Biology subjects

Malkin, D.

Publications and source records attributed to Malkin, D..

6 recordsLinked to original sources

bioRxiv2026

A microbiota-dependent gut bile acid axis links germline mutant p53 to distal tumour growth.

The gut microbiome can influence cancer development, but its contribution to cancer predisposition in Li-Fraumeni Syndrome (LFS) remains poorly understood. Here, we investigated whether germline mutant p53 alters the intestinal environment using Trp53R172H/+ mice. Tumour-naive mutant mice exhibited increased intestinal permeability and a pro-inflammatory colonic phenotype, including elevated inflammatory cytokines and enhanced NF-{kappa}B signalling. Untargeted metabolomic profiling revealed a striking alteration in bile acid metabolism, with targeted analysis identifying increases in multiple primary and microbiota-derived secondary bile acids. Notably, these changes occurred despite no detectable differences in overall microbial community composition, suggesting altered host-microbial metabolic output in the absence of broad compositional change. Several faecal bile acids were positively associated with distal tumour burden, with the microbiota-derived secondary bile acid deoxycholic acid (DCA) showing the strongest relationship. Microbiota depletion reduced distal tumour growth in mutant mice and attenuated intestinal barrier dysfunction, colonic inflammation and the altered faecal bile acid profile, including DCA. Conversely, transfer of cell-free faecal filtrate from mutant donors to WT recipients induced colonic inflammation and enhanced distal tumour growth. Together, these findings identify a mutant p53-gut axis in which germline mutant p53 establishes a microbiota-shaped metabolic and inflammatory intestinal environment capable of promoting tumour growth beyond the gut.

cancer biology↗

bioRxiv2024

tp53 R217H and R242H Mutant Zebrafish Exhibit Dysfunctional p53 Hallmarks and Recapitulate Li-Fraumeni Syndrome Phenotypes

Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with a highly penetrant cancer spectrum characterized by germline TP53 mutations. We characterized the first LFS zebrafish hotspot mutants, tp53 R217H and R242H (human R248H and R273H), and found these mutants exhibit partial-to-no activation of p53 target genes, have a defective G1 cell-cycle checkpoint, and are resistant to apoptosis. Spontaneous tumor development histologically resembling human sarcomas developed as early as 6 months. tp53 R242H mutants had a higher lifetime tumor incidence compared to tp53 null and R217H mutants, suggesting it is a more aggressive mutation. We observed mutation-specific tumor phenotypes across tp53 mutants with associated diverse transcriptomic and DNA methylome profiles, impacting metabolism, cell signalling, and biomacromolecule synthesis and degradation. These tp53 zebrafish mutants demonstrate fidelity to their human counterparts and provide new insights into underlying tumorigenesis mechanisms and kinetics, which will guide targeted therapeutics for LFS.

cancer biology↗

bioRxiv2022

Transcriptional immunogenomic analysis reveals distinct immunological clusters in pediatric nervous system tumours

To inform immunotherapy approaches in children, we performed an immunogenomic analysis of RNA-seq data from 925 treatment-naive pediatric nervous system tumours (pedNST) spanning 12 cancer types from three public data sets. Within pedNST, we uncovered four broad immune clusters: Pediatric Inflamed (10%), Myeloid Predominant (30%), Immune Neutral (43%) and Immune Excluded (17%). We validated these clusters using immunohistochemistry, methylation immune inference, and segmentation analysis of tissue images. We report shared biology of these immune clusters within and across cancer types, and characterization of specific immune-cell frequencies as well as T- and B-cell repertoires. We found no associations between immune infiltration levels and tumour mutational burden, although molecular cancer entities were enriched within specific immune clusters. Given the heterogeneity within pedNST, our findings suggest personalized immunogenomic profiling is needed to guide selection of immunotherapeutic strategies.

genomics↗

bioRxiv2021

CanDIG: Secure Federated Genomic Queries and Analyses Across Jurisdictions

Rapid expansions of bioinformatics and computational biology have broadened the collection and use of -omics data including genomic, transcriptomic, methylomic and a myriad of other health data types, in the clinic and the laboratory. Both clinical and research uses of such data require co-analysis with large datasets, for which participant privacy and the need for data custodian controls must remain paramount. This is particularly challenging in multi-jurisdictional settings, such as Canada, where health privacy and security requirements are often heterogeneous. Data federation presents a solution to this, allowing for integration and analysis of large datasets from various sites while abiding by local policies. The Canadian Distributed Infrastructure for Genomics platform (CanDIG) enables federated querying and analysis of -omics and health data while keeping that data local and under local control. It builds upon existing infrastructures to connect five health and research institutions across Canada, relies heavily on standards and tooling brought together by the Global Alliance for Genomics and Health (GA4GH), implements a clear division of responsibilities among its participants and adheres to international data sharing standards. Participating researchers and clinicians can therefore contribute to and quickly access a critical mass of -omics data across a national network in a manner that takes into account the multi-jurisdictional nature of our privacy and security policies. Through this, CanDIG gives medical and research communities the tools needed to use and analyze the ever-growing amount of -omics data available to them in order to improve our understanding and treatment of various conditions and diseases. CanDIG is being used to make genomic and phenotypic data available for querying across Canada as part of data sharing for five leading pan-Canadian projects including the Terry Fox Comprehensive Cancer Care Centre Consortium Network (TF4CN) and Terry Fox PRecision Oncology For Young peopLE (PROFYLE), and making data from provincial projects such as POG (Personalized Onco- Genomics) more widely available.

genomics↗

bioRxiv2018

Childhood cerebellar tumors mirror conserved fetal transcriptional programs

The study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. We used single-cell transcriptomics to study >60,000 cells from the developing murine cerebellum, and show that different molecular subgroups of childhood cerebellar tumors mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. Sonic Hedgehog medulloblastoma transcriptionally mirrors the granule cell hierarchy as expected, whereas Group 3 medulloblastoma resemble Nestin+ve stem cells, Group 4 medulloblastomas resemble unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumors demonstrates that many bulk tumors contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumors as a disorder of early brain development, and provide a proximate explanation for the peak incidence of cerebellar tumors in early childhood.

cancer biology↗

bioRxiv2017

Successful optimization of CRISPR/Cas9-mediated defined point mutation knock-in using allele-specific PCR assays in zebrafish

Single-stranded oligodeoxynucleotides (ssODN) are donor templates for homology-directed repair-based knock-in of point mutations using CRISPR/Cas9. To optimize the efficiency of ssODN-based knock-ins in zebrafish, we developed allele-specific PCR (AS-PCR) assays for introducing point mutations in tp53, cdh5 and lmna as case studies. In these point mutation strategies we introduced the codon mutations, sgRNA site mutations and restriction sites which can be detected by AS-PCR with the primers matching their respective alleles in combination with a common primer. We employed the anti-sense asymmetric oligo design as the main optimization as well as phosphorothioate oligo modification and also observed that proximity of the mutation site to the Cas9 cut site improves the efficiency when knock-ins into different genes were compared. We improved the efficiencies of two tp53 knock-ins using anti-sense asymmetric ultramer oligos (126-nt in length with homology arms of 36 and 90 nucleotides, anti-sense to the sgRNA) by 3-10 fold, the optimizations which resulted in successful founders for both tp53 knock-ins with transmission rates of 20-40 %. The initially low knock-in efficiency for tp53 mutants was likely due to the distance between the Cas9 cut site and mutations since cdh5 G767S knock-in located at the cut site had much higher founder identification and germline transmission rates. The phosphorothioate oligo modifications was used for a lamin A/C (lmna) knock-in strategy and it resulted in 40 % overall improvement in knock-in efficiency and greater knock-in consistency. We also determined that AS-PCR detected false-positive knock-ins which constituted 25-80 % of total in different strategies and developed a workflow to screen out the founders and F1 zebrafish carrying these undesirable modifications. In summary, we provide a complementary set of optimizations for CRISPR/Cas9-based ssODN knock-ins in zebrafish using a novel combination of methods.

molecular biology↗