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Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

The cyclin-cdk (cyclin-dependent kinase) inhibitor p27Kip1 (p27) has a crucial negative role on cell cycle progression. In addition to its classical role as a cyclin-cdk inhibitor, it also performs cyclin-cdk-independent functions as the regulation of cytoskeleton rearrangements and cell motility. p27 deficiency has been associated with tumor aggressiveness and poor clinical outcome, although the mechanisms underlying this participation still remain elusive. We report here a new cellular function of p27 as a transcriptional regulator in association with p130/E2F4 complexes that could be relevant for tumorigenesis. We observed that p27 associates with specific promoters of genes involved in important cellular functions as processing and splicing of RNA, mitochondrial organization and respiration, translation and cell cycle. On these promoters p27 co-localizes with p130, E2F4 and co-repressors as histone deacetylases (HDACs) and mSIN3A. p27 co-immunoprecipitates with these proteins and by affinity chromatography, we demonstrated a direct interaction of p27 with p130 and E2F4 through its carboxyl-half. We have also shown that p130 recruits p27 on the promoters, and there p27 is needed for the subsequent recruitment of HDACs and mSIN3A. Expression microarrays and luciferase assays revealed that p27 behaves as transcriptional repressor of these p27-target genes (p27-TGs). Finally, in human tumors, we established a correlation with overexpression of p27-TGs and poor survival. Thus, this new function of p27 as a transcriptional repressor could have a role in the major aggressiveness of tumors with low levels of p27. [ABSTRACT FROM AUTHOR]

Abstract Background Cells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli. Results Here, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFα and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38α SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38α the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38α deficient (p38α-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress. Conclusions Our genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.

Abstract Background Melon (Cucumis melo) is a horticultural specie of significant nutritional value, which belongs to the Cucurbitaceae family, whose economic importance is second only to the Solanaceae. Its small genome of approx. 450 Mb coupled to the high genetic diversity has prompted the development of genetic tools in the last decade. However, the unprecedented existence of a transcriptomic approaches in melon, highlight the importance of designing new tools for high-throughput analysis of gene expression. Results We report the construction of an oligo-based microarray using a total of 17,510 unigenes derived from 33,418 high-quality melon ESTs. This chip is particularly enriched with genes that are expressed in fruit and during interaction with pathogens. Hybridizations for three independent experiments allowed the characterization of global gene expression profiles during fruit ripening, as well as in response to viral and fungal infections in plant cotyledons and roots, respectively. Microarray construction, statistical analyses and validation together with functional-enrichment analysis are presented in this study. Conclusion The platform validation and enrichment analyses shown in our study indicate that this oligo-based microarray is amenable for future genetic and functional genomic studies of a wide range of experimental conditions in melon.

Abstract Background Different regions in a genome evolve at different rates depending on structural and functional constraints. Some genomic regions are highly conserved during metazoan evolution, while other regions may evolve rapidly, either in all species or in a lineage-specific manner. A strong or even moderate change in constraints in functional regions, for example in coding regions, can have significant evolutionary consequences. Results Here we discuss a novel framework, 'BaseDiver', to classify groups of genes in humans based on the patterns of evolutionary constraints on polymorphic positions in their coding regions. Comparing the nucleotide-level divergence among mammals with the extent of deviation from the ancestral base in the human lineage, we identify patterns of evolutionary pressure on nonsynonymous base-positions in groups of genes belonging to the same functional category. Focussing on groups of genes in functional categories, we find that transcription factors contain a significant excess of nonsynonymous base-positions that are conserved in other mammals but changed in human, while immunity related genes harbour mutations at base-positions that evolve rapidly in all mammals including humans due to strong preference for advantageous alleles. Genes involved in olfaction also evolve rapidly in all mammals, and in humans this appears to be due to weak negative selection. Conclusion While recent studies have identified genes under positive selection in humans, our approach identifies evolutionary constraints on Gene Ontology groups identifying changes in humans relative to some of the other mammals.

Abstract Background Global analyses of human disease genes by computational methods have yielded important advances in the understanding of human diseases. Generally these studies have treated the group of disease genes uniformly, thus ignoring the type of disease-causing mutations (dominant or recessive). In this report we present a comprehensive study of the evolutionary history of autosomal disease genes separated by mode of inheritance. Results We examine differences in protein and coding sequence conservation between dominant and recessive human disease genes. Our analysis shows that disease genes affected by dominant mutations are more conserved than those affected by recessive mutations. This could be a consequence of the fact that recessive mutations remain hidden from selection while heterozygous. Furthermore, we employ functional annotation analysis and investigations into disease severity to support this hypothesis. Conclusion This study elucidates important differences between dominantly- and recessively-acting disease genes in terms of protein and DNA sequence conservation, paralogy and essentiality. We propose that the division of disease genes by mode of inheritance will enhance both understanding of the disease process and prediction of candidate disease genes in the future.

Abstract Background One of the main goals of cancer genetics is to identify the causative elements at the molecular level leading to cancer. Results We have conducted an analysis of a set of genes known to be involved in cancer in order to unveil their unique features that can assist towards the identification of new candidate cancer genes. Conclusion We have detected key patterns in this group of genes in terms of the molecular function or the biological process in which they are involved as well as sequence properties. Based on these features we have developed an accurate Bayesian classification model with which human genes have been scored for their likelihood of involvement in cancer.

Analyzes the frequency of the R32W variant in Connexin 26 and Connexin 31 genes in a family suffering from a skin disease and profound deafness. Mutation; Genetic polymorphism.

Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

Summary: Advances in high-throughput DNA sequencing technologies and decreasing costs have fueled the identification of small genetic variants (such as single nucleotide variants and indels) across tumors. Despite efforts to standardize variant formats and vocabularies, many sources of variability persist across databases and computational tools that annotate variants, hindering their integration within cancer genomic analyses. In this context, we present OpenVariant, an easily extendable Python package that facilitates seamless reading, parsing and refinement of diverse input file formats in a customizable structure, all within a single process., Availability and Implementation: OpenVariant is an open-source package available at https://github.com/bbglab/openvariant. Documentation may be found at https://openvariant.readthedocs.io., (© The Author(s) 2024. Published by Oxford University Press.)

Clonal hematopoiesis (CH) is a phenomenon of clonal expansion of hematopoietic stem cells driven by somatic mutations affecting certain genes. Recently, CH has been linked to the development of hematologic malignancies, cardiovascular diseases, and other conditions. Although the most frequently mutated CH driver genes have been identified, a systematic landscape of the mutations capable of initiating this phenomenon is still lacking. In this study, we trained machine learning models for 12 of the most recurrent CH genes to identify their driver mutations. These models outperform expert-curated rules based on prior knowledge of the function of these genes. Moreover, their application to identify CH driver mutations across almost half a million donors of the UK Biobank reproduces known associations between CH driver mutations and age, and the prevalence of several diseases and conditions. We thus propose that these models support the accurate identification of CH across healthy individuals. Significance: We developed and validated gene-specific machine learning models to identify CH driver mutations, showing their advantage with respect to expert-curated rules. These models can support the identification and clinical interpretation of CH mutations in newly sequenced individuals. See related commentary by Arends and Jaiswal, p. 1581., (©2024 The Authors; Published by the American Association for Cancer Research.)

Insulinomas are rare neuroendocrine tumors arising from pancreatic β cells, characterized by aberrant proliferation and altered insulin secretion, leading to glucose homeostasis failure. With the aim of uncovering the role of noncoding regulatory regions and their aberrations in the development of these tumors, we coupled epigenetic and transcriptome profiling with whole-genome sequencing. As a result, we unraveled somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development, and epigenetic modifying genes, including polycomb complex components. Chromatin remodeling is apparent in insulinoma-selective domains shared across patients, containing a specific set of regulatory sequences dominated by the SOX17 binding motif. Moreover, many of these regions are H3K27me3 repressed in β cells, suggesting that tumoral transition involves derepression of polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements affecting the function and fate of β cells in their progression to insulinomas and a framework to identify coding and noncoding driver mutations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pediatric cancers are rare diseases, and children without known germline predisposing conditions who develop a second malignancy during developmental ages are extremely rare. We present four such clinical cases and, through whole-genome and error-correcting ultra-deep duplex sequencing of tumor and normal samples, we explored the origin of the second malignancy in four children, uncovering different routes of development. The exposure to cytotoxic therapies was linked to the emergence of a secondary acute myeloid leukemia. A common somatic mutation acquired early during embryonic development was the driver of two solid malignancies in another child. In two cases, the two tumors developed from completely independent clones diverging during embryogenesis. Importantly, we demonstrate that platinum-based therapies contributed at least one order of magnitude more mutations per day of exposure than aging to normal tissues in these children., Significance: Using whole-genome and error-correcting ultra-deep duplex sequencing, we uncover different origins for second neoplasms in four children. We also uncover the presence of platinum-related mutations across 10 normal tissues of exposed individuals, highlighting the impact that the use of cytotoxic therapies may have on cancer survivors. See related commentary by Pacyna and Nangalia, p. 900. This article is featured in Selected Articles from This Issue, p. 897., (©2024 The Authors; Published by the American Association for Cancer Research.)

DNA base damage is a major source of oncogenic mutations 1 . Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation 2 . Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication 3,4 , we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts 5 . The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution., (© 2024. The Author(s).)

Resistance to MAPK inhibitors (MAPKi), the main cause of relapse in BRAF-mutant melanoma, is associated with the production of alternative BRAF mRNA isoforms (altBRAFs) in up to 30% of patients receiving BRAF inhibitor monotherapy. These altBRAFs have been described as being generated by alternative pre-mRNA splicing, and splicing modulation has been proposed as a therapeutic strategy to overcome resistance. In contrast, we report that altBRAFs are generated through genomic deletions. Using different in vitro models of altBRAF-mediated melanoma resistance, we demonstrate the production of altBRAFs exclusively from the BRAF V600E allele, correlating with corresponding genomic deletions. Genomic deletions are also detected in tumor samples from melanoma and breast cancer patients expressing altBRAFs. Along with the identification of altBRAFs in BRAF wild-type and in MAPKi-naive melanoma samples, our results represent a major shift in our understanding of mechanisms leading to the generation of BRAF transcripts variants associated with resistance in melanoma., Competing Interests: Declaration of interests F.A. received honoraria from MSD and Novartis. J.V. is a member of the scientific advisory boards of Remix Therapeutics, Stoke Therapeutics and IntronX., (Copyright © 2024 Center for Genomic Regulation. Published by Elsevier Inc. All rights reserved.)

Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines., (© 2024. The Author(s).)

The COVID-19 pandemic has brought significant changes and advances in the field of vaccination, including the implementation and widespread use of encapsidated mRNA vaccines in general healthcare practice. Here, we present two new mRNAs expressing antigenic parts of the SARS-CoV-2 spike protein and provide data supporting their functionality. The first mRNA, called RBD-mRNA, encodes a trimeric form of the virus spike protein receptor binding domain (RBD). The other mRNA, termed T-mRNA, codes for the relevant HLA I and II spike epitopes. The two mRNAs (COVARNA mRNAs) were designed to be used for delivery to cells in combination, with the RBD-mRNA being the primary source of antigen and the T-mRNA working as an enhancer of immunogenicity by supporting CD4 and CD8 T-cell activation. This innovative approach substantially differs from other available mRNA vaccines, which are largely directed to antibody production by the entire spike protein. In this study, we first show that both mRNAs are functionally transfected into human antigen-presenting cells (APCs). We obtained peripheral blood mononuclear cell (PBMC) samples from three groups of voluntary donors differing in their immunity against SARS-CoV-2: non-infected (naïve), infected-recovered (convalescent), and vaccinated. Using an established method of co-culturing autologous human dendritic cells (hDCs) with T-cells, we detected proliferation and cytokine secretion, thus demonstrating the ability of the COVARNA mRNAs to activate T-cells in an antigen-specific way. Interestingly, important differences in the intensity of the response between the infected-recovered (convalescent) and vaccinated donors were observed, with the levels of T-cell proliferation and cytokine secretion (IFNγ, IL-2R, and IL-13) being higher in the vaccinated group. In summary, our data support the further study of these mRNAs as a combined approach for future use as a vaccine.

Clonal hematopoiesis (CH) is a phenomenon of clonal expansion of hematopoietic stem cells driven by somatic mutations affecting certain genes. Recently, CH has been linked to the development of a number of hematologic malignancies, cardiovascular diseases and other conditions. Although the most frequently mutated CH driver genes have been identified, a systematic landscape of the mutations capable of initiating this phenomenon is still lacking. Here, we train high-quality machine-learning models for 12 of the most recurrent CH driver genes to identify their driver mutations. These models outperform an experimental base-editing approach and expert-curated rules based on prior knowledge of the function of these genes. Moreover, their application to identify CH driver mutations across almost half a million donors of the UK Biobank reproduces known associations between CH driver mutations and age, and the prevalence of several diseases and conditions. We thus propose that these models support the accurate identification of CH across healthy individuals., Competing Interests: The authors declare no potential conflicts of interest.

Genetic information has been crucial to understand the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL) at diagnosis and at relapse, but still nowadays has a limited value in a clinical context. Few genetic markers are associated with the outcome of T-ALL patients, independently of measurable residual disease (MRD) status after therapy. In addition, the prognostic relevance of genetic features may be modulated by the specific treatment used. We analyzed the genetic profile of 145 T-ALL patients by targeted deep sequencing. Genomic information was integrated with the clinicalbiological and survival data of a subset of 116 adult patients enrolled in two consecutive MRD-oriented trials of the Spanish PETHEMA (Programa Español de Tratamientos en Hematología) group. Genetic analysis revealed a mutational profile defined by DNMT3A/ N/KRAS/ MSH2/ U2AF1 gene mutations that identified refractory/resistant patients. Mutations in the DMNT3A gene were also found in the non-leukemic cell fraction of patients with T-ALL, revealing a possible mutational-driven clonal hematopoiesis event to prime T-ALL in elderly. The prognostic impact of this adverse genetic profile was independent of MRD status on day +35 of induction therapy. The combined worse-outcome genetic signature and MRD on day +35 allowed risk stratification of T-ALL into standard or high-risk groups with significantly different 5- year overall survival (OS) of 52% (95% confidence interval: 37-67) and 17% (95% confidence interval: 1-33), respectively. These results confirm the relevance of the tumor genetic profile in predicting patient outcome in adult T-ALL and highlight the need for novel gene-targeted chemotherapeutic schedules to improve the OS of poor-prognosis T-ALL patients.

Richter transformation (RT) is a paradigmatic evolution of chronic lymphocytic leukemia (CLL) into a very aggressive large B cell lymphoma conferring a dismal prognosis. The mechanisms driving RT remain largely unknown. We characterized the whole genome, epigenome and transcriptome, combined with single-cell DNA/RNA-sequencing analyses and functional experiments, of 19 cases of CLL developing RT. Studying 54 longitudinal samples covering up to 19 years of disease course, we uncovered minute subclones carrying genomic, immunogenetic and transcriptomic features of RT cells already at CLL diagnosis, which were dormant for up to 19 years before transformation. We also identified new driver alterations, discovered a new mutational signature (SBS-RT), recognized an oxidative phosphorylation (OXPHOS) high -B cell receptor (BCR) low -signaling transcriptional axis in RT and showed that OXPHOS inhibition reduces the proliferation of RT cells. These findings demonstrate the early seeding of subclones driving advanced stages of cancer evolution and uncover potential therapeutic targets for RT., (© 2022. The Author(s).)

In celebration of the 20th anniversary of Nature Reviews Genetics, we asked 12 leading researchers to reflect on the key challenges and opportunities faced by the field of genetics and genomics. Keeping their particular research area in mind, they take stock of the current state of play and emphasize the work that remains to be done over the next few years so that, ultimately, the benefits of genetic and genomic research can be felt by everyone.