Biology subjects

Zhabotynsky, V.

Publications and source records attributed to Zhabotynsky, V..

8 recordsLinked to original sources

bioRxiv2026

Folate metabolism in tumor-associated macrophages drives immunosuppressive function to promote tumor growth

Tumor-associated macrophages (TAMs) promote tumor growth, inhibit effector lymphocytes, and induce resistance to immune checkpoint inhibitors (ICI). Therapies designed to deplete TAMs have had little success in the clinic, and strategies aimed to repolarize immunosuppressive TAMs to pro-inflammatory states have struggled with systemic toxicity. We have identified that the combination of low folate in the tumor microenvironment and TAM dependency on the high affinity folate receptor beta (FR{beta}) for folate uptake results in a unique metabolic dependency of TAMs necessary for their immunosuppressive function. FR{beta} (encoded by Folr2) is uniquely expressed on myeloid cells and upregulated on TAMs. FR{beta}+ TAMs exhibit an immunosuppressive phenotype in melanoma and are associated with worse clinical outcomes and resistance to ICI in melanoma patients. We generated a novel Folr2-/- mouse model to study why TAMs express a unique folate receptor and showed that tumor growth was slowed in the absence of Folr2 in a T-cell dependent manner. We observed a dramatic repolarization of Folr2-/- TAMs in vivo to pro-inflammatory states, resulting in increased cytotoxic T cell and NK cell infiltration into tumors. Importantly, we show that folate is low in the tumor microenvironment by performing metabolomics on melanoma tumors and adjacent normal tissue from warm autopsy patient specimens, and that we could increase tumor growth in Folr2-/- mice by increasing serum folate to supraphysiologic concentrations with a high folate diet. Integrated metabolomics and transcriptomic analysis demonstrated that Folr2-/- macrophages have impaired folate uptake and 1C metabolism-based reduction of oxidized glutathione in low folate conditions, resulting in increased mitochondrial reactive oxygen species (ROS) in Folr2-/- cells. Excess ROS results in leakage of mitochondrial DNA into the cytoplasm, activating cGAS-STING signaling and promoting pro-inflammatory macrophage polarization through TBK1 and NF-kB. Together, our data demonstrate that TAM expression of FR{beta} promotes their immunosuppressive functions by maintaining folate uptake in the low folate tumor microenvironment, suggesting that FR{beta} is a novel metabolic checkpoint on TAMs.

immunology↗

bioRxiv2026

Harnessing Inflammatory Monocytes to Overcome Resistance to Anti-PD-1 Immunotherapy

BackgroundResistance to immune checkpoint inhibitors represents a major therapeutic challenge, as less than 50% of patients with melanoma achieve long-term response to immune checkpoint inhibitor therapy. One mechanism of acquired resistance involves somatic mutations, such as loss of beta-2 microglobulin (B2m), that enable tumor cells to evade T cell-mediated killing. MethodsThis study used single-cell RNA-seq, flow cytometry, and ex vivo functional assays to characterize tumor-infiltrating immune cells in antigen presentation-deficient tumors. Tumor-bearing mice were treated with anti-PD-1 or CD40 agonist antibodies and cell depletion or cytokine blocking antibodies to define mechanisms of action. Analysis of published human RNA-seq datasets was performed to dissect the contributions of inflammatory monocytes to patient outcomes. ResultsWe found an increase in immunosuppressive macrophages in B2m-null tumors. We hypothesized that repolarizing myeloid cells may restore control of tumor growth. Treatment with CD40 agonist antibody, which promotes differentiation of monocytes and macrophages towards a proinflammatory phenotype, reduced tumor growth and improved survival in B2m-null melanoma and colorectal cancer models. Unexpectedly, both CD8+ T cells and NK cells, but not CD4+ T cells, were required for the efficacy of CD40 agonist, even though CD8+ T cells cannot directly recognize antigen presentation-deficient tumor cells. Instead, these lymphocytes control tumor growth via secretion of IFN{gamma}, as depletion of IFN{gamma} inhibited the therapeutic effect of CD40 agonist. IFN{gamma} receptor (Ifngr1) expression was required on host cells, not tumor cells, for CD40 agonist-mediated tumor control. Single-cell analysis identified a distinct population of inflammatory monocytes that were enriched for an IFN{gamma} response signature in CD40 agonist-treated tumors, suggesting that these cells may be important for tumor control. Analysis of human bulk and single-cell RNA-seq datasets demonstrated that an inflammatory monocyte signature derived from our data was associated with improved patient outcomes and response to immune checkpoint inhibitors. ConclusionsThese data demonstrate that CD8+ T cells contribute to tumor control even in the absence of direct antigen presentation by tumor cells. More broadly, our work suggests that strategies to activate the effector functions of inflammatory monocytes may limit tumor growth and overcome acquired resistance to immune checkpoint inhibitors.

immunology↗

bioRxiv2026

Latent Regulatory Programs Generate Synthetic T Cell States with Enhanced Therapeutic Potential

Transcription factors (TFs) govern cell fate through coordinated gene-regulatory networks, yet the full potential of these networks to generate non-native, therapeutically advantageous cell states in vivo remains largely unexplored. We hypothesized that systematic gain-of-function (GOF) overexpression of TFs in CD8 T cells, central mediators of immune protection, could reveal latent, or "hidden," regulatory programs capable of generating synthetic T cell states with therapeutic utility. To test this, we developed single-cell GOF sequencing (scGOF-seq), a multiplexed platform for unbiased, in vivo mapping of GOF effects on T cell fate in immunocompetent mouse models of infection and cancer. scGOF-seq uncovered unexpected regulators of T cell differentiation and accumulation, including SOX2, OCT4, and GATA2, which are normally silenced during T cell differentiation. Notably, outside its native regulatory context, supraphysiologic cMyc GOF reprogrammed CD8 T cells into a synthetic stem-effector hybrid state, enabling >5,000-fold antigen-dependent expansion and antitumor activity, contrasting sharply with its native function in driving terminal differentiation. scGOF-seq further identified TF modules that cooperate with cMyc GOF to promote robust CD8 T cell responses in solid tumors. Together, these findings establish GOF perturbation as a powerful strategy for revealing latent immune regulatory programs and engineering synthetic immune states with therapeutic potential. One-Sentence SummaryIn vivo single-cell gain-of-function screening reveals latent transcriptional programs that can reprogram T cells into highly functional synthetic states.

immunology↗

bioRxiv2022

Cell type-specific Expression Quantitative Trait Loci

Mapping cell type-specific gene expression quantitative trait loci (ct-eQTLs) is a powerful way to investigate the genetic basis of complex traits. A popular method for ct-eQTL mapping is to assess the interaction between the genotype of a genetic locus and the abundance of a specific cell type using a linear model. However, this approach requires transforming RNA-seq count data, which distorts the relation between gene expression and cell type proportions and results in reduced power and/or inflated type I error. To address this issue, we have developed a statistical method called CSeQTL that allows for ct-eQTL mapping using bulk RNA-seq count data while taking advantage of allele-specific expression. We validated the results of CSeQTL through simulations and real data analysis, comparing CSeQTL results to those obtained from purified bulk RNA-seq data or single cell RNA-seq data. Using our ct-eQTL findings, we were able to identify cell types relevant to 21 categories of human traits.

genomics↗

bioRxiv2021

eQTL mapping using allele-specific gene expression

Using information from allele-specific gene expression (ASE) can sub-stantially improve the power to map gene expression quantitative trait loci (eQTLs). However, such practice has been limited, partly due to high computational cost and the requirement to access raw data that can take a large amount of storage space. To address these computational challenges, we have developed a computational framework that uses a statistical method named TReCASE as its computational engine, and it is computationally feasible for large scale analysis. We applied it to map eQTLs in 28 human tissues using the data from the Genotype-Tissue Expression (GTEx) project. Compared with a popular linear regression method that does not use ASE data, TReCASE can double the number of eGenes (i.e., genes with at least one significant eQTL) when sample size is relatively small, e.g., n = 200. We also demonstrated how to use the ASE data that we have collected to study dynamic eQTLs whose effect sizes vary with respect to another variable, such as age. We find the majority of such dynamic eQTLs are due to some underlying latent factors, such as cell type proportions. We further compare TReCASE versus another method RASQUAL. TReCASE is ten times or more faster than RASQUAL and it provides more robust type I error control.

genomics↗

bioRxiv2021

Distribution and vulnerability of transcriptional outputs across the genome in Myc-amplified medulloblastoma cells

Myc plays a central role in tumorigenesis by orchestrating the expression of genes essential to numerous cellular processes1-4. While it is well established that Myc functions by binding to its target genes to regulate their transcription5, the distribution of the transcriptional output across the human genome in Myc-amplified cancer cells, and the susceptibility of such transcriptional outputs to therapeutic interferences remain to be fully elucidated. Here, we analyze the distribution of transcriptional outputs in Myc-amplified medulloblastoma (MB) cells by profiling nascent total RNAs within a temporal context. This profiling reveals that a major portion of transcriptional action in these cells was directed at the genes fundamental to cellular infrastructure, including rRNAs and particularly those in the mitochondrial genome (mtDNA). Notably, even when Myc protein was depleted by as much as 80%, the impact on transcriptional outputs across the genome was limited, with notable reduction mostly only in genes involved in ribosomal biosynthesis, genes residing in mtDNA or encoding mitochondria-localized proteins, and those encoding histones. In contrast to the limited direct impact of Myc depletion, we found that the global transcriptional outputs were highly dependent on the activity of Inosine Monophosphate Dehydrogenases (IMPDHs), rate limiting enzymes for de novo guanine nucleotide synthesis and whose expression in tumor cells was positively correlated with Myc expression. Blockage of IMPDHs attenuated the global transcriptional outputs with a particularly strong inhibitory effect on infrastructure genes, which was accompanied by the abrogation of MB cells proliferation in vitro and in vivo. Together, our findings reveal a real time action of Myc as a transcriptional factor in tumor cells, provide new insight into the pathogenic mechanism underlying Myc-driven tumorigenesis, and support IMPDHs as a therapeutic vulnerability in cancer cells empowered by a high level of Myc oncoprotein.

cancer biology↗

bioRxiv2020

Skewed X inactivation in genetically diverse mice is associated with recurrent copy number changes at the Xce locus

Female mammals are functional mosaics of their parental X-linked gene expression due to X chromosome inactivation (XCI). This process inactivates one copy of the X chromosome in each cell during embryogenesis and that state is maintained clonally through mitosis. In mice, the choice of which parental X chromosome remains active is determined by the X chromosome controlling element (Xce), which has been mapped to a 176 kb candidate interval. A series of functional Xce alleles has been characterized or inferred for classical inbred strains based on biased, or skewed, inactivation of the parental X chromosomes in crosses between strains. To further explore the function-structure basis and location of the Xce, we measured allele-specific expression of X-linked genes in a large population of F1 females generated from Collaborative Cross strains. Using published sequence data and applying a Bayesian "Polya urn" model of XCI skew, we report two major findings. First, inter-individual variability in XCI suggests mouse epiblasts contain on average 20-30 cells contributing to brain. Second, NOD/ShiLtJ has a novel and unique functional allele, Xcef, that is the weakest in the Xce allelic series. Despite phylogenetic analysis confirming that NOD/ShiLtJ carries a haplotype almost identical to the well-characterized C57BL/6J (Xceb), we observed unexpected patterns of XCI skewing in females carrying the NOD/ShiLtJ haplotype within the Xce. Copy number variation is common at the Xce locus and we conclude that the observed allelic series is a product of independent and recurring duplications shared between weak Xce alleles.

genetics↗

bioRxiv2019

Antipsychotic behavioral phenotypes in the mouse Collaborative Cross recombinant inbred inter-crosses (RIX)

Schizophrenia is an idiopathic disorder that affects approximately 1% of the human population, and presents with persistent delusions, hallucinations, and disorganized behaviors. Antipsychotics are the standard pharmacological treatment for schizophrenia, but are frequently discontinued by patients due to inefficacy and/or side effects. Chronic treatment with the typical antipsychotic haloperidol causes tardive dyskinesia (TD), which manifests as involuntary and often irreversible orofacial movements in around 30% of patients. Mice treated with haloperidol develop many of the features of TD, including jaw tremors, tongue protrusions, and vacuous chewing movements (VCMs). In this study, we used genetically diverse Collaborative Cross (CC) recombinant inbred inter-cross (RIX) mice to elucidate the genetic basis of antipsychotic-induced adverse drug reactions (ADRs). We performed a battery of behavioral tests in 840 mice from 73 RIX lines (derived from 62 CC strains) treated with haloperidol or placebo in order to monitor the development of ADRs. We used linear mixed models to test for strain and treatment effects. We observed highly significant strain effects for almost all behavioral measurements investigated (p<0.001). Further, we observed strong strain-by-treatment interactions for most phenotypes, particularly for changes in distance traveled, vertical activity, and extrapyramidal symptoms (EPS). Estimates of overall heritability ranged from 0.21 (change in body weight) to 0.4 (VCMs and change in distance traveled) while the portion attributable to the interactions of treatment and strain ranged from 0.01 (for change in body weight) to 0.15 (for change in EPS). Interestingly, close to 30% of RIX mice exhibited VCMs, a sensitivity to haloperidol exposure, approximately similar to the rate of TD in humans chronically exposed to haloperidol. Understanding the genetic basis for the susceptibility to antipsychotic ADRs may be possible in mouse, and extrapolation to humans could lead to safer therapeutic approaches for schizophrenia.

systems biology↗