Biology subjects

Kholodenko, B. N.

Publications and source records attributed to Kholodenko, B. N..

10 recordsLinked to original sources

bioRxiv2026

SMAD3-driven cell state transitions underpin immunoresistance in bladder cancer

Despite the clinical success of anti-PD-1/PD-L1 immunotherapy in a subset of patients with high-risk bladder cancer (BLCA), most tumors remain refractory to treatment. We hypothesized that immunotherapy resistance is encoded within the intrinsic signaling state of BLCA cells and arises through specific cell-state transitions that precede therapeutic exposure. To test this hypothesis, we applied a cell State Transition Assessment and Regulation (cSTAR) framework to identify key signaling modules and aberrant causal network connections that (i) drive oncogenic progression and acquisition of aggressive phenotypes and (ii) govern BLCA cell susceptibility to T cell-mediated cytotoxicity. cSTAR analysis identified TGF{beta}/SMAD3 signaling as the principal driver of both oncogenic transformation and immunoresistance in BLCA cells. Inhibiting SMAD3 sensitized BLCA cells to PD-1-expressing Jurkat cells and primary human CD4+ T cell-mediated cytotoxicity through suppression of autophagy and activation of multiple cell death pathways. By integrating cell state transition analysis with functional immune co-culture assays, our work demonstrates that tumor cell state transitions control sensitivity to T cell killing, complementing established immune-intrinsic mechanisms of immunotherapy response.

systems biology↗

bioRxiv2026

IFN and IFN mimetics prevent IFN-I-mediated TB susceptibility by regulating iron metabolism and lipid peroxidation

Type I interferons (IFN-I) and IFN{gamma} exert divergent effects during tuberculosis, but the mechanisms that determine whether macrophage activation promotes host defense or inflammatory pathology remain incompletely understood. Here, we dissect the interplay between IFN-I and IFN{gamma} in macrophage activation using genetically susceptible B6.Sst1S macrophages. We show that, during tumor necrosis factor (TNF) stimulation, susceptible macrophages enter a persistent pathological activation state (pPAS) characterized by sustained lipid peroxidation and super-induction of IFN-I responses. This pathological state is maintained by autocrine IFN-I signaling. In contrast, IFN{gamma} priming prevents pPAS development by enhancing macrophage resilience to oxidative stress, in part through regulation of iron metabolism and induction of ferritin expression. Computational cell state transition assessment and regulation (cSTAR) analysis identified pathways and small molecules predicted to promote the transition of susceptible macrophages toward an IFN{gamma}-induced, Mtb-resistant state. Consistent with these predictions, the CDK4/6 inhibitor trilaciclib reduced lipid peroxidation by regulating iron metabolism, whereas retinoic acid signaling enhanced GPX4 expression and lipid biosynthesis programs. Combined CDK4/6 inhibition and retinoic acid receptor activation efficiently prevented the pathological activation state. Together, these findings delineate a mechanism of IFN-I/IFN{gamma} crosstalk during macrophage activation and identify pharmacologic strategies to prevent IFN-I-dominant, lipid peroxidation-driven macrophage pathology.

immunology↗

bioRxiv2025

A structure-based modelling approach identifies effective drug combinations for RAS-mutant acute myeloid leukemia

Mutations activating RAS/RAF/MEK/ERK signaling are associated with poor outcome in acute myeloid leukemia (AML), but therapeutic targeting of this pathway is challenging. Here, we employ a structure-based, dynamic RAS pathway model to successfully predict RAF inhibitor (RAFi) combinations which synergistically suppress ERK signaling in RAS-mutant AML. Our in silico models predicted therapeutic synergy of two iterations of conformation-specific RAF inhibitors: Type I[1/2] + Type II and Type I + Type II. Predictions were validated in vitro in AML cell lines and patient samples, with synergy verified by the Loewe Additivity model. Lifirafenib (Type II) + encorafenib (Type I[1/2]) was highly synergistic against both NRAS- and KRAS-mutant lines, while synergy of lifirafenib + SB590885 (Type I) was specific to NRAS-mutants. Immunoblotting confirmed that combination efficacy correlated strongly with decreased RAS pathway activation. Leveraging the pharmacokinetic predictions of our in silico model, both combinations were then assessed in a pre-clinical NRAS-mutant AML patient-derived xenograft (PDX) model, showing significantly improved leukaemia growth delay and event-free survival compared with single agent approaches. Assessment of leukemia burden in bone marrow and spleen during treatment further showed site-specific efficacy against circulating and spleen-resident blasts for both combinations. In summary, we report that our structure based-modelling approach can effectively identify novel, non-obvious, and well-tolerated RAFi combinations that are highly effective against in vitro and in vivo models, thereby suggesting alternative potential therapeutic strategies for high-risk RAS-mutant AML.

cancer biology↗

bioRxiv2024

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop pulmonary TB. We developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: the development of necrotic lung granulomas and determined that the sst1-susceptible phenotype was driven by the aberrant macrophage activation. This study demonstrates that the aberrant response of the sst1-susceptible macrophages to prolonged stimulation with TNF is primarily driven by conflicting Myc and antioxidant response pathways leading to a coordinated failure 1) to properly sequester intracellular iron and 2) to activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled Ifn{beta} superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. The upregulation of Myc pathway in peripheral blood cells of human TB patients was significantly associated with poor outcomes of TB treatment. Thus, Myc dysregulation in activated macrophages results in an aberrant macrophage activation and represents a novel target for host-directed TB therapies.

immunology↗

bioRxiv2024

Cell state transition analysis reveals a causal network that controls critical limb ischemia

Increased endothelial cell (EC) permeability significantly contributes to peripheral arterial disease and its severe manifestation, critical limb ischemia (CLI). CLI remains refractory to current pharmacological interventions, often requiring limb amputation and showing how limited our knowledge is about signaling pathways that control EC states. Here, we utilize a two-pronged approach, employing cell State Transition Assessment and Regulation (cSTAR) analyses to both the published knowledge (e.g., LINCS data portal) and our own transcriptomics data collected from CLI patients and healthy adult (HA) control. We identified CLI and HA transcriptomics patterns and reconstructed causal network that controls transitions between EC states associated with CLI and HA. Contrary to general belief, our study demonstrates that VEGF administration slightly promotes the CLI EC states, indicating VEGF ineffectiveness in enhancing perfusion of CLI tissue. Based on the unveiled control network, we suggest targets whose inhibition would reverse CLI EC states closer to HA states.

bioinformatics↗

bioRxiv2024

VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling.

Postnatal vascular morphogenesis is a dynamic process that initially involves angiogenesis to form the capillary bed, followed by its partial remodeling into arteries. Vascular endothelial growth factor A (VEGF hereafter), the primary inducer of angiogenesis, is also implicated in arterial specification in developmental contexts. However, it is unclear why arterial patterning is spatiotemporally segregated from angiogenesis, while postnatal arterial specification in animal models with blocked VEGF signaling remains unstudied. Here, we report that VEGF does not induce arterial fate in the capillaries, instead serving as a physiological brake to attenuate fluid shear stress (FSS)-driven capillary-to-arterial cell fate transition. Mouse models with disrupted VEGF signaling reveal impaired angiogenesis but intact and ectopic arterialization. Mechanistically, mechanosensitive transcription factor Sox17 determines the FSS-arterial program, while VEGF suppresses Sox17 transcription activity at arterial promoters and enhancers sites. Angiogenic signaling, while essential for the initial capillary morphogenesis, inhibits arterial specification, thereby protecting the capillary bed from premature arterialization driven by flow. These findings establish a new paradigm in which precise spatiotemporal coordination of environmental stimuli orchestrates angiogenesis, arterial patterning and capillary maintenance during vascular morphogenesis.

developmental biology↗

bioRxiv2023

cSTAR analysis identifies endothelial cell cycle as a key regulator of flow-dependent artery remodeling

Fluid shear stress (FSS) from blood flow is sensed by vascular endothelial cells (ECs) to determine vessel stability, remodeling and susceptibility to atherosclerosis and other inflammatory diseases but the regulatory networks that govern these behaviors are only partially understood. We used cSTAR, a powerful new computational method, to define EC transcriptomic states under low shear stress (LSS) that triggers vessel inward remodeling, physiological shear stress (PSS) that stabilizes vessels, high shear stress (HSS) that triggers outward remodeling, and oscillatory shear stress (OSS) that confers disease susceptibility, all in comparison to cells under static conditions (STAT). We combined these results with the LINCS database where EC transcriptomic responses to drug treatments to define a preliminary regulatory network in which the cyclin-dependent kinases CDK1/2 play a central role in promoting vessel stability. Experimental analysis showed that PSS induced a strong late G1 cell cycle arrest in which CDK2 was activated. EC deletion of CDK2 in mice resulted in inward artery remodeling and both pulmonary and systemic hypertension. These results validate use of cSTAR to determine EC state and in vivo vessel behavior, reveal unexpected features of EC phenotype under different FSS conditions, and identify CDK2 as a key element within the EC regulatory network that governs artery remodeling.

cell biology↗

bioRxiv2020

Maladaptive oxidative stress cascade drives type I interferon hyperactivity in TNF activated macrophages promoting necrosis in murine tuberculosis granulomas

Tuberculosis remains a critical infectious disease world-wide. The development of novel therapeutic strategies requires greater understanding of host factors that contribute to disease susceptibility. A major unknown in TB pathogenesis is the mechanism of necrosis in TB granulomas that leads to the massive lung tissue damage and cavity formation necessary for the pathogen transmission. In humans, TB progression has been linked to hyperactivity of type I IFN (IFN-I) pathway, the primary cause of which remains elusive. We studied the mechanistic drivers of pulmonary TB progression using a unique model B6J.C3-Sst1C3HeB/Fej Krmn mice that develop human-like necrotic TB granulomas and IFN-I hyperactivity. We established that IFN{beta} super-induction occurred in the susceptible macrophages in response to continuous TNF stimulation in the context of a dysregulated antioxidant defense. We observed that unresolving oxidative stress amplified the induction of IFN{beta} through JNK activation and induced the Integrated Stress Response via PKR activation as a compensatory pathway. Subsequently, PKR amplifies IFN{beta} upregulation, forming a positive feedback loop, maintaining the hyperinflammatory state in susceptible macrophages and leading to mitochondrial dysfunction. Thus, within the inflammatory milieu, a cell-intrinsic mechanism of chronic regulatory dysfunction and unresolved stress gradually weakens the macrophage and ultimately promotes the necrotization of TB granulomas. The aberrant macrophage response to TNF can be prevented by an iron chelator and inhibitor of lipid peroxidation, ferrostatin-1. Moreover, ferrostatin treatment increased macrophage survival and boosted bacterial control in the TNF-stimulated macrophages infected with virulent Mtb. These findings identify targets for host-directed therapeutics to interrupt necrotization in TB granulomas.

immunology↗

bioRxiv2020

Periodic propagating activation waves coordinate Rho GTPase network dynamics at the leading and trailing edges during cell migration

Migrating cells need to coordinate distinct leading and trailing edge dynamics but the underlying mechanisms are unclear. Here, we combine experiments and mathematical modeling to elaborate the minimal autonomous biochemical machinery necessary and sufficient for this dynamic coordination and cell movement. RhoA activates Rac1 via DIA and inhibits Rac1 via ROCK, while Rac1 inhibits RhoA through PAK. Our data suggest that in motile, polarized cells, RhoA-ROCK interactions prevail at the rear whereas RhoA-DIA interactions dominate at the front where Rac1/Rho oscillations drive protrusions and retractions. At the rear, high RhoA and low Rac1 activities are maintained until a wave of oscillatory GTPase activities from the cell front reaches the rear, inducing transient GTPase oscillations and RhoA activity spikes. After the rear retracts, the initial GTPase pattern resumes. Our findings show how periodic, propagating GTPase waves coordinate distinct GTPase patterns at the leading and trailing edge dynamics in moving cells.

systems biology↗

bioRxiv2018

Transcriptional regulatory mechanisms of fibrosis development in mouse lung tissue exposed to carbon nanotubes

BackgroundCarbon nanotubes (CNTs) usage has rapidly increased in the last few decades due to their unique properties, exploited in various industrial and commercial products. Certain types of CNTs cause adverse health effects, including chronic inflammation and fibrosis. Despite the large number of in vitro and in vivo studies evaluating these effects, many important questions remain unanswered due to a lack of mechanistic understanding of how CNTs induce cellular stress responses. In order to predict CNT toxicity, it is important to understand which transcriptional programs are specifically activated in response to CNTs, and what similarities and differences exist in relation to other toxic inducers exerting similar adverse effects.\n\nResultsA systems biology approach was applied to reveal complex interactions at the molecular level in mouse lung tissue in response to different fibrosis inducers: two types of multi-walled CNTs, NM-401 and NRCWE-26, and bleomycin (BLM). Based on mRNA gene expression profiles, we inferred gene regulatory networks (GRNs) to capture functional hierarchical regulatory structures between genes and their regulators. We found that activities of the transcription factors (TFs) Myc, Arid5a and Mxd1 were associated with the regulation of cytokine transcription in response to CNTs, while in response to BLM treatment, Myc was associated with p53 signaling. TF Litaf was identified as the essential regulator for noncanonical signaling of TLR2/4 driven by CNTs. Despite the different nature of the lung injury caused by CNTs and BLM, we identified common stress response modules, that included DNA damage (TFs: E2f8, E2f1, Foxm1), M1/M2 macrophage polarization (TF: Mafb), Interferon response (TFs: Irf7, Stat2 and Irf9) for all agents.\n\nConclusionsThese results suggest that the reconstruction and analysis of TF-centric gene interaction networks can reveal key targets and regulators of cellular stress responses to toxic agents.

pharmacology and toxicology↗