Mikko Myllymäki, Gerd Wohlfahrt, Elina Mattila, Stefan Karlsson, Mari Björkman, Reetta Riikonen, Tarja Ikonen, Laura Leimu, Chira Mälmström, Timo Korjamo, Anu Moilanen, Murali Ramachandra, Susanta Samajdar. Targeting cancer with selective cbp/p300 bromodomain inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1753.

MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (MP51)1 Apr 2020MP51-09 THE NOVEL ROLE OF SPOP IN REGULATING TOPOISOMERASE 2A IN PROSTATE CANCER CELLS AS A POTENTIAL THERAPEUTIC MARKER FOR DNA REPAIR TARGETED THERAPY Ryuta Watanabe*, Masashi Maekawa, Miki Hieda, Tomohiko Taguchi, Noriyoshi Miura, Tadahiko Kikugawa, Shigeki Higashiyama, and Takashi Saika Ryuta Watanabe*Ryuta Watanabe* More articles by this author , Masashi MaekawaMasashi Maekawa More articles by this author , Miki HiedaMiki Hieda More articles by this author , Tomohiko TaguchiTomohiko Taguchi More articles by this author , Noriyoshi MiuraNoriyoshi Miura More articles by this author , Tadahiko KikugawaTadahiko Kikugawa More articles by this author , Shigeki HigashiyamaShigeki Higashiyama More articles by this author , and Takashi SaikaTakashi Saika More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000913.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: SPOP, speckle-type POZ protein, is a substrate recognizing receptor of the Cullin-3/RING ubiquitin E3 complex. Heterozygous point mutations in the substrate-binding domain of SPOP have been found in 10-15% of human prostate cancer patients. Although SPOP is essential for a DNA repair in exogenous DNA damage stresses, it still remains unclear whether SPOP is involved in the process of a DNA repair in endogenous DNA damage stresses. In this study, we focus on relationships the functions of SPOP, especially those involved in topoisomerase 1 (TOP1) and topoisomerase 2 (TOP2), to the DNA-protein crosslink repair from endogenous DNA damage in prostate cancer cell lines. METHODS: We first examined the level of γH2AX, a marker of DNA breaks in various prostate cancer cell lines; C4-2, LNCaP, PC3, DU145 upon SPOP knockdown (KD). To elucidate the site of action of SPOP during DNA repair, we treated control or SPOP-KD C4-2 cells with topoisomerase (TOP) inhibitors, which inhibit DNA repair. To investigate functions of SPOP in the regulation of TOP, we analyzed protein expression and cellular localization of TOPs by western blotting and immunofluorescence staining. We also measured the amounts of TOPs which covalently bound on DNA by cesium chloride-density gradient centrifugation. We finally detected the level of enzymes (TDP1, TDP2 and MRE11) which liberate TOPs from the TOP-DNA cleavage complex. RESULTS: We found that SPOP KD in androgen receptor (AR)-positive prostate cancer cells increased the amount of γH2AX. Because the level of γH2AX in SPOP-knockdown cells was not additive by treatment of TOP inhibitors, it was suggested that SPOP could act upstream of TOP1 and TOP2-dependent pathways. Immunofluorescence staining indicated that TOP2A, but not TOP1, was accumulated in the nuclei by SPOP knockdown. By cesium chloride-density gradient centrifugation, we found that SPOP KD increased the amount of TOP2A on genomic DNA. The amount of TDP1 and TDP2, which liberate TOP2A from the TOP2A-DNA complex was decreased by SPOP knockdown. We also confirmed that the increase of γH2AX in SPOP-KD C4-2 cells was dependent on both AR and TOP2A. CONCLUSIONS: Our results suggest that SPOP is involved in the DNA-protein crosslink repair process through the elimination of TOP2A from the TOP2A cleavage complex in prostate cancer cell lines. These results imply that TOP inhibitor might be effective to AR-positive, SPOP-wild type prostate cancer. Also, PARP inhibitor might be effective to SPOP-mutated prostate cancer. Source of Funding: JSPS KAKENHI Grant Number 19K18613 © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e767-e767 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ryuta Watanabe* More articles by this author Masashi Maekawa More articles by this author Miki Hieda More articles by this author Tomohiko Taguchi More articles by this author Noriyoshi Miura More articles by this author Tadahiko Kikugawa More articles by this author Shigeki Higashiyama More articles by this author Takashi Saika More articles by this author Expand All Advertisement PDF downloadLoading ...

Prostate Cancer-associated SPOP mutations enhance cancer cell survival and docetaxel resistance by upregulating Caprin1-dependent stress granule assembly

BackgroundThe gene encoding the E3 ubiquitin ligase substrate-binding adaptor SPOP is frequently mutated in primary prostate cancer, but how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. Stress granules (SG) assembly is an evolutionarily conserved strategy for survival of cells under stress, and often upregulated in human cancers. We investigated the role of SPOP mutations in aberrant activation of the SG in prostate cancer and explored the relevanve of the mechanism in therapy resistance.MethodsWe identified SG nucleating protein Caprin1 as a SPOP interactor by using the yeast two hybrid methods. A series of functional analyses in cell lines, patient samples, and xenograft models were performed to investigate the biological significance and clinical relevance of SPOP regulation of SG signaling in prostate cancer.ResultsThe cytoplasmic form of wild-type (WT) SPOP recognizes and triggers ubiquitin-dependent degradation of Caprin1. Caprin1 abundance is elevated in SPOP-mutant expressing prostate cancer cell lines and patient specimens. SPOP WT suppresses SG assembly, while the prostate cancer-associated mutants enhance SG assembly in a Caprin1-dependent manner. Knockout of SPOP or expression of prostate cancer-associated SPOP mutants conferred resistance to death caused by SG inducers (e.g. docetaxel, sodium arsenite and H2O2) in prostate cancer cells.ConclusionsSG assembly is aberrantly elevated in SPOP-mutated prostate cancer. SPOP mutations cause resistance to cellular stress induced by chemtherapeutic drug such as docetaxel in prostate cancer.

Activity of NEO2734, a novel dual inhibitor of both BET and CBP-P300, in SPOP-mutated prostate cancer.

62 Background: We have previously reported that mutations in SPOP promote bromodomain and extra-terminal motif (BET) protein upregulation and BET inhibitor resistance in prostate cancer (Zhang et al., Nature Medicine 23(9):1055-1062, 2017). Other studies indicate that histone acetyltransferases (HATs) such as P300 are also upregulated in SPOP-mutated prostate cancer (Blattner et al., Cancer Cell 31: 436-451, 2017). Methods: We examined the anti-cancer effect of NEO2734 (Epigene Therapeutics Inc), a novel dual inhibitor of both BET and CBP-P300, in prostate cancer using cell line, organoid and patient-derived xenografts (PDX) as working models. We examined the anti-cancer effect of NEO2734 (Epigene Therapeutics Inc), a novel dual inhibitor of both BET and CBP-P300, in prostate cancer using cell line, organoid and patient-derived xenografts (PDX) as working models. Results: We demonstrated that while prostate cancer cell lines expressing F133V, the most frequent SPOP mutant, are very resistant to both BET inhibitor JQ1 and CBP/P300 inhibitor CPI-637, they are very sensitive to simultaneous cotreatment with JQ1 and CPI-637. Similarly, we demonstrated that F133V mutant cells are also very sensitive to NEO2734. We established PDX and organoid models from a patient with prostate cancer expressing a novel SPOP mutation Q165P. Computer simulation analysis reveals that the Q165P mutation causes larger conformational fluctuation, indicating that the structure of Q165P is less stable than that of wild type SPOP. Accordingly, co-immunoprecipitation assay showed that Q165P mutation impaired the dimerization ability of SPOP. We further showed that the Q165P mutant organoid was very sensitive to NEO2734 in vitro and the Q165P mutant PDX was very sensitive to NEO2734 in mice. Conclusions: These PDX-based pre-clinical studies indicate that the inhibition of both BET and CBP-P300, either by co-administration of two agents, or by treatment with the dual inhibitor, NEO2734, is effective in SPOP-mutated prostate cancer. These data provide a strong rationale for the inclusion of patients with SPOP-mutated prostate cancer in clinical studies of NEO2734.

Abstract 2259: Prostate cancer molecular subtype impacts interpretation of clinical parameters

Abstract Background: Molecular characterization of prostate cancer has revealed distinct subtypes with underlying genomic alterations. One of these core subtypes (SPOP mutant prostate cancer) has previously only been identifiable through DNA sequencing, making the impact on prognosis and routinely utilized risk stratification parameters unclear. We aimed to identify prostate cancer molecular subtypes from transcriptional data alone and determine associations with clinicopathologic outcomes and long-term prognosis. Methods: We developed a novel gene expression signature, classifier (SCaPT), and decision tree to predict SPOP mutant subclass from RNA gene expression data and classify common prostate cancer molecular subtypes. We then validated and further interrogated the association of prostate cancer molecular subtypes with pathologic and clinical outcomes in retrospective and prospective cohorts of 8,158 patients. Associations with clinicopathologic variables, metastasis-free and prostate cancer-specific survival were the main outcomes. Findings: The SCaPT model prediction showed high sensitivity and specificity in multiple cohorts across both RNA-seq and microarray gene expression platforms. We predicted 8~9% of cases to be SPOP mutant from both retrospective and prospective cohorts. Surprisingly, we found routine clinical risk factors were disconjugate when molecular subtype was considered. The SPOP mutant subclass was associated with lower frequency of positive margins, extraprostatic extension, and seminal vesicle invasion at prostatectomy. However, SPOP mutant cancers were associated with higher pretreatment serum prostate-specific antigen (PSA). The association between SPOP mutant status and higher PSA level was validated in three independent cohorts. Despite high pre-treatment PSA, the SPOP mutant subtype was associated with a favorable prognosis with improved metastasis-free survival, particularly in patients with “high-risk” preoperative PSA levels. Interpretation: Using a novel gene expression signature to assign the SPOP mutant subclass and a decision tree algorithm to define prostate cancer molecular subclasses, we found the SPOP mutant subclass associated with higher preoperative PSA, less adverse pathological features, and favorable prognosis. These findings suggest a paradigm where interpretation of common risk stratification parameters, particularly PSA, must be modified to account for the molecular subtype of prostate cancer. Citation Format: Deli Liu. Prostate cancer molecular subtype impacts interpretation of clinical parameters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2259.

Dual inhibition of AKT ‐m TOR and AR signaling by targeting HDAC 3 in PTEN ‐ or SPOP ‐mutated prostate cancer

AKT‐mTOR and androgen receptor (AR) signaling pathways are aberrantly activated in prostate cancer due to frequent PTEN deletions or SPOP mutations. A clinical barrier is that targeting one of them often activates the other. Here, we demonstrate that HDAC3 augments AKT phosphorylation in prostate cancer cells and its overexpression correlates with AKT phosphorylation in patient samples. HDAC3 facilitates lysine‐63‐chain polyubiquitination and phosphorylation of AKT, and this effect is mediated by AKT deacetylation at lysine 14 and 20 residues and HDAC3 interaction with the scaffold protein APPL1. Conditional homozygous deletion of Hdac3 suppresses prostate tumorigenesis and progression by concomitant blockade of AKT and AR signaling in the Pten knockout mouse model. Pharmacological inhibition of HDAC3 using a selective HDAC3 inhibitor RGFP966 inhibits growth of both PTEN‐deficient and SPOP‐mutated prostate cancer cells in culture, patient‐derived organoids and xenografts in mice. Our study identifies HDAC3 as a common upstream activator of AKT and AR signaling and reveals that dual inhibition of AKT and AR pathways is achievable by single‐agent targeting of HDAC3 in prostate cancer.

SPOP mutation drives prostate neoplasia without stabilizing oncogenic transcription factor ERG.

Nearly 50% of prostate cancers harbor gene fusions that lead to overexpression of the transcription factor ERG, while a mutually exclusive 10% of prostate cancers harbor recurrent mutations in the gene encoding the E3 ubiquitin ligase SPOP. Recent reports suggest that SPOP acts as a ubiquitin ligase for ERG and propose that ERG stabilization is the oncogenic effector of SPOP mutation. Here, we used human prostate cancer samples and showed that the vast majority of human SPOP-mutant cancers do not express ERG. Comparison of SPOP-mutant and ERG-fusion organoid models showed evidence of divergent, rather than common, transcriptional programs. Furthermore, expression of prostate cancer–associated SPOP mutations in genetically engineered mouse models of SPOP-mutant prostate cancer did not result in the expression of ERG protein in histologically normal prostate glands, high-grade prostatic intraepithelial neoplasia, invasive adenocarcinoma, or prostate organoids. In summary, we found no evidence that ERG is an effector of SPOP mutation in human prostate cancer or mouse models.

Prostate cancer: BET inhibitors - SPOP right there!

Although speckle-type POZ protein (SPOP) is the most frequently mutated gene in primary prostate cancer, its therapeutic implications are incompletely understood. Now, three studies describe mechanisms of resistance to bromodomain and extraterminal (BET) protein inhibitors in SPOP-mutated prostate cancer.

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

Bromodomain and extraterminal domain (BET) protein inhibitors are emerging as promising anti-cancer therapies. The gene encoding the E3 ubiquitin ligase substrate-binding adaptor speckle-type POZ protein (SPOP) is most frequently mutated in 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 common degron motif. In contrast, prostate cancer-associated SPOP mutants impair binding and proteasomal degradation of BET proteins, thus inducing their accumulation in prostate cancer cells and patient specimens. Transcriptome and BRD4 cistrome analyses reveal that SPOP mutation enhances BRD4-dependent expression of GTPase RAC1 and cholesterol biosynthesis genes and AKT-mTORC1 activation. SPOP mutant expression confers BET inhibitor resistance and this effect can be overcome by AKT inhibitors. Thus, SPOP mutations promote AKT-mTORC1 activation and intrinsic BET inhibitor resistance by stabilizing BET proteins, suggesting that SPOP mutation can be an effective biomarker to guide BET inhibitor-oriented therapy of prostate cancer.

Prostate cancer–associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4

The bromodomain and extra-terminal (BET) family of proteins, comprised of four members including BRD2, BRD3, BRD4 and the testis-specific isoform BRDT, largely function as transcriptional co-activators 1–3 and play critical roles in various cellular processes, including cell cycle, apoptosis, migration and invasion 4,5. As such, BET proteins enhance the oncogenic functions of major cancer drivers by either elevating their expression such as c-Myc in leukemia 6,7 or by promoting transcriptional activities of oncogenic factors such as AR and ERG in the prostate cancer setting 8. Pathologically, BET proteins are frequently overexpressed and clinically linked to various types of human cancers 5,9,10, therefore pursued as attractive therapeutic targets for selective inhibition in patients. To this end, a number of bromodomain inhibitors, including JQ1 and I-BET, have been developed 11,12 and shown promising outcomes in early clinical trials. Despite resistance to BET inhibitor has been documented in pre-clinical models 13–15 the molecular mechanisms underlying acquired resistance are largely unknown. Here, we report that Cullin 3SPOP earmarks BET proteins including BRD2, BRD3 and BRD4 for ubiquitination-mediated degradation. Pathologically, prostate cancer-associated SPOP mutants fail to interact with and promote the destruction of BET proteins, leading to their elevated abundance in SPOP-deficient prostate cancer. As a result, prostate cancer cells and prostate cancer patient-derived organoids harboring SPOP mutations are more resistant to BET inhibitor-induced cell growth arrest and apoptosis. Therefore, our results elucidate the tumor suppressor role of SPOP in prostate cancer by negatively controlling BET protein stability, and also provide a molecular mechanism for BET inhibitor resistance in prostate cancer patients bearing SPOP mutations.

Dysregulation of INF2-mediated mitochondrial fission in SPOP-mutated prostate cancer.

Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alternations. SPOP (Speckle-type POZ Protein) was one of the most frequently mutated genes in primary prostate cancer, suggesting SPOP is a potential driver of prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex that generally recruits substrates for ubiquitination and subsequent degradation. ER-localized isoform of the formin protein inverted formin 2 (INF2) mediates actin polymerization at ER-mitochondria intersections and facilitates DRP1 recruitment to mitochondria, which is a critical step in mitochondrial fission. Here, we revealed that SPOP recognizes a Ser/Thr (S/T)-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. These ubiquitination modifications do not lead to INF2 instability, but rather reduces INF2 localization in ER and mitochondrially associated DRP1 puncta formation, therefore abrogates its ability to facilitate mitochondrial fission. INF2 mutant escaping from SPOP-mediated ubiquitination is more potent in prompting mitochondrial fission. Moreover, prostate cancer-associated SPOP mutants increase INF2 localization in ER and promote mitochondrial fission, probably through a dominant-negative effect to inhibit endogenous SPOP. Moreover, INF2 is important for SPOP inactivation-induced prostate cancer cell migration and invasion. These findings reveal novel molecular events underlying the regulation of INF2 function and localization, and provided insights in understanding the relationship between SPOP mutations and dysregulation of mitochondrial dynamics in prostate cancer.

Abstract 1108: SPOP mutation leads to genomic instability in prostate cancer

Abstract Background: Genomic instability is a fundamental feature of human cancer, and DNA repair defects resulting in impaired genome maintenance promote pathogenesis of many types of cancers. In prostate cancer, structural genomic rearrangements, including translocations and copy number aberrations, are a key mechanism driving tumorigenesis. Recently, whole genome sequencing revealed a striking abundance, complexity, and heterogeneity of genomic rearrangements, potentially suggesting distinct mechanisms of instability in different molecular classes of prostate cancer. However, the somatic alterations underlying these phenomena remain largely undefined. Recurrent mutations in SPOP, the substrate-recognition component of an E3-ubiquitin ligase, represent the most common point mutations in primary prostate cancer, occurring in about 10% of tumors. SPOP mutations define a distinct molecular class of prostate cancer; they are mutually exclusive with TMPRSS2-ERG fusions, but harbor distinct patterns of copy number aberrations. Here, we report that SPOP mutant prostate cancers also harbor increased numbers of genomic rearrangements, and functional data suggest that SPOP mutation alters repair of DNA double strand breaks (DSB). Methods: We systematically investigated somatic alterations associated with genomic rearrangements, using a composite data set of 402 clinically localized prostate cancers. Functional analyses in vitro and in vivo were used to define pathways affected, and interrogate DNA repair phenotypes. Results: In human prostate cancers, SPOP mutation is an early event specifically associated with increased intrachromosomal genomic rearrangements. Using a zebrafish model, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation, implicating altered repair of DSB. In vitro data suggest that SPOP participates in repair of DSB, and SPOP mutation impairs homology-directed repair (HDR), instead promoting error-prone non-homologous end joining (NHEJ). Finally, SPOP mutation sensitizes prostate cancer cells to DNA damaging therapeutic agents such as PARP inhibitors. Conclusions: These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that SPOP mutant prostate cancer may be selectively responsive to DNA damaging therapeutics. Citation Format: Gunther Boysen, Christopher E. Barbieri, Davide Prandi, Sung-Suk Chae, Srilakshmi Nataraj, Mirjam Blattner, Clarisse Marotz, Limei Xu, Paola Lecca, Sagar Chhangawala, Pengbo Zou, Andrea Sboner, Francesca Demichelis, Yariv Houvras, Mark A. Rubin. SPOP mutation leads to genomic instability in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1108. doi:10.1158/1538-7445.AM2015-1108