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Salazar, R.

Publications and source records attributed to Salazar, R..

14 recordsLinked to original sources

Pharmacologic decoupling of IRBC activation from anabolic collapse redefines ribosome biogenesis inhibition as a selective tumor suppressive strategy

Ribosome biogenesis (Ri-Bi) is widely targeted in cancer therapy, yet its inhibition is generally viewed as a broadly anti-anabolic intervention. In colorectal cancer, frontline treatments such as FOLFOX partly disrupt Ri-Bi, eliciting two biologically distinct outputs: an early p53-dependent checkpoint activation, known as the impaired ribosome biogenesis checkpoint (IRBC), and a later global anti-anabolic collapse associated with toxicity and limited durability. At clinically relevant doses, these outputs have been considered pharmacologically inseparable. Here we demonstrate that Ri-Bi inhibition can be functionally dissociated and selectively tuned toward checkpoint engagement. Using a genome-engineered Venus-RPL11 reporter and TP53 isogenic colorectal cancer models, we show that combining sub-effective doses of mechanistically distinct Ri-Bi inhibitors reprograms the cellular response toward dominant IRBC-mediated p53 activation while minimizing p53-independent cytotoxicity. This dose architecture induces profound growth suppression exclusively in TP53-proficient cells and prevents adaptive outgrowth during prolonged treatment. Importantly, pharmacologic rescue of mutant p53 (R175H) with arsenic trioxide restores IRBC responsiveness, extending this framework to genetically advanced disease. Together, our findings establish that ribosome biogenesis inhibition can be selectively directed toward nucleolar surveillance activation, redefining Ri-Bi targeting as a checkpoint-based therapeutic principle.

cancer biology↗

LARP1 integrates MYC and mTOR signaling to enable anabolic growth during tumor initiation

Tumor initiation requires the integration of oncogenic signals with environmental cues to enable anabolic growth. Among oncogenic drivers, MYC is central to tumorigenesis, with its deregulation observed in more than 60% of human cancers. Oncogenic MYC profoundly rewires transcription, enabling cells to bypass cell cycle checkpoints and resetting metabolism. A cornerstone of this rewiring is the up regulation of biomass-producing pathways, particularly ribosome biogenesis. How and when MYC oncogenic program is translationally executed, either immediately or until a favorable metabolic context emerges, remains a central unanswered question in tumor initiation, limiting our understanding of tumor latency and early intervention windows. Here, we identify the RNA-binding protein LARP1 as a critical effector of MYC-driven transformation, connecting MYC oncogenic activity with mTOR signaling. Mechanistically MYC represses miR-26a/b, relieving post-transcriptional repression of LARP1 and leading to its up regulation. LARP1 associates with the translational machinery loading it with the anabolic translatome induced by MYC in a translationally poised state. Upon permissive mTOR signaling, and dependent on the phosphorylation of LARP1 at serines 689 and 697, this program is rapidly translated, fueling the biosynthetic processes essential for tumor development. Importantly, genetic deletion of LARP1 or pharmacological mTOR inhibition completely abrogates tumor initiation in a genetically engineered colorectal organoid model of MYC-driven tumorigenesis, underscoring the physiological relevance of this two-step mechanism. These findings reveal a fundamental mechanism underlying MYC oncogenesis, whereby LARP1 bridges the anabolic translatome primed by MYC with its metabolic execution controlled by mTOR. By temporally uncoupling transformation from metabolic permissiveness, this mechanism defines a critical checkpoint in early tumorigenesis and reveals a potential vulnerability for intercepting MYC-driven cancer before biomass expansion.

cancer biology↗

The Blueprint for Survival: The Blue Dasher Dragonfly as a Model for Urban Adaptation

Human alteration of natural environments and habitats is a major driver of species decline. However, a handful of species thrive in human altered environments. The biology, distribution, population structure, and molecular adaptations enabling certain species to thrive in human-altered habitats are not well understood. Here, we evaluate the population and functional genomics, ecological niche and distributions, and geometric morphometrics of the blue dasher (Pachydiplax longipennis), one of the most ubiquitously observed insects in human altered habitats. Using resequencing data we identify a number of genes involved with the success of the blue dasher in human altered habitats, including loci contributing to immune function and response to oxidative stress. Some genes related to these functions are found in regions of strong population structure, while others are not, potentially indicating both regional and widespread adaptations to urban environments within this species. Using one of the most robust locality datasets for any species to date, we also generate habitat suitability predictions which show that P. longipennis has spread with urbanization, suggesting humans have created suitable habitat for this species. These results complement morphological and genomic data showing P. longipennis (particularly East of the Rocky Mountains) has the capacity to rapidly disperse to newly suitable habitats. Given the shared barriers to colonizing an urban habitat, we expect that many of the adaptations we have identified in P. longipennis can be used for predicting what animals might succeed in urban habitats more generally.

evolutionary biology↗

Genomic Characterization of a Dog-Mediated Rabies Outbreak in El Pedregal, Arequipa, Peru

BackgroundRabies, a re-emerging zoonosis with the highest known human case fatality rate, has been largely absent from Peru, except for endemic circulation in the Puno region on the Bolivian border and re-emergence in Arequipa City in 2015, where it has persisted. In 2021, an outbreak occurred in the rapidly expanding city of El Pedregal near Arequipa, followed by more cases in 2022 after nearly a year of epidemiological silence. While currently under control, questions persist regarding the origin of the El Pedregal outbreak and implications for maintaining rabies control in Peru. MethodsWe sequenced 25 dog rabies virus (RABV) genomes from the El Pedregal outbreak (n=11) and Arequipa City (n=14) from 2021-2023 using Nanopore sequencing in Peru. Historical genomes from Puno (n=4, 2010-2012) and Arequipa (n=5, 2015-2019), were sequenced using an Illumina approach in the UK. In total, 34 RABV genomes were analyzed, including archived and newly obtained samples. The genomes were analyzed phylogenetically to understand the outbreaks context and origins. ResultsPhylogenomic analysis identified two genetic clusters in El Pedregal: 2021 cases stemmed from a single introduction unrelated to Arequipa cases, while the 2022 sequence suggested a new introduction from Arequipa rather than persistence. In relation to canine RABV diversity in Latin America, all new sequences belonged to a new minor clade, Cosmopolitan Am5, sharing relatives from Bolivia, Argentina, and Brazil. ConclusionGenomic insights into the El Pedregal outbreak revealed multiple introductions over a 2-year window. Eco-epidemiological conditions, including migratory worker patterns, suggest human-mediated movement drove introductions. Despite outbreak containment, El Pedregal remains at risk of dog-mediated rabies due to ongoing circulation in Arequipa, Puno, and Bolivia. Human-mediated movement of dogs presents a major risk for rabies re-emergence in Peru, jeopardizing regional dog-mediated rabies control. Additional sequence data is needed for comprehensive phylogenetic analyses.

genomics↗

ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart

BackgroundMetabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear. MethodsWe utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate Acly in cardiomyocytes of MyH6-Cas9 mice. In vivo, positron emission tomography and ex vivo stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level. ResultsHere, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. Ex vivo isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate. ConclusionsThis study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.

molecular biology↗

hkb is required for DIP-α expression and target recognition in the Drosophila neuromuscular circuit

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins (CSPs). In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth and cell survival. However, the upstream regulation and downstream signaling mechanisms of Dprs and DIPs are not clear. In the Drosophila larval neuromuscular system, DIP- is expressed in the dorsal and ventral type-Is motor neurons (MNs). We conducted an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We found that the transcription factor, huckebein (hkb), genetically interacts with DIP- and is important for target recognition specifically in the dorsal Is MN, but not the ventral Is MN. Loss of hkb led to complete removal of DIP- expression. We then confirmed that this specificity is through the dorsal Is MN specific transcription factor, even-skipped (eve), which acts downstream of hkb. Genetic interaction between hkb and eve revealed that they act in the same pathway to regulate dorsal Is MN connectivity. Our study provides insight into the transcriptional regulation of DIP- and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons.

neuroscience↗

S100A1ct: a synthetic peptide derived from human S100A1 protein improves cardiac contractile performance and survival in pre-clinical heart failure models

BackgroundThe EF-hand Ca2+ sensor protein S100A1 has been identified as a molecular regulator and enhancer of cardiac performance. S100A1s ability to recognize and modulate the activity of targets such as SERCA2a and RyR2 in cardiomyocytes has mostly been ascribed to its hydrophobic C-terminal -helix (residues 75-94). Objective: We therefore hypothesized that a synthetic peptide consisting of residues 75-94 of S100A1 and an N-terminal solubilization tag (S100A1ct) could mimic the performance enhancing effects of S100A1 and may be suitable as a peptide therapeutic to improve the function of diseased hearts. Methods and Results: Applying an integrative translational research pipeline, ranging from computational molecular modeling to large animal cardiac disease models, we characterize S100A1ct as a cell-penetrating peptide with positive inotropic and antiarrhythmic properties in normal and failing myocardium in vitro and in vivo. This activity translates into improved contractile performance and survival in pre-clinical heart failure models with reduced ejection fraction after S100A1ct systemic administration. Mechanistically, S100A1ct exerts a fast and sustained dose-dependent enhancement of cardiomyocyte Ca2+ cycling and prevents {beta}-adrenergic receptor triggered Ca2+ imbalances by targeting SERCA2a and RyR2 activity. Modeling suggests that S100A1ct may stimulate SERCA2a by interacting with the sarcoplasmic transmembrane segments of the multi-span integral membrane Ca2+ pump. Incorporation of a cardiomyocyte targeting peptide tag into S100A1ct (cor-S100A1ct) further enhanced its biological and therapeutic potency in vitro and in vivo. Conclusion: S100A1ct peptide is a promising lead for the development of a novel peptide-based therapeutic against heart failure with reduced ejection fraction.

pharmacology and toxicology↗

Paradoxical activation of oncogenic signaling as a cancer treatment strategy

Cancer homeostasis depends on a balance between activated oncogenic pathways driving tumorigenesis and engagement of stress-response programs that counteract the inherent toxicity of such aberrant signaling. While inhibition of oncogenic signaling pathways has been explored extensively, there is increasing evidence that overactivation of the same pathways can also disrupt cancer homeostasis and cause lethality. We show here that inhibition of Protein Phosphatase 2A (PP2A) hyperactivates multiple oncogenic pathways and engages stress responses in colon cancer cells. Genetic and compound screens identify combined inhibition of PP2A and WEE1 as synergistic in multiple cancer models by collapsing DNA replication and triggering premature mitosis followed by cell death. This combination also suppressed the growth of patient-derived tumors in vivo. Remarkably, acquired resistance to this drug combination suppressed the ability of colon cancer cells to form tumors in vivo. Our data suggest that paradoxical activation of oncogenic signaling can result in tumor suppressive resistance.

cancer biology↗

MmCMS: Mouse models' Consensus Molecular Subtypes of colorectal cancer

BACKGROUNDColorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODSUsing transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n=577) and mouse (n=57 across n=8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTSWe developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONSWhen applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package with three options helps researchers select suitable mouse models of human CRC subtype for their experimental testing.

cancer biology↗

Discovery of an unusual high number of de novo mutations in sperm of older men using duplex sequencing

De novo mutations (DNMs) are an important player in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultra-deep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined [~]4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant frequencies of 10-4 to 10-5, with an overall mutation frequency of the region of [~]6x10-7. Some of the substitutions were re-current and were found at a higher variant frequency in older donors than in younger ones, or exclusively, in older donors. Also, older donors harbored more mutations associated with congenital disorders. Other mutations were present in both age groups suggesting that these might result from a different mechanism (e.g., post-zygotic mosaicism). We also observed that independent of age, the frequency and deleteriousness of the mutational spectra was more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain-of-function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

genetics↗

Chemotherapy induces a YAP1-dependent fetal conversion to human Colorectal Cancer cells that is predictive of poor patient outcome

Current therapy against colorectal cancer is based on DNA-damaging agents that eradicate highly proliferative malignant cells. Whether sublethal chemotherapy affects tumor cell behavior and impacts on patient outcome is primarily unstudied. We now show that sublethal chemotherapy imposes a quiescent-like state to p53 wildtype human colorectal cancer (CRC) cells that is linked to the acquisition of a fetal phenotype downstream of YAP1, similar to that observed after intestinal damage. CRC cells displaying this fetal phenotype exhibit tumor- initiating activity comparable to untreated cells but superior metastatic capacity. Notably, nuclear YAP1 accumulation, or detection of the fetal signature in tumors predict poor prognosis in CRC patients carrying p53 wildtype tumors. Collectively, our results uncover a potential adverse response of tumor cells to suboptimal chemotherapy, and identify nuclear YAP1 and fetal conversion of colorectal tumors as biomarkers for prognosis and therapy prescription. Statement of significanceChemotherapy induces a quiescent-like phenotype to colorectal cancer cells that is linked to the acquisition of a YAP1-dependent fetal signature. Notably, this signature is predictive of patient outcome in different cohorts of human colorectal cancer.

cancer biology↗

The claustrum-medial prefrontal cortex network controls attentional set-shifting

In various mental disorders, dysfunction of the prefrontal cortex contributes to cognitive deficits. Here we studied how the claustrum (CLA), a nucleus sharing reciprocal connections with the cortex, may participate in these cognitive impairments. We show that specific ensembles of CLA and of medial prefrontal cortex (mPFC) neurons are activated during a task requiring cognitive control such as attentional set-shifting, i.e. the ability to shift attention towards newly relevant stimulus-reward associations while disengaging from irrelevant ones. CLA neurons exert a direct excitatory input on mPFC pyramidal cells, and chemogenetic inhibition of CLA neurons suppresses the formation of specific mPFC assemblies during attentional set-shifting. Furthermore, impairing the recruitment of specific CLA assemblies through opto/chemogenetic manipulations prevents attentional set-shifting. In conclusion, we propose that the CLA controls the reorganization of mPFC ensembles to enable attentional set-shifting, emphasizing a potential role of the CLA-mPFC network in attentional dysfunctions.

neuroscience↗

Increased yields of duplex sequencing data by a series of quality control tools

Duplex sequencing is currently the most reliable method to identify ultra-low frequency DNA variants by grouping sequence reads derived from the same DNA molecule into families with information on the forward and reverse strand. However, only a small proportion of reads are assembled into duplex consensus sequences, and reads with potentially valuable information are discarded at different steps of the bioinformatics pipeline, especially reads without a family. We developed a bioinformatics tool-set that analyses the tag and family composition with the purpose to understand data loss and implement modifications to maximize the data output for the variant calling. Specifically, our tools show that tags contain PCR and sequencing errors that contribute to data loss and lower DCS yields. Our tools also identified chimeras, which result in unpaired families that do not form DCS. Finally, we also developed a tool called Variant Analyzer that re-examines variant calls from raw reads and provides different summary data that categorizes the confidence level of a variant call by a tier-based system. We demonstrate that this tool identified false positive variants tagged by the tier-based classification. Furthermore, with this tool we can include reads without a family and check the reliability of the call, which increases substantially the sequencing depth for variant calling, a particular important advantage for low-input samples or low-coverage regions.

genomics↗

Family reunion via error correction: An efficient analysis of duplex sequencing data

Duplex sequencing is the most accurate approach for identification of sequence variants present at very low frequencies. Its power comes from pooling together multiple descendants of both strands of original DNA molecules, which allows distinguishing true nucleotide substitutions from PCR amplification and sequencing artifacts. This strategy comes at a cost--sequencing the same molecule multiple times increases dynamic range but significantly diminishes coverage, making whole genome duplex sequencing prohibitively expensive. Furthermore, every duplex experiment produces a substantial proportion of singleton reads that cannot be used in the analysis and are, technically, thrown away. In this paper we demonstrate that a significant fraction of these reads contains PCR or sequencing errors within duplex tags. Correction of such errors allows \"reuniting\" these reads with their respective families increasing the output of the method and making it more cost effective. Additionally, we combine error correction strategy with a number of algorithmic improvements in a new version of the duplex analysis software, Du Novo 2.0, readily available through Galaxy, Bioconda, and as the source code.

bioinformatics↗