Biology subjects

Wahl, G.

Publications and source records attributed to Wahl, G..

7 recordsLinked to original sources

bioRxiv2025

Oncogene Silencing via ecDNA Micronucleation

Extrachromosomal DNA (ecDNA) contributes to cancer genome instability by enabling high-copy oncogene amplification, intratumoural heterogeneity and rapid genetic change. Micronuclei (MN) are frequently observed in chromosomally unstable cancers, yet their origins and relevance in ecDNA-driven tumours remain incompletely understood. Here, we investigate the relationship between ecDNA segregation errors and MN formation. We find that ecDNA frequently localizes to MN and represents a prominent source of MN content in ecDNA-positive cancer cells. Mitotic clustering of oncogene-bearing ecDNAs is associated with asymmetric inheritance and mis-segregation into MN. Transfer of oncogenes from ecDNA into MN is accompanied by reduced transcriptional output. Single-MN sequencing shows that individual MN are enriched for ecDNA to a degree that exceeds expectations from stochastic mis-segregation and that in MN oncogenes originating from multiple distinct ecDNAs coalesce. Using live-cell imaging, we observe that cells inheriting ecDNA-positive MN show limited proliferative capacity and an increased likelihood of cell death. In neuroblastoma patients with MYCN-amplified ecDNA, higher frequencies of ecDNA-positive MN at diagnosis are associated with improved event-free and overall survival. Together, these findings link ecDNA mis-segregation to MN formation and reduced cellular fitness, suggesting that ecDNA-positive MN may reflect a state of impaired oncogenic ecDNA function with potential relevance for clinical outcome.

cancer biology↗

bioRxiv2025

Pcdh20 is a POU2F3 target gene required for proper tuft cell microvillus formation

BACKGROUND & AIMSTuft cells play protective roles in infection, inflammation, and tumorigenesis through the secretion of cytokines and eicosanoids. Tuft cells are known for their tall, blunt microvilli, thought to be analogous to mechanosensory hair cell stereocilia; however, a functional role for the microvillar apparatus has not been identified. POU2F3 is the master regulator transcription factor for tuft cells, yet how POU2F3 drives formation of this unique structure is unknown. Here, we aimed to identify POU2F3 target genes and commonalities between tuft and hair cells to better understand this unique structure. METHODSPOU2F3 ChIP-seq was performed on tuft cells and compared to the hair cell transcriptome. Tuft cell RNA-seq datasets were interrogated for hair cell structural and mechanosensory genes; expression was validated. Intestinal and gallbladder tuft cells were examined using multiple light and electron microscopy (EM) modalities. PCDH20 was knocked down in mouse models and ultrastructural analyses were performed. The tuft cell cytoskeleton was modeled using AlphaFold3 prediction. RESULTSGenes encoding structural and mechanosensory proteins common to both tuft and hair cells, including Pcdh20, were identified. Imaging localized PCDH20 to tuft cell microvilli and hair cell stereocilia. Genetic ablation of Pcdh20 in mice resulted in structural defects in tuft cell microvilli, including loss of rigidity and organization. Molecular modeling suggests PCDH20 homodimers link adjacent microvilli. CONCLUSIONSPcdh20 is a POU2F3 target gene in tuft cells, critical to maintain the rigid microvillar apparatus. These findings, together with the shared expression of mechanosensory components like TMC1, support the hypothesis that tuft cells could have mechanosensory capabilities analogous to cochlear hair cells.

cell biology↗

bioRxiv2022

Illuminating histidine phosphorylation in the pancreatic tumor microenvironment

Development of phosphohistidine (pHis) antibodies has significantly advanced our understanding of pHis contributions to tumor biology, including a tumor suppressive role for a pHis phosphatase, a metastasis suppressive role for His kinases, and pHis regulation of T cell receptor signaling. Using these antibodies, we investigated pHis pathway regulation in the mouse pancreatic tumor microenvironment. We identified deregulated expression of pHis and pHis phosphatases that correlated with mouse pancreatic tumor progression. We developed a protocol to circumvent the acid and heat-sensitivity of pHis signals, enabling their co-staining with other proteins in FFPE tissue, identifying a significant enrichment of 1-pHis and a subtype of 3-pHis signals (Gly-3-pHis) in the stroma. We discovered increased Gly-3-pHis levels in tumor-associated myeloid cells mainly resulting from elevated ATP citrate lyase 3-pHis levels and predicted the existence of pHis in cell-cell adhesion proteins. We provide evidence that mitochondrial delocalization of PGAM5, a pHis phosphatase with increased expression during pancreatic tumorigenesis, occurs in tumor cells as compared to stromal cells, enabling access to PGAM5s known cytoplasmic substrate, pHis-NME (Non-MEtastatic), and two potential Gly-3-pHis substrates, SCS (Succinyl CoA Synthetase) and {beta}-catenin. Overall, we introduce a new method and possible targets for future studies of pHis pathway deregulation during tumorigenesis.

cancer biology↗

bioRxiv2021

Eicosanoids in the pancreatic tumor microenvironment: a multicellular, multifaceted progression

Eicosanoids, oxidized fatty acids that serve as cell-signaling molecules, have been broadly implicated in tumorigenesis. To identify eicosanoids relevant to pancreatic tumorigenesis, we profiled normal pancreas and pancreatic ductal adenocarcinoma (PDAC) in mouse models and patient samples using mass spectrometry. We interrogated RNA sequencing datasets for eicosanoid synthase or receptor expression. Findings were confirmed by immunostaining. In murine models, we identified elevated levels of PGD2, prostacyclin, and thromboxanes in neoplasia while PGE2, 12-HHTre, HETEs, and HDoHEs are elevated specifically in tumors. Analysis of scRNA-seq datasets suggests that PGE2 and prostacyclins are derived from fibroblasts, PGD2 and thromboxanes from myeloid cells, and PGD2 and 5-HETE from tuft cells. In patient samples, we identified a transition from PGD2 to PGE2-producing enzymes in the epithelium during the transition to PDAC, fibroblast/tumor expression of PTGIS, and myeloid/tumor cell expression of TBXAS1. Altogether, our analyses identify key changes in eicosanoid species during pancreatic tumorigenesis and the cell types responsible for their synthesis.

cancer biology↗

bioRxiv2021

Multiplexed identification of RAS paralog imbalance as a driver of lung cancer growth

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the positive and negative regulators of RAS signaling is incomplete. To uncover the functional impact of diverse KRAS-interacting proteins on lung cancer growth in vivo, we used multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumor barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung tumors, we identified HRAS and NRAS as key suppressors of KRASG12D-driven tumor growth in vivo and confirmed these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogs interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signaling. HRAS mutations identified in KRAS-driven human tumors partially abolished this effect. Comparison of the tumor-suppressive effects of HRAS and NRAS in KRAS- and BRAF-driven lung cancer models confirmed that RAS paralogs are specific suppressors of oncogenic KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role of RAS paralog imbalance in oncogenic KRAS-driven lung cancer.

cancer biology↗

bioRxiv2021

Single-cell transcriptomics reveals a conserved metaplasia program in pancreatic injury

BACKGROUND & AIMSAcinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression. METHODSAcinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate upon injury. Transcripts of over 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared to gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTSscRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison to KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype. CONCLUSIONSUnder conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia from injury-induced ADM and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

cell biology↗

bioRxiv2019

Tuft cells restrain pancreatic tumorigenesis through paracrine eicosanoid signaling

Despite numerous advances in our understanding of pancreatic ductal adenocarcinoma (PDA) genetics and biology, this disease is expected to become the second leading cause of cancer-related U.S. deaths within the next few years. Incomplete understanding of how it arises precludes development of early detection and interception strategies to improve therapeutic outcomes. Acinar to ductal metaplasia involving genesis of tuft cells is one early step in PDA formation, but their functional significance has remained obscure due to their rarity and a lack of methods and relevant animal models for their molecular and functional analysis. Here, we show that deletion of tuft cell master regulator Pou2f3 eliminates pancreatic tuft cells and increases fibrosis, alters immune cell activation, and accelerates disease progression. We demonstrate that tuft cell expression of the prostaglandin D2 synthase Hpgds restrains pancreatic disease progression in early stages by inhibiting stromal activation. Analyses of human data sets are consistent with mouse studies. We propose that tuft cells and, by inference, the associated metaplastic lesions, play a protective role early in pancreatic tumorigenesis. SignificanceWe find that tuft cell formation in response to oncogenic Kras is protective and restrains tumorigenesis through local production of anti-inflammatory substances, including paracrine prostaglandin D2 signaling to the stroma. Our findings establish tuft cells as a metaplasia-induced tumor suppressive cell type.

cancer biology↗