Biology subjects

Mikolajewicz, N.

Publications and source records attributed to Mikolajewicz, N..

5 recordsLinked to original sources

bioRxiv2025

Integrated genetic screening reveals FEN1 as a driver of stemness and temozolomide resistance in glioblastoma

Glioblastoma (GBM) is a lethal brain tumor with limited response to standard of care chemoradiotherapy. In this study, we conducted genome-wide CRISPR knockout screening in patient-derived glioblastoma stem cells (GSCs) to identify genetic dependencies of cell survival and therapy resistance. Our screening identified flap endonuclease 1 (FEN1) as a key driver of GSC survival, with enhanced dependency under temozolomide (TMZ) treatment. Genetic perturbation of FEN1 reduced GSC self-renewal and proliferation in vitro, and prolonged survival in a patient-derived xenograft model of GBM. FEN1 inhibition (FEN1i) preferentially affected highly aggressive or recurrent GBM models compared with less aggressive GBMs and healthy neural stem cells. Moreover, FEN1 inhibition synergized with TMZ only in these aggressive FEN1i-sensitive GSCs, providing cancer-selective killing and TMZ sensitization in the most untreatable of GBMs. Mechanistically, FEN1i-sensitive GSCs exhibited greater proliferation and sphere formation, while stalling their proliferation conferred resistance to FEN1 inhibition. Single-cell transcriptomics further linked FEN1 expression to stemness and the DNA damage response, elucidating broader determinants of FEN1 dependency. These findings establish FEN1 as a promising therapeutic target in GBM, offering a strategy for both selective targeting and enhancement of TMZ efficacy in aggressive cancers. Statement of SignificanceThis study identifies FEN1 as a key vulnerability of glioblastoma stem cells, revealing its role in therapy resistance and stemness, and proposes FEN1 inhibition as a strategy to enhance temozolomide efficacy.

cancer biology↗

bioRxiv2022

Multi-level cellular and functional annotation of single-cell transcriptomes

Single-cell RNA-sequencing (scRNA-seq) offers unprecedented insight into heterogenous biology, allowing for the interrogation of cellular populations and gene expression programs at single-cell resolution. Here, we introduce scPipeline, a single-cell analytic toolbox that offers modular workflows for multi-level cellular annotation and user-friendly analysis reports. Novel methods that are introduced to facilitate scRNA-seq annotation include: (i) co-dependency index (CDI)-based differential expression; (ii) cluster resolution optimization using a marker-specificity criterion; (iii) marker-based cell-type annotation with Miko scoring; and (iv) gene program discovery using scale-free shared nearest neighbor network (SSN) analysis. Our unsupervised and supervised procedures were validated using a diverse collection of scRNA-seq datasets and we provide illustrative examples of cellular and transcriptomic annotation of developmental and immunological scRNA-seq atlases. Overall, scPipeline provides a flexible computational framework for in-depth scRNA-seq analysis.

bioinformatics↗

bioRxiv2022

Characterization of the minimal residual disease state reveals distinct evolutionary trajectories of human glioblastoma

Recurrence of solid tumors renders patients vulnerable to a distinctly advanced, highly treatment-refractory disease state that has an increased mutational burden and novel oncogenic drivers not detected at initial diagnosis. Improving outcomes for recurrent cancers requires a better understanding of cancer cell populations that expand from the post-therapy, minimal residual disease (MRD) state. We profiled barcoded tumor stem cell populations through therapy at tumor initiation/engraftment, MRD and recurrence in our therapy-adapted, patient-derived xenograft models of glioblastoma (GBM). Tumors showed distinct patterns of recurrence in which clonal populations exhibited either an a priori, pre-existing fitness advantage, or a priori equipotency fitness acquired through therapy. Characterization of the MRD state by single-cell and bulk RNA sequencing revealed a tumor-intrinsic immunomodulatory signature with strong prognostic significance at the transcriptomic level and in proteomic analysis of cerebrospinal fluid (CSF) collected from GBM patients at all stages of disease. Our results provide insight into the innate and therapy-driven dynamics of human GBM, and the prognostic value of interrogating the MRD state in solid cancers.

cancer biology↗

bioRxiv2021

High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

1P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consist of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10-4 and 10-2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses, and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naive state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanitransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation. 2 Author SummaryATP-sensitive purinergic receptors comprise a network of cell-surface receptors that activate upon ATP binding, allowing them to transmit information in a tissue- and context-dependent manner. In bone, mechanically-stimulated osteoblasts release ATP that stimulates low- and high-affinity P2 receptors in neighboring cellular populations, inducing appropriate physiological responses. P2 receptor signaling is characterized by elevations in intracellular calcium levels. When simultaneously stimulated by their common ligand, ATP, the contribution of each P2 receptor subtype gives rise to a complex calcium response, exhibiting oscillatory characteristics and biphasic dose-dependent behaviours. Here we used experimental and computational modeling approaches to determine the underlying dynamics of ATP-mediated calcium signaling in osteoblasts. The latter was done by developing a mathematical model that was comprised of a subset of low-(P2X7) and high-(P2Y2) affinity P2 receptors, reflecting the conserved P2 expression observed across different osteoblast models. We demonstrated that this model recapitulates experimental recordings of ATP-induced calcium signaling in osteoblasts and describes the dynamic interplay between P2Y2 and P2X7 receptors in the P2 receptor network.

cell biology↗

bioRxiv2018

Transmission of mechanical information by purinergic signaling

1The human skeleton constantly interacts and adapts to the physical world. We have previously reported that physiologically-relevant mechanical forces lead to small, repairable membrane injuries in bone-forming osteoblasts, resulting in the release of ATP and stimulation of purinergic (P2) calcium responses in neighbouring cells. The goal of this study was to develop a theoretical model describing injury-related ATP and ADP release, extracellular diffusion and degradation, and purinergic responses in neighboring cells. The model was validated using experimental data obtained by measuring intracellular free calcium ([Ca2+]i) elevations following mechanical stimulation of a single osteoblast. The validated single-cell injury model was then scaled to a tissue-level injury to investigate how purinergic responses communicate information about injuries with varying geometries. We found that total ATP released, peak extracellular ATP concentration and the ADP-mediated signaling component contributed complementary information regarding the mechanical stimulation event. The total amount of ATP released governed the maximal distance from the injury at which purinergic responses were stimulated, as well as the overall number of responders. The peak ATP concentration reflected the severity of an individual cell injury and determined signal propagation velocity and temporal synchrony of responses. Peak ATP concentrations also discriminated between minor and severe injuries that led to the release of similar total amounts of ATP due to differences in injury repair dynamics. The third component was ADP-mediated signaling which became relevant only in larger tissue-level injuries, and it conveyed information about the distance to the injury site and its geometry. Taken together, this study identified specific features of extracellular ATP/ADP spatiotemporal signals that encode the severity of the mechanical stimulus, the distance from the stimulus, as well as the mechano-resilient status of the tissue.

biophysics↗