Biology subjects

Karakoc, E.

Publications and source records attributed to Karakoc, E..

15 recordsLinked to original sources

bioRxiv2026

Structure-function analysis of PI3K signalling cascade base editing screens in cancer cells

Knowledge of protein structure and function underpins rational drug discovery, yet many targets lack known selectively druggable sites. Furthermore, the identification of secondary druggable sites offers a strategy to overcome drug resistance. Fragment-based drug discovery (FBDD) can identify new ligandable binding pockets, though how to triage those with the ability to exert biologically relevant effects can be unclear. Systematic approaches to identify novel functionally-important protein sites for therapeutic intervention, such as allosteric pockets or protein-protein interaction (PPI) interfaces, has the potential to accelerate drug discovery, particularly when combined with structure-based hit-finding modalities. The phosphoinositide-3 kinase (PI3K) signalling pathway is frequently altered in human cancer and resistance to approved inhibitors is an ongoing challenge. Here, we performed large-scale CRISPR base editing mutagenesis screens across 30 PI3K pathway proteins in three disease-relevant cancer cell models to systematically map functional residues. Integration of base editing data with structural information identified residues corresponding to known catalytic sites, fragment-binding pockets and PPI interfaces, providing validation for the approach. Additionally, we identified putative allosteric pockets near regions of undefined function. Together, these findings establish high-throughput base editing mutagenesis combined with structural analysis as a scalable strategy to delineate structure-function relationships and inform drug development.

genomics↗

bioRxiv2025

On-target mutations confer resistance to WRN helicase inhibitors in Microsatellite Unstable Cancer Cells.

Werner helicase inhibitors (WRNi) are in clinical development for microsatellite-unstable (MSI) tumors with defective DNA mismatch repair. Here, we investigate how cancer cell evolution shapes response to WRN inhibition and informs potential resistance mechanisms. Genome-wide CRISPR screens combined with WRN knockout did not identify bypass mechanisms, underscoring WRNs essential, non-redundant function in MSI cells. Pharmacogenomic screens identified modulators of WRNi sensitivity, including SMARCAL1, which links it to WRN-MSI synthetic lethality. Semi-saturation mutagenesis of WRN and prolonged drug treatment identified on-target WRN mutations driving acquired resistance to multiple WRNi in vitro and in vivo, which was mitigated by combination with standard chemotherapies. Some resistance mutations conferred broad cross-resistance, whereas others preserved sensitivity to alternative clinical-grade WRNi with distinct mechanism of action. Our findings could inform clinical trial design by suggesting the feasibility of real-time tracking of emerging resistance and enabling early therapeutic adaptations. SignificanceWe present the first exploration of how MSI cancer cells evolve under the selective pressure of WRN helicase inhibition, providing a framework for understanding adaptive responses to this newly identified synthetic-lethal dependency. This study identifies on-target WRN mutations as key drivers of resistance in MSI cancers, supporting the use of combination strategies with other standard-of-care treatments to prevent resistance. It highlights how mutation tracking can guide therapeutic switching to clinically available WRN inhibitors with distinct mechanisms of action, thereby refining clinical development and potentially improving biomarker-informed patient outcomes.

cancer biology↗

bioRxiv2025

Foraging competence and scrounging tolerance enhance social relationships in a socially tolerant wild primate

Social interactions are crucial for individual health and ultimately fitness, making the choice of social partners particularly important. Previous research has shown that individuals who succeed in foraging tasks often receive increased affiliation from group members. Similarly, in a social learning context, individuals who possess valuable information become more attractive social partners. Thus, an individuals role in a foraging context-specifically, whether it is a successful producer-can influence its social relationships. Therefore, we examined the interplay between social learning, producing and scrounging behavior, and social relationships in four groups of wild redfronted lemurs (Eulemur rufifrons). We conducted an open diffusion experiment with food boxes that required animals to learn one of two techniques to open them. 27 out of 29 individuals participated in the experiment, 24 interacted with the boxes and 16 learned to open them. Initial success was better predicted by use of individual than social information, i.e., manipulating the food boxes vs. time spent watching successful individuals or scrounging. Older males were less successful than females. Scrounging occurred in about 26% of events, with on average 1.3 individuals scrounging. The technique used, age and sex of the producer did not predict scrounging frequency. Learners and males scrounged more often than non-learners and females. Among learners, less successful individuals scrounged more often and this effect was more pronounced in males. More successful individuals and those that were scrounged more often received more affiliative behavior. Thus, cognitive skills and scrounging tolerance may strengthen social relationships in this primate species.

ecology↗

bioRxiv2025

CRISPR screens in the context of immune selection identify CHD1 and MAP3K7 as mediators of cancer immunotherapy resistance

Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR/Cas9 knock-out screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 potentiates the transcriptional response to IFN-{gamma}, thereby creating an acquired vulnerability through sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade was more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and was associated with elevated intra-tumoral CD8+ T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.

cancer biology↗

bioRxiv2024

PriMAT: A robust multi-animal tracking model for primates in the wild

O_LIDetection and tracking of animals is an important first step for automated behavioral studies using videos. Animal tracking is currently done mostly using deep learning frameworks based on keypoints, which show remarkable results in lab settings with fixed cameras, backgrounds, and lighting. However, multi-animal tracking in the wild presents several challenges such as high variability in background and lighting conditions, complex motion, and occlusion. C_LIO_LIWe propose a multi-animal tracking model, PriMAT, for nonhuman primates in the wild. The model learns to detect and track primates and other objects of interest from labeled videos or single images using bounding boxes instead of keypoints. Using bounding boxes significantly facilitates data annotation and robustness. Our one-stage model is conceptually simple but highly flexible, and we add a classification branch that allows us to train individual identification. C_LIO_LITo evaluate the performance of our model, we applied it in two case studies with Assamese macaques (Macaca assamensis) and redfronted lemurs (Eulemur rufifrons) in the wild. We show that with only a few hundred frames labeled with bounding boxes, we can achieve robust tracking results. Combining these results with the classification branch for the lemur videos, our model shows an accuracy of 84% in predicting lemur identities. C_LIO_LIOur approach presents a promising solution for accurately tracking and identifying animals in the wild, offering researchers a tool to study animal behavior in their natural habitats. Our code, models, training images, and evaluation video sequences are publicly available1, facilitating their use for animal behavior analyses and future research in this field. C_LI

animal behavior and cognition↗

bioRxiv2024

Genetic interaction library screening with a next-generation dual guide CRISPR system

Pairwise perturbation of gene function using the CRISPR/Cas9 system has huge potential in screening for genetic interactions and synthetic lethal gene pairs to identify novel combination therapies for cancer. However, existing dual guide expression systems are cumbersome to clone, often result in a large proportion of undesired guide pairs and have imbalance of guide expression from the two positions. Here, we demonstrate a next-generation system for dual guide delivery based around a tRNA spacer that allows a single step cloning strategy, as little as 2% of undesired guide pairs, and highly balanced expression of the two guides. This system allows efficient library-scale screening for hundreds of thousands of genetic interactions using the well understood Streptococcus pyogenes Cas9 (SpCas9) system. We use this to screen a 100,136 guide pair library in colorectal cancer cells and successfully identify synthetic lethal genetic interactions between paralogs or other known interacting genes, establishing our method for performing efficient large scale genetic interaction screens. This system is versatile and can be used with most guide RNA vector systems, and for other uses of paired guide delivery such as improving single gene knockout efficiency or improving guide detection in single cell or optical CRISPR screens.

molecular biology↗

bioRxiv2023

Transcriptomics and chromatin accessibility in multiple African population samples

Mapping the functional human genome and impact of genetic variants is often limited to European-descendent population samples. To aid in overcoming this limitation, we measured gene expression using RNA sequencing in lymphoblastoid cell lines (LCLs) from 599 individuals from six African populations to identify novel transcripts including those not represented in the hg38 reference genome. We used whole genomes from the 1000 Genomes Project and 164 Maasai individuals to identify 8,881 expression and 6,949 splicing quantitative trait loci (eQTLs/sQTLs), and 2,611 structural variants associated with gene expression (SV-eQTLs). We further profiled chromatin accessibility using ATAC-Seq in a subset of 100 representative individuals, to identity chromatin accessibility quantitative trait loci (caQTLs) and allele-specific chromatin accessibility, and provide predictions for the functional effect of 78.9 million variants on chromatin accessibility. Using this map of eQTLs and caQTLs we fine-mapped GWAS signals for a range of complex diseases. Combined, this work expands global functional genomic data to identify novel transcripts, functional elements and variants, understand population genetic history of molecular quantitative trait loci, and further resolve the genetic basis of multiple human traits and disease.

genomics↗

bioRxiv2023

Integrative ensemble modelling of cetuximab sensitivity in colorectal cancer PDXs

AO_SCPLOWBSTRACTC_SCPLOWPatient-derived xenografts (PDXs) are tumour fragments engrafted into mice for preclinical studies. PDXs offer clear advantages over simpler in vitro cancer models - such as cancer cell lines (CCLs) and organoids - in terms of structural complexity, heterogeneity, and stromal interactions. We characterised 231 colorectal cancer PDXs at the genomic, transcriptomic, and epigenetic level and measured their response to cetuximab, an EGFR inhibitor in clinical use for metastatic colorectal cancer. After assessing PDXs quality, stability, and molecular concordance with publicly available patient cohorts, we trained, interpreted, and validated an integrated ensemble classifier (CeSta) which takes in input the PDXs multi-omic characterisation and predicts their sensitivity to cetuximab treatment (AUROC > 0.9). Our study shows that large PDX collections can be used to train accurate, interpretable models of drug sensitivity, which 1) better recapitulate patient-derived therapeutic biomarkers than other models trained on CCL data, 2) can be robustly validated across independent PDX cohorts, and 3) can be used for the development of novel therapeutic biomarkers.

bioinformatics↗

bioRxiv2022

Hexokinase 1b is a novel target for non-small-cell lung cancer

Deregulation of glycolysis is common in non-small cell lung cancer (NSCLC). Hexokinase (HK) enzymes catalyze the phosphoryl-group-transfer in glucose metabolism. There are a very few studies that have begun to reveal the connections between glucose metabolism and splicing programs. Unlike HK2 gene, which is expressed as a single transcript, there are several transcripts of the HK1 gene due to alternative splicing. However, the functional differential roles of HK1 isoforms in glucose metabolism and tumor progression are still elusive. Here, we show that primary NSCLC patient tumor cells metabolically differ from the normal lung epithelium where they display predominant expression of one of the HK1 transcripts, hexokinase1b (HK1b). We utilized CRISPR-Cas9 system to selectively target specific HK1b isoform in NSCLC and show that silencing HK1b in NSCLC cells inhibits tumorigenesis through diminishing glycolysis and proliferation. Our findings constitute the first demonstration of the first biochemical distinction between the HK1 splice variants. Finally, HK1b deletion sensitizes NSCLC cells to standard-of-care, cisplatin treatment, and the combination therapy synergistically increases both apoptotic cell death by cisplatin and autophagic cell death by increased formation of LC3-II associated autophagic vesicles and myelinoid bodies. Notably, loss of HK1b leads to cellular DNA damage, further combination with cisplatin therapy showed significantly increased levels of DNA damage. Importantly, we showed that glycolysis and cisplatin resistance can be restored by adding-back HK1b in HK1b knock-out cells. Our findings reveal that targeting HK1b isoform alone or in combination with cisplatin may represent a novel strategy for NSCLC patients.

cancer biology↗

bioRxiv2022

Base editing screens map mutations affecting IFNγ signalling in cancer

IFN{gamma}signalling underpins host responses to infection, inflammation and anti-tumour immunity. Mutations in the IFN{gamma}signalling pathway cause immunological disorders, haematological malignancies, and resistance to immune checkpoint blockade (ICB) in cancer, however the function of most clinically observed variants remain unknown. Here, we systematically investigate the genetic determinants of IFN{gamma}response in colorectal cancer cells using CRISPR-Cas9 screens and base editing mutagenesis. Deep mutagenesis of JAK1 with cytidine and adenine base editors, combined with pathway-wide screens, reveal loss-of-function and gain-of-function mutations with clinical precedence, including causal variants in haematological malignancies and mutations detected in patients refractory to ICB. We functionally validate variants of uncertain significance in primary tumour organoids, where engineering missense mutations in JAK1 enhanced or reduced sensitivity to autologous tumour-reactive T cells. By classifying > 300 missense variants altering IFN{gamma} pathway activity, we demonstrate the utility of base editing for mutagenesis at scale, and generate a resource to inform genetic diagnosis.

cancer biology↗

bioRxiv2022

CRISPRcleanR WebApp: an interactive web application for processing genome-wide pooled CRISPR-Cas9 viability screen

A limitation of pooled CRISPR-Cas9 viability screens is the high false-positive rate in detecting essential genes arising from copy number-amplified (CNA) regions of the genome. To solve this issue, we developed CRISPRcleanR: a computational method implemented as R/python package and in a dockerized version. CRISPRcleanR detects and corrects biased responses to CRISPR-Cas9 targeting in an unsupervised fashion, accurately reducing false-positive signals, while maintaining sensitivity in identifying relevant genetic dependencies. Here, we present CRISPRcleanRWebApp, a web-based application enabling access to CRISPRcleanR through an intuitive graphical web-interface. CRISPRcleanRWebApp removes the complexity of low-level R/python-language user interactions; it provides a user-friendly access to a complete analytical pipeline, not requiring any data pre-processing, and generating gene-level summaries of essentiality with associated statistical scores; it offers a range of interactively explorable plots, while supporting a wider range of CRISPR guide RNAs libraries with respect to the original package. CRISPRcleanRWebApp is freely available at: https://crisprcleanr-webapp.fht.org/. HighlightsO_LICRISPR-Cas9 screens are widely used for the identification of cancer dependencies C_LIO_LIIn such screens, false-positives arise from targeting copy number amplified genes C_LIO_LICRISPRcleanR corrects this bias in an unsupervised fashion C_LIO_LICRISPRcleanRWebApp is a web user-friendly front-end for CRISPRcleanR C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/483924v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@739480org.highwire.dtl.DTLVardef@1a6c0aorg.highwire.dtl.DTLVardef@ab2b8eorg.highwire.dtl.DTLVardef@1b8a623_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2021

CoRe: A robustly benchmarked R package for the identification of core fitness genes in genome-wide pooled CRISPR knock-out screens

CRISPR-Cas9 genome-wide screens are being increasingly performed, allowing systematic explorations of cancer dependencies at unprecedented accuracy and scale. Identifying from these screens the genes that are essential for cell survival invariantly across tissues, conditions, and genomic-contexts (core-fitness genes), is of paramount importance to assess the safety profile of candidate therapeutic targets and for elucidating mechanisms involved in tissue-specific genetic diseases. We present CoRe: An R package implementing novel methods for identifying core-fitness genes from joint analyses of multiple CRISPR-Cas9 screens. We demonstrate that CoRe outperforms state-of-the-art tools, yielding more reliable sets of core-fitness genes than existing and widely used reference sets.

bioinformatics↗

bioRxiv2020

Whole genome CRISPR screens identify LRRK2-regulated endocytosis as a major mechanism for extracellular tau uptake by human neurons

Extracellular release and cellular uptake of pathogenic forms of the microtubule-associated protein tau contribute to the pathogenesis of several neurodegenerative diseases, including Alzheimers disease. Defining the cellular mechanisms and pathways for tau entry to human neurons is essential to understanding tauopathy pathogenesis and the rational design of disease-modifying therapeutics. Whole genome CRISPR loss-of-function screens in human iPSC-derived excitatory neurons, the major neuronal cell type affected in these diseases, enabled the delineation of the different cellular pathways for uptake of extracellular monomeric and fibrillar tau. Monomeric and fibrillar tau are both taken up by human neurons by receptor-mediated endocytosis, but involve different routes of entry at the neuronal surface: the low-density lipoprotein LRP1 is the primary receptor for monomeric tau, but contributes less to fibrillar tau entry. Similarly, endocytosis of monomeric tau is dependent on the familial Parkinsons disease gene LRRK2, but not required for endocytosis of fibrillar tau. These findings implicate LRP1 and LRRK2 in the pathogenesis of tauopathies and Parkinsons disease and identify LRRK2 as a potential therapeutic target for altering progression of these diseases.

neuroscience↗

bioRxiv2020

Integrated cross-study datasets of genetic dependencies in cancer

CRISPR-Cas9 viability screens are increasingly performed at a genome-wide scale across large panels of cell lines to identify new therapeutic targets for precision cancer therapy. Integrating the datasets resulting from these studies is necessary to adequately represent the heterogeneity of human cancers and to assemble a comprehensive map of cancer genetic vulnerabilities. Here, we integrated the two largest public independent CRISPR-Cas9 screens performed to date (at the Broad and Sanger institutes) by assessing, comparing, and selecting methods for correcting biases due to heterogeneous single guide RNA efficiency, gene-independent responses to CRISPR-Cas9 targeting originated from copy number alterations, and experimental batch effects. Our integrated datasets recapitulate findings from the individual datasets, provide greater statistical power to cancer- and subtype-specific analyses, unveil additional biomarkers of gene dependency, and improve the detection of common essential genes. We provide the largest integrated resources of CRISPR-Cas9 screens to date and the basis for harmonizing existing and future functional genetics datasets.

bioinformatics↗

bioRxiv2019

Engineering recombination between diverged yeast species reveals genetic incompatibilities

The major cause of the sterility of F1 hybrids formed between Saccharomyces cerevisiae and Saccharomyces paradoxus is anti-recombination. The failure of homologous chromosomes from the different species to recombine causes them to mis-segregate, resulting in aneuploid gametes, most of which are inviable. These effects of anti-recombination have previously impeded the search for other forms of incompatibility, such as negative genetic interactions (Bateson-Dobzhoansky-Muller incompatibilities). By suppressing the meiotic expression of MSH2 and SGS1, we could increase recombination and improve hybrid fertility seventy-fold. This allowed us to recover meiotic tetrads in which all four gametes were viable, ensuring that segregation had occurred properly to produce perfectly haploid, not aneuploid, recombinant hybrid gametes. We sequenced the genomes of 84 such tetrads, and discovered that some combinations of alleles from different species were significantly under-represented, indicating that there are incompatible genes contributing to reproductive isolation.

evolutionary biology↗