Biology subjects

Slack, F. J.

Publications and source records attributed to Slack, F. J..

9 recordsLinked to original sources

bioRxiv2026

Tumor suppressor-like mutations mark a dosage-constrained dependency in leukemia

Cancer genes are classically categorized as oncogenes or tumor suppressors based on whether gain or loss of function promotes malignancy. Here we show that oncogene-imposed dosage constraints can produce mutations that mimic tumor suppressor loss yet mark essential genes. In acute promyelocytic leukemia (APL), the transcription factor EBF3 gene harbors recurrent heterozygous loss-of-function mutations, a tumor suppressor signature, and is essential for APL survival. We resolve this paradox by showing that while PML::RARA, the fusion oncoprotein defining APL, drives EBF3 expression, excess EBF3 suppresses proliferation. Patient mutations reduce EBF3 activity through heterozygous inactivation, optimizing its expression within a corridor bounded by fitness constraints at both extremes. PML::RARA activates EBF3 via a p300-dependent intronic enhancer, and p300 inhibition exploits this constraint by suppressing EBF3 below the viability threshold. Our findings reveal that oncogenes can impose dosage constraints that drive adaptive mutations conventionally misclassified as tumor suppressor loss, with implications for interpreting cancer genomes.

cancer biology↗

bioRxiv2026

African Green Monkey Cerebrospinal Fluid miRNome Captures Conserved miRNAs Relevant to Human Neurodegenerative Disease

BackgroundThe African green monkey (AGM) is increasingly used as a model for early-stage Alzheimers disease (AD), with cerebrospinal fluid (CSF) targeted for biomarker discovery and longitudinal disease monitoring of shifts in the central nervous system. MicroRNAs (miRNAs) are particularly informative indicators of early neuropathological change. Despite the complementary value of an early-stage disease model and a molecular marker capable of capturing early change, the miRNA composition (miRNome) of AGM remains undefined. We established the AGM CSF miRNome from antemortem samples using miRNA sequencing and a qRT-PCR-based array. We also developed a hierarchical annotation pipeline to classify miRNAs as either family-conserved or unclassified and to assess sequence alignment across humans and other species. ResultsWe used untargeted miRNA sequencing to characterize the AGM CSF miRNome and identified 205 miRNAs that could be classified into three family-conserved categories: canonical, noncanonical, and 3'-terminal variants. Of these, 150 were also detected using a human-targeted qRT-PCR array, providing independent support for the sequence-derived miRNome. Sequencing abundance and qRT-PCR array Ct values showed significant cross-platform concordance overall, although concordance was lower for 3'-terminal isomiRs than for canonical miRNAs. Comparison with human GTEx tissue-expression data indicated that several human homologs of AGM CSF miRNAs exhibited brain-preferential expression. Notably, predicted targets of many of these miRNAs were enriched for pathways implicated in neurodegenerative disease. Finally, we identified 20 unclassified candidates that could not be assigned to established miRNA families, two of which we propose as putatively novel miRNAs. ConclusionThe AGM CSF miRNome is substantially conserved with the human miRNome but also contains 3'-terminal isomiRs and unclassified miRNA candidates. AGM CSF contains miRNAs homologous to human miRNAs associated with AD and other neuropathologies, highlighting the translational potential of this model. However, our study also reveals challenges related to species-specific sequence variation and reduced cross-platform concordance for isomiRs. Thus, comparative studies will be needed to validate the functional and biomarker relevance of these miRNAs across species. More generally, this initial miRNome provides a reference resource for future studies of miRNAs in AGM across disease-related, physiological, experimental, and evolutionary contexts.

genomics↗

bioRxiv2026

MicroRNA correlates of resilience to Alzheimer's disease identify candidate therapeutic targets for neuroprotection

Many aged individuals accumulate advanced Alzheimers disease (AD) neuropathology without cognitive decline, signifying potent endogenous resilience mechanisms. Mimicking resilience to AD may offer new therapeutic opportunities by emulating endogenous neuroprotection, but the molecular pathways that underpin resilience remain largely unknown. MicroRNAs (miRNAs)--small noncoding RNA molecules that post-transcriptionally regulate gene expression--influence neuronal and glial processes associated with development, aging, and AD neurodegeneration but their role in resilience has not been characterized. We investigated resilience-associated miRNAs and their targeted programs in the dorsolateral prefrontal cortex of post-mortem human brains. We analyzed matched miRNA and messenger RNA (mRNA) expression across resilient, AD, and control subjects from the Religious Order Study and Memory and Aging Project (ROSMAP) cohort. Nine miRNAs were differentially expressed in resilience compared with AD, including previously unreported miR-362-3p and miR-433-3p and new resilience-associated roles for known AD-related miRNAs, including neuronal miR-132-3p and miR-129-5p. By analyzing miRNA activity across AD progression, we found additional cognitive-associated miRNAs (e.g., miR-335-5p and miR-19b) and one plaque-restricted miRNA (miR-199a-5p). Sex-specific miRNA dysregulation was observed: miR-7-5p showed male-specific upregulation in AD versus resilience and suggested sex-specific differences in AD patients. Integrated co-expression and target-enrichment analyses linked resilience-associated miRNAs to pathways that were associated with cognitive decline, including transforming growth factor {beta}, Rho guanosine triphosphatases, and neurotransmitter receptor regulation. We also report restricted co-activity in AD subjects for miR-362-3p with inflammatory pathways, including tumor necrosis factor and Toll-like receptor signaling. These results demonstrate systematic involvement of miRNAs across neuronal and glial programs of AD resilience, cognitive decline, and sex-specific regulation. Our study provides an important resource for discovery of actionable regulatory programs that could lead to new therapies, based on endogenous molecules, that emulate natural resilience to AD. Key pointsO_LINine distinct cortical microRNA (miRNA) signatures correlate with resilience to Alzheimers disease (AD)-related cognitive decline, pointing to endogenous regulatory programs that may help preserve cognitive function. C_LIO_LIAt the miRNA level, resilient brains show minimal or no detectable differences to healthy individuals, despite the profound pathological differences, supporting the idea that resilience reflects preserved molecular homeostasis rather than a separate disease state. C_LIO_LISome miRNA signatures are specific to a certain pathology (e.g., one linked to amyloid-{beta} plaques independently of tau pathology and cognitive decline), helping to disentangle the regulation of these processes. C_LIO_LISex-specific miRNA dysregulation in AD suggests that resilience-linked pathways may be regulated differently in males and females, reinforcing the value of sex-stratified analyses and the potential for differential therapeutic strategies. C_LIO_LIIntegrative miRNA-pathway analysis highlights candidate regulatory networks involved in inflammation, matrisome, and cellular stress responses, providing a framework for resilience-linked therapeutic targets. C_LI

neuroscience↗

bioRxiv2026

A microRNA atlas of the human prefrontal cortex across the adult lifespan

Aging of the human brain is characterized by widespread changes in gene expression regulated in part by microRNAs (miRNAs). We present a lifespan miRNA atlas of the human dorsolateral prefrontal cortex generated from small RNA sequencing of 113 postmortem samples spanning 18 to 100 years of age. Differential expression analysis revealed progressive age-associated remodeling of miRNA expression, with the strongest differences observed between old and young individuals. Among the significantly altered miRNAs, miR-34a-5p emerged as one of the most robustly upregulated miRNAs in the aged cortex, alongside additional aging-associated miRNAs including miR-155-5p, miR-132-3p, miR-212-3p, miR-449a, and members of the miR-302 family. This atlas provides a resource for investigating miRNA dysregulation and small RNA regulatory networks during human cortical aging.

neuroscience↗

bioRxiv2025

tRNA modifications are required for stress granule formation and melanoma metastasis

Metastasis is the leading cause of cancer related deaths, however therapies specifically targeting metastasis are lacking and remain a dire therapeutic need in the clinic. Metastasis is a highly inefficient process that is inhibited by extracellular stress. Therefore, metastasizing cells that ultimately survive and successfully colonize distant organs must undergo molecular rewiring to mitigate stress. Wobble uridine modifications, especially 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), have been implicated in stress response and poor prognosis of cancer patients. We use a patient derived xenograft (PDX) model of melanoma metastasis to study the role of the mcm5s2U34 modification in the stress response of metastasizing cells. We find that upon depletion of elongator acetyltransferase complex subunit 1 (ELP1)-- a component of the mcm5s2U34 pathway on [Formula], and [Formula] --codon-biased translation, migration, invasion, and metastatic burden in vivo is reduced. Further, we observe that stress granule components are enriched in a subset of codon-biased genes that are exclusively upregulated at the protein level in metastatic nodules compared to the primary tumor in our PDX model. Additionally, upon knockdown of ELP1, stress granule components have decreased protein expression with no significant change to their mRNA levels. Efficient translation, mediated by the carboxy-methylation arm of the mcm5s2U34 modification, is required for metastasizing cancer cells to withstand stress via stress granule formation and increase survival throughout the metastatic cascade. This makes the mcm5s2U34 machinery a potentially actionable therapeutic target, specific to metastatic disease.

cancer biology↗

bioRxiv2025

Host microRNA-31-5p represses oncogenic herpesvirus lytic reactivation by restricting the RNA-binding protein KHDRBS3-mediated viral gene expression

Oncogenic Kaposis sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposis sarcoma and primary effusion lymphoma, employs a biphasic life cycle consisting of latency and lytic replication to achieve lifelong infection. Despite its essential role in KSHV persistence and tumorigenicity, much remains unknown about how KSHV lytic reactivation is regulated. Leveraging high-throughput transcriptomics, we identify microRNA-31-5p (miR-31-5p) as a key regulator of KSHV lytic reactivation capable of restricting KSHV entry into the lytic replication cycle. Ectopic expression of miR-31-5p impairs KSHV lytic gene transcription and production of lytic viral proteins, culminating in dramatic reduction of infectious virion production during KSHV reactivation. miR-31-5p overexpression also markedly reduces the expression of critical viral early genes, including the master regulator of the latent-lytic switch, KSHV replication and transcription activator (RTA) protein. Through mechanistic studies, we demonstrate that miR-31-5p represses KSHV lytic reactivation by directly targeting the KH domain protein KHDRBS3, an RNA-binding protein known to regulate RNA processing including alternative splicing. Our study highlights KHDRBS3 as an essential proviral host factor that is key to the successful completion of KSHV lytic replication and suggests its novel function in viral lytic gene transcription during KSHV reactivation. Taken together, these findings reveal a previously unrecognized role for the miR-31-5p/KHDRBS3 axis in regulating the KSHV latency-lytic replication switch and provide insights into gene expression regulation of lytic KSHV, which may be leveraged for lytic cycle-targeted therapeutic strategies against KSHV-associated malignancies.

molecular biology↗

bioRxiv2024

Drug resistant pancreatic cancer cells exhibit altered biophysical interactions with stromal fibroblasts in imaging studies of 3D co-culture models

Interactions between tumor and stromal cells are well known to play a prominent roles in progression of pancreatic ductal adenocarcinoma (PDAC). As knowledge of stromal crosstalk in PDAC has evolved, it has become clear that cancer associated fibroblasts can play both tumor promoting and tumor suppressive roles through a combination of paracrine crosstalk and juxtacrine interactions involving direct physical contact. Another major contributor to dismal survival statistics for PDAC is development of resistance to chemotherapy drugs. Though less is known about how the acquisition of chemoresistance impacts upon tumor-stromal crosstalk. Here, we use 3D co-culture geometries to recapitulate juxtacrine interactions between epithelial and stromal cells. In particular, extracellular matrix (ECM) overlay cultures in which stromal cells (pancreatic stellate cells, or normal human fibroblasts) are placed adjacent to PDAC cells (PANC1), result in direct heterotypic cell adhesions accompanied by dramatic fibroblast contractility which leads to highly condensed macroscopic multicellular aggregates as detected using particle image velocimetry (PIV) analysis to quantify cell velocities over the course of time lapse movie sequences. To investigate how drug resistance impacts these juxtacrine interactions we contrast cultures in which PANC1 are substituted with a drug resistant subline (PANC1-OR) previously established in our lab. We find that heterotypic cell-cell interactions are highly suppressed in drug-resistant cells relative to the parental PANC1 cells. To investigate further we conduct RNA-seq and bioinformatics analysis to identify differential gene expression in PANC1 and PANC1-OR, which shows that negative regulation of cell adhesion molecules, consistent with increased epithelial mesenchymal transition (EMT), is also consistent with loss of hetrotypic cell-cell contact necessary for the contractile behavior observed in drug naive cultures. Overall these findings elucidate the role of drug-resistance in inhibiting an avenue of stromal crosstalk which is associated with tumor suppression and also help to establish cell culture conditions useful for further mechanistic investigation.

biophysics↗

bioRxiv2023

Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

COVID-19 remains a significant public health threat due to the ability of SARS-CoV-2 variants to evade the immune system and cause breakthrough infections. Although pathogenic coronaviruses such as SARS-CoV-2 and MERS-CoV lead to severe respiratory infections, how these viruses affect the chromatin proteomic composition upon infection remains largely uncharacterized. Here we used our recently developed integrative DNA And Protein Tagging (iDAPT) methodology to identify changes in host chromatin accessibility states and chromatin proteomic composition upon infection with pathogenic coronaviruses. SARS-CoV-2 infection induces TP53 stabilization on chromatin, which contributes to its host cytopathic effect. We mapped this TP53 stabilization to the SARS-CoV-2 spike and its propensity to form syncytia, a consequence of cell-cell fusion. Differences in SARS-CoV-2 spike variant-induced syncytia formation modify chromatin accessibility, cellular senescence, and inflammatory cytokine release via TP53. Our findings suggest that differences in syncytia formation alter senescence-associated inflammation, which varies among SARS-CoV-2 variants.

microbiology↗

bioRxiv2021

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.

molecular biology↗