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Fletcher, A.

Publications and source records attributed to Fletcher, A..

5 recordsLinked to original sources

Truncation and Motif Based Pan-Cancer Analysis Highlights Novel Tumor Suppressing Kinases.

A major challenge in cancer genomics is identifying driver mutations from the large number of neutral passenger mutations within a given tumor. Here, we utilize motifs critical for kinase activity to functionally filter genomic data to identify driver mutations that would otherwise be lost within mutational noise. In the first step of our screen, we define a putative tumor suppressing kinome by identifying kinases with truncation mutations occurring within or before the kinase domain. We aligned these kinase sequences and, utilizing data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas databases, identified amino acids that represent predicted hotspots for loss-of-function mutations. The functional consequences of new LOF mutations were validated and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7 as a candidate tumor suppressor in gastric cancer, despite the mutational frequency of MAP2K7 falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished catalytic activity compared to the wild type kinase, consistent with a tumor suppressive role for MAP2K7 in gastric cancer. Furthermore, reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or JNK1 suppresses clonogenicity and growth in soft agar, demonstrating the functional importance of inactivating the JNK pathway in gastric cancer. In summary, our data highlights a broadly applicable strategy to identify functional cancer driver mutations leading us to define the JNK pathway as tumor suppressive in gastric cancer.\n\nSummaryA unique computational pan-cancer analysis pinpoints novel tumor suppressing kinases, and highlights the power of functional genomics by defining the JNK pathway as tumor suppressive in gastric cancer.

cancer biology

Chronic inflammation delays cell migration to villi in the intestinal epithelium

The intestinal epithelium is a single layer of cells which provides the first line of defence of the intestinal mucosa to bacterial infection. Cohesion of this physical barrier is supported by renewal of epithelial stem cells, residing in invaginations called crypts, and by crypt cell migration onto protrusions called villi; dysregulation of such mechanism may render the gut susceptible to chronic inflammation. The impact that excessive or misplaced epithelial cell death may have on villus cell migration is currently unknown. We integrated cell-tracking methods with computational models to determine how epithelial homeostasis is affected by acute and chronic inflammatory cell death. Parameter inference reveals that acute inflammatory cell death has a transient effect on epithelial cell dynamics, whereas cell death caused by chronic inflammation causes a delay in the accumulation of labelled cells onto the villus compared to control. Such a delay may be reproduced by using a cell-based model to simulate the dynamics of each cell in a crypt-villus geometry, showing that a prolonged increase in cell death slows the migration of cells from the crypt to the villus. This investigation highlights which injuries (acute or chronic) may be regenerated and which cause disruption of healthy epithelial homeostasis.

systems biology

Bimodal Spindle Orientation Drives Tissue Regularity in a Proliferating Epithelium

We investigated the relationship between proliferation and tissue topology in an epithelial tissue undergoing elongation. We found that cell division is not required for elongation of the early Drosophila follicular epithelium, but does drive the tissue towards optimal geometric packing. To increase tissue regularity, cell divisions are oriented in the planar axis, along the direction of tissue expansion. Planar division orientation is governed by apico-cortical tension, which aligns with tissue expansion but not with interphase cell shape elongation. Hertwigs Rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. We tested whether this observation could be explained by anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis. We found that Pins/Mud does not participate in planar division orientation. Rather, tension translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin to adherens junctions. These findings demonstrate that division orientation in different axes - apical-basal and planar - is controlled by distinct, independent mechanisms in a proliferating epithelium.\n\nSummary StatementRegularity in a proliferating epithelium requires cells to divorce division orientation from interphase shape, which they accomplish by using distinct mechanisms to orient divisions in the apical-basal and planar axes.

developmental biology

Harnessing the lymphocyte meta-phenotype to optimize adoptive cell therapy

There is an urgent need for reliable effective therapy for patients with metastatic sarcoma. Approaches that manipulate the immune system have shown promise for patients with advanced, widely disseminated malignancies. One of these approaches is adoptive cell therapy (ACT), where tumor-infiltrating lymphocytes (TIL) are isolated from the tumor, expanded ex vivo, and then transferred back to the patient. This approach has shown great promise in melanoma, leading to an objective response in approximately half of treated patients [14]. Standard protocols involve characterization of TIL populations with respect to adaptive CD4+ and CD8+ T-lymphocytes, but neglect the possible role of the innate lymphoid repertoire. Due to toxicity and the high cost associated with ACT, the IFN-{gamma} release assay is currently used as a proxy to identify suitable TIL isolates for ACT. Efforts in TIL-ACT for sarcoma, which are pre-clinical and pioneered at Moffitt Cancer Center, have shown that only a minority of the TIL cultures show tumor specific activity in ex vivo IFN-{gamma} assays. Surprisingly, internal melanoma trial data reveal a lack of correlation between IFN-{gamma} assay and clinical outcomes, highlighting the need for a more reliable proxy. We hypothesize the existence of a predictable TIL meta-phenotype that leads to optimal tumor response. Here, we describe preliminary efforts to integrate prospective and existing patient data with mathematical models to optimize the TIL meta-phenotype prior to re-injection.

cancer biology

QuickStitch for seamless stitching of confocal mosaics through high-pass filtering and recursive normalization

Fluorescence micrographs naturally exhibit darkening around their edges (vignetting), which makes seamless stitching challenging. If vignetting is not corrected for, a stitched image will have visible seams where the individual images (tiles) overlap, introducing a systematic error into any quantitative analysis of the image. Although multiple vignetting correction methods exist, there remains no open-source tool that robustly handles large 2D immunofluorescence-based mosaic images. Here, we develop and validate QuickStitch, a tool that applies a recursive normalization algorithm to stitch large-scale immunofluorescence-based mosaics without incurring vignetting seams. We demonstrate how the tool works successfully for tissues of differing size, morphology, and fluorescence intensity. QuickStitch requires no specific information about the imaging system. It is provided as an open-source tool that is both user friendly and extensible, allowing straightforward incorporation into existing image processing pipelines. This enables studies that require accurate segmentation and analysis of high-resolution datasets when parameters of interest include both cellular-level phenomena and larger tissue-level regions of interest.

bioinformatics