Biology subjects

Alvarado, J.

Publications and source records attributed to Alvarado, J..

10 recordsLinked to original sources

bioRxiv2025

Development and Characterization of Triazole-Based WDR5 Inhibitors for the Treatment of Glioblastoma

Glioblastoma (GBM) cancer stem cells (CSCs) contribute to tumor recurrence, treatment resistance, and dismal clinical outcomes. Genetic and pharmacological evidence suggests that the nuclear scaffolding protein WD-repeat containing protein 5 (WDR5) is a therapeutic vulnerability of the CSC population. However, previously reported WDR5 inhibitors display low permeability and are unable to penetrate the blood-brain barrier (BBB), limiting their utility in GBM. Herein, we report the structure-guided development of a novel series of triazole-based WDR5 WIN-site inhibitors designed to increase passive brain penetration. We identified triazole-based WDR5 inhibitors that are potent, passively permeable, and in some cases more brain penetrant than other scaffolds. We phenotypically assessed our novel WDR5 inhibitors in a panel of patient-derived CSC models and uncovered unique WDR5-regulated metabolic genes in GBM. We also evaluated their antiproliferative activity against CSCs both in vitro and in vivo. Finally, to identify novel combination opportunities, we screened a 2,100-compound chemical probe library and identified that the ATAD2 inhibitor BAY-850 synergizes with WDR5 inhibitors to enhance CSC killing. Our work diversifies the chemical matter targeting WDR5, clarifies the in vitro consequences of WIN-site inhibition in CSCs, and encourages the future development of next-generation WDR5 inhibitors with the potential to achieve in vivo efficacy in the brain.

cancer biology↗

bioRxiv2025

A balance between nucleating and elongating actin filaments controls deformation of protein condensates

Protein condensates use multivalent binding and surface tension to assemble actin filaments into diverse architectures, reminiscent of filopodia and stress fibers. During this process, nucleation of new filaments and elongation of existing filaments inherently compete for a shared pool of actin monomers. Here we show that a balance between these competing processes is required to deform condensates of VASP, an actin binding protein, into structures with high aspect ratios. Addition of magnesium, which promotes filament nucleation, helped actin to deform condensates into high aspect ratio structures. In contrast, addition of profilin, which inhibits filament nucleation, allowing existing filaments to elongate, caused actin to assemble into ring-like bundles that failed to substantially increase condensate aspect ratio. Computational modeling helped to explain these results by predicting that a group of short linear filaments, which can apply asymmetric pressure to the condensate boundary, is needed to increase condensate aspect ratio. In contrast, a small number of long filaments with the same total actin content should fail to overcome the droplet surface tension, forming a ring-like bundle. To test these predictions, we introduced gelsolin, which severed log filaments within rings, creating new barbed ends. The resulting set of shorter filaments regained the ability to deform condensates into high aspect ratio structures. Collectively, these results suggest that a balance of actin filament nucleation and elongation is required to deform protein condensates. More broadly, these findings illustrate how protein condensates can balance multiple kinetic processes to direct the assembly of diverse cytoskeletal architectures.

biophysics↗

bioRxiv2025

The mutational and clonality profile of HGSOC is established early in tumor development and conserved throughout therapy resistance

High grade serous ovarian cancer (HGSOC) is the most lethal gynecologic malignancy, killing more than 9,000 women each year in the United States alone. Nearly 80% of patients with HGSOC tumors will experience recurrence within 5 years, but little is known about the mechanisms that drive this process. Intratumor heterogeneity is believed to be a key feature of recurrence and resistance in HGSOC tumors, with early studies reporting diverse and complex mechanisms of the seeding of metastatic sites, including metastasis reseeding the primary tumor. Few studies have investigated temporal changes to clonality and structural variants through disease recurrence and the development of chemoresistance as surgical debulking is not frequently performed at this later stage. We performed multi-omic profiling in paired chemo-naive and chemoresistant tumors from 32 HGSOC patients to investigate the mutational and genomic landscape of disease progression. Somatic mutation profiles were largely conserved through disease progression. Mutational burdens did not significantly differ across recurrence but were driven by homologous recombination repair deficiency status. Clonal composition and dynamics were measured through variant allele frequency alterations as tumors progressed from primary chemo-naive to recurrent chemo-resistant tumors. A novel candidate driver gene, MDC1, from the homologous recombination repair pathway, was significantly mutated and over-represented in patients with homologous recombination proficient tumors, with somatic mutations clustered in a single exon. Tumor evolution and phylogeny revealed that few changes in clonal abundance and complexity occur across the disease course in these patients. Taking structural variants into account, homologous recombination repair proficient (HRP) tumors tend to be polyclonal while homologous recombination repair deficient (HRD) tumors tend to be monoclonal, accompanied by a longer progression-free survival than the HRP patients. Three distinct classes of tumors were identified by structural variant signature analysis: tumors defined by DNA losses, tumors defined by DNA gains, and tumors defined by copy number neutral changes, which were largely defined by HRD status. Each class displayed distinct regions of the chromosome that were frequently affected by large scale SV events (>5Mb). Although no regions were frequently altered in recurrent tumors, GO analysis revealed that recurrent tumors have a significantly reduced immune response, which was not seen in the primary tumors. Ultra long read sequencing validated a majority of the SVs identified in short read sequencing and identified additional SVs undetected by short reads. These analyses identify that the phenotype of high grade serous ovarian tumors as defined by mutation and clonality profiles is established early in disease development and remain largely unchanged through chemotherapy and recurrence. This, when considered with the significant inter-patient heterogeneity identified in HGSOC, demonstrates the need for personalized therapies based on tumor profiling.

genomics↗

bioRxiv2024

Integration of an invasive plant in hummingbird and flower mite networks is driven by ecological fitting and generalization

Most plant communities worldwide include exotic plants, which did not evolve with local organisms. The central goal of this study is to test if native organisms expanding to novel hosts are usually generalists or specialists. Here we studied new association between hummingbirds, flower mites and Musa velutina (Musaceae), an exotic plant native to Asia currently invading lowland forests in Costa Rica. Hummingbirds are pollinators, but flower mites feed on nectar without contributing to pollen transfer. Flower mites hitch rides on hummingbird beaks to colonize new flowers. To determine the original diet breadth of hummingbird and flower mite species, we assembled interaction networks including 33 native host plants, 14 hummingbird and 19 flower mite species. Transportation analyses show that flower mites colonization of native or exotic hosts is not constrained by hummingbird flight connections. We identified four hummingbird species visiting Musa velutina. DNA barcodes analyses identified only one species of flower mite colonizing flowers of M. velutina. All new associations with M. velutina involved generalist hummingbird and flower mite species. Musa velutina displays both male and female flowers. Although flowers of both sexes were equally visited by hummingbirds, mites were 15 times more abundant in male than in female flowers. This might be the result of constant immigration coupled with population growth. Only half of the mites hitching rides on hummingbird beaks emigrate to newly open flowers. Our results show that M. velutina integration to a plant community occurs mainly by establishing interactions with generalists.

ecology↗

bioRxiv2024

Inbreeding and gallbladder cancer risk: Homozygosity associations adjusted for indigenous American ancestry, BMI and genetic risk of gallstone disease

Latin Americans have a rich genetic make-up that translates into heterogeneous fractions of the autosomal genome in runs of homozygosity (FROH), and heterogeneous types and proportions of indigenous American ancestry. While autozygosity has been linked to several human diseases, very little is known about the relationship between inbreeding, genetic ancestry and cancer risk in Latin Americans. Chile has one of the highest incidences of gallbladder cancer (GBC) in the world, and here we investigated the association between inbreeding, GBC, gallstone disease (GSD) and body mass index (BMI) in 4029 genetically admixed Chileans. We calculated individual FROH above 1.5 Mb and weighted polygenic risk scores for GSD, and applied multiple logistic regression to assess the association between homozygosity and GBC risk. We found that homozygosity was due to a heterogeneous mixture of genetic drift and consanguinity in the study population. Although we found no association between homozygosity and overall GBC risk, we detected interactions between FROH and sex, age, and genetic risk of GSD on GBC risk. Specifically, the increase in GBC risk per 1% FROH was 19% in men (P-value = 0.002), 30% in those under 60 years of age (P-value = 0.001), and 12% in those with a genetic risk of GSD above the median (P-value = 0.01). The present study highlights the complex interplay between inbreeding, genetic ancestry and genetic risk of GSD in the development of GBC. The applied methodology and our findings underscore the importance of considering the population-specific genetic architecture, along with sex- and age specific-effects, when investigating the genetic basis of complex traits in Latin Americans.

genetics↗

bioRxiv2023

New tools reveal PCP-dependent polarized mechanics in the cortex and cytoplasm of single cells during convergent extension

Understanding biomechanics of biological systems is crucial for unraveling complex processes like tissue morphogenesis. However, current methods for studying cellular mechanics in vivo are limited by the need for specialized equipment and often provide limited spatiotemporal resolution. Here we introduce two new techniques, Tension by Transverse Fluctuation (TFlux) and in vivo microrheology, that overcome these limitations. They both offer time-resolved, subcellular biomechanical analysis using only fluorescent reporters and widely available microscopes. Employing these two techniques, we have revealed a planar cell polarity (PCP)-dependent mechanical gradient both in the cell cortex and the cytoplasm of individual cells engaged in convergent extension. Importantly, the non-invasive nature of these methods holds great promise for its application for uncovering subcellular mechanical variations across a wide array of biological contexts. SummaryNon-invasive imaging-based techniques providing time-resolved biomechanical analysis at subcellular scales in developing vertebrate embryos.

developmental biology↗

bioRxiv2022

PCP and Septins govern the polarized organization and mechanics of the actin cytoskeleton during convergent extension.

Actin is generally required for cell movement but must be brought under the control of distinct developmental systems to drive specific morphogenetic processes, such as convergent extension (CE)1. This evolutionarily conserved collective cell movement elongates the body axis of all vertebrates and is governed by the Planar Cell Polarity (PCP) signaling system 1,2. Here, we sought to understand the interplay of PCP and actin during Xenopus CE. Our data provide new insights into the organization and emergence of polarity in this network and reveal new roles for the actin organizing Septins 3, thereby shedding light on the mechanisms by which developmental signaling systems exploit the ubiquitous machinery of cells to drive morphogenesis.

developmental biology↗

bioRxiv2021

The WRAD complex represents a therapeutically exploitable target for cancer stem cells in glioblastoma

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1-family-mediated Lys4 methylation of histone H3, associated with positive regulation of transcription. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment diminished reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSC in vitro growth and self-renewal as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers. SignificanceIn this study, we perform an epigenetic-focused functional genomics screen in glioblastoma organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=135 HEIGHT=200 SRC="FIGDIR/small/461125v2_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@bc5c5eorg.highwire.dtl.DTLVardef@545a2borg.highwire.dtl.DTLVardef@1ebd3c9org.highwire.dtl.DTLVardef@19d5d30_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Virtual partition digital PCR for high precision chromosomal counting applications

Digital PCR (dPCR) is the gold standard analytical platform for rapid high precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platforms ability to address some clinical applications that require the simultaneous monitoring of 20 - 50 analytes per sample. Here we present Virtual Partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescent intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for non-invasive fetal aneuploidy testing. This demonstration assay - tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNA - is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers variance of chromosome ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding >98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultra-high-precision quantitation.

molecular biology↗

bioRxiv2020

Twinfilin1 controls lamellipodial protrusive activity and actin turnover during vertebrate gastrulation

The dynamic control of the actin cytoskeleton is a key aspect of essentially all animal cell movements. Experiments in single migrating cells and in vitro systems have provided an exceptionally deep understanding of actin dynamics. However, we still know relatively little of how these systems are tuned in cell-type specific ways, for example in the context of collective cell movements that sculpt the early embryo. Here, we provide an analysis of the actin severing and depolymerization machinery during vertebrate gastrulation, with a focus on Twinfilin1. We confirm previous results on the role of Twf1 in lamellipodia and extend those findings by linking Twf1, actin turnover, and cell polarization required for convergent extension during vertebrate gastrulation.

cell biology↗