SWI/SNF complexes in hematological malignancies: biological implications and therapeutic opportunities.

Hematological malignancies are a highly heterogeneous group of diseases with varied molecular and phenotypical characteristics. SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complexes play significant roles in the regulation of gene expression, being essential for processes such as cell maintenance and differentiation in hematopoietic stem cells. Furthermore, alterations in SWI/SNF complex subunits, especially in ARID1A/1B/2, SMARCA2/4, and BCL7A, are highly recurrent across a wide variety of lymphoid and myeloid malignancies. Most genetic alterations cause a loss of function of the subunit, suggesting a tumor suppressor role. However, SWI/SNF subunits can also be required for tumor maintenance or even play an oncogenic role in certain disease contexts. The recurrent alterations of SWI/SNF subunits highlight not only the biological relevance of SWI/SNF complexes in hematological malignancies but also their clinical potential. In particular, increasing evidence has shown that mutations in SWI/SNF complex subunits confer resistance to several antineoplastic agents routinely used for the treatment of hematological malignancies. Furthermore, mutations in SWI/SNF subunits often create synthetic lethality relationships with other SWI/SNF or non-SWI/SNF proteins that could be exploited therapeutically. In conclusion, SWI/SNF complexes are recurrently altered in hematological malignancies and some SWI/SNF subunits may be essential for tumor maintenance. These alterations, as well as their synthetic lethal relationships with SWI/SNF and non-SWI/SNF proteins, may be pharmacologically exploited for the treatment of diverse hematological cancers.

Multi-omic alterations of the SWI/SNF complex define a clinical subgroup in lung adenocarcinoma.

SWI/SNF complexes are major targets of mutations in cancer. Here, we combined multiple "-omics" methods to assess SWI/SNF composition and aberrations in LUAD. Mutations in lung SWI/SNF subunits were highly recurrent in our LUAD cohort (41.4%), and over 70% of the mutations were predicted to have functional impact. Furthermore, SWI/SNF expression in LUAD suffered an overall repression that could not be explained exclusively by genetic alterations. Finally, SWI/SNF mutations were associated with poorer overall survival in TCGA-LUAD. We propose SWI/SNF-mutant LUAD as a separate clinical subgroup with practical implications.

PKP1 and MYC create a feedforward loop linking transcription and translation in squamous cell lung cancer.

PURPOSE: Plakophilin 1 (PKP1) is well-known as an important component of the desmosome, a cell structure specialized in spot-like cell-to-cell adhesion. Although desmosomes have generally been associated with tumor suppressor functions, we recently found that PKP1 is recurrently overexpressed in squamous cell lung cancer (SqCLC) to exert an oncogenic role by enhancing the translation of MYC (c-Myc), a major oncogene. In this study, we aim to further characterize the functional relationship between PKP1 and MYC. METHODS: To determine the functional relationship between PKP1 and MYC, we performed correlation analyses between PKP1 and MYC mRNA expression levels, gain/loss of function models, chromatin immunoprecipitation (ChIP) and promoter mutagenesis followed by luciferase assays. RESULTS: We found a significant correlation between the mRNA levels of MYC and PKP1 in SqCLC primary tumor samples. In addition, we found that MYC is a direct transcription factor of PKP1 and binds to specific sequences within its promoter. In agreement with this, we found that MYC knockdown reduced PKP1 protein expression in different SqCLC models, which may explain the PKP1-MYC correlation that we found. Conversely, we found that PKP1 knockdown reduced MYC protein expression, while PKP1 overexpression enhanced MYC expression in these models. CONCLUSIONS: Based on these results, we propose a feedforward functional relationship in which PKP1 enhances MYC translation in conjunction with the translation initiation complex by binding to the 5'-UTR of MYC mRNA, whereas MYC promotes PKP1 transcription by binding to its promoter. These results suggest that PKP1 may serve as a therapeutic target for SqCLC.

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency.

Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

The SWI/SNF complex regulates the expression of miR-222, a tumor suppressor microRNA in lung adenocarcinoma.

SWitch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes are key epigenetic regulators that are recurrently mutated in cancer. Most studies of these complexes are focused on their role in regulating protein-coding genes. However, here, we show that SWI/SNF complexes control the expression of microRNAs. We used a SMARCA4-deficient model of lung adenocarcinoma (LUAD) to track changes in the miRNome upon SMARCA4 restoration. We found that SMARCA4-SWI/SNF complexes induced significant changes in the expression of cancer-related microRNAs. The most significantly dysregulated microRNA was miR-222, whose expression was promoted by SMARCA4-SWI/SNF complexes, but not by SMARCA2-SWI/SNF complexes via their direct binding to a miR-222 enhancer region. Importantly, miR-222 expression decreased cell viability, phenocopying the tumor suppressor role of SMARCA4-SWI/SNF complexes in LUAD. Finally, we showed that the miR-222 enhancer region resides in a topologically associating domain that does not contain any cancer-related protein-coding genes, suggesting that miR-222 may be involved in exerting the tumor suppressor role of SMARCA4. Overall, this study highlights the relevant role of the SWI/SNF complex in regulating the non-coding genome, opening new insights into the pathogenesis of LUAD.

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-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.

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.

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.

Expression inactivation of SMARCA4 by microRNAs in lung tumors.

SMARCA4 is the catalytic subunit of the SWI/SNF chromatin-remodeling complex, which alters the interactions between DNA and histones and modifies the availability of the DNA for transcription. The latest deep sequencing of tumor genomes has reinforced the important and ubiquitous tumor suppressor role of the SWI/SNF complex in cancer. However, although SWI/SNF complex plays a key role in gene expression, the regulation of this complex itself is poorly understood. Significantly, an understanding of the regulation of SMARCA4 expression has gained in importance due to recent proposals incorporating it in therapeutic strategies that use synthetic lethal interactions between SMARCA4-MAX and SMARCA4-SMARCA2. In this report, we found that the loss of expression of SMARCA4 observed in some primary lung tumors, whose mechanism was largely unknown, can be explained, at least partially by the activity of microRNAs (miRNAs). We reveal that SMARCA4 expression is regulated by miR-101, miR-199 and especially miR-155 through their binding to two alternative 3'UTRs. Importantly, our experiments suggest that the oncogenic properties of miR-155 in lung cancer can be largely explained by its role inhibiting SMARCA4. This new discovered functional relationship could explain the poor prognosis displayed by patients that independently have high miR-155 and low SMARCA4 expression levels. In addition, these results could lead to application of incipient miRNA technology to the aforementioned synthetic lethal therapeutic strategies.