Biology subjects

Magnuson, B.

Publications and source records attributed to Magnuson, B..

10 recordsLinked to original sources

bioRxiv2024

Isoform and pathway-specific post-transcriptional RNA processing in human cells

Steady-state levels of RNA transcripts are controlled by their rates of synthesis and degradation. Here we used nascent RNA Bru-seq and BruChase-seq to profile RNA dynamics across 16 human cell lines as part of ENCODE4 Deeply Profiled Cell Lines collection. We show that RNA turnover dynamics differ widely between transcripts of different genes and between different classes of RNA. Gene set enrichment analysis (GSEA) revealed that transcripts encoding proteins belonging to the same pathway often show similar turnover dynamics. Furthermore, transcript isoforms show distinct dynamics suggesting that RNA turnover is important in regulating mRNA isoform choice. Finally, splicing across newly made transcripts appears to be cooperative with either all or none type splicing. These data sets generated as part of ENCODE4 illustrate the intricate and coordinated regulation of RNA dynamics in controlling gene expression to allow for the precise coordination of cellular functions.

molecular biology↗

bioRxiv2024

Characterizing nascent transcription patterns of PROMPTs, eRNAs, and readthrough transcripts in the ENCODE4 deeply profiled cell lines

Arising as co-products of canonical gene expression, transcription-associated lincRNAs, such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and readthrough (RT) transcripts, are often regarded as byproducts of transcription, although they may be important for the expression of nearby genes. We identified regions of nascent expression of these lincRNA in 16 human cell lines using Bru-seq techniques, and found distinctly regulated patterns of PROMPT, eRNA, and RT transcription using the diverse biochemical approaches in the ENCODE4 deeply profiled cell lines collection. Transcription of these lincRNAs was influenced by sequence-specific features and the local or 3D chromatin landscape. However, these sequence and chromatin features do not describe the full spectrum of lincRNA expression variability we identify, highlighting the complexity of their regulation. This may suggest that transcription-associated lincRNAs are not merely byproducts, but rather that the transcript itself, or the act of its transcription, is important for genomic function.

molecular biology↗

bioRxiv2024

Targeting PIKfyve-driven lipid homeostasis as a metabolic vulnerability in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism1 2. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes3-5. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development6. Here, we characterize PIKfyve, a lipid kinase integral to lysosomal functioning7, as a novel and targetable vulnerability in PDAC. In human patient and murine PDAC samples, we discovered that PIKFYVE is overexpressed in PDAC cells compared to adjacent normal cells. Employing a genetically engineered mouse model, we established the essential role of PIKfyve in PDAC progression. Further, through comprehensive metabolic analyses, we found that PIKfyve inhibition obligated PDAC to upregulate de novo lipid synthesis, a relationship previously undescribed. PIKfyve inhibition triggered a distinct lipogenic gene expression and metabolic program, creating a dependency on de novo lipid metabolism pathways, by upregulating genes such as FASN and ACACA. In PDAC, the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Accordingly, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC.

cancer biology↗

bioRxiv2023

Modeling Molecular Pathogenesis of Idiopathic Pulmonary Fibrosis-Associated Lung Cancer in Mice

Idiopathic Pulmonary Fibrosis (IPF) is characterized by progressive, often fatal loss of lung function due to overactive collagen production and tissue scarring. IPF patients have a sevenfold-increased risk of developing lung cancer. The COVID-19 pandemic has increased the number of patients with lung diseases, and infection can worsen prognoses for those with chronic lung diseases and disease-associated cancer. Understanding the molecular pathogenesis of IPF-associated lung cancer is imperative for identifying diagnostic biomarkers and targeted therapies that will facilitate prevention of IPF and progression to lung cancer. To understand how IPF-associated fibroblast activation, matrix remodeling, epithelial-mesenchymal transition, and immune modulation influences lung cancer predisposition, we developed a mouse model to recapitulate the molecular pathogenesis of pulmonary fibrosis-associated lung cancer using the bleomycin and the Lewis Lung Carcinoma models. Models of pulmonary fibrosis, particularly bleomycin-induced fibrosis, do not recapitulate all aspects of human disease; however, to simplify nomenclature, we refer to our bleomycin-induced fibrosis model as IPF. We demonstrate that development of pulmonary fibrosis-associated lung cancer is linked to increased recruitment or reprogramming of tumor-associated macrophages and a unique gene signature that supports an immune-suppressive microenvironment through secreted factors. Not surprisingly, pre-existing fibrosis provides a pre-metastatic niche and results in augmented tumor growth. Tumors associated with bleomycin-induced fibrosis are characterized by an epithelial-to-mesenchymal transition characterized by dramatic loss of cytokeratin expression. ImplicationsWe provide new therapeutic targets that may aid the characterization of tumors associated with lung diseases and development of treatment paradigms for lung cancer patients with pre-existing pulmonary diseases.

cancer biology↗

bioRxiv2023

Cdc73 protects Notch-induced T-cell leukemia cells from DNA damage and mitochondrial stress

Activated Notch signaling is highly prevalent in T-cell acute lymphoblastic leukemia (T-ALL) but pan-Notch inhibitors were toxic in clinical trials. To find alternative ways to target Notch signals, we investigated Cell division cycle 73 (Cdc73), which is a Notch cofactor and component of transcriptional machinery, a potential target in T-ALL. While we confirmed previous work that CDC73 interacts with NOTCH1, we also found that the interaction in T-ALL was context-dependent and facilitated by the lymphoid transcription factor ETS1. Using mouse models, we showed that Cdc73 is important for Notch-induced T-cell development and T-ALL maintenance. Mechanistically, Cdc73, Ets1, and Notch intersect chromatin at promoters and enhancers to activate oncogenes and genes that are important for DNA repair and oxidative phosphorylation. Consistently, Cdc73 deletion in T-ALL cells induced DNA damage and impaired mitochondrial function. Our data suggests that Cdc73 might promote a gene expression program that was eventually intersected by Notch to mitigate the genotoxic and metabolic stresses of elevated Notch signaling. We also provide mechanistic support for testing inhibitors of DNA repair, oxidative phosphorylation, and transcriptional machinery. Inhibiting pathways like Cdc73 that intersect with Notch at chromatin might constitute a strategy to weaken Notch signals without directly targeting the Notch complex.

cancer biology↗

bioRxiv2022

Therapeutic targeting of differentiation state-dependent metabolic vulnerabilities in DIPG

H3K27M diffuse intrinsic pontine gliomas (DIPG) exhibit cellular heterogeneity comprising less-differentiated, stem-like glioma cells that resemble oligodendrocyte precursors (OPC) and more differentiated astrocyte (AC)-like cells. H3K27M DIPG stem-like cells exhibit tumor-seeding capabilities in vivo, a feature lost or greatly diminished in the more differentiated AC-like cells. In this study, we established isogenic in vitro models of DIPG that closely recapitulated the OPC-like and AC-like phenotypes of DIPG cells. Using these tools, we performed transcriptomics, metabolomics, and bioenergetic profiling to identify metabolic programs operative in the different cellular states. From this, we defined new strategies to selectively target metabolic vulnerabilities within the specific tumor populations. Namely, we showed that the AC-like cells exhibited a more mesenchymal phenotype and were thus sensitized to ferroptotic cell death. In contrast, OPC-like cells upregulated cholesterol metabolism and mitochondrial oxidative phosphorylation (OXPHOS) and were accordingly more sensitive to statins and OXPHOS inhibitors. Additionally, statins and OXPHOS inhibitors showed efficacy and extended survival in preclinical orthotopic models established with stem-like H3K27M DIPG cells. Together, this study demonstrates that cellular subtypes within DIPGs harbor distinct metabolic vulnerabilities that can be uniquely and selectively targeted for therapeutic gain.

cancer biology↗

bioRxiv2021

OXPHOS Promotes Apoptotic Resistance and Persistence in TH17 cells

Apoptotic cell death is a cell-intrinsic, immune tolerance mechanism that regulates the magnitude and resolution of T cell-mediated responses. Evasion of apoptosis is critical for the generation of memory T cells, as well as autoimmune T cells, and knowledge of the mechanisms that enable resistance to apoptosis will provide insight into ways to modulate their activity during protective and pathogenic responses. IL-17-producing CD4 T cells (TH17s) are long-lived, memory cells. These features enable their role in host defense, chronic inflammatory disorders, and anti-tumor immunity. A growing number of reports now indicate that TH17s in vivo require mitochondrial oxidative phosphorylation (OXPHOS), a metabolic phenotype that is poorly induced in vitro. To elucidate the role of OXPHOS in TH17 processes, we developed a system to polarize TH17s that metabolically resembled their in vivo counterparts. We discovered that directing TH17s to use OXPHOS promotes mitochondrial fitness, glutamine anaplerosis, and an anti-apoptotic phenotype marked by high BCL-XL and low BIM. Through competitive co-transfer experiments and tumor studies, we further revealed how OXPHOS protects TH17s from cell death while enhancing their persistence in the periphery and tumor microenvironment. Together, our work demonstrates a non-classical role of metabolism in regulating TH17 cell fate and highlights the potential for therapies that target OXPHOS in TH17-driven diseases.

immunology↗

bioRxiv2020

Principles of mRNA control by human PUM proteins elucidated from multi-modal experiments and integrative data analysis

The human PUF-family proteins, PUM1 and PUM2, post-transcriptionally regulate gene expression by binding to a PUM recognition element (PRE) in the 3 UTR of target mRNAs. Hundreds of PUM1/2 targets have been identified from changes in steady state RNA levels; however, prior studies could not differentiate between the contributions of changes in transcription and RNA decay rates. We applied metabolic labeling to measure changes in RNA turnover in response to depletion of PUM1/2, showing that human PUM proteins regulate expression almost exclusively by changing RNA stability. We also applied an in vitro selection workflow to precisely identify the binding preferences of PUM1 and PUM2. By integrating our results with prior knowledge, we developed a rulebook of key contextual features that differentiate functional vs. non-functional PREs, allowing us to train machine learning models that accurately predict the functional regulation of RNA targets by the human PUM proteins.

molecular biology↗

bioRxiv2019

Replication Timing and Transcription Identifies a Novel Fragility Signature Under Replication Stress

Common fragile sties (CFSs) are regions susceptible to replication stress and are hotspots for chromosomal instability in cancer. Several features characterizing CFSs have been associated with their instability, however, these features are prevalent across the genome and do not account for all known CFSs. Therefore, the molecular mechanism underlying CFS instability remains unclear. Here, we explored the transcriptional profile and temporal order of DNA replication (replication timing, RT) of cells under replication stress conditions. We show that the RT of only a small portion of the genome is affected by replication stress, and that CFSs are enriched for delayed RT. We identified a signature for chromosomal fragility, comprised of replication stress-induced delay in RT of early/mid S-phase replicating regions within actively transcribed large genes. This fragility signature enabled precise mapping of the core fragility region. Furthermore, the signature enabled the identification of novel fragile sites that were not detected cytogenetically, highlighting the improved sensitivity of our approach for identifying fragile sites. Altogether, this study reveals a link between altered DNA replication and transcription of large genes underlying the mechanism of CFS expression. Thus, investigating the RT and transcriptional changes in cancer may contribute to the understanding of mechanisms promoting genomic instability in cancer.

genetics↗

bioRxiv2017

HNF1A is a Novel Oncogene and Central Regulator of Pancreatic Cancer Stem Cells

The biological properties of pancreatic cancer stem cells (PCSCs) remain incompletely defined and the central regulators are unknown. By bioinformatic analysis of a PCSC-enriched gene signature, we identified the transcription factor HNF1A as a putative central regulator of PCSC function. Levels of HNF1A and its target genes were found to be elevated in PCSCs and tumorspheres, and depletion of HNF1A resulted in growth inhibition, apoptosis, impaired tumorsphere formation, PCSC depletion, and downregulation of OCT4 expression. Conversely, HNF1A overexpression increased PCSC numbers and tumorsphere formation in pancreatic cancer cells and drove PDA cell growth. Importantly, depletion of HNF1A in primary tumor xenografts impaired tumor growth and depleted PCSCs in vivo. Finally, we established an HNF1A-dependent gene signature in PDA cells that significantly correlated with reduced survivability in patients. These findings identify HNF1A as a central transcriptional regulator of the PCSC state and novel oncogene in pancreatic ductal adenocarcinoma.

cancer biology↗