Citations

Abstract Background Atypical teratoid rhabdoid tumor (ATRT) is a deadly central nervous system embryonal tumor caused by loss of SMARCB1, a core subunit of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes. SMARCB1-deficient cancers are defined by loss of cell differentiation-associated enhancers, but how SWI/SNF interacts with other arbiters of cell differentiation (specifically lineage-specific transcription factors [TFs]) remains poorly understood. Methods We leveraged a multi-omics approach, patient-derived ATRT cells, and patient-derived orthotopic xenografts to investigate the interplay of SWI/SNF with lineage-specific TFs in a clinically relevant setting. Results We observe that an activating protein 1 (AP-1)-dependent transcriptional regulatory network is lost in ATRT, and AP-1 and lineage-specific TFs TEAD1 and ZIC2 require SMARCB1 for enhancer binding. SMARCB1-dependent SWI/SNF integrates transcriptional functions of lineage-specific TFs into a core regulatory circuit that depends on the AP-1 subunit c-JUN, whose expression is determined by a SMARCB1-dependent super-enhancer that is lost in ATRT-MYC. In the absence of SMARCB1, lineage-specific TFs are sequestered to promoters, where they maintain core transcriptional programs necessary for cell survival. Targeting residual, promoter-proximal TF activity by a protein degrader of the SWI/SNF ATPase SMARCA4 or small-molecule inhibitors that indirectly inhibit AP-1 and TEAD activity abrogates expression of these networks, reducing cell viability in vitro and prolonging survival in an orthotopic patient-derived xenograft model. Conclusions These results demonstrate SWI/SNF complexes are critical for lineage-specific TF binding and activity at both promoters and enhancers. In the context of ATRT, these findings reveal a previously underappreciated therapeutic vulnerability in targeting residual promoter-proximal TF function in ATRT.

(1) Background: Prostate cancer treatment efficacy is significantly influenced by androgen receptor (AR) signaling pathways. SLC22A3, a membrane transporter, has been linked to SNP rs9364554 risk loci for drug efficacy in prostate cancer. (2) Methods: We examined the location of SNP rs9364554 in the genome and utilized TCGA and other publicly available datasets to analyze the association of this SNP with SLC22A3 transcription levels. We verified onco-mining findings in prostate cancer cell lines using quantitative PCR and Western blots. Additionally, we employed electrophoretic mobility shift assay (EMSA) to detect the binding affinity of transcription factors to this SNP. The ChIP-Seq was used to analyze the enrichment of H3K27ac on the SLC22A3 promoter. (3) Results: In this study, we revealed that SNP rs9364554 resides in the SLC22A3 gene and affects its transcription. The downregulation of SLC22A3 is associated with drug resistance. More importantly, we found that this SNP has different binding affinities with transcription factors, specifically FOXA1 and AR, which significantly affects their regulation of SLC22A3 transcription. (4) Conclusions: Our findings highlight the potential of using this SNP as a biomarker for predicting chemotherapeutic outcomes and uncover possible mechanisms underlying drug resistance in advanced prostate cancers. More importantly, it provides a clinical foundation for targeting FOXA1 to enhance drug efficacy in prostate cancer patients.

Abstract Background: Diffuse midline glioma (DMG) is a highly aggressive pediatric brain cancer with a poor prognosis [1]. Mechanistically, DMG harbors a lysine-to-methionine substitution at position 27 of histone H3.3 (H3.3K27M), which leads to global hypomethylation and a dramatically altered epigenetic landscape [2, 3]. Aim: This study aims to explore the H3.3K27M mutation from a novel perspective by investigating its effects on H3.3 ubiquitination, with the goal of identifying effective, novel therapeutic targets. Methods: Mass spectrometry was used to identify potential E3 ligases for H3.3, while immunoprecipitation to verify this interaction. Cell viability and survival were determined by MTS and clonogenic survival assays, respectively. Nucleosome fractionation was performed to detect the distribution of wild-type (WT) or K27M mutant H3.3 histones. Results: Mass spectrometry identified UBR7, an E3 ligase, as exhibiting a stronger binding affinity with WT H3.3 than its K27M mutant counterpart. Further nucleosome fractionation assay revealed that K27M histones are more likely deposited in nucleosomes than WT H3.3. Further analysis demonstrated that a non-phosphorylation mimic mutation (serine to alanine) at amino acid 28 (S28A) restored the binding affinity of K27M mutant H3.3 with UBR7. Importantly, this mutation disrupted H3.3 phosphorylation and significantly diminished cell survival, whereas the constitutively activated phosphorylation mutation (S to glutamine (E)) on S28 (S28E) mimics K27M mutation in promoting cell proliferation and survival. Conclusion: These findings suggest that S28ph impairs UBR7-mediated K27M ubiquitination and promotes DMG cell growth, suggesting that targeting S28ph is an effective therapeutic strategy. However, the underlying mechanisms by which K27M interplays with S28ph in promoting tumorigenesis require further investigation. Citation Format: Yuqian Yan. Targeting H3.3 serine 28 phosphorylation as a novel therapeutic strategy for diffuse midline glioma [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 1719.

Abstract Retinoblastoma (RB) protein can exert tumor suppressor functions even when it becomes phosphorylated. It is thus essential to understand how phosphorylated RB (p-RB) expression and function are regulated. Here, we demonstrated that RING finger domain protein TRIM28 bound and promoted ubiquitination and degradation of CDK4/6-phosphorylated RB protein. SETDB1, a known TRIM28 binding partner, protected p-RB from degradation through the binding of methylated RB by its Tudor domain independent of its methyltransferase activity. SETDB1 was found to be frequently overexpressed due to gene amplification and positively correlated with p-RB in prostate cancer patient specimens. Inhibition of SETDB1 expression using a gene-specific antisense oligonucleotide (ASO) reduced tumor growth but accelerated RB protein degradation, limiting the therapeutic efficacy. However, coadministration of the CDK4/6 inhibitor palbociclib blocked ASO-induced RB degradation and resulted in a much greater cancer-inhibitory effect than each inhibitor alone both in vitro and in vivo. This study identified CDK4/6-dependent, TRIM28-mediated proteasomal degradation as a mechanism of RB inactivation and reveals SETDB1 as a key inhibitor of this process. Our findings suggest that combined targeting of SETDB1 and CDK4/6 represents a viable approach for the treatment of cancers with SETDB1 gene amplification or overexpression. Significance: The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.

In this study, we discovered 3-aminophthalic acid as a new ligand of cereblon (CRBN) E3 ubiquitin ligase and developed a phthalic acid-based O’PROTAC for ERG destruction, expanding the pool of ligands for development of PROTACs, especially O’PROTACs.

Abstract Although the Sonic hedgehog (SHH) signaling pathway has been implicated in promoting malignant phenotypes of prostate cancer, details on how it is activated and exerts its oncogenic role during prostate cancer development and progression is less clear. Here, we show that GLI3, a key SHH pathway effector, is transcriptionally upregulated during androgen deprivation and posttranslationally stabilized in prostate cancer cells by mutation of speckle-type POZ protein (SPOP). GLI3 is a substrate of SPOP-mediated proteasomal degradation in prostate cancer cells and prostate cancer driver mutations in SPOP abrogate GLI3 degradation. Functionally, GLI3 is necessary and sufficient for the growth and migration of androgen receptor (AR)–positive prostate cancer cells, particularly under androgen-depleted conditions. Importantly, we demonstrate that GLI3 physically interacts and functionally cooperates with AR to enrich an AR-dependent gene expression program leading to castration-resistant growth of xenografted prostate tumors. Finally, we identify an AR/GLI3 coregulated gene signature that is highly correlated with castration-resistant metastatic prostate cancer and predictive of disease recurrence. Together, these findings reveal that hyperactivated GLI3 promotes castration-resistant growth of prostate cancer and provide a rationale for therapeutic targeting of GLI3 in patients with castration-resistant prostate cancer (CRPC). Implications: We describe two clinically relevant mechanisms leading to hyperactivated GLI3 signaling and enhanced AR/GLI3 cross-talk, suggesting that GLI3-specific inhibitors might prove effective to block prostate cancer development or delay CRPC.

AbstractDNA‐binding proteins, including transcription factors (TFs), play essential roles in various cellular processes and pathogenesis of diseases, deeming to be potential therapeutic targets. However, these proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand‐binding pocket. Proteolysis‐targeting chimera (PROTAC) technology has been developed by engineering a bifunctional molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through the proteasome pathway. Here, the development of oligonucleotide‐based PROTAC (O'PROTACs), a class of noncanonical PROTACs in which a TF‐recognizing double‐stranded oligonucleotide is incorporated as a binding moiety of POI is reported. It is demonstrated that O'PROTACs of lymphoid enhancer‐binding factor 1 (LEF1) and ETS‐related gene (ERG), two highly cancer‐related transcription factors, successfully promote degradation of these proteins, impede their transcriptional activity, and inhibit cancer cell growth in vitro and in vivo. The programmable nature of O'PROTACs indicates that this approach is also applicable to destruct other TFs. O'PROTACs not only can serve as a research tool but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.

SPOP mutation disables 53BP1 ubiquitination and exclusion from DNA double-strand break sites and causes genome instability.

Abstract 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.

Abstract The tumor-suppressor protein RB acts as a transcription repressor via interaction of its pocket domain with an LXCXE motif in histone deacetylase (HDAC) proteins such as HDAC1. Here, we demonstrate that HDAC5 deficient for the LXCXE motif interacts with both RB-N (via an FXXXV motif) and RB-C segments, and such interactions are diminished by phosphorylation of RB serine-249/threonine-252 and threonine-821. HDAC5 was frequently downregulated or deleted in human cancers such as prostate cancer. Loss of HDAC5 increased histone H3 lysine 27 acetylation (H3K27-ac) and circumvented RB-mediated repression of cell-cycle–related pro-oncogenic genes. HDAC5 loss also conferred resistance to CDK4/6 inhibitors such as palbociclib in prostate and breast cancer cells in vitro and prostate tumors in vivo, but this effect was overcome by the BET-CBP/p300 dual inhibitor NEO2734. Our findings reveal an unknown role of HDAC5 in RB-mediated histone deacetylation and gene repression and define a new mechanism modulating CDK4/6 inhibitor therapeutic sensitivity in cancer cells. Significance: This study defines a previously uncharacterized role of HDAC5 in tumor suppression and provides a viable strategy to overcome CDK4/6 inhibitor resistance in HDAC5-deficent cancer.