Splicing modulators impair DNA damage response and induce killing of cohesin-mutant MDS and AML.

Splicing modulation is a promising treatment strategy pursued to date only in splicing factor-mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we showed that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing factor 3B subunit 1 (SF3B1) H3B-8800 and E-7107. We identified drug-induced alterations in splicing, and corresponding reduced gene expression, of a number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We found that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators due to their long length and large number of exons per transcript. Furthermore, we demonstrated that treatment of cohesin-mutant cells with SF3B1 modulators not only resulted in impaired DNA damage response and accumulation of DNA damage, but it sensitized cells to subsequent killing by poly(ADP-ribose) polymerase (PARP) inhibitors and chemotherapy and led to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML.

Global identification of SWI/SNF targets reveals compensation by EP400.

Mammalian SWI/SNF chromatin remodeling complexes move and evict nucleosomes at gene promoters and enhancers to modulate DNA access. Although SWI/SNF subunits are commonly mutated in disease, therapeutic options are limited by our inability to predict SWI/SNF gene targets and conflicting studies on functional significance. Here, we leverage a fast-acting inhibitor of SWI/SNF remodeling to elucidate direct targets and effects of SWI/SNF. Blocking SWI/SNF activity causes a rapid and global loss of chromatin accessibility and transcription. Whereas repression persists at most enhancers, we uncover a compensatory role for the EP400/TIP60 remodeler, which reestablishes accessibility at most promoters during prolonged loss of SWI/SNF. Indeed, we observe synthetic lethality between EP400 and SWI/SNF in cancer cell lines and human cancer patient data. Our data define a set of molecular genomic features that accurately predict gene sensitivity to SWI/SNF inhibition in diverse cancer cell lines, thereby improving the therapeutic potential of SWI/SNF inhibitors.

Oncogenic CDK13 mutations impede nuclear RNA surveillance.

RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.

Screening thousands of transcribed coding and non-coding regions reveals sequence determinants of RNA polymerase II elongation potential.

Precise regulation of transcription by RNA polymerase II (RNAPII) is critical for organismal growth and development. However, what determines whether an engaged RNAPII will synthesize a full-length transcript or terminate prematurely is poorly understood. Notably, RNAPII is far more susceptible to termination when transcribing non-coding RNAs than when synthesizing protein-coding mRNAs, but the mechanisms underlying this are unclear. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in non-coding RNAs, rather than specific sequence motifs, drives RNAPII termination. Further, we demonstrate that 5' splice sites autonomously stimulate processive transcription, even in the absence of polyadenylation signals. Our results reveal a potent role for the transcribed sequence in dictating gene output and demonstrate the power of INSERT-seq toward illuminating these contributions.

Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells.

Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 µM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

Pterostilbene leads to DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in breast cancer cells.

Transcription factor (TF)-mediated regulation of genes is often disrupted during carcinogenesis. The DNA methylation state of TF-binding sites may dictate transcriptional activity of corresponding genes. Stilbenoid polyphenols, such as pterostilbene (PTS), have been shown to exert anticancer action by remodeling DNA methylation and gene expression. However, the mechanisms behind these effects still remain unclear. Here, the dynamics between oncogenic TF OCT1 binding and de novo DNA methyltransferase DNMT3B binding in PTS-treated MCF10CA1a invasive breast cancer cells has been explored. Using chromatin immunoprecipitation (ChIP) followed by next generation sequencing, we determined 47 gene regulatory regions with decreased OCT1 binding and enriched DNMT3B binding in response to PTS. Most of those genes were found to have oncogenic functions. We selected three candidates, PRKCA, TNNT2, and DANT2, for further mechanistic investigation taking into account PRKCA functional and regulatory connection with numerous cancer-driving processes and pathways, and some of the highest increase in DNMT3B occupancy within TNNT2 and DANT2 enhancers. PTS led to DNMT3B recruitment within PRKCA, TNNT2, and DANT2 at loci that also displayed reduced OCT1 binding. Substantial decrease in OCT1 with increased DNMT3B binding was accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, DNA hypermethylation of the genes was not detected in response to PTS in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells. It indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anticancer action of stilbenoid polyphenols.

Strength is in engagement: The rise of an online scientific community during the COVID-19 pandemic.

Many scientists, confined to home office by COVID-19, have been gathering in online communities, which could become viable alternatives to physical meetings and conferences.

ecDNA party bus: Bringing the enhancer to you.

Recent work from Zhu et al. (2021) reveals that extrachromosomal DNA circles harboring enhancers can serve as mobile regulatory elements that interact with chromosomal oncogenes, stimulating high-level gene activity and contributing to tumor heterogeneity and cancer progression.

Transcription shapes genome-wide histone acetylation patterns.

Histone acetylation is a ubiquitous hallmark of transcription, but whether the link between histone acetylation and transcription is causal or consequential has not been addressed. Using immunoblot and chromatin immunoprecipitation-sequencing in S. cerevisiae, here we show that the majority of histone acetylation is dependent on transcription. This dependency is partially explained by the requirement of RNA polymerase II (RNAPII) for the interaction of H4 histone acetyltransferases (HATs) with gene bodies. Our data also confirms the targeting of HATs by transcription activators, but interestingly, promoter-bound HATs are unable to acetylate histones in the absence of transcription. Indeed, HAT occupancy alone poorly predicts histone acetylation genome-wide, suggesting that HAT activity is regulated post-recruitment. Consistent with this, we show that histone acetylation increases at nucleosomes predicted to stall RNAPII, supporting the hypothesis that this modification is dependent on nucleosome disruption during transcription. Collectively, these data show that histone acetylation is a consequence of RNAPII promoting both the recruitment and activity of histone acetyltransferases.

Transcription Promotes the Interaction of the FAcilitates Chromatin Transactions (FACT) Complex with Nucleosomes in Saccharomyces cerevisiae.

The FACT (FAcilitates Chromatin Transactions) complex is a conserved complex that maintains chromatin structure on transcriptionally active genes. Consistent with this, FACT is enriched on highly expressed genes, but how it is targeted to these regions is unknown. In vitro, FACT binds destabilized nucleosomes, supporting the hypothesis that FACT is targeted to transcribed chromatin through recognition of RNA polymerase (RNAP)-disrupted nucleosomes. In this study, we used high-resolution analysis of FACT occupancy in Saccharomyces cerevisiae to test this hypothesis. We demonstrate that FACT interacts with nucleosomes in vivo and that its interaction with chromatin is dependent on transcription by any of the three RNAPs. Deep sequencing of micrococcal nuclease-resistant fragments shows that FACT-bound nucleosomes exhibit differing nuclease sensitivity compared to bulk chromatin, consistent with a modified nucleosome structure being the preferred ligand for this complex. Interestingly, a subset of FACT-bound nucleosomes may be "overlapping dinucleosomes," in which one histone octamer invades the ∼147-bp territory normally occupied by the adjacent nucleosome. While the differing nuclease sensitivity of FACT-bound nucleosomes could also be explained by the demonstrated ability of FACT to alter nucleosome structure, transcription inhibition restores nuclease resistance, suggesting that it is not due to FACT interaction alone. Collectively, these results are consistent with a model in which FACT is targeted to transcribed genes through preferential interaction with RNAP-disrupted nucleosomes.

Viral proteins as a potential driver of histone depletion in dinoflagellates.

Within canonical eukaryotic nuclei, DNA is packaged with highly conserved histone proteins into nucleosomes, which facilitate DNA condensation and contribute to genomic regulation. Yet the dinoflagellates, a group of unicellular algae, are a striking exception to this otherwise universal feature as they have largely abandoned histones and acquired apparently viral-derived substitutes termed DVNPs (dinoflagellate-viral-nucleoproteins). Despite the magnitude of this transition, its evolutionary drivers remain unknown. Here, using Saccharomyces cerevisiae as a model, we show that DVNP impairs growth and antagonizes chromatin by localizing to histone binding sites, displacing nucleosomes, and impairing transcription. Furthermore, DVNP toxicity can be relieved through histone depletion and cells diminish their histones in response to DVNP expression suggesting that histone reduction could have been an adaptive response to these viral proteins. These findings provide insights into eukaryotic chromatin evolution and highlight the potential for horizontal gene transfer to drive the divergence of cellular systems.

Histone Acetylation, Not Stoichiometry, Regulates Linker Histone Binding in Saccharomyces cerevisiae.

Linker histones play a fundamental role in shaping chromatin structure, but how their interaction with chromatin is regulated is not well understood. In this study, we used a combination of genetic and genomic approaches to explore the regulation of linker histone binding in the yeast, Saccharomyces cerevisiae We found that increased expression of Hho1, the yeast linker histone, resulted in a severe growth defect, despite only subtle changes in chromatin structure. Further, this growth defect was rescued by mutations that increase histone acetylation. Consistent with this, genome-wide analysis of linker histone occupancy revealed an inverse correlation with histone tail acetylation in both yeast and mouse embryonic stem cells. Collectively, these results suggest that histone acetylation negatively regulates linker histone binding in S. cerevisiae and other organisms and provide important insight into how chromatin structure is regulated and maintained to both facilitate and repress transcription.

Translation and Clinical Development of Bispecific T-cell Engaging Antibodies for Cancer Treatment.

Bispecific T-cell Engagers (BiTE®) antibody constructs enable a polyclonal T-cell response to cell-surface tumor-associated antigens, bypassing the narrow specificities of T-cell receptors and the need for antigen presentation through the major histocompatibility complex pathways. Blinatumomab, a CD19xCD3 BiTE® antibody construct, received accelerated approval for the treatment of relapsed/refractory Philadelphia chromosome negative acute lymphoblastic leukemia. Herein we review the pharmacology, safety, and efficacy observed in studies of blinatumomab and other BiTE® antibody constructs. Quantitative systems pharmacology is envisioned as a means to optimize dosing decisions for trials in which BiTE® antibody constructs are administered as monotherapy or in combination with other immunotherapies.

Histone H3K4 and H3K36 Methylation Independently Recruit the NuA3 Histone Acetyltransferase in Saccharomyces cerevisiae.

Histone post-translational modifications (PTMs) alter chromatin structure by promoting the interaction of chromatin-modifying complexes with nucleosomes. The majority of chromatin-modifying complexes contain multiple domains that preferentially interact with modified histones, leading to speculation that these domains function in concert to target nucleosomes with distinct combinations of histone PTMs. In Saccharomyces cerevisiae, the NuA3 histone acetyltransferase complex contains three domains, the PHD finger in Yng1, the PWWP domain in Pdp3, and the YEATS domain in Taf14; which in vitro bind to H3K4 methylation, H3K36 methylation, and acetylated and crotonylated H3K9, respectively. While the in vitro binding has been well characterized, the relative in vivo contributions of these histone PTMs in targeting NuA3 is unknown. Here, through genome-wide colocalization and by mutational interrogation, we demonstrate that the PHD finger of Yng1, and the PWWP domain of Pdp3 independently target NuA3 to H3K4 and H3K36 methylated chromatin, respectively. In contrast, we find no evidence to support the YEATS domain of Taf14 functioning in NuA3 recruitment. Collectively our results suggest that the presence of multiple histone PTM binding domains within NuA3, rather than restricting it to nucleosomes containing distinct combinations of histone PTMs, can serve to increase the range of nucleosomes bound by the complex. Interestingly, however, the simple presence of NuA3 is insufficient to ensure acetylation of the associated nucleosomes, suggesting a secondary level of acetylation regulation that does not involve control of HAT-nucleosome interactions.

Divergent Residues Within Histone H3 Dictate a Unique Chromatin Structure in Saccharomyces cerevisiae.

Histones are among the most conserved proteins known, but organismal differences do exist. In this study, we examined the contribution that divergent amino acids within histone H3 make to cell growth and chromatin structure in Saccharomyces cerevisiae. We show that, while amino acids that define histone H3.3 are dispensable for yeast growth, substitution of residues within the histone H3 α3 helix with human counterparts results in a severe growth defect. Mutations within this domain also result in altered nucleosome positioning, both in vivo and in vitro, which is accompanied by increased preference for nucleosome-favoring sequences. These results suggest that divergent amino acids within the histone H3 α3 helix play organismal roles in defining chromatin structure.

Allogeneic hematopoietic cell transplantation after failed autologous transplant for lymphoma using TLI and anti-thymocyte globulin conditioning.

We describe 47 patients with lymphoma and failed prior autologous hematopoietic cell transplantation (HCT) who received TLI-ATG (anti-thymocyte globulin) conditioning followed by allogeneic HCT. Thirty-two patients had non-Hodgkin lymphoma (NHL; diffuse large B-cell lymphoma (n=19), T-cell NHL (n=6), mantle cell lymphoma (n=4) or other B-cell subtypes (n=3)), and 15 had Hodgkin lymphoma. The median follow-up was 4.9 (range, 2.1-11.9) years. The cumulative incidence of grade II-IV acute GvHD at day +100 was 12%, and the cumulative incidence of extensive chronic GvHD at 1 year was 36%. The 3-year cumulative incidences of overall survival (OS), PFS and non-relapse mortality (NRM) were 81%, 44% and 7%, respectively. Fifteen patients died (relapse, n=10; NRM, n=5). Among the 25 patients with relapse after allogeneic HCT, 11 (44%) achieved durable (>1 year) CRs following donor lymphocyte infusion or chemoradiotherapy. The majority of surviving patients (75%; n=24) were able to discontinue all immunosuppression. For patients with relapsed lymphoma after autologous HCT, allogeneic HCT using TLI-ATG conditioning is a well-tolerated, predominantly outpatient therapy with low NRM (7% at 3 years), a low incidence of GvHD, durable disease control and excellent OS (81% at 3 years).

Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae.

Histone H3 lysine 4 trimethylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady-state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation (H3K14ac), which colocalizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 serve opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that H3K14ac negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression.