Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCC) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The first group shows decreased proliferation, genome instability, and increased sensitivity to genotoxic agents, such as PARP1/2 inhibitors. In contrast, the H3K36M HNSCC models with steady H3K27me3 levels do not exhibit these characteristics unless H3K27me3 levels are elevated, either by DNA hypomethylating agents or by inhibiting the H3K27me3 demethylases KDM6A/B. Mechanistically, we found that H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, we found that aberrant H3K27me3 levels induced by H3K36M expression is not a bona fide epigenetic mark in HNSCC since it requires continuous expression of H3K36M to be inherited. Moreover, increased sensitivity of H3K36M HNSCC models to PARP1/2 inhibitors solely depends on the increased H3K27me3 levels. Indeed, aberrantly high H3K27me3 levels decrease BRCA1 and FANCD2-dependent DNA repair, resulting in higher sensitivity to DNA breaks and replication stress. Finally, in support of our in vitro findings, a PARP1/2 inhibitor alone reduce tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a H3K36M HNSCC xenograft model with consistent H3K27me3 levels, a combination of PARP1/2 inhibitors and agents that upregulate H3K27me3 proves to be successful. In conclusion, our findings underscore a delicate balance between H3K36 and H3K27 methylation, essential for maintaining genome stability. This equilibrium presents promising therapeutic opportunities for patients with H3K36me-deficient tumors.

Endocrine resistance and breast cancer plasticity are controlled by CoREST.

Resistance to cancer treatment remains a major clinical hurdle. Here, we demonstrate that the CoREST complex is a key determinant of endocrine resistance and ER+ breast cancer plasticity. In endocrine-sensitive cells, CoREST is recruited to regulatory regions co-bound to ERα and FOXA1 to regulate the estrogen pathway. In contrast, during temporal reprogramming towards a resistant state, CoREST is recruited to AP-1 sites. In reprogrammed cells, CoREST favors chromatin opening, cJUN binding to chromatin, and gene activation by controlling SWI/SNF recruitment independently of the demethylase activity of the CoREST subunit LSD1. Genetic and pharmacological CoREST inhibition reduces tumorigenesis and metastasis of endocrine-sensitive and endocrine-resistant xenograft models. Consistently, CoREST controls a gene signature involved in invasiveness in clinical breast tumors resistant to endocrine therapies. Our studies reveal CoREST functions that are co-opted to drive cellular plasticity and resistance to endocrine therapies and tumorigenesis, thus establishing CoREST as a potential therapeutic target for the treatment of advanced breast cancer.

The Polycomb protein RING1B enables estrogen-mediated gene expression by promoting enhancer-promoter interaction and R-loop formation.

Polycomb complexes have traditionally been prescribed roles as transcriptional repressors, though increasing evidence demonstrate they can also activate gene expression. However, the mechanisms underlying positive gene regulation mediated by Polycomb proteins are poorly understood. Here, we show that RING1B, a core component of Polycomb Repressive Complex 1, regulates enhancer-promoter interaction of the bona fide estrogen-activated GREB1 gene. Systematic characterization of RNA:DNA hybrid formation (R-loops), nascent transcription and RNA Pol II activity upon estrogen administration revealed a key role of RING1B in gene activation by regulating R-loop formation and RNA Pol II elongation. We also found that the estrogen receptor alpha (ERα) and RNA are both necessary for full RING1B recruitment to estrogen-activated genes. Notably, RING1B recruitment was mostly unaffected upon RNA Pol II depletion. Our findings delineate the functional interplay between RING1B, RNA and ERα to safeguard chromatin architecture perturbations required for estrogen-mediated gene regulation and highlight the crosstalk between steroid hormones and Polycomb proteins to regulate oncogenic programs.

Epigenetic mechanisms in breast cancer therapy and resistance.

The majority of breast cancers express the estrogen receptor (ERα) and agents targeting this pathway represent the main treatment modality. Endocrine therapy has proven successful in the treatment of hormone-responsive breast cancer since its early adoption in the 1940s as an ablative therapy. Unfortunately, therapeutic resistance arises, leading to disease recurrence and relapse. Recent studies increased our understanding in how changes to the chromatin landscape and deregulation of epigenetic factors orchestrate the resistant phenotype. Here, we will discuss how the epigenome is an integral determinant in hormone therapy response and why epigenetic factors are promising targets for overcoming clinical resistance.

Estrogen induces dynamic ERα and RING1B recruitment to control gene and enhancer activities in luminal breast cancer.

RING1B, a core Polycomb repressive complex 1 subunit, is a histone H2A ubiquitin ligase essential for development. RING1B is overexpressed in patients with luminal breast cancer (BC) and recruited to actively transcribed genes and enhancers co-occupied by the estrogen receptor α (ERα). Whether ERα-induced transcriptional programs are mediated by RING1B is not understood. We show that prolonged estrogen administration induces transcriptional output and chromatin landscape fluctuations. RING1B loss impairs full estrogen-mediated gene expression and chromatin accessibility for key BC transcription factors. These effects were mediated, in part, by RING1B enzymatic activity and nucleosome binding functions. RING1B is recruited in a cyclic manner to ERα, FOXA1, and GRHL2 cobound sites and regulates estrogen-induced enhancers and ERα recruitment. Last, ChIP exo revealed multiple binding events of these factors at single-nucleotide resolution, including RING1B occupancy approximately 10 base pairs around ERα bound sites. We propose RING1B as a key regulator of the dynamic, liganded-ERα transcriptional regulatory circuit in luminal BC.

Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.