Speckle-type POZ Protein Research Articles

N6-methyladenosine modification of SPOP relieves ferroptosis and diabetic cardiomyopathy by enhancing ubiquitination of VDAC3.

Understanding the pathogenesis of diabetic cardiomyopathy (DCM), a common microvascular complication affecting the heart, is crucial for identifying new therapeutic targets and intervention strategies for DCM. Our study revealed a significant downregulation in Speckle-type POZ protein (SPOP) expression in DCM, while the overexpression of SPOP improved DCM-induced myocardial dysfunction, injury, fibrosis, hypertrophy, and ferroptosis. Mechanistically, SPOP facilitated the degradation of voltage-dependent anion channel 3 (VDAC3) by enhancing its ubiquitination. M6A demethylase AlkB homolog 5 (ALKBH5) reduced the mRNA stability of SPOP by decreasing m6A modification in its 3'UTR. The m6A reader insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced the stability of SPOP mRNA through recognition of m6A-modified SPOP 3'UTR. Furthermore, ALKBH5 promoted ferroptosis by inhibiting SPOP-induced VDAC3 degradation, while IGF2BP2 inhibited ferroptosis via activation of SPOP-induced VDAC3 degradation in high glucose-treated neonatal mouse ventricular cardiomyocytes (NMVCs). Overall, our study has unveiled a novel role of SPOP in the pathogenesis of ferroptosis and DCM, thereby significantly advancing our understanding of the involvement of ferroptosis during the progression of DCM. Moreover, this discovery offers promising potential therapeutic interventions targeting DCM.

SPOP expression is associated with tumor-infiltrating lymphocytes in pancreatic cancer.

Speckle Type POZ Protein (SPOP), despite its tumor type-dependent role in tumorigenesis, primarily as a tumor suppressor gene is associated with a variety of different cancers. However, its function in pancreatic cancer remains uncertain. SPOP expression and the association between its expression and patient prognosis and immune function were evaluated using The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), The Tumor Immune Estimation Resource 2.0 (TIMER2.0) database, cBioportal, and various bioinformatic databases. Enrichment analysis of SPOP and the association between SPOP expression with clinical stage and grade were analyzed using the R software package. Then immunohistochemistry (IHC) was used to estimate the correlation between SPOP and tumor-infiltrating lymphocytes (TILs) in patients with pancreatic cancer. As part of our study, we assessed that SPOP was anomalously expressed in kinds of cancers, associated with clinical stage and outcomes. Meanwhile, SPOP also played a crucial role in the tumor microenvironment (TME). The expression level of SPOP was significantly correlated to tumor-infiltrating immune cells (TICs) in pancreatic cancer. Our study uncovered the potential corrections in SPOP with TICs, suggesting that SPOP may act as a biomarker for immunotherapy in pancreatic cancer.

Targeting Nuclear Receptor Coactivator SRC-1 Prevents Colorectal Cancer Immune Escape by Reducing Transcription and Protein Stability of PD-L1.

Programmed death-ligand 1 (PD-L1) is overexpressed in multiple cancers and critical for their immune escape. It has previously shown that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability, yet its role in CRC immune escape is unclear. Here, we demonstrate that SRC-1 is positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibits PD-L1 expression in CRC cells and retards murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration of CD8+ T cells. Genetic ablation of SRC-1 in mice also decreases PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, these findings highlight a crucial role of SRC-1 in regulating PD-L1 expression and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment.

ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance.

Accumulating evidence demonstrates that the activity regulation of ELK3, a member of the E26 transformation-specific oncogene family, is critical to regulating cell proliferation, migration, and survival in human cancers. However, the molecular mechanisms of how ELK3 induces chemoresistance in prostate cancer (PCa) have not been elucidated. In this study, we found that SPOP and ELK3 are an interacting partner. The interaction between SPOP and ELK3 resulted in increased ELK3 ubiquitination and destruction, assisted by checkpoint kinase-mediated ELK3 phosphorylation. Notably, the modulation of SPOP-mediated ELK3 protein stability affected the c-Fos-induced cell proliferation and invasion of PCa cells. The clinical involvement of the SPOP-ELK3 axis in PCa development was confirmed by an immunohistochemical assay on 123 PCa tissues, with an inverse correlation between increased ELK3 and decreased SPOP being present in ~80% of the specimens. This observation was supported by immunohistochemistry analysis using a SPOP-mutant PCa specimen. Finally, docetaxel treatment induced cell death by activating checkpoint kinase- and SPOP-mediated ELK3 degradation, while SPOP-depleted or SPOP-mutated PCa cells showed cell death resistance. Notably, this observation was correlated with the protein levels of ELK3. Taken together, our study reveals the precise mechanism of SPOP-mediated degradation of ELK3 and provides evidence that SPOP mutations contribute to docetaxel resistance in PCa.

SPOP attenuates the proliferation, invasion, and migration of choriocarcinoma JAR cells by promoting KIF23 degradation

Abstract Objectives Choriocarcinoma is a highly malignant and aggressive trophoblastic tumor. In our previous study, we discovered that the speckle-type POZ protein (SPOP), which recognizes substrates of E3 ubiquitin ligase, plays a crucial role in trophoblast-derived choriocarcinoma cell lines. Therefore, we investigated the correlation between SPOP and the substrate kinesin-like protein KIF23, as well as the role of KIF23 in choriocarcinoma cells. Methods We constructed JAR cells overexpressing SPOP using lentiviral vectors and subsequently screened the related proteins through ubiquitination-modified quantitative proteomic analysis. The relationship between KIF23 and SPOP was determined using western blotting, and CCK-8, plate cloning, flow cytometry, and Transwell assays were used to investigate the effects of KIF23 and SPOP/KIF23. Results We identified the KIF23 protein and observed that SPOP promoted its degradation. The abundance of KIF23 increased after the addition of the protease inhibitor MG132. KIF23 was highly expressed in choriocarcinoma cells. Compared with JAR cells transfected with NC–small-interfering RNA (siRNA), the proliferation, invasion, migration, and percentage of G0/G1 cells in the KIF23-siRNA group were significantly lower, and the activation of the Akt/GSK3β signaling pathway was markedly attenuated. Additionally, the sh-SPOP+KIF23-siRNA group exhibited significantly inhibited JAR cell proliferation, invasion, and migration, along with clearly attenuated activation of the Akt/GSK3β signaling pathway. Conclusions SPOP attenuates the proliferation, invasion, and migration of choriocarcinoma JAR cells by promoting KIF23 degradation.

Abstract 1688: Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability

Abstract Programmed death-ligand 1 (PD-L1), one of the immunosuppressive molecules, overexpresses in multiple cancers and is critical for their immune escape. We previously showed that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability; however, the role of SRC-1 in CRC immune escape is unclear. Here, we demonstrated that SRC-1 was positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibited PD-L1 expression in both human and murine CRC cells and retarded murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration and antitumor activity of effector CD8+ T cells. Genetic ablation of SRC-1 in mice also decreased PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, our findings highlight a crucial role of SRC-1 in facilitating CRC immune escape and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment. Citation Format: Yilin Hong, Qiang Chen, Zinan Wang, Yong Zhang, Bei Li, Xu Kong, Pingli Mo, Nengming Xiao, Jianming Xu, Yunbin Ye, Chundong Yu. Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1688.

CSN6-SPOP-HMGCS1 Axis Promotes Hepatocellular Carcinoma Progression via YAP1 Activation.

Cholesterol metabolism has important roles in maintaining membrane integrity and countering the development of diseases such as obesity and cancers. Cancer cells sustain cholesterol biogenesis for their proliferation and microenvironment reprograming even when sterols are abundant. However, efficacy of targeting cholesterol metabolism for cancer treatment is always compromised. Here it is shown that CSN6 is elevated in HCC and is a positive regulator of hydroxymethylglutaryl-CoA synthase 1 (HMGCS1) of mevalonate (MVA) pathway to promote tumorigenesis. Mechanistically, CSN6 antagonizes speckle-type POZ protein (SPOP) ubiquitin ligase to stabilize HMGCS1, which in turn activates YAP1 to promote tumor growth. In orthotopic liver cancer models, targeting CSN6 and HMGCS1 hinders tumor growth in both normal and high fat diet. Significantly, HMGCS1 depletion improves YAP inhibitor efficacy in patient derived xenograft models. The results identify a CSN6-HMGCS1-YAP1 axis mediating tumor outgrowth in HCC and propose a therapeutic strategy of targeting non-alcoholic fatty liver diseases- associated HCC.

Synthesis and Biological Evaluation of β-Lactam Derivatives Targeting Speckle-Type POZ Protein (SPOP).

Speckle-type POZ protein (SPOP) acts as a cullin3-RING ubiquitin ligase adaptor, which facilitates the recognition and ubiquitination of substrate proteins. Previous research suggests that targeting SPOP holds promise in the treatment of clear cell renal cell carcinoma (ccRCC). On the basis of the reported SPOP inhibitor 230D7, a series of β-lactam derivatives were synthesized in this study. The biological activity assessment of these compounds revealed E1 as the most potent inhibitor, which can disrupt the SPOP-substrate interactions in vitro and suppress the colony formation of ccRCC cells. Taken together, this study provided compound E1 as a potent inhibitor against ccRCC and offered insight into the development of the β-lactam SPOP inhibitor.

The Mutational and Transcriptional Landscapes of Speckle-Type POZ Protein (SPOP) and Androgen Receptor (AR) in a Single-Center pT3 Prostatectomy Cohort.

Prostate cancer (PCa) is a heterogeneous disease both clinically and genetically. According to The Cancer Genome Atlas (TCGA), the speckle‑type POZ protein (SPOP) mutant form is one of the significant core subtypes of PCa. However, the prognostic value of SPOP variations remains unknown. As a critical PCa driver and an SPOP-targeted protein, androgen receptor (AR) also plays a role in PCa initiation and progression. Thus, we aimed to analyze the mutational status of SPOP and AR with their transcriptional levels in a pathological stage 3 (pT3) prostatectomy cohort consisting of 89 Turkish PCa patients. Targeted sequence analysis and RT-qPCR were performed for SPOP and AR in the benign and malign prostate tissue samples. Our results introduced the two novel pathogenic SPOP variations, C203Y and S236R, in the BTB/POZ domain and a novel pathogenic variant in the ligand-binding domain of AR, R789W. Their predicted pathogenicities and effects on protein features were evaluated by web-based in silico analysis. The overall frequency of SPOP and AR variations for pT3 patients in our population was 3.4% (3/89) and 4.5% (4/89), respectively. The mutational results represented a possible subgroup characterized by carrying the novel variants in SPOP and AR in pT3 PCa patients. In addition to the significant clinicopathological parameters, the mutational results provide a better understanding of the molecular structure of pathologically advanced PCa in the SPOP and AR aspects.

Addressing the Binding Mechanism of the Meprin and TRAF-C Homology Domain of the Speckle-Type POZ Protein Using Protein Engineering.

Protein-protein interactions play crucial roles in a wide range of biological processes, including metabolic pathways, cell cycle progression, signal transduction, and the proteasomal system. For PPIs to fulfill their biological functions, they require the specific recognition of a multitude of interacting partners. In many cases, however, protein-protein interaction domains are capable of binding different partners in the intracellular environment, but they require precise regulation of the binding events in order to exert their function properly and avoid misregulation of important molecular pathways. In this work, we focused on the MATH domain of the E3 Ligase adaptor protein SPOP in order to decipher the molecular features underlying its interaction with two different peptides that mimic its physiological partners: Puc and MacroH2A. By employing stopped-flow kinetic binding experiments, together with extensive site-directed mutagenesis, we addressed the roles of specific residues, some of which, although far from the binding site, govern these transient interactions. Our findings are compatible with a scenario in which the binding of the MATH domain with its substrate is characterized by a fine energetic network that regulates its interactions with different ligands. Results are briefly discussed in the context of previously existing work regarding the MATH domain.

Co-occurring BRCA2/SPOP Mutations Predict Exceptional Poly (ADP-ribose) Polymerase Inhibitor Sensitivity in Metastatic Castration-Resistant Prostate Cancer

Background and objectiveBRCA2 mutations in metastatic castration-resistant prostate cancer (mCRPC) confer sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors. However, additional factors predicting PARP inhibitor efficacy in mCRPC are needed. Preclinical studies support a relationship between speckle-type POZ protein (SPOP) inactivation and PARP inhibitor sensitivity. We hypothesized that SPOP mutations may predict enhanced PARP inhibitor response in BRCA2-altered mCRPC. MethodsWe conducted a multicenter retrospective study involving 13 sites. We identified 131 patients with BRCA2-altered mCRPC treated with PARP inhibitors, 14 of which also carried concurrent SPOP mutations. The primary efficacy endpoint was prostate-specific antigen (PSA) response rate (≥50% PSA decline). The secondary endpoints were biochemical progression-free survival (PSA-PFS), clinical/radiographic progression-free survival (PFS), and overall survival (OS). These were compared by multivariable Cox proportional hazard models adjusting for age, tumor stage, baseline PSA level, Gleason sum, prior therapies, BRCA2 alteration types, and co-occurring mutations. Key findings and limitationsBaseline characteristics were similar between groups. PSA responses were observed in 60% (70/117) of patients with BRCA2mut/SPOPwt disease and in 86% (12/14) of patients with BRCA2mut/SPOPmut disease (p = 0.06). The median time on PARP inhibitor treatment was 24.0 mo (95% confidence interval [CI] 19.2 mo to not reached) in this group versus 8.0 mo (95% CI 6.1–10.9 mo) in patients with BRCA2 mutation alone (p = 0.05). In an unadjusted analysis, patients with BRCA2mut/SPOPmut disease experienced longer PSA-PFS (hazard ratio [HR] 0.33 [95% CI 0.15–0.72], p = 0.005) and clinical/radiographic PFS (HR 0.4 [95% CI 0.18–0.86], p = 0.02), and numerically longer OS (HR 0.4 [95% CI 0.15–1.12], p = 0.08). In a multivariable analysis including histology, Gleason sum, prior taxane, prior androgen receptor pathway inhibitor, stage, PSA, BRCA2 alteration characteristics, and other co-mutations, patients with BRCA2mut/SPOPmut disease experienced longer PSA-PFS (HR 0.16 [95% CI 0.05–0.47], adjusted p = 0.001), clinical/radiographic PFS (HR 0.28 [95% CI 0.1–0.81], adjusted p = 0.019), and OS (HR 0.19 [95% CI 0.05–0.69], adjusted p = 0.012). In a separate cohort of patients not treated with a PARP inhibitor, there was no difference in OS between patients with BRCA2mut/SPOPmut versus BRCA2mut/SPOPwt disease (HR 0.97 [95% CI 0.40–2.4], p = 0.94). In a genomic signature analysis, Catalog of Somatic Mutations in Cancer (COSMIC) SBS3 scores predictive of homologous recombination repair (HRR) defects were higher for BRCA2mut/SPOPmut than for BRCA2mut/SPOPwt disease (p = 0.04). This was a retrospective study, and additional prospective validation cohorts are needed. Conclusions and clinical implicationsIn this retrospective analysis, PARP inhibitors appeared more effective in patients with BRCA2mut/SPOPmut than in patients with BRCA2mut/SPOPwt mCRPC. This may be related to an increase in HRR defects in coaltered disease. Patient summaryIn this study, we demonstrate that co-alteration of both BRCA2 and SPOP predicts superior clinical outcomes to treatment with poly (ADP-ribose) polymerase (PARP) inhibitors than BRCA2 alteration without SPOP mutation.

Abstract A027: Exploring autoantibody response to proteins encoded by prostate cancer driver genes

Abstract Prostate cancer (PCa) is a complex disease driven by genomic alterations, and understanding the immune response to these alterations can provide valuable insights into the pathogenesis. This study aimed to explore the immunogenicity of proteins encoded by PCa driver genes and to further identify corresponding autoantibodies as potential biomarkers. Through a comprehensive screening process involving bioinformatics analysis based on published studies, a set of PCa driver genes was identified for further investigation. Three strategies were employed to identify candidate driver genes: (1) utilizing the IntOGen database (Martínez-Jiménez et.al, Nature Reviews Cancer 2020), nine cohorts comprising 1503 primary PCa patient samples were analyzed, resulting in the identification of 63 PCa driver genes, with the top 20 genes exhibiting high mutation rates and frequencies in these cohorts; (2) employing the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, 16 driver genes that were enriched in prostate cancer pathways were identified from the initial 63 genes; (3) referring to a published article (Dietlein et.al, Nature Genetics 2020), the top 17 driver genes specific to prostate cancer across 28 tumor types were selected. A final set of 15 PCa driver genes, which appeared in at least two of the screening strategies, were included in subsequent analyses. To assess the presence of autoantibodies targeting proteins encoded by these 15 PCa driver genes, an enzyme-linked immunosorbent assay (ELISA) was performed. Autoantibodies to proteins encoded by the selected PCa driver genes were evaluated using serum samples from PCa patients and normal controls. The results revealed that seven out of the 15 autoantibodies showed significantly elevated levels in the sera of PCa patients compared to normal controls. Notably, the autoantibody targeting SPOP (speckle-type POZ protein) exhibited the highest frequency of 42.4%, followed by the autoantibody to PTEN (phosphatase and tensin homolog) at 37.4% in the PCa group. These findings suggest that proteins encoded by PCa driver genes may possess immunogenic properties, leading to the production of corresponding autoantibodies. The identification of specific autoantibodies associated with PCa driver genes could offer novel opportunities for the discovery of autoantibody-based biomarkers in prostate cancer. Further validation and characterization of these autoantibodies are warranted to evaluate their diagnostic or prognostic potential and elucidate their functional roles in PCa pathogenesis. Citation Format: Jian-Ying Zhang, Cuipeng Qiu, Xiao Wang, Bofei Wang. Exploring autoantibody response to proteins encoded by prostate cancer driver genes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A027.

SPOP inhibits HBV transcription and replication by ubiquitination and degradation of HNF1α.

Hepatitis B virus (HBV) infection remains a significant public health burden worldwide. The persistence of covalently closed circular DNA (cccDNA) within the nucleus of infected hepatocytes is responsible for the failure of antiviral treatments. The ubiquitin proteasome system (UPS) has emerged as a promising antiviral target, as it can regulate HBV replication by promoting critical protein degradation in steps of viral life cycle. Speckle-type POZ protein (SPOP) is a critical adaptor for Cul3-RBX1 E3 ubiquitin ligase complex, but the effect of SPOP on HBV replication is less known. Here, we identified SPOP as a novel host antiviral factor against HBV infection. SPOP overexpression significantly inhibited the transcriptional activity of HBV cccDNA without affecting cccDNA level in HBV-infected HepG2-NTCP and primary human hepatocyte cells. Mechanism studies showed that SPOP interacted with hepatocyte nuclear factor 1α (HNF1α), and induced HNF1α degradation through host UPS pathway. Moreover, the antiviral role of SPOP was also confirmed in vivo. Together, our findings reveal that SPOP is a novel host factor which inhibits HBV transcription and replication by ubiquitination and degradation of HNF1α, providing a potential therapeutic strategy for the treatment of HBV infection.

SPOP promotes CREB5 ubiquitination to inhibit MET signaling in liver cancer

Liver cancer is ranked as the sixth most prevalent from of malignancy globally and stands as the third primary contributor to cancer-related mortality. Metastasis is the main reason for liver cancer treatment failure and patient deaths. Speckle-type POZ protein (SPOP) serves as a crucial substrate junction protein within the cullin-RING E3 ligase complex, acting as a significant tumor suppressor in liver cancer. Nevertheless, the precise molecular mechanism underlying the role of SPOP in liver cancer metastasis remain elusive. In the current study, we identified cAMP response element binding 5 (CREB5) as a novel SPOP substrate in liver cancer. SPOP facilitates non-degradative K63-polyubiquitination of CREB5 on K432 site, consequently hindering its capacity to activate receptor tyrosine kinase MET. Moreover, liver cancer-associated SPOP mutant S119N disrupts the SPOP-CREB5 interactions and impairs the ubiquitination of CREB5.This disruption ultimately leads to the activation of the MET signaling pathway and enhances metastatic properties of hepatoma cells both in vitro and in vivo. In conclusion, our findings highlight the functional significance of the SPOP-CREB5-MET axis in liver cancer metastasis.

Reduction of oligomer size modulates the competition between cluster formation and phase separation of the tumor suppressor SPOP

Phase separation compartmentalizes many cellular pathways. Given that the same interactions that drive phase separation mediate the formation of soluble complexes below the saturation concentration, the contribution of condensates vs complexes to function is sometimes unclear. Here, we characterized several new cancer-associated mutations of the tumor suppressor Speckle-type POZ protein (SPOP), a substrate recognition subunit of the Cullin3-RING ubiquitin ligase (CRL3). These pointed to a strategy for generating separation-of-function mutations. SPOP self-associates into linear oligomers and interacts with multivalent substrates, and this mediates the formation of condensates. These condensates bear the hallmarks of enzymatic ubiquitination activity. We characterized the effect of mutations in the dimerization domains of SPOP on its linear oligomerization, binding to its substrate DAXX, and phase separation with DAXX. We showed that the mutations reduce SPOP oligomerization and shift the size distribution of SPOP oligomers to smaller sizes. The mutations therefore reduce the binding affinity to DAXX but unexpectedly enhance the poly-ubiquitination activity of SPOP towards DAXX. Enhanced activity may be explained by enhanced phase separation of DAXX with the SPOP mutants. Our results provide a comparative assessment of the functional role of complexes versus condensates and support a model in which phase separation is an important factor in SPOP function. Our findings also suggest that tuning of linear SPOP self-association could be used by the cell to modulate activity and provide insights into the mechanisms underlying hypermorphic SPOP mutations. The characteristics of cancer-associated SPOP mutations suggest a route for designing separation-of-function mutations in other phase-separating systems.

The Spop/BRD4/c-Myc Axis Modulates IMiDs Sensitivity in Multiple Myeloma

The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has led to improved patient outcomes in multiple myeloma (MM); however, MM patients inevitably experience relapse and drug resistance. The expression level of CRBN, CRBN-binding proteins, and CRL4 CRBN ubiquitin ligase is considered to be associated with IMiDs resistance. Clinical studies have shown that MM patients can exhibit IMiDs resistance even without CRBN abnormalities, suggesting the molecular mechanisms of intrinsic resistance to IMiDs are not fully understood. To delineate the molecular mechanisms underlying IMiDs resistance, we performed genome-wide knockout (KO) screening in IMiDs-sensitive MM cells. For in vitro work, we used the MM cell lines MM1S, and H929. We used sgRNA to KO SPOP and BRD4 in MM cells. After performing a CRISPR KO screen in MM cells, we discovered that in addition to CRBN and its associated genes, sgRNAs targeting SPOP (speckle-type POZ protein, a speckled zinc finger protein) were highly enriched after treatment with Poma. Importantly, SPOP KO MM cell lines acquired significant resistance to Pom and Len treatment. To examine whether SPOP KO-induced IMiDs resistance was CRBN-pathway dependent, we assessed CRBN and its downstream interacting protein levels. SPOP KO showed no effect on CRBN expression; moreover, IMiDs can still trigger IKZF1 and IKZF3 degradation, associated with the downregulation of IRF4, suggesting that SPOP mediates sensitivity to IMiDs in a mechanism independent of CRBN-IKZF1/3 axis.Compared to plasma cells from normal donors, we found that the expression levels of SPOP were significantly decreased in MGUS patients, smoldering myeloma patients, newly diagnosed MM patients, and refractory relapsed MM patients (p-values: 2.4e-2, 2.0e-4, 9.9e-3, 3.17e-6, respectively). SPOP is the adaptor protein of the Cullin3-RING ubiquitin ligase complex. Therefore, the function of SPOP is determined by the characteristics of the substrates it recruits. We performed immunoprecipitation with overexpressed SPOP in MM cells, followed by mass spectrometry analysis, and identified BRD4 as a candidate protein binding to SPOP in MM, which was confirmed by Co-IP. KO of SPOP led to an upregulation of BRD4 protein levels in MM cells. As a downstream regulator of BRD4, the oncogene c-Myc showed a significant increase in protein expression in IMiDs-resistant MM patients. Furthermore, the KO of SPOP notably enhanced c-Myc expression in MM cells, while the KO of BRD4 in SPOP KO MM cells decreaed c-Myc expression and restored MM cell sensitivity to IMiD treatment. These findings suggest that SPOP modulates c-Myc expression through BRD4 in a CRBN-IKZF1/3 independent manner, thereby mediating MM cell sensitivity to IMiDs. BRD4 plays a significant role in the development and progression of various tumors. The BRD4 inhibitor JQ1 has been shown to exhibit anti-tumor effects in multiple types of cancer. We found that combination treatment with JQ-1 and IMiDs reversed IMiDs resistance of SPOP KO MM cell lines and primary MM cells from relapsed patients. Our data show that SPOP is a CRBN-independent modulator of IMiDs sensitivity by regulating the BRD4/c-Myc axis and provides the preclinical rationale for combining IMiDs with BRD4 inhibitors to overcome IMiDs resistance and improve patient outcomes.

Ubiquitin E3 ligase SPOP is a host negative regulator of enterovirus 71-encoded 2A protease.

EV71 poses a significant health threat to children aged 5 and below. The process of EV71 infection and replication is predominantly influenced by ubiquitination modifications. Our previous findings indicate that EV71 prompts the activation of host deubiquitinating enzymes, thereby impeding the host interferon signaling pathway as a means of evading the immune response. Nevertheless, the precise mechanisms by which the host employs ubiquitination modifications to hinder EV71 infection remain unclear. The present study demonstrated that the nonstructural protein 2Apro, which is encoded by EV71, exhibits ubiquitination and degradation mediated by the host E3 ubiquitin ligase SPOP. In addition, it is the first report, to our knowledge, that SPOP is involved in the host antiviral response.

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.

SPOP Mutations Target STING1 Signaling in Prostate Cancer and Create Therapeutic Vulnerabilities to PARP Inhibitor-Induced Growth Suppression.

Speckle-type POZ protein (SPOP) is important in DNA damage response (DDR) and maintenance of genomic stability. Somatic heterozygous missense mutations in the SPOP substrate-binding cleft are found in up to 15% of prostate cancers. While mutations in SPOP predict for benefit from androgen receptor signaling inhibition (ARSi) therapy, outcomes for patients with SPOP-mutant (SPOPmut) prostate cancer are heterogeneous and targeted treatments for SPOPmut castrate-resistant prostate cancer (CRPC) are lacking. Using in silico genomic and transcriptomic tumor data, proteomics analysis, and genetically modified cell line models, we demonstrate mechanistic links between SPOP mutations, STING signaling alterations, and PARP inhibitor vulnerabilities. We demonstrate that SPOP mutations are associated with upregulation of a 29-gene noncanonical (NC) STING (NC-STING) signature in a subset of SPOPmut, treatment-refractory CRPC patients. We show in preclinical CRPC models that SPOP targets and destabilizes STING1 protein, and prostate cancer-associated SPOP mutations result in upregulated NC-STING-NF-κB signaling and macrophage- and tumor microenvironment (TME)-facilitated reprogramming, leading to tumor cell growth. Importantly, we provide in vitro and in vivo mechanism-based evidence that PARP inhibitor (PARPi) treatment results in a shift from immunosuppressive NC-STING-NF-κB signaling to antitumor, canonical cGAS-STING-IFNβ signaling in SPOPmut CRPC and results in enhanced tumor growth inhibition. We provide evidence that SPOP is critical in regulating immunosuppressive versus antitumor activity downstream of DNA damage-induced STING1 activation in prostate cancer. PARPi treatment of SPOPmut CRPC alters this NC-STING signaling toward canonical, antitumor cGAS-STING-IFNβ signaling, highlighting a novel biomarker-informed treatment strategy for prostate cancer.

Identification of novel and potent dual-targeting HDAC1/SPOP inhibitors using structure-based virtual screening, molecular dynamics simulation and evaluation of in vitro and in vivo antitumor activity.

Cancer is one of the important factors threatening human health. Hence, it is essential to create novel potent drugs to treat it. Due to the strong correlation among histone deacetylase1 (HDAC1), speckle-type POZ protein (SPOP) and cancers, dual inhibition of HDAC1 and SPOP may be a promising strategy for cancer treatment. In this study, we successfully identified four potential dual-targeting HDAC1/SPOP candidate compounds with structure-based virtual screening. In vitro inhibition experiments confirmed that the four compounds had dual inhibitory effects on HDAC1 and SPOP. Among them, compound HS-2 had a stronger inhibitory effect on HDAC1 and SPOP than the positive controls. Further molecular dynamics simulations indicated that HS-2 could stably bind to HDAC1 and SPOP. In addition, MTT assay indicated that HS-2 inhibited the growth of tumor cells in the micromolar range. In vivo evaluation showed that HS-2 could obviously inhibit the growth of tumor in nude mice without obvious toxicity. These findings suggest that HS-2 is a novel and potent dual-targeting HDAC1/SPOP inhibitor for cancer treatment.