Biology subjects

Winton, D. J.

Publications and source records attributed to Winton, D. J..

4 recordsLinked to original sources

bioRxiv2024

PTEN and ARID1A haploinsufficiency equip colonic epithelium for oncogenic transformation

Normal aged tissues are thought to exist as a patchwork of mutant clones. However, the relevance of driver mutations in normal tissue in terms of cancer initiation has not been well described. Here, we sought a quantitative understanding of how different cancer drivers achieve an age-related mutational footprint in the human colonic epithelium and to relate the clonal behaviours they generate to cancer risk. Metanalysis of contemporary multiregional sampling studies of colorectal tumours revealed many of the weak or moderate cancer drivers are trunk mutations present in the last common ancestor from which cancers arise. To study the processes by which such driver mutations could contribute to cancer predisposition, immunohistochemistry was used to detect PTEN, SMAD4 and ARID1A deficient clones in normal colon FFPE surgical resection samples (N=182 patients). Age-related changes in clone size and frequency identified positive biases in clone dynamics that acted to increase the mutational footprint for ARID1A and PTEN but not SMAD4. In vitro engineered monoallelic loss of PTEN and ARID1A implicated specific altered downstream pathways and acquired pro-oncogenic cellular fates corresponding to haploinsufficiency for these genes. In situ analysis confirmed enhanced proliferation in both PTEN and ARID1A deficient clones and creation of an immune exclusive microenvironment associated with ARID1A deficiency. The behaviours resulting from haploinsufficiency of PTEN and ARID1A exemplify how priming of the tissue through somatic mosaicism could contribute alternative combinations of genetic events leading to transformation.

cancer biology↗

bioRxiv2023

Temporal recording of mammalian development and precancer

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo, while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

developmental biology↗

bioRxiv2023

Efficient genetic editing of human intestinal organoids using ribonucleoprotein-based CRISPR

Organoids are currently one of the most widely used ex vivo models in epithelial biology. Combined with genetic editing strategies, organoids offer a promise of rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has been only up to 30%, with implications for the subsequent need for selection including including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method of intestinal organoid editing using a Ribonucleoprotein CRISPR-based approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different anatomical gut locations and developmental timepoints from multiple patient samples with no off-target editing. The method allowed us to study the effect of the loss of the tumour suppressor gene, PTEN, in normal human intestinal cells. Analysis of PTEN deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss including mTORC1 activation.

cell biology↗

bioRxiv2021

BMP pathway antagonism by Grem1 regulates epithelial cell fate in intestinal regeneration

In the intestine, the homeostatic effect of Bone Morphogenetic Protein (BMP) on cell fate has predominantly been inferred through pathway inactivation. Here, we assessed the impact of autocrine Bmp4 expression on secretory cell fate. Ligand exposure reduced proliferation, expedited terminal differentiation, abrogated long-term secretory cell survival and prevented dedifferentiation. As stem cell plasticity is required for regenerative adaptive reprogramming, we spatiotemporally mapped and functionally explored BMPs role in epithelial restitution. Following ulceration, physiological attenuation of BMP signalling arose through upregulation of the secreted antagonist, Grem1, from topographically distinct populations of stromal cells. Concomitant expression supported functional compensation, following Grem1 deletion from tissue-resident fibroblasts. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory, but functionally sub-maximal, as regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1 respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming, despite a convergent impact of YAP/TAZ on cell fate in remodelled wounds.

cell biology↗