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Tumour addicted to oncomiR MicroRNAs (miRNAs) — small RNA molecules that regulate gene expression and have an important role in establishing cell identity — have been linked to human cancers, where they are referred to as oncomiRs. One model of cancer development proposes that proliferating cells become 'addicted' to activating mutations in an oncogene, and it has been suggested that tumours may also become dependent on oncomiRs. Work in mice that were engineered to conditionally express microRNA-21 (miR-21), which is overexpressed in most tumour types so far analysed, now shows that miR-21 induces pre-B-cell lymphoma. In the absence of miR-21, malignant cells undergo apoptosis and regress, as would be expected if they were addicted to its presence. The pharmacological inactivation of 'oncomiR-21' and other similar miRNAs may therefore be of therapeutic benefit. One model for cancer development posits that the proliferating cells in a tumour can become 'addicted' to activating mutations in an oncogene. With the realization that certain microRNAs promote tumorigenesis, it has been proposed that tumours may also become dependent on such 'oncomiRs'. Here, evidence is provided that the gene encoding microRNA-21 is an oncogene, and that in its absence, tumours undergo apoptosis and regress. Thus tumours can indeed become addicted to oncomiRs. MicroRNAs (miRNAs) belong to a recently discovered class of small RNA molecules that regulate gene expression at the post-transcriptional level. miRNAs have crucial functions in the development and establishment of cell identity, and aberrant metabolism or expression of miRNAs has been linked to human diseases, including cancer 1 . Components of the miRNA machinery and miRNAs themselves are involved in many cellular processes that are altered in cancer, such as differentiation, proliferation and apoptosis. Some miRNAs, referred to as oncomiRs 2 , show differential expression levels in cancer and are able to affect cellular transformation, carcinogenesis and metastasis, acting either as oncogenes or tumour suppressors. The phenomenon of ‘oncogene addiction’ reveals that despite the multistep nature of tumorigenesis, targeting of certain single oncogenes can have therapeutic value 3 , 4 , and the possibility of oncomiR addiction has been proposed but never demonstrated 3 . MicroRNA-21 (miR-21) is a unique miRNA in that it is overexpressed in most tumour types analysed so far. Despite great interest in miR-21, most of the data implicating it in cancer have been obtained through miRNA profiling and limited in vitro functional assays. To explore the role of miR-21 in cancer in vivo , we used Cre and Tet-off technologies to generate mice conditionally expressing miR-21. Here we show that overexpression of miR-21 leads to a pre-B malignant lymphoid-like phenotype, demonstrating that mir-21 is a genuine oncogene. When miR-21 was inactivated, the tumours regressed completely in a few days, partly as a result of apoptosis. These results demonstrate that tumours can become addicted to oncomiRs and support efforts to treat human cancers through pharmacological inactivation of miRNAs such as miR-21.

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.

Despite the genetic inactivation of SMARCA4 , a core component of the SWI/SNF-complex commonly found in cancer, there are no therapies that effectively target SMARCA4-deficient tumours. Here, we show that, unlike the cells with activated MYC oncogene, cells with SMARCA4 inactivation are refractory to the histone deacetylase inhibitor, SAHA, leading to the aberrant accumulation of H3K27me3. SMARCA4 -mutant cells also show an impaired transactivation and significantly reduced levels of the histone demethylases KDM6A/UTX and KDM6B/JMJD3, and a strong dependency on these histone demethylases, so that its inhibition compromises cell viability. Administering the KDM6 inhibitor GSK-J4 to mice orthotopically implanted with SMARCA4 -mutant lung cancer cells or primary small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT), had strong anti-tumour effects. In this work we highlight the vulnerability of KDM6 inhibitors as a characteristic that could be exploited for treating SMARCA4- mutant cancer patients. SMARCA4 is commonly inactivated in lung and ovarian cancers. Here the authors show that SMARCA4-deficient tumours have significantly reduced levels of the histone demethylases KDM6s and a strong dependency on these demethylases for tumour growth, so that they are vulnerable to KDM6s inhibition.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma. A major mutagenic process in DLBCL is aberrant somatic hypermutation (aSHM) by activation-induced cytidine deaminase (AID), which occurs preferentially at R C H/ T W sequence motifs proximal to transcription start sites. Splice sequences are highly conserved, rich in R C H/ T W motifs, and recurrently mutated in DLBCL. Therefore, we hypothesized that aSHM may cause recurrent splicing mutations in DLBCL. In a meta-cohort of > 1,800 DLBCLs, we found that 77.5% of splicing mutations in 29 recurrently mutated genes followed aSHM patterns. In addition, in whole-genome sequencing (WGS) data from 153 DLBCLs, proximal mutations in splice sequences, especially in donors, were significantly enriched in R C H/ T W motifs ( p  < 0.01). We validated this enrichment in two additional DLBCL cohorts (N > 2,000; p  < 0.0001) and confirmed its absence in 12 cancer types without aSHM (N > 6,300). Comparing sequencing data from mouse models with and without AID activity showed that the splice donor sequences were the top genomic feature enriched in AID-induced mutations ( p  < 0.0001). Finally, we observed that most AID-related splice site mutations are clonal within a sample, indicating that aSHM may cause early loss-of-function events in lymphomagenesis. Overall, these findings support that AID causes an overrepresentation of clonal splicing mutations in DLBCL. Graphical Abstract

Missense mutations in the 12 th codon of KRAS are key drivers of lung cancer, with glycine-to-cysteine (G12C) and glycine-to-aspartic acid (G12D) substitutions being among the most prevalent. These mutations are strongly associated with poor survival outcomes. Given the critical role of KRAS in lung cancer and other cancers, it remains as a major target for the development of new and complementary treatments. We have developed a CRISPR-High Fidelity (HiFi)-Cas9-based therapy strategy that can effectively and specifically target KRAS G12C and KRAS G12D mutants, avoiding KRAS WT off-targeting and affecting KRAS downstream pathways, thereby significantly reducing tumorgenicity. The delivery of HiFiCas9 components via ribonucleoprotein particles (RNPs) and adenovirus (AdV) effectively abrogates cell viability in KRAS -mutant Non-Small Cell Lung Cancer (NSCLC) preclinical models, including 2D and 3D cell cultures, cell-derived xenografts (CDX), and patient-derived xenograft organoids (PDXO). Our in vitro studies demonstrate that HiFiCas9-based therapy achieves superior KRAS inhibition compared to Sotorasib and effectively circumvents certain resistance mechanisms associated with Sotorasib treatment. Moreover, in vivo delivery using adenoviral particles significantly suppresses tumor growth in preclinical NSCLC models. Collectively, our findings establish HiFiCas9 as an effective therapeutic strategy with promising clinical applications, especially if in vivo delivery methods are further optimized. Missense mutations in the twelfth codon of KRAS are key drivers of lung cancer. Here, the authors develop a CRISPR-High Fidelity-Cas9-based strategy to target KRAS-G12C and KRAS-G12D mutants, reducing tumourigenicity without wild type KRAS off-targeting and circumventing certain therapy resistance mechanisms in preclinical models.

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.

Circular RNAs (circRNAs) are emerging regulators in cancer biology, yet the mechanisms underlying their biogenesis remain incompletely defined. RBM10, a splicing regulator frequently mutated in lung adenocarcinoma (LUAD), modulates RNA processing, but its involvement in circRNA regulation has not yet been addressed. Transcriptomic profiling of RBM10-restored LUAD cells, followed by RT-qPCR validation, identified circHIPK3 and circSMARCA5 as consistently RBM10-dependent circRNAs. Subcellular fractionation confirmed nuclear confinement of RBM10 and cytoplasmic enrichment of circRNAs, supporting a nuclear role for RBM10 in circRNA biogenesis. PAR-CLIP and RNA pulldown assays demonstrated direct RBM10 binding to intronic flanking regions of these circRNAs. Using a splicing reporter assay, we found that RBM10 binding to the 3' flanking region promotes exon skipping and circularization more efficiently than 5' binding, revealing a position-dependent mechanism controlling circRNA output. Analysis of RBM10 point mutants showed impaired regulation of circHIPK3 and circSMARCA5, linking defective exon skipping to disrupted circRNA formation. Functionally, modulation of circHIPK3 and circSMARCA5 phenocopied RBM10 restoration in mutant LUAD cell lines and rescued the tumorigenic phenotype driven by RBM10 loss. In two independent LUAD cohorts, circHIPK3 was consistently downregulated, particularly in RBM10-mutant tumors, and strongly correlated with RBM10 expression. Proteomic analyses further identified RBM10–SF3B1 interaction as a key upstream event governing circHIPK3 biogenesis. Together, these findings uncover a previously unrecognized mechanism through which RBM10 exerts tumor-suppressive functions via circRNA regulation and highlight circHIPK3 as a promising biomarker and potential therapeutic target in RBM10-deficient LUAD.

Lung cancer is the leading cause of cancer-related deaths worldwide, with lung squamous cell carcinoma (LUSC) lacking effective targeted therapies. Recent studies have identified Plakophilin-1 (PKP1) as one of the most differentially overexpressed genes in LUSC. This is particularly intriguing given that PKP1 is primarily known as a desmosomal component involved in cell adhesion, typically regarded as a tumor suppressor. To elucidate its biological role, we performed a genome-wide CRISPR knockout screening in PKP1-deficient models, revealing a strong dependence on mitochondrial metabolism. Metabolic assays further demonstrated that PKP1 loss significantly disrupts both mitochondrial function and glycolytic activity. In contrast, cells expressing PKP1 display a metabolically hyperactive phenotype, characterized by elevated oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Building on these findings, we found that PKP1 depletion selectively reduces platelet-type phosphofructokinase (PFKP) levels, a key rate-limiting enzyme in glycolysis, by enhancing its ubiquitination and subsequent degradation. Functional rescue experiments confirmed that PFKP mediates the proliferative role of PKP1. These findings suggest that PKP1 overexpression in LUSC promotes a hyperactive metabolic state binding to TRIM21 and preventing PFKP degradation, facilitating tumor progression. These effects were consistently observed across multiple LUSC cell lines, underscoring the robustness of the mechanism. These findings highlight a potential therapeutic vulnerability in LUSC metabolic regulation. Graphical Abstract

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.

Maslinic acid (MA) is a natural triterpene present in high concentrations in the waxy skin of olives. We have previously reported that MA induces apoptotic cell death via the mitochondrial apoptotic pathway in HT29 colon cancer cells. Here, we show that MA induces apoptosis in Caco-2 colon cancer cells via the extrinsic apoptotic pathway in a dose-dependent manner. MA triggered a series of effects associated with apoptosis, including the cleavage of caspases -8 and -3, and increased the levels of t-Bid within a few hours of its addition to the culture medium. MA had no effect on the expression of the Bax protein, release of cytochrome-c or on the mitochondrial membrane potential. This suggests that MA triggered the extrinsic apoptotic pathway in this cell type, as opposed to the intrinsic pathway found in the HT29 colon-cancer cell line. Our results suggest that the apoptotic mechanism induced in Caco-2 may be different from that found in HT29 colon-cancer cells, and that in Caco-2 cells MA seems to work independently of p53. Natural antitumoral agents capable of activating both the extrinsic and intrinsic apoptotic pathways could be of great use in treating colon-cancer of whatever origin.