EZH2 mutations in follicular lymphoma distort H3K27me3 profiles and alter transcriptional responses to PRC2 inhibition.

Mutations in chromatin regulators are widespread in cancer. Among them, the histone H3 lysine 27 methyltransferase Polycomb Repressive Complex 2 (PRC2) shows distinct alterations according to tumor type. This specificity is poorly understood. Here, we model several PRC2 alterations in one isogenic system to reveal their comparative effects. Focusing then on lymphoma-associated EZH2 mutations, we show that Ezh2Y641F induces aberrant H3K27 methylation patterns even without wild-type Ezh2, which are alleviated by partial PRC2 inhibition. Remarkably, Ezh2Y641F rewires the response to PRC2 inhibition, leading to induction of antigen presentation genes. Using a unique longitudinal follicular lymphoma cohort, we further link EZH2 status to abnormal H3K27 methylation. We also uncover unexpected variability in the mutational landscape of successive biopsies, pointing to frequent co-existence of different clones and cautioning against stratifying patients based on single sampling. Our results clarify how oncogenic PRC2 mutations disrupt chromatin and transcription, and the therapeutic vulnerabilities this creates.

BCL7A is silenced by hypermethylation to promote acute myeloid leukemia.

BACKGROUND: Recent massive sequencing studies have revealed that SWI/SNF complexes are among the most frequently altered functional entities in solid tumors. However, the role of SWI/SNF in acute myeloid leukemia is poorly understood. To date, SWI/SNF complexes are thought to be oncogenic in AML or, at least, necessary to support leukemogenesis. However, mutation patterns in SWI/SNF genes in AML are consistent with a tumor suppressor role. Here, we study the SWI/SNF subunit BCL7A, which has been found to be recurrently mutated in lymphomas, but whose role in acute myeloid malignancies is currently unknown. METHODS: Data mining and bioinformatic approaches were used to study the mutational status of BCL7A and the correlation between BCL7A expression and promoter hypermethylation. Methylation-specific PCR, bisulfite sequencing, and 5-aza-2'-deoxycytidine treatment assays were used to determine if BCL7A expression was silenced due to promoter hypermethylation. Cell competition assays after BCL7A expression restoration were used to assess the role of BCL7A in AML cell line models. Differential expression analysis was performed to determine pathways and genes altered after BCL7A expression restoration. To establish the role of BCL7A in tumor development in vivo, tumor growth was compared between BCL7A-expressing and non-expressing mouse xenografts using in vivo fluorescence imaging. RESULTS: BCL7A expression was inversely correlated with promoter methylation in three external cohorts: TCGA-LAML (N = 160), TARGET-AML (N = 188), and Glass et al. (2017) (N = 111). The AML-derived cell line NB4 silenced the BCL7A expression via promoter hypermethylation. Ectopic BCL7A expression in AML cells decreased their competitive ability compared to control cells. Additionally, restoration of BCL7A expression reduced tumor growth in an NB4 mouse xenograft model. Also, differential expression analysis found that BCL7A restoration altered cell cycle pathways and modified significantly the expression of genes like HMGCS1, H1-0, and IRF7 which can help to explain its tumor suppressor role in AML. CONCLUSIONS: BCL7A expression is silenced in AML by promoter methylation. In addition, restoration of BCL7A expression exerts tumor suppressor activity in AML cell lines and xenograft models.

LncRNA-mRNA Co-Expression Analysis Identifies AL133346.1/CCN2 as Biomarkers in Pediatric B-Cell Acute Lymphoblastic Leukemia.

Pediatric acute B-cell lymphoblastic leukemia (B-ALL) constitutes a heterogeneous and aggressive neoplasia in which new targeted therapies are required. Long non-coding RNAs have recently emerged as promising disease-specific biomarkers for the clinic. Here, we identified pediatric B-ALL-specific lncRNAs and associated mRNAs by comparing the transcriptomic signatures of tumoral and non-tumoral samples. We identified 48 lncRNAs that were differentially expressed between pediatric B-ALL and healthy bone marrow samples. The most relevant lncRNA/mRNA pair was AL133346.1/CCN2 (previously known as RP11-69I8.3/CTGF), whose expression was positively correlated and increased in B-ALL samples. Their differential expression pattern and their strong correlation were validated in external B-ALL datasets (Therapeutically Applicable Research to Generate Effective Treatments, Cancer Cell Line Encyclopedia). Survival curve analysis demonstrated that patients with "high" expression levels of CCN2 had higher overall survival than those with "low" levels (p = 0.042), and this gene might be an independent prognostic biomarker in pediatric B-ALL. These findings provide one of the first detailed descriptions of lncRNA expression profiles in pediatric B-ALL and indicate that these potential biomarkers could help in the classification of leukemia subtypes and that CCN2 expression could predict the survival outcome of pediatric B-cell acute lymphoblastic leukemia patients.

Comprehensive Analysis of SWI/SNF Inactivation in Lung Adenocarcinoma Cell Models.

Mammalian SWI/SNF (SWitch/Sucrose Non-Fermentable) complexes are ATP-dependent chromatin remodelers whose subunits have emerged among the most frequently mutated genes in cancer. Studying SWI/SNF function in cancer cell line models has unveiled vulnerabilities in SWI/SNF-mutant tumors that can lead to the discovery of new therapeutic drugs. However, choosing an appropriate cancer cell line model for SWI/SNF functional studies can be challenging because SWI/SNF subunits are frequently altered in cancer by various mechanisms, including genetic alterations and post-transcriptional mechanisms. In this work, we combined genomic, transcriptomic, and proteomic approaches to study the mutational status and the expression levels of the SWI/SNF subunits in a panel of 38 lung adenocarcinoma (LUAD) cell lines. We found that the SWI/SNF complex was mutated in more than 76% of our LUAD cell lines and there was a high variability in the expression of the different SWI/SNF subunits. These results underline the importance of the SWI/SNF complex as a tumor suppressor in LUAD and the difficulties in defining altered and unaltered cell models for the SWI/SNF complex. These findings will assist researchers in choosing the most suitable cellular models for their studies of SWI/SNF to bring all of its potential to the development of novel therapeutic applications.

LncRNA DLG2-AS1 as a Novel Biomarker in Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) are a heterogeneous class of non-coding RNAs whose biological roles are still poorly understood. LncRNAs serve as gene expression regulators, frequently interacting with epigenetic factors to shape the outcomes of crucial biological processes, and playing roles in different pathologies including cancer. Over the last years, growing scientific evidence supports the key role of some lncRNAs in tumor development and proposes them as valuable biomarkers for the clinic. In this study, we aimed to characterize lncRNAs whose expression is altered in tumor samples from patients with lung adenocarcinoma (LUAD) compared to adjacent normal tissue samples. On an RT-qPCR survey of 90 cancer-related lncRNAs, we found one lncRNA, DLG2-AS1, which was consistently downregulated in 70 LUAD patients. To gain insight into its biological function, DLG2-AS1 was cloned and successfully re-expressed in LUAD cancer cell lines. We determined that DLG2-AS1 is not a cis-regulatory element of its overlapping gene DLG2, as their transcription levels were not correlated, nor did DLG2-AS1 restoration modify the expression of DLG2 protein. Furthermore, after generating a receiver operating curve (ROC) and calculating the area under curve (AUC), we found that DLG2-AS1 expression showed high sensitivity and specificity (AUC = 0.726) for the classification of LUAD and normal samples, determining its value as a potential lung cancer biomarker.

Frequent mutations in the amino-terminal domain of BCL7A impair its tumor suppressor role in DLBCL.

Mutations in genes encoding subunits of the SWI/SNF chromatin remodeling complex are frequently found in different human cancers. While the tumor suppressor function of this complex is widely established in solid tumors, its role in hematologic malignancies is largely unknown. Recurrent point mutations in BCL7A gene, encoding a subunit of the SWI/SNF complex, have been reported in diffuse large B-cell lymphoma (DLBCL), but their functional impact remains to be elucidated. Here we show that BCL7A often undergoes biallelic inactivation, including a previously unnoticed mutational hotspot in the splice donor site of intron one. The splice site mutations render a truncated BCL7A protein, lacking a portion of the amino-terminal domain. Moreover, restoration of wild-type BCL7A expression elicits a tumor suppressor-like phenotype in vitro and in vivo. In contrast, splice site mutations block the tumor suppressor function of BCL7A by preventing its binding to the SWI/SNF complex. We also show that BCL7A restoration induces transcriptomic changes in genes involved in B-cell activation. In addition, we report that SWI/SNF complex subunits harbor mutations in more than half of patients with germinal center B-cell (GCB)-DLBCL. Overall, this work demonstrates the tumor suppressor function of BCL7A in DLBCL, and highlights that the SWI/SNF complex plays a relevant role in DLBCL pathogenesis.

Correction: Plakophilin 1 enhances MYC translation, promoting squamous cell lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Plakophilin 1 enhances MYC translation, promoting squamous cell lung cancer.

Plakophilin 1 (PKP1) is a member of the arm-repeat (armadillo) and plakophilin gene families and it is an essential component of the desmosomes. Although desmosomes have generally been associated with tumor suppressor functions, we have consistently observed that PKP1 is among the top overexpressed proteins in squamous cell lung cancer. To explore this paradox, we developed in vivo and in vitro functional models of PKP1 gain/loss in squamous cell lung cancer. CRISPR-Cas9 PKP1 knockout severely impaired cell proliferation, but it increased cell dissemination. In addition, PKP1 overexpression increased cell proliferation, cell survival, and in vivo xenograft engraftment. We further investigated the molecular mechanism of the mainly oncogenic function of PKP1 by combining transcriptomics, proteomics, and protein-nucleic acid interaction assays. Interestingly, we found that PKP1 enhances MYC translation in collaboration with the translation initiation complex by binding to the 5'-UTR of MYC mRNA. We propose PKP1 as an oncogene in SqCLC and a novel posttranscriptional regulator of MYC. PKP1 may be a valuable diagnostic biomarker and potential therapeutic target for SqCLC. Importantly, PKP1 inhibition may indirectly target MYC, a primary anticancer target.