Biology subjects

Parham, L. R.

Publications and source records attributed to Parham, L. R..

6 recordsLinked to original sources

bioRxiv2025

Leveraging autophagy and pyrimidine metabolism to target pancreatic cancer

Autophagy inhibitors are promising compounds to treat pancreatic ductal adenocarcinoma (PDA) but their efficacy in patients is unclear, highlighting a need to understand mechanisms of resistance. We used a novel approach to uncover metabolic adaptations that bypass autophagy inhibition. Utilizing PDA cells with acquired resistance to different autophagy inhibitors, we found that severe autophagy depletion induces metabolic rewiring to sustain TCA intermediates and nucleotides for biosynthesis. Long-term autophagy inhibition results in altered pyruvate metabolism likely regulated by lower pyrimidine pools. Cells adapting to loss of autophagy preferentially salvage pyrimidines to replenish these pools instead of synthesizing them de novo. Exploiting this metabolic vulnerability, we found that acquired resistance to autophagy inhibition promotes increased salvage and therefore sensitivity to pyrimidine analogues, including gemcitabine and trifluridine/tipiracil leading to combinatory effects with autophagy inhibitors and pyrimidine analogs. These studies provide mechanistic insight defining how autophagy inhibition can be leveraged to treat pancreatic cancer.

cancer biology↗

bioRxiv2024

TNFSF13 insufficiency disrupts human colonic epithelial cell-mediated B cell differentiation

Cytokines mediating epithelial and immune cell interactions modulate mucosal healing-a process that goes awry with chronic inflammation as in inflammatory bowel disease. TNFSF13 is a cytokine important for B cell maturation and function, but roles for epithelial TNFSF13 and putative contribution to inflammatory bowel disease are poorly understood. We evaluated functional consequences of a novel monoallelic TNFSF13 variant using biopsies, tissue-derived colonoids and induced pluripotent stem cell (iPSC)-derived colon organoids. TNFSF13 variant colonoids exhibited a >50% reduction in secreted TNFSF13, increased epithelial proliferation, and reduced apoptosis, which was confirmed in iPSC-derived colon organoids. Single cell RNA-sequencing, flow cytometry, and co-immunoprecipitation identified FAS as the predominant colonic epithelial receptor for TNFSF13. Imaging mass cytometry revealed an increase in epithelial-associated B cells in TNFSF13 variant colon tissue sections. Finally, TNFSF13 variant colonoids co-cultured with memory B cells demonstrated a reduction in the production of IgA+ plasma cells compared to control colonoid co-cultures. Our findings support a role for epithelial TNFSF13 as a regulator of colonic epithelial growth and epithelial crosstalk with B cells. SUMMARYEpithelial TNFSF13 regulates colonic epithelial growth and epithelial-B cell interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/614260v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1e5b892org.highwire.dtl.DTLVardef@b8857forg.highwire.dtl.DTLVardef@448b95org.highwire.dtl.DTLVardef@2820e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Autophagy contributes to homeostasis in esophageal epithelium where high autophagic vesicle content marks basal cells with limited proliferation and enhanced self-renewal potential

Background & AimsAutophagy has been demonstrated to play roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelium under homeostatic conditions. MethodsWe generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histological and biochemical analyses. We FACS sorted esophageal basal cells based upon fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID, then subjected these cells to transmission electron microscopy, image flow cytometry, 3D organoid assays, RNA-Sequencing (RNA-Seq), and cell cycle analysis. 3D organoids were subjected to passaging, single cell (sc) RNA-Seq, cell cycle analysis, and immunostaining. ResultsGenetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability upon initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA-Seq suggested increased autophagy in Cyto- IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. scRNA-Seq of 3D organoids generated by Cyto-IDLow and Cyto- IDHigh cells identified expansion of 3 cell populations, enrichment of G2/M-associated genes, and aberrant localization of cell cycle-associated genes beyond basal cell populations in the Cyto- IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in cells beyond the basal cell layer. Squamous epithelial-specific autophagy inhibition induced significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. ConclusionsHigh AV level identifies esophageal epithelium with limited proliferation and enhanced self-renewal capacity that contributes to maintenance of the esophageal proliferation- differentiation gradient in vivo.

physiology↗

bioRxiv2023

METTL3 is essential for small intestinal epithelial proliferation via regulation of growth factor signaling including KRAS

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

cell biology↗

bioRxiv2022

Autophagic state prospectively identifies facultative stem cells in the intestinal epithelium

The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic activity predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineageagnostic marker for the prospective identification of facultative stem cells.

cell biology↗

bioRxiv2022

IGF2BP1/IMP1 contributes to autophagy modulation directly via MAP1LC3B

Homeostatic tissue maintenance requires coordinated regulation of metabolic processes including macroautophagy/autophagy. Autophagy dysregulation underlies numerous human diseases. Our prior work revealed that the RNA binding protein IGF2BP1/IMP1 binds transcripts encoding autophagy-related proteins. Furthermore, Imp1 deletion in gastrointestinal epithelial cells in mice was associated with enhanced autophagy flux and improved recovery from tissue injury. In the current study, we evaluated molecular mechanisms underlying IMP1 modulation of autophagy. We provide a mechanism of direct IMP1 regulation of MAP1LC3B that is dependent upon IMP1 phosphorylation or cell stress, suggesting dynamic modulation of Imp1-mediated autophagy repression that facilitates tissue regeneration. More broadly, our study supports a new mechanism by which tissue regeneration is modulated post-transcriptionally via cell state rather than changes in stem or other cell lineages. This new mechanism may be particularly important in gastrointestinal epithelial cells, where autophagy is essential for tissue recovery following injury, or in diseases such as inflammatory bowel disease where defective autophagy is implicated.

cell biology↗