Biology subjects

Levchenko, A.

Publications and source records attributed to Levchenko, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineered CTLA-4 targeting chimeras for targeted protein degradation and immune synapse reprogramming

Despite the success of immune checkpoint blockade in cancer therapy, overcoming the immunosuppressive tumor microenvironment remains a major barrier to durable responses. Here, we introduce cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4)-targeting chimeras (cTACs), a class of synthetic molecules that harness CTLA-4-mediated trans-endocytosis (TE) for targeted protein degradation across cellular boundaries. The lead candidate, cTAC4.0, is composed of the extracellular domain of CD80 fused to an anti-programmed death-ligand 1 (PD-L1) fragment via a human immunoglobulin G4 (IgG4) heavy chain. Unlike existing protein degraders that function within cancer cells via house-keeping pathways, cTAC4.0 operates in trans between immune and tumor cells. It redirects the natural CD80-CTLA-4 internalization to capture and degrade PD-L1 from tumor cells into T cells, while simultaneously converting PD-L1-mediated suppression into CD28-driven co-stimulation, depending on the relative availability of CTLA-4 or CD28. Mechanistically, we demonstrate that cTAC4.0 promotes CTLA-4-dependent internalization and lysosomal degradation of both membrane-bound and soluble PD-L1, reduces surface PD-L1 levels on target cells, and activates T cells in a CD28-dependent manner. Functionally, cTAC4.0 enhances the cytotoxicity of both bispecific T cell engagers and T cell receptor (TCR)-engineered T cells in vitro, and when combined with these engineered T cells, it suppresses tumor growth in vivo in a glioblastoma xenograft model. By coupling immune checkpoint degradation with T cell activation in an immune synapse-enriched manner, cTACs establish a paradigm for intercellular protein degradation systems that simultaneously deplete suppressive ligands and enhance T cell function to overcome resistance in cancer immunotherapy.

immunology↗

bioRxiv2025

A Neyman-Pearson Framework for Modeling Cellular Decision Making Using Single-Cell TNF-NF-κB Signaling Data

Cells make hard calls under noise. When signaling is abnormal, those calls can go wrong and drive pathological conditions and diseases. In this research, we develop a Neyman-Pearson (NP) detection-theory framework that maximizes probability of detection (PD) for a chosen false alarm probability (PFA), without requiring prior probabilities, using experimental single-cell measurements of NF-{kappa}B responses to tumor necrosis factor (TNF), a critical pathway involved in cell survival, apoptosis, immune signaling, and stress response, in wild-type and A20-deficient fibroblasts. We model log-responses as (multi)variate Gaussian and compute optimal thresholds, PD-PFA trade-offs, and ROC curves at 30 minutes and 4 hours. The NP framework captures expected biology: PD increases with TNF dose; wild-type cells outperform A20-/- at matched conditions; and combining two time points (bivariate analysis) improves detection (e.g., for 0.0052 vs. 0.2 ng/mL, PD rises from 0.71 (30 minutes) and 0.42 (4 hours) to 0.80 at PFA = 0.1). The analysis recovers expected biology (higher TNF causes higher detectability; negative feedback lowers late responses) and flags cases where decision quality degrades (e.g., perturbations that blunt separation between conditions). The same recipe extends to multivariate readouts without changing the logic. Overall, the NP detection framework provides a compact, quantitative score of pathway performance and failure. It turns noisy single-cell readouts into actionable decision metrics that compare doses, time points, and perturbations, and ultimately, can help explain when and how cellular decisions drift toward pathology.

systems biology↗

bioRxiv2025

Analysis of the phosphorylation networks characterizing distinct phenotypic states in glioblastoma cell Populations

Cancer cells, including the most aggressive brain cancer, glioblastoma, can exhibit a high degree of phenotypic plasticity. However, it is not well understood whether and how distinct phenotypic states might be associated with or driven by specific signaling processes. Here, we use a novel approach to the identification of phenotype-specific signaling networks in cells undergoing the Go-or-Grow switch in populations of GBM cell lines. We find that the transition to invasive spread may be associated with the onset of DNA damage response, cell cycle arrest, and activation of the AKT-mTOR signaling. We reconstructed the large-scale signaling networks, revealing integration of diverse pathways and possible feedback interactions mediating phenotypic stability. We further show that phosphorylation outcomes may be preferentially mediated by 14-3-3 proteins, previously implicated in stress response and control of the cell cycle. Finally, we demonstrate that the phenotype-specific phospho-site signatures have predictive power for disease-free patient survival. This study paves the way for a more comprehensive understanding of phenotypic plasticity in GBM and other aggressive cancers.

systems biology↗

bioRxiv2025

Oscillatory signal decoding within the ERK cascade

Extracellular-signal-regulated kinase (ERK) integrates multiple growth factor and hormone stimuli to control essential cellular processes such as proliferation, survival, and migration. In electrically excitable cells, the ERK pathway also interfaces with intracellular Ca2+ dynamics to achieve non-canonical, cell-type specific functions, having been implicated in neuronal synaptic plasticity, cardiac hypertrophy, and pancreatic insulin secretion. Yet how the classical Ras/MEK/ERK cascade responds to and decodes dynamic Ca2+ signals at its multiple levels to regulate cellular function is poorly understood. Here, we investigated the dynamics of Ca2+-induced ERK pathway activation in a pancreatic {beta}-cell line using genetically encoded fluorescent biosensors. By carefully manipulating Ca2+ input signals and directly monitoring the activity dynamics of individual ERK pathway components, we reveal that {beta}-cell Ca2+ oscillations undergo sequential signal processing along the ERK cascade, mediated by the characteristic response kinetics at each pathway step. We further demonstrate that the ERK cascade and possibly other Ca2+-responsive pathways operate within a hybrid network architecture to achieve both hierarchical and parallel processing of {beta}-cell Ca2+ oscillations, providing important insights into dynamic signal decoding by this crucial signaling network.

cell biology↗

bioRxiv2025

SPINK1-COL18A1 Crosstalk Shapes Epigenome and Drives Cancer Stemness in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer with an increasing incidence and extremely dismal prognosis. Discovery and mechanistic understanding of the genetic and epigenetic drivers of PDAC is therefore of critical importance. Here, we uncover serine protease inhibitor Kazal type 1 (SPINK1) as a putative determinant of clinical progression and demonstrate that it can have a non-canonical function as a regulator of epigenomic states of PDAC cells and associated extensive changes in gene expression. We show that SPINK1 expression, which varies in PDAC, is associated with key aggressive phenotypic cancer states and is correlated with the expression of stemness markers both in vitro and in patient samples. Mechanistically, our results strongly suggest that SPINK1 acts through a new signaling axis, by interacting with COL18A1 in the Golgi apparatus, promoting endostatin release and eventually inducing extensive histone H3 modifications. These results reveal a new function of SPINK1 in PDAC and suggest a potential therapeutic route to combat PDAC aggressiveness by targeting the SPINK1-COL18A1-endostatin signaling.

cancer biology↗

bioRxiv2025

Interplay between cytokine and FGF2 signaling in induction of entosis and vasculogenic mimicry response in glioblastoma

Tumor vascularization is critical to survival of cancer cells, but is frequently perturbed leading to disorganized angiogenesis and emergence of alternative means of delivery of oxygen and nutrients, such as vasculogenic mimicry (VM). Understanding of VM and its relationship to endothelial vascularization has been hampered by the lack of comprehensive combination of in vivo clinical data and relevant in vitro models. We address this challenge by analyzing glioblastoma (GBM) tumors and clinically isolated cancer cells. This analysis strongly suggests a key role of macrophage-induced controlled cell death in emergence of VM. The results further point to entosis of cancer cells as a critical intermediate state in this process, enabled by mechano-chemical cell heterogeneity. We find evidence that macrophages can regulate endothelial angiogenesis and VM as two alternative vascularization mechanisms. These results reveal mechanistic underpinnings of VM and pave the way to predictive analysis of tumor progression.

cancer biology↗

bioRxiv2024

Tunability of calcium dynamics by signaling inputs and cell-cell communication in pancreatic beta cells

In addition to frequently occurring stable all-or-none responses, live cells can display more complex response dynamics, e.g., oscillations in the activity/concentration of biomolecules. While the emergence and function of oscillatory dynamics have been heavily investigated, fewer efforts have been spent on whether cells can fine-tune different aspects of an oscillatory signal (e.g., peakwidth, duty cycle, and frequency) and whether this fine-tuning can allow oscillatory signals to convey different information. In this study, we investigate glucose-induced calcium (Ca2+) oscillation in MIN6 cells, finding that the spontaneous or induced changes in the phosphatidylinositol 4,5-bisphosphate (PIP2) level during Ca2+ oscillation can modulate the peakwidth of individual Ca2+ spikes. Additionally, using a combination of optogenetics and Ca2+ imaging, we demonstrate that variation of the widths and frequencies of Ca2+ spikes can exert strong influence on coupling between neighboring MIN6 cells.

systems biology↗

bioRxiv2024

Specification of human regional brain lineages using orthogonal gradients of WNT and SHH in organoids

The repertory of neurons generated by progenitor cells depends on their location along antero-posterior and dorso-ventral axes of the neural tube. To understand if recreating those axes was sufficient to specify human brain neuronal diversity, we designed a mesofluidic device termed Duo-MAPS to expose induced pluripotent stem cells (iPSC) to concomitant orthogonal gradients of a posteriorizing and a ventralizing morphogen, activating WNT and SHH signaling, respectively. Comparison of single cell transcriptomes with fetal human brain revealed that Duo-MAPS-patterned organoids generated the major neuronal lineages of the forebrain, midbrain, and hindbrain. Morphogens crosstalk translated into early patterns of gene expression programs predicting the generation of specific brain lineages. Human iPSC lines from six different genetic backgrounds showed substantial differences in response to morphogens, suggesting that interindividual genomic and epigenomic variations could impact brain lineages formation. Morphogen gradients promise to be a key approach to model the brain in its entirety.

neuroscience↗

bioRxiv2024

Organ Boundary Circuits Regulate Sox9+ Alveolar Tuft Cells During Post-Pneumonectomy Lung Regeneration

Tissue homeostasis is controlled by cellular circuits governing cell growth, organization, and differentation. In this study we identify previously undescribed cell-to-cell communication that mediates information flow from mechanosensitive pleural mesothelial cells to alveolar-resident stem-like tuft cells in the lung. We find mesothelial cells to express a combination of mechanotransduction genes and lineage-restricted ligands which makes them uniquely capable of responding to tissue tension and producing paracrine cues acting on parenchymal populations. In parallel, we describe a large population of stem-like alveolar tuft cells that express the endodermal stem cell markers Sox9 and Lgr5 and a receptor profile making them uniquely sensitive to cues produced by pleural Mesothelium. We hypothesized that crosstalk from mesothelial cells to alveolar tuft cells might be central to the regulation of post-penumonectomy lung regeneration. Following pneumonectomy, we find that mesothelial cells display radically altered phenotype and ligand expression, in a pattern that closely tracks with parenchymal epithelial proliferation and alveolar tissue growth. During an initial pro-inflammatory stage of tissue regeneration, Mesothelium promotes epithelial proliferation via WNT ligand secretion, orchestrates an increase in microvascular permeability, and encourages immune extravasation via chemokine secretion. This stage is followed first by a tissue remodeling period, characterized by angiogenesis and BMP pathway sensitization, and then a stable return to homeostasis. Coupled with key changes in parenchymal structure and matrix production, the cumulative effect is a now larger organ including newly-grown, fully-functional tissue parenchyma. This study paints Mesothelial cells as a key orchestrating cell type that defines the boundary of the lung and exerts critical influence over the tissue-level signaling state regulating resident stem cell populations. The cellular circuits unearthed here suggest that human lung regeneration might be inducible through well-engineered approaches targeting the induction of tissue regeneration and safe return to homeostasis.

systems biology↗

bioRxiv2023

Solid tumor growth depends on an intricate equilibrium of malignant cell states

Control of cell identity and number is central to tissue function, yet principles governing organization of malignant cells in tumor tissues remain poorly understood. Using mathematical modeling and candidate-based analysis, we discover primary and metastatic pancreatic ductal adenocarcinoma (PDAC) organize in a stereotypic pattern whereby PDAC cells responding to WNT signals (WNT-R) neighbor WNT-secreting cancer cells (WNT-S). Leveraging lineage-tracing, we reveal the WNT-R state is transient and gives rise to the WNT-S state that is highly stable and committed to organizing malignant tissue. We further show that a subset of WNT-S cells expressing the Notch ligand DLL1 form a functional niche for WNT-R cells. Genetic inactivation of WNT secretion or Notch pathway components, or cytoablation of the WNT-S state disrupts PDAC tissue organization, suppressing tumor growth and metastasis. This work indicates PDAC growth depends on an intricately controlled equilibrium of functionally distinct cancer cell states, uncovering a fundamental principle governing solid tumor growth and revealing new opportunities for therapeutic intervention.

cancer biology↗

bioRxiv2023

Dynamic decoding of VEGF signaling and coordinated control of multiple phenotypes by the Src-TEM4-YAP pathway

Responses of endothelial cells to elevated levels of Vascular Endothelial Growth Factor (VEGF), frequently accompanying local decrease in oxygen supply, include loosening of cell contacts, rearrangement of cells in the process of vessel remodeling and ultimately, angiogenic growth. How these complex processes, occurring on diverse time scales, are coordinated and how they are guided by a single key signaling input, is still incompletely understood. Here we show that the various phenotypic responses associated with VEGF signaling are controlled at different steps of a pathway involving sequential activation of Src, TEM4, YAP and components of pro-angiogenic Notch signaling. Strikingly, due to feedback regulation at different pathway levels, the functional outcomes are controlled by oscillations of the pathway components occurring on distinct time scales. Deeper pathway layers integrate faster upstream responses and control progressively slower phenotypic outcomes. This signal decoding pathway organization can ensure a high degree of complexity in a vital physiological process.

systems biology↗

bioRxiv2023

Single Cell Measurements and Modeling and Computation of Decision Making Errors in a Molecular Signaling System with Two Output Molecules

A cell constantly receives signals and takes different fates accordingly. Given the uncertainty rendered by signal transduction noise, the cell may incorrectly perceive the signals. It may mistakenly behave as if there is a signal - although there is none, or may miss the presence of a signal that actually exists. In this paper, we consider a signaling system that has two outputs, and introduce and develop methods to model and compute key cell decision making parameters based on the two outputs, and in response to the input signal. In the considered system, the tumor necrosis factor (TNF) regulates the two transcription factors, the nuclear factor {kappa}B (NF{kappa}B) and the activating transcription factor-2 (ATF-2). These two system outputs are involved in important physiological functions such as cell death and survival, viral replication, and pathological conditions such as autoimmune diseases and different types of cancer. Using the introduced methods, we compute and show what the decision thresholds are, based on the single cell measured concentration levels of NF{kappa}B and ATF-2. We also define and compute the decision error probabilities, i.e., false alarm and miss probabilities, based on the concentration levels of the two outputs. By considering the joint response of the two outputs of a signaling system, one can learn more about complex cellular decision making processes, the corresponding decision error rates, and their possible involvement in the development of some pathological conditions.

systems biology↗

bioRxiv2023

Experimental and Phylogenetic Evidence for Correlated Gene Expression Evolution between Dermal and Endometrial Fibroblasts: implications for the evolution of cancer malignancy

Changes in transcriptional gene expression is a dominant mode of evolution, mostly driven by mutations at cis-regulatory regions. Mutations can affect gene expression in multiple cell types if the same cis-regulatory elements are used by different cell types. As a consequence, changes in gene expression in one cell type may be associated with similar gene expression changes in another cell type. Correlated gene expression change can explain correlated character evolution, as for instance the correlation between placental invasion and vulnerability to cancer malignancy. Here we test this hypothesis using a comparative and an experimental data set. Specifically, we investigate gene expression in dermal skin fibroblasts (SF) and uterine endometrial stomal fibroblasts (ESF). The comparative dataset consists of transcriptomes from cultured SF and ESF from 9 mammalian species. We calculated the independent phylogenetic contrasts (PIC) for each gene and cell type. We find that evolutionary changes in gene expression in SF and ESF are highly correlated, supporting the hypothesis that the correlated gene expression changes are a prevalent feature of gene expression evolution. The experimental data set derives from a SCID mouse strain that was selected for slow cancer growth which led to substantial changes in the SF compared to wild type SCID mice. We isolated SF and ESF from wild type and evolved SCID mice and compared their gene expression profiles. We find a significant correlation between the gene expression contrasts of SF and ESF, which supports the hypothesis that gene expression variation in SF and ESF is correlated. We discuss the implications of these findings for the hypothesized correlation between placental invasiveness and vulnerability to metastatic cancer.

evolutionary biology↗

bioRxiv2023

CTLA-4 tail fusion enhances CAR-T anti-tumor immunity

Chimeric antigen receptor (CAR) T cells are powerful therapeutics; however, their efficacy is often hindered by critical hurdles. Here, utilizing the endocytic feature of the cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) cytoplasmic tail (CT), we reprogram CAR function and substantially enhance CAR-T efficacy in vivo. CAR-T cells with monomeric, duplex, or triplex CTLA-4 CTs (CCTs) fused to the C-terminus of CAR exhibit a progressive increase in cytotoxicity under repeated stimulation, accompanied by reduced activation and production of pro-inflammatory cytokines. Further characterization reveals that CARs with increasing CCT fusion show a progressively lower surface expression, regulated by their constant endocytosis, recycling and degradation under steady state. The molecular dynamics of reengineered CAR with CCT fusion results in reduced CAR-mediated trogocytosis, loss of tumor antigen, and improved CAR-T survival. CARs with either monomeric (CAR-1CCT) or duplex CCTs (CAR-2CCT) have superior anti-tumor efficacy in a relapsed leukemia model. Single-cell RNA sequencing and flow cytometry analysis reveal that CAR-2CCT cells retain a stronger central memory phenotype and exhibit increased persistence. These findings illuminate a unique strategy for engineering therapeutic T cells and improving CAR-T function through synthetic CCT fusion, which is orthogonal to other cell engineering techniques.

immunology↗

bioRxiv2022

Spatial-temporal order-disorder transition in angiogenic NOTCH signaling controls cell fate specification

Angiogenesis is a morphogenic process resulting in the formation of new blood vessels from pre-existing ones, usually in hypoxic micro-environments. The initial steps of angiogenesis depend on robust differentiation of oligopotent endothelial cells into the Tip and Stalk phenotypic cell fates, controlled by NOTCH-dependent cell-cell communication. The dynamics of spatial patterning of this cell fate specification are only partially understood. Here, by combining a controlled experimental angiogenesis model with mathematical and computational analyses, we find that the regular spatial Tip-Stalk cell patterning can undergo an order-disorder transition at a relatively high input level of a pro-angiogenic factor VEGF. The resulting differentiation is robust but temporally unstable for most cells, with only a subset of presumptive Tip cells leading sprout extensions. We further find that sprouts form in a manner maximizing their mutual distance, consistent with a Turing-like model that may depend on local enrichment and depletion of fibronectin. Together, our data suggest that NOTCH signaling mediates a robust way of cell differentiation enabling but not instructing subsequent steps in angiogenic morphogenesis, which may require additional cues and self-organization mechanisms. This analysis can assist in further understanding of cell plasticity underlying angiogenesis and other complex morphogenic processes. Significance StatementWe investigate the spatial and temporal patterns of Tip/Stalk specification and the ensuing angiogenic sprouting by using a novel controlled micro-engineered experimental model of angiogenesis and a set of mathematical models of the spatially resolved, cell population-level VEGF-NOTCH signaling. Our analysis provides a dynamic view of the initial step of angiogenesis, revealing fluctuations in its onset, and features suggesting transitions between order and disorder in cell organization. These findings suggest how a potentially very restrictive patterning mechanism can become sensitive to a variety of environmental cues. This sensitivity can be crucial for proper vascularization of a damaged organ, and may suggest new ways of analyzing angiogenesis in the context of cancer and other pathologies. This analysis also suggests a framework for understanding of other instances of NOTCH-mediated patterning processes.

systems biology↗

bioRxiv2022

SARS-CoV-2 leverages airway epithelial protective mechanism for viral infection

Despite much concerted effort to better understand SARS-CoV-2 viral infection, relatively little is known about the dynamics of early viral entry and infection in the airway. Here we analyzed a single-cell RNA sequencing dataset of early SARS-CoV-2 infection in a humanized in vitro model, to elucidate key mechanisms by which the virus triggers a cell-systems-level response in the bronchial epithelium. We find that SARS-CoV-2 virus preferentially enters the tissue via ciliated cell precursors, giving rise to a population of infected mature ciliated cells, which signal to basal cells, inducing further rapid differentiation. This feed-forward loop of infection is mitigated by further cell-cell communication, before interferon signaling begins at three days post-infection. These findings suggest hijacking by the virus of potentially beneficial tissue repair mechanisms, possibly exacerbating the outcome. This work both elucidates the interplay between barrier tissues and viral infections, and may suggest alternative therapeutic approaches targeting non-immune response mechanisms.

systems biology↗

bioRxiv2021

Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

Kinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner. We further demonstrate that superposition of gradients of PKA-R abundance across single cells can control the directionality of cell migration, reversing it at high enough input levels. Thus complex in vivo patterns of PKA-R localization can drive complex phenotypes, including cell migration.

systems biology↗

bioRxiv2021

Connectome: computation and visualization of cell-cell signaling topologies in single-cell systems data

Single-cell RNA-sequencing data can revolutionize our understanding of the patterns of cell-cell and ligand-receptor connectivity that influence the function of tissues and organs. However, the quantification and visualization of these patterns are major computational and epistemological challenges. Here, we present Connectome, a software package for R which facilitates rapid calculation, and interactive exploration, of cell-cell signaling network topologies contained in single-cell RNA-sequencing data. Connectome can be used with any reference set of known ligand-receptor mechanisms. It has built-in functionality to facilitate differential and comparative connectomics, in which complete mechanistic networks are quantitatively compared between systems. Connectome includes computational and graphical tools designed to analyze and explore cell-cell connectivity patterns across disparate single-cell datasets. We present approaches to quantify these topologies and discuss some of the biologic theory leading to their design.

systems biology↗