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.

Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia.

TET2 haploinsufficiency is a driving event in myeloid cancers and is associated with a worse prognosis in patients with acute myeloid leukemia (AML). Enhancing residual TET2 activity using vitamin C increases oxidized 5-methylcytosine (mC) formation and promotes active DNA demethylation via base excision repair (BER), which slows leukemia progression. We utilize genetic and compound library screening approaches to identify rational combination treatment strategies to improve use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of several US Food and Drug Administration (FDA)-approved drugs, vitamin C treatment with poly-ADP-ribosyl polymerase inhibitors (PARPis) elicits a strong synergistic effect to block AML self-renewal in murine and human AML models. Vitamin-C-mediated TET activation combined with PARPis causes enrichment of chromatin-bound PARP1 at oxidized mCs and γH2AX accumulation during mid-S phase, leading to cell cycle stalling and differentiation. Given that most AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant.

The Trypanosoma brucei RNA-Binding Protein TbRRM1 is Involved in the Transcription of a Subset of RNA Pol II-Dependent Genes.

It has been long thought that RNA Polymerase (Pol) II transcriptional regulation does not operate in trypanosomes. However, recent reports have suggested that these organisms could regulate RNA Pol II transcription by epigenetic mechanisms. In this paper, we investigated the role of TbRRM1 in transcriptional regulation of RNA Pol II-dependent genes by focusing both in genes located in a particular polycistronic transcription unit (PTU) and in the monocistronic units of the SL-RNA genes. We showed that TbRRM1 is recruited throughout the PTU, with a higher presence on genes than intergenic regions. However, its depletion leads both to the decrease of nascent RNA and to chromatin compaction only of regions located distal to the main transcription start site. These findings suggest that TbRRM1 facilitates the RNA Pol II transcriptional elongation step by collaborating to maintain an open chromatin state in particular regions of the genome. Interestingly, the SL-RNA genes do not recruit TbRRM1 and, after TbRRM1 knockdown, nascent SL-RNAs accumulate while the chromatin state of these regions remains unchanged. Although it was previously suggested that TbRRM1 could regulate RNA Pol II-driven genes, we provide here the first experimental evidence which involves TbRRM1 to transcriptional regulation.

TbRRM1 knockdown produces abnormal cell morphology and apoptotic-like death in the bloodstream form of T. brucei.

TbRRM1, an SR-related protein, is involved in transcriptional and post-transcriptional gene expression regulation in procyclic T. brucei. In previous work, we found that TbRRM1 is essential and its depletion leads to cell cycle impairment, aberrant phenotypes and cell loss by apoptotic-like death. Here, we report the findings obtained after TbRRM1 knockdown in bloodstream parasites. Depletion of TbRRM1 in this cell stage led also to growth arrest and cell loss by apoptosis-like death. However, microscopic analysis showed aberrant cell morphology with parasites displaying flagellum detachment and cytokinesis impairment after RNAi induction, suggesting that TbRRM1 could play different roles depending on parasite stage.

Depletion of the SR-Related Protein TbRRM1 Leads to Cell Cycle Arrest and Apoptosis-Like Death in Trypanosoma brucei.

Arginine-Serine (RS) domain-containing proteins are RNA binding proteins with multiple functions in RNA metabolism. In mammalian cells this group of proteins is also implicated in regulation and coordination of cell cycle and apoptosis. In trypanosomes, an early branching group within the eukaryotic lineage, this group of proteins is represented by 3 members, two of them are SR proteins and have been recently shown to be involved in rRNA processing as well as in pre-mRNA splicing and stability. Here we report our findings on the 3rd member, the SR-related protein TbRRM1. In the present study, we showed that TbRRM1 ablation by RNA-interference in T. brucei procyclic cells leads to cell-cycle block, abnormal cell elongation compatible with the nozzle phenotype and cell death by an apoptosis-like mechanism. Our results expand the role of the trypanosomal RS-domain containing proteins in key cellular processes such as cell cycle and apoptosis-like death, roles also carried out by the mammalian SR proteins, and thus suggesting a conserved function in this phylogenetically conserved protein family.