In the original publication [...].

Chromatin immunoprecipitation (ChIP) has been a cornerstone for epigenetic analyses over the last decades, but even coupled to sequencing approaches (ChIP-seq), it is ultimately limited to one protein at a time. In a complementary effort, we here combine ChIP with label-free quantitative mass spectrometry (qChIP-MS) to interrogate local chromatin compositions. We demonstrate the versatility of our approach at telomeres, with transcription factors, in tissue and by dCas9-mediated locus-specific enrichment.

INTRODUCTION:Amplification of chromosome 12q13-15 spanning MDM2 and CDK4 genes serves as a molecular diagnostic hallmark of dedifferentiated liposarcoma (DDLPS), an aggressive soft-tissue sarcoma. Epigenetic activation of master transcription factors (RUNX proteins, FOSL2, and MYC) establishes a self-reinforcing oncogenic transcriptional circuitry in DDLPS. Nevertheless, the collaborative interplay between genomic alterations and epigenetic dysregulation in defining DDLPS cell identity remains elusive. OBJECTIVES:This work aimed to elucidate the primary genetic drivers and mechanistic basis of DDLPS-specific core transcriptional regulatory circuitry. METHODS:We performed integrative chromatin profiling analysis of DDLPS clinical specimens and cell lines to map cis-regulatory landscapes. Cistromes of MDM2, JUN, and E2F1 were delineated through chromatin immunoprecipitation sequencing in two DDLPS models. Essential driver functions and transcriptional regulatory effects of key regulators were assessed via various genetic manipulation approaches. Synergistic interactions between BET-targeting agents and MDM2/p53 or CDK4 inhibitors were quantified by cell viability assays. In vivo xenograft assays evaluated the oncogenic potential of key regulators and the therapeutic efficacy of novel strategies. RESULTS:Co-amplification of MDM2, CDK4, and JUN during sarcomagenesis converges with BET protein-dependent chromatin remodeling to fuel feed-forward transcriptional circuits among master transcription factors. Mechanistically, excessively expressed MDM2 stabilizes the core regulatory circuitry by forming chromatin-bound complexes with JUN/FOSL2 at cis-regulatory elements, especially super-enhancers across DDLPS genome. Concurrently, CDK4 maintains expression of E2F1 which further fosters transcriptional output of master transcription factors in DDLPS cells. Leveraging DDLPS-selective overexpression of MDM2 and its E3 ligase activity, targeted degradation of BET proteins by MDM2-recruiting proteolysis targeting chimera selectively disrupted the core regulatory circuitry, suppressing DDLPS growth and exhibiting strong synergy with CDK4 inhibitor. CONCLUSION:DDLPS-associated genomic lesions collaborate with BET-dependent chromatin regulation to establish disease-sustaining transcriptional circuitry. Our findings also provide a mechanistic rationale for harnessing MDM2's E3 ligase activity to therapeutically degrade oncoproteins in MDM2-amplified malignancies.

Dedifferentiated liposarcoma (DDLPS) is a rare and aggressive subtype of liposarcoma, driven by a core transcriptional regulatory circuitry (CRC) that sustains tumor proliferation. This malignancy poses considerable clinical challenges, marked by high postoperative recurrence and metastatic potential, alongside a lack of effective targeted therapies. In this study, we establish that KPT-330 (Selinexor), a selective inhibitor of exportin 1 (XPO1), effectively compromises DDLPS cell viability by perturbing CRC homeostasis. Mechanistically, we demonstrate that KPT-330 attenuates the cellular translation machinery in a biphasic manner: initially, it disrupts translation initiation by suppressing eukaryotic translation initiation factor 4E phosphorylation and eukaryotic translation initiation factor 4 F complex assembly; subsequently, it impedes translation elongation by inhibiting the nuclear export of ribosomal large subunit proteins. Furthermore, we identify a synergistic antitumor effect between KPT-330 and translation inhibitors, including everolimus and homoharringtonine. Notably, the disruptive impact of KPT-330 on CRC homeostasis extends to other cancer cell lineages, underscoring its broad mechanistic relevance. Collectively, our findings elucidate a novel mechanism through which KPT-330 destabilizes CRC via translational dysregulation and highlight its potential therapeutic utility in combination regimens for DDLPS.

Despite considerable pathological diversity, pediatric sarcomas lack molecularly targeted treatments, demanding deeper pathobiological insights and innovative therapeutic strategies. Here, we demonstrate that overexpressed MDM2 functions as an important pathogenic driver in these malignancies, rewiring oncogenic programs through both p53-independent chromatin occupancy to regulate active transcription and conventional proteasome-mediated p53 degradation leading to pathway suppression. To leverage this dependency for targeted eradication of pediatric sarcomas with MDM2 overexpression, we develop MDM2-recruiting proteolysis-targeting chimeras that selectively degrade the CDK9/Cyclin T complex (P-TEFb). Among the lead compounds, dCDK9-010 demonstrates superior activity compared to its parental CDK9 inhibitor or MDM2 antagonist either alone or in combination, by coordinatedly disrupting the MDM2-p53 axis and super-enhancer-driven transcription. Remarkably, the transcriptional effects of P-TEFb degradation by dCDK9-010 are phenocopied by MDM2-mediated BET degradation, resulting in potent anti-sarcoma efficacy alongside a favorable therapeutic index and minimal toxicity in nonmalignant cells. Moreover, these MDM2-recruiting transcriptional/epigenetic machinery degraders (termed MDM2-TEMADs) consistently impair the homologous recombination repair pathway and confer synthetic lethality with PARP inhibitors. Together, this work elucidates MDM2's central role in pediatric sarcoma pathogenesis and presents dCDK9-010 as a first-in-class, MDM2-recruiting P-TEFb degrader and an exemplary MDM2-TEMAD that enables precise targeting of MDM2-dependent oncogenic transcriptional addiction.

Cancer is characterized by abnormal cell proliferation. Cyclins and cyclin-dependent kinases (CDKs) have been recognized as essential regulators of the intricate cell cycle, orchestrating DNA replication and transcription, RNA splicing, and protein synthesis. Dysregulation of the CDK pathway is prevalent in the development and progression of human cancers, rendering cyclins and CDKs attractive therapeutic targets. Several CDK4/6 inhibitors have demonstrated promising anti-cancer efficacy and have been successfully translated into clinical use, fueling the development of CDK-targeted therapies. With this enthusiasm for finding novel CDK-targeting anti-cancer agents, there have also been exciting advances in the field of targeted protein degradation through innovative strategies, such as using proteolysis-targeting chimera, heat shock protein 90 (HSP90)-mediated targeting chimera, hydrophobic tag-based protein degradation, and molecular glue. With a focus on the translational potential of cyclin- and CDK-targeting strategies in cancer, this review presents the fundamental roles of cyclins and CDKs in cancer. Furthermore, it summarizes current strategies for the proteasome-dependent targeted degradation of cyclins and CDKs, detailing the underlying mechanisms of action for each approach. A comprehensive overview of the structure and activity of existing CDK degraders is also provided. By examining the structure–activity relationships, target profiles, and biological effects of reported cyclin/CDK degraders, this review provides a valuable reference for both CDK pathway-targeted biomedical research and cancer therapeutics.

Ferroptosis is a unique modality of regulated cell death induced by excessive lipid peroxidation, playing a crucial role in tumor suppression and providing potential therapeutic strategy for cancer treatment. Here, we find that aldehyde dehydrogenase-ALDH3A1 tightly links to ferroptosis in squamous cell carcinomas (SCCs). Functional assays demonstrate the enzymatic activity-dependent regulation of ALDH3A1 in protecting SCC cells against ferroptosis through catalyzing aldehydes and mitigating lipid peroxidation. Furthermore, a specific covalent inhibitor of ALDH3A1-EN40 significantly enhances the ferroptosis sensitivity induced by the ferroptosis inducer. The combination of EN40 and a ferroptosis inducer exhibits a synergistic effect, effectively inhibiting the proliferation of SCC cells/organoids and suppressing tumor growth both in vitro and in vivo. On mechanism, high expression of ALDH3A1 is transcriptionally governed by TP63, which binds to super-enhancer of ALDH3A1. Collectively, our findings reveal a yet-unrecognized function of ALDH3A1 exploited by SCC cells to evade ferroptosis, and targeting ALDH3A1 may enhance the effect of ferroptosis-induced therapy in SCCs.

Supplementary Figures S1-S8 with legends.

BackgroundProstate cancer (PCa) is a heterogeneous disease affecting over 14% of the male population worldwide. Although patients often respond positively to initial treatments within the first 2-3 years, many eventually develop a more lethal form of the disease known as castration-resistant PCa (CRPC). At present, no biomarkers that predict the onset of CRPC are available. This study aims to provide insights into the diagnosis and prediction of CRPC emergence.MethodsProtein expression dynamics were analysed in drug (androgen receptor inhibitor)-tolerant persister (DTP) and drug withdrawal cells using proteomics to identify potential biomarkers. These biomarkers were subsequently validated using a mouse model, 180-paired carcinoma/benign tissues, and 482 serum samples. Five machine learning algorithms were employed to build clinical prediction models, wherein the SHapley Additive exPlanation (SHAP) framework was used to interpret the best-performing model. Moreover, three regression models were developed to determine the Time from initial PCa diagnosis to CRPC development (TPC) in patients.ResultsWe identified that the protein expression levels of GPX4, NDUFS4, PRDX5, and TXNRD2 were significantly upregulated in PCa patients, particularly in those with CRPC. Among the tested machine learning models, the random forest and extreme gradient boosting models performed best on tissue and serum cohorts, achieving AUCs of 0.958 and 0.988, respectively. In addition, a significant inverse correlation was observed between TPC and serum levels of these four biomarkers. This correlation was formulated in three regression models, which achieved the smallest mean absolute error of 1.903 on independent datasets for predicting CRPC emergence.ConclusionOur study provides new insights into the role of DTP cells in CRPC development. The quad protein panel identified in our study, along with the post hoc and intrinsically explainable prediction models, may serve as a convenient and real-time prognostic tool, addressing the current lack of clinical biomarkers for CRPC.

Upper aerodigestive squamous cell carcinoma (UASCC) presents significant challenges in clinical management due to its aggressive nature. Here, we elucidate the role of MLL3 mutations as early, clonal genomic events in UASCC tumorigenesis, highlighting their role as foundational drivers of cancer development. Utilizing CRISPR-edited, cross-species organoid modeling, we demonstrate that loss of MLL3 contributes to early squamous neoplastic evolution. Furthermore, we identify an MLL3/GRHL2 protein complex that regulates the UASCC epigenome, particularly impacting immune response pathways. Notably, a novel MLL3/GRHL2-IRF1 axis promotes the expression of Th1 chemokines, enhancing anti-tumor immunity by facilitating T cell infiltration into the tumor microenvironment. Consequently, MLL3 regulates the in vivo efficacy of immune checkpoint blockade (ICB) therapy, corroborated by the strong association between MLL3 expression and human patients' clinical response to ICB therapy. Our work underscores the significance of MLL3 in UASCC pathogenesis and highlights the interplay between MLL3/GRHL2 and immune response pathways as potential therapeutic targets for UASCC treatment.

This file contains supplementary methods, Figs. S1 to S6 and Tables S1 to S5.