Biology subjects

Rich, J.

Publications and source records attributed to Rich, J..

14 recordsLinked to original sources

bioRxiv2026

Adenosylhomocysteinase drives epigenetic and metabolic reprogramming in ZFTA-RELA ependymoma

Supratentorial ependymomas (ST-EPNs) are aggressive pediatric brain tumors predominantly driven by the recurrent ZFTA-RELA fusion, an oncogenic chromatin regulator that establishes an aberrant transcriptional program. Although metabolic pathways modulate epigenetic regulation by controlling the availability of metabolic cofactors, how ZFTA-RELA coordinates metabolic reprogramming with epigenetic regulation is not well understood. Here, we show that ZFTA-RELA-positive ependymomas exhibit enhanced methionine dependency, elevated adenosyl homocysteinase (AHCY) expression, and epigenetically control aspartate metabolism for tumor growth. Mechanistically, ZFTA-RELA upregulates AHCY, increasing cellular methylation potential which promotes H3K4me3-dependent expression of CAD, the first enzyme complex of de novo pyrimidine biosynthesis, while simultaneously enforcing H3K27me3 mediated repression of ASNS, which encodes asparagine synthetase. Through this coordinated epigenetic program, aspartate utilization is directed toward pyrimidine biosynthesis while endogenous asparagine production is limited. Restricting S-adenosylmethionine availability or genetically or pharmacologically inhibiting AHCY reverses these epigenetic states, leading to CAD downregulation, ASNS upregulation, impaired nucleotide synthesis, and suppression of tumor growth in vitro and in vivo. Similarly, EZH2 inhibition also restores ASNS expression and suppresses tumor proliferation. These findings suggest that ZFTA-RELA EPN maintain ASNS-low phenotype which conditionally make them dependent on extracellular asparagine. We further show that enhanced de novo pyrimidine synthesis not only supports nucleotide production but also provides UDP-sugars required for N-glycosylation and stabilization of the asparagine transporter SLC1A5, thereby promoting extracellular asparagine uptake and tumor proliferation. Collectively, our findings identify AHCY as a critical metabolic regulator of epigenetic and metabolic reprogramming that drives pyrimidine synthesis and establishes extracellular asparagine dependency, revealing actionable metabolic vulnerabilities in ZFTA-RELA-positive ST-EPNs.

cancer biology↗

bioRxiv2026

fastQpick: scalable bootstrap and subsampling of FASTQ reads

Summary: fastQpick is a command-line tool and Python library for sampling FASTQ reads with replacement. Sampling with replacement turns a single FASTQ file into an arbitrary number of bootstrap replicates, which enables uncertainty quantification and statistical analysis at the level of raw reads. This process answers questions such as how much an abundance estimate would change if the library were resequenced, or whether a low-abundance call is robust to the particular reads that were sequenced. fastQpick works efficiently on large libraries by streaming files in two passes by default: first to count reads and create a hash-based counter, and then to write the sample. It generates a full-size bootstrap replicate of a 500-million-read library in under 30 minutes with 9.4 GB of peak memory, with a low-memory mode that reduces the peak to 1.4 GB. A single-pass mode draws samples in a single read through the file, using O(1) working memory and producing an output size that is exact in expectation but not fixed. In a real yeast RNA-seq experiment, bootstrap replicates generated by fastQpick recover the sampling uncertainty of transcript abundance estimates, matching the analytic multinomial standard errors to within a few percent. Availability and implementationfastQpick is open source and freely available under the MIT license on GitHub at https://github.com/pachterlab/fastQpick and on PyPI (pip install fastQpick).

bioinformatics↗

bioRxiv2026

Single-Cell Genomics Decontamination with CellSweep

Single-cell genomics technologies enable high-throughput cell profiling, but technical contamination remains an obstacle to accurate downstream analysis. Free-floating ambient molecules released from lysed cells and global bulk contamination introduced during library preparation can distort molecular profiles. These artifacts can obscure cellular identities and reduce the reliability of differential analysis or clustering results. We present an efficient and effective approach to removing ambient and bulk contamination that can be applied to data generated from a wide variety of technologies. We show that our tool, CellSweep, outperforms other methods to remove artifacts using numerous benchmarks.

bioinformatics↗

bioRxiv2025

AAV Kills Dividing Cells by Depleting PARP1 and Other DNA Damage Response Proteins

Recombinant adeno-associated virus (rAAV) is a replication-defective viral vector used in hundreds of human gene therapy trials, resulting in five FDA-approved therapies. Despite this success, rAAV-based gene therapies suffer from dose-limiting toxicities, resulting in several severe adverse reactions, including death. Previously, we discovered that rAAV rapidly kills mouse NPCs in vitro and in vivo. This vector contains a minimal genome comprised of 145-base pair inverted terminal repeats (ITRs) with a T-shaped hairpin structure that appears to be necessary and sufficient for this toxicity. However, the mechanism for AAV ITR toxicity is not known, and there have been few attempts to engineer ITRs to attenuate rAAV toxicity. In the current study, we explore the molecular mechanisms that drive dose-dependent rAAV toxicity in dividing human NPCs (hNPCs) and test whether disrupting these mechanisms mitigates this toxicity. Recombinant AAV infection induces aberrant cell cycle progression with activation of the ATM /CHK1/CHK2 pathway and expression of the DNA damage markers {gamma}H2AX and 53BP1. Affinity-based proteomics indicate that AAV ITRs bind to Poly-(ADP-Ribose)polymerase 1 (PARP1) and other DNA damage response (DDR) proteins involved in single-strand break repair (SSBR). Recombinant AAV infection attenuates poly-(ADP-ribose) (PAR) formation and mimics the antiproliferative effects of pharmacological PARP inhibitors used in cancer therapy. Moreover, treatment of hNPCs with PARP inhibitors is sufficient to reproduce many features of rAAV-induced toxicity. Finally, we demonstrate that eliminating the T-shaped hairpin within the AAV ITR reduces binding to SSBR proteins and the resulting rAAV toxicity. These findings suggest that rAAV infection induces replication stress and cell death in dividing hNPCs by functionally depleting PARP1 and other DDR proteins that are essential for DNA replication. This work fills substantial gaps in the understanding of the mechanisms of rAAV toxicity and has important implications for the development of safer rAAV-based human gene therapies. One Sentence SummaryThe rAAV genome binds to and depletes PARP1 and other SSBR proteins that are essential for DNA replication, resulting in DNA double stranded breaks, checkpoint activation, and cell death in dividing cells.

cell biology↗

bioRxiv2025

Geospatially informed representation of spatial genomics data with SpatialFeatureExperiment

SpatialFeatureExperiment is a Bioconductor package that leverages the versatility of Simple Features for spatial data analysis and SpatialExperiment for single-cell -omics to provide an expansive and convenient S4 class for working with spatial -omics data. SpatialFeatureExperiment can be used to store and analyze a variety of spatial -omics data types, including data from the Visium, Xenium, MERFISH, SeqFish, and Slide-seq platforms, bringing spatial operations to the SingleCellExperiment ecosystem.

bioinformatics↗

bioRxiv2023

Timp2 loss-of-function mutation and TIMP2 treatment in murine model of NSCLC: modulation of immunosuppression and oncogenic signaling

Mounting evidence suggests that the tissue inhibitor of metalloproteinases-2 (TIMP2) can reduce tumor burden and metastasis. However, the demonstration of such anti-tumor activity and associated mechanisms using in vivo tumor models is lacking. The effects of a Timp2 functional mutation and administration of recombinant TIMP2 were examined in both orthotopic and heterotopic murine models of lung cancer using C57Bl/6 syngeneic Lewis Lung 2-luciferase 2 cells (LL2-luc2) cells. Mice harboring a functional mutation of TIMP2 (mT2) display markedly increased primary lung tumor growth, increased mortality, enriched vasculature, and enhanced infiltration of pro-tumorigenic, immunosuppressive myeloid cells. Treatment with recombinant TIMP2 reduced primary tumor growth in both mutant and wild-type (wt) mice. Comparison of transcriptional profiles of lung tissues from tumor-free, wt versus mT2 mice reveals only minor changes. However, lung tumor-bearing mice of both genotypes demonstrate significant genotype-dependent changes in gene expression following treatment with TIMP. In tumor-bearing wt mice, TIMP2 treatment reduced the expression of upstream oncogenic mediators, whereas treatment of mT2 mice resulted in an immunomodulatory phenotype. A heterotopic subcutaneous model generating metastatic pulmonary tumors demonstrated that daily administration of recombinant TIMP2 significantly downregulates the expression of heat shock proteins, suggesting a reduction of cell-stress responses. In summary, we describe how TIMP2 exerts novel, anti-tumor effects in a murine model of lung cancer and that rTIMP2 treatment supports a normalizing effect on the tumor microenvironment. Our findings show that TIMP2 treatment demonstrates significant potential as an adjuvant in the treatment of NSCLC. One Sentence SummaryTIMP2 treatment compensates for TIMP2 mutation to reveal immunomodulatory and oncogene regulation that inhibits tumor growth and metastatic niche formation.

cancer biology↗

bioRxiv2023

Targeted proteomic quantitation of NRF2 signaling and predictive biomarkers in HNSCC

The NFE2L2/NRF2 oncogene and transcription factor drives a gene expression program that promotes cancer progression, metabolic reprogramming, immune evasion and chemoradiation resistance. Patient stratification by NRF2 activity may guide treatment decisions to improve outcome. Here, we developed a mass spectrometry (MS)-based targeted proteomics assay based on internal standard triggered parallel reaction monitoring (IS-PRM) to quantify 69 NRF2 pathway components and targets as well as 21 proteins of broad clinical significance in head and neck squamous cell carcinoma (HNSCC). We improved the existing IS-PRM acquisition algorithm, called SureQuant, to increase throughput, sensitivity, and precision. Testing the optimized platform on 27 lung and upper aerodigestive cancer cell models revealed 35 NRF2 responsive proteins. In formalin-fixed paraffin-embedded (FFPE) HNSCCs, NRF2 signaling intensity positively correlated with NRF2 activating mutations and with SOX2 protein expression. PD-L2/CD273 and protein markers of T-cell infiltration correlated positively with one another and with human papilloma virus (HPV) infection status. p16/CDKN2A protein expression positively correlated with the HPV oncogenic E7 protein, and confirmed the presence of translationally active virus. This work establishes a clinically actionable HNSCC protein biomarker assay capable of quantifying over 600 peptides from frozen or FFPE archived tissues in under 90 minutes.

cancer biology↗

bioRxiv2021

The landscape of extrachromosomal circular DNA in medulloblastoma

Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive tumor growth, promoting high oncogene copy number, intratumoral heterogeneity, accelerated evolution of drug resistance, enhancer rewiring, and poor outcome. ecDNA has been reported in medulloblastoma (MB), the most common malignant pediatric brain tumor, but the ecDNA landscape and its association with specific MB subgroups, its impact on enhancer rewiring, and its potential clinical implications, are not known. We assembled a retrospective cohort of 468 MB patient samples with available whole genome sequencing (WGS) data covering the four major MB subgroups WNT, SHH, Group 3 and Group 4. Using computational methods for the detection and reconstruction of ecDNA1, we find ecDNA in 82 patients (18%) and observe that ecDNA+ MB patients are more than twice as likely to relapse and three times as likely to die of disease. In addition, we find that individual medulloblastoma tumors often harbor multiple ecDNAs, each containing different amplified oncogenes along with co-amplified non-coding regulatory enhancers. ecDNA was substantially more prevalent among 31 analyzed patient-derived xenograft (PDX) models and cell lines than in our patient cohort. By mapping the accessible chromatin and 3D conformation landscapes of MB tumors that harbor ecDNA, we observe frequent candidate "enhancer rewiring" events that spatially link oncogenes with co-amplified enhancers. Our study reveals the frequency and diversity of ecDNA in a subset of highly aggressive tumors and suggests enhancer rewiring as a frequent oncogenic mechanism of ecDNAs in MB. Further, these results demonstrate that ecDNA is a frequent and potent driver of poor outcome in MB patients.

cancer biology↗

bioRxiv2020

Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase

N6-methyladenosine (m6A) is the most common modification on endogenous RNA transcripts in mammalian cells. Currently, the lack of precise single-nucleotide RNA modification tools makes it challenging to understand the relationship between site-specific RNA methylation and the corresponding phenotypic outcomes. Here, we developed a bidirectional dCasRx epitranscriptome editing platform composed of a nucleus-localized dCasRx conjugated with either a methyltransferase, METTL3, or a demethylase, ALKBH5, to manipulate methylation events at targeted m6A sites in HEK293T and glioma stem cells. This platform specifically and efficiently edited m6A modifications at targeted sites, modulating both gene expression and cell proliferation. We then employed the dCasRx epitranscriptomic editor to further elucidate the molecular function of m6A-binding proteins YTH (DF1, DF2, DF3) family and found that the YTH paralogs act together to mediate RNA degradation. These findings collectively demonstrate that the dCasRx epitranscriptome perturbation platform reported in this study can be employed as site-specific m6A editors for delineating the functional roles of individual m6A modifications in the mammalian epitranscriptome.

molecular biology↗

bioRxiv2020

Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches

BackgroundGlioblastoma (GBM) is marked by cellular heterogeneity, including metabolic heterogeneity, that varies among cellular microenvironments in the same tumor. Altered cellular metabolism in cancer is well-established, but how lipid metabolism is altered to suit different microenvironmental conditions and cellular states within a tumor remains unexplored. MethodsWe assessed GBM organoid models that mimic the transition zone between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones and performed spatial RNA-sequencing of cells to interrogate lipid metabolism. Using targeted lipidomic analysis, we assessed differences in acutely enriched cancer stem cells (CSCs) and non-CSCs from multiple patient-derived models to explore the link between the stem cell state and lipid metabolism. ResultsSpatial analysis revealed a striking difference in lipid content between microenvironments, with lipid enrichment in the hypoxic organoid cores and the perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and in clinical GBM specimens, but not lower-grade brain tumors, that was specifically localized to pseudopalisading regions of patient tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis revealed that CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression. ConclusionsOur data demonstrate that lipid metabolism is differentially altered across GBM microenvironments and cellular hierarchies, providing guidance for targeting of these altered lipid metabolic pathways. Key pointsO_LIGBM cells in nutrient-poor tumor regions have increased accumulation of lipid droplets. C_LIO_LICSCs have reduced lipid content compared to non-CSCs. C_LIO_LIGBM CSCs and non-CSCs have disparate lipid metabolisms that may be uniquely targetable. C_LI Importance of the StudyMetabolic targeting has long been advocated as a therapy against many tumors including GBM, and it remains an outstanding question whether cancer stem cells (CSCs) have altered lipid metabolism. We demonstrated striking differences in lipid metabolism between diverse cell populations from the same patient. These spatially and phenotypically distinct lipid phenotypes occur clinically in the majority of patients and can be recapitulated in laboratory models. Lipidomic analysis of multiple patient-derived models shows a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. Our results suggest that therapeutic targeting of GBM lipid metabolism must consider multiple separate tumor cell populations to be effective, and we provide a methodologic framework for studying these metabolically diverse cellular populations.

cancer biology↗

bioRxiv2020

Field-based body temperatures reveal behavioralthermoregulation strategies of the Atlantic marsh fiddler crab Minuca pugnax

Behavioral thermoregulation is an important defense against the negative impacts of climate change for ectotherms. In this study we examined the use of burrows by a common intertidal crab, Minuca pugnax, to control body temperature. To understand how body temperatures respond to changes in the surface temperature and explore how efficiently crabs exploit the cooling potential of burrows to thermoregulate, we measured body, surface, and burrow temperature data during low tide on Sapelo Island, GA in March, May, August, and September of 2019. We found that an increase in 1{degrees}C in the surface temperature led to a 0.70-0.71{degrees}C increase in body temperature for females and an increase in 0.75-0.77{degrees}C in body temperature for males. Body temperatures of small females were 0.3{degrees}C warmer than large females for the same surface temperature. Female crabs used burrows more efficiently for thermoregulation compared to the males. Specifically, an increase of 1{degrees}C in the cooling capacity (the difference between the burrow temperature and the surface temperature) led to an increase of 0.42-0.50{degrees}C for females and 0.34-0.35{degrees}C for males in the thermoregulation capacity (the difference between body temperature and surface temperature). The body temperature that crabs began to use burrows to thermoregulate was estimated to be around 24{degrees}C, which is far below the critical body temperatures that could lead to death. Many crabs experience body temperatures of 24{degrees}C early in the reproductive season, several months before the hottest days of the year. Because the use of burrows involves fitness trade-offs, these results suggest that warming temperatures could begin to impact crabs far earlier in the year than expected.

animal behavior and cognition↗

bioRxiv2018

Genetic data and cognitively-defined late-onset Alzheimer’s disease subgroups

Categorizing people with late-onset Alzheimers disease into biologically coherent subgroups is important for personalized medicine. We evaluated data from five studies (total n=4 050, of whom 2 431 had genome-wide single nucleotide polymorphism (SNP) data). We assigned people to cognitively-defined subgroups on the basis of relative performance in memory, executive functioning, visuospatial functioning, and language at the time of Alzheimers disease diagnosis. We compared genotype frequencies for each subgroup to those from cognitively normal elderly controls. We focused on APOE and on SNPs with p<10-5 and odds ratios more extreme than those previously reported for Alzheimers disease (<0.77 or >1.30). There was substantial variation across studies in the proportions of people in each subgroup. In each study, higher proportions of people with isolated substantial relative memory impairment had [≥]1 APOE e4 allele than any other subgroup (overall p= 1.5 x 10-27). Across subgroups, there were 33 novel suggestive loci across the genome with p<10-5 and an extreme OR compared to controls, of which none had statistical evidence of heterogeneity and 30 had ORs in the same direction across all datasets. These data support the biological coherence of cognitively-defined subgroups and nominate novel genetic loci.

genetics↗

bioRxiv2018

A novel interplay between GEFs orchestrates Cdc42 activation in cell polarity and cytokinesis

Cdc42, a conserved regulator of cell polarity, is activated by two GEFs, Gef1 and Scd1, in fission yeast. While gef1 and scd1 mutants exhibit distinct phenotypes, how they do so is unclear given that they activate the same GTPase. Using the GEF localization pattern during cytokinesis as a paradigm, we report a novel interplay between Gef1 and Scd1 that spatially modulates Cdc42. We find that Gef1 promotes Scd1 localization to the division site during cytokinesis and to the new end during polarized growth through the recruitment of the scaffold Scd2 via a Cdc42 feedforward pathway. Gef1-mediated Scd1 recruitment at the new end enables the transition from monopolar to bipolar growth. Reciprocally, Scd1 restricts Gef1 localization to prevent ectopic Cdc42 activation during cytokinesis to promote cell separation and during interphase to maintain cell shape. Our findings reveal an elegant regulatory pattern in which Gef1 establishes new sites of Scd1-mediated Cdc42 activity, while Scd1 restricts Gef1 to functional sites. We propose that crosstalk between GEFs is a conserved mechanism that orchestrates Cdc42 activation during complex cellular processes.\n\nSummary StatementCdc42 GEFs Gef1 and Scd1 crosstalk to fine-tune Cdc42 activity. This crosstalk promotes bipolar growth and maintains cell shape in fission yeast.

cell biology↗

bioRxiv2017

Mapping DNA damage-dependent genetic interactions in yeast via orgy mating and barcode fusion genetics

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double mutant strains, does not scale readily to multi-condition studies. Here we describe Barcode Fusion Genetics to map Genetic Interactions (BFG-GI), by which double mutant strains generated via en masse party mating can also be monitored en masse for growth and genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to a decrease in the activation or activity of the checkpoint protein kinase Rad53.

systems biology↗