Biology subjects

Lobanov, A.

Publications and source records attributed to Lobanov, A..

12 recordsLinked to original sources

bioRxiv2026

Dynamic Schlafen 11 (SLFN11) expression revealed by single-cell RNA sequencing of clonal human cancer cells

Schlafen 11 (SLFN11) is a key determinant of sensitivity to DNA-damaging therapies in multiple cancers including leukemia, Ewing sarcoma, head and neck cancers, mesothelioma and small cell lung cancer (SCLC). Here, utilizing immunofluorescence microscopy and single-cell RNA sequencing we characterized SLFN11 expression across eight different human cancer cell lines and found cell-to-cell heterogenous expression both at the protein and RNA levels. Gene set enrichment and pathway analyses show that SLFN11 is coexpressed with a distinct stress-response state and enhanced genome maintenance programs while being inversely correlated with protein translation pathways. Publicly available single-cell RNA-sequencing datasets of human SCLC tumors confirmed the heterogeneous distribution of SLFN11 expression and confirmed its association with replication-related transcriptional programs. These findings indicate that heterogeneous SLFN11 expression reflects dynamic regulation at the single-cell level. They also underscore the limitations of bulk proteomic and transcriptome profiling and point to a potential reservoir of intrinsic chemotherapy resistance.

cancer biology↗

bioRxiv2025

CRISPR-Based Gene Dependency Screens reveal Mechanism of BRAF Inhibitor Resistance in Anaplastic Thyroid Cancer

Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by the WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC. TAZ is overexpressed in ATC compared to well-differentiated thyroid tumors. We demonstrate that TAZ-deficient ATC cells display heightened sensitivity to BRAF inhibitors both in vitro and in vivo. Using gene essentiality score across a large panel of cancer cell lines, we found that BRAFV600E-driven cancers are highly sensitive to TAZ loss, unlike their counterparts with wild-type BRAF and non-BRAFV600E. Mechanistically, we demonstrate that dabrafenib triggers the Unfolded Protein Response (UPR) under ER stress and suppresses protein synthesis. TAZ loss represses the UPR, reverses the inhibition of protein synthesis, and triggers increased cell death by ferroptosis in dabrafenib-treated ATC. Collectively, our findings unveil TAZ as a new target to overcome resistance to BRAF inhibitors in undifferentiated thyroid cancer.

cancer biology↗

bioRxiv2025

SRSF3 is oncogenic in breast but tumor-suppressive in liver by differential regulation of gene expression

SRSF3 (SRp20) is an essential splicing factor. We discovered Srsf3 plays an oncogenic role in breast cancer and Srsf3 knockout (KO) in mammary glands delays the development of breast cancer in an Erbb2 mouse model. In contrast, Srsf3 is tumor-suppressive in mouse liver tissues. Srsf3 KO in hepatocytes enhances DEN-induced liver cancer and disrupts the sex disparity in DEN-induced liver cancer. Comparing to Srsf3 WT liver cancer, Srsf3 KO significantly increases Sox4, E2f1, Trpv4, Trim6, and Myc expression, but does not so in Erbb2 breast cancer. Srsf3 KO inhibits expression of Mfsd4a and Eif4a2 in breast cancer but enhances Mfsd4a and Eif4a2 expression in liver cancer. Moreover, Srsf3 KO suppresses the expression of ER and Foxa genes to reduce Lifr and Egfr but induce Myc expression and promote liver cancer in female mice. Together, our data highlight a new functional paradigm of SRSF3 at its physiological level in tissue context-dependent gene regulation.

cancer biology↗

bioRxiv2024

Spatially resolved rewiring of mitochondria-lipid droplet interactions in hepatic lipid homeostasis.

Hepatic lipid accumulation, or Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD), is a significant risk factor for liver cancer. Despite the rising incidence of MASLD, the underlying mechanisms of steatosis and lipotoxicity remain poorly understood. Interestingly, lipid accumulation also occurs during fasting, driven by the mobilization of adipose tissue-derived fatty acids into the liver. However, how hepatocytes adapt to increased lipid flux during nutrient deprivation and what occurs differently in MASLD is not known. To investigate the differences in lipid handling in response to nutrient deficiency and excess, we developed a novel single-cell tissue imaging (scPhenomics) technique coupled with spatial proteomics. Our investigation revealed extensive remodeling of lipid droplet (LD) and mitochondrial topology in response to dietary conditions. Notably, fasted mice exhibited extensive mitochondria-LD interactions, which were rarely observed in Western Diet (WD)-fed mice. Spatial proteomics showed an increase in PLIN5 expression, a known mediator of LD-mitochondria interaction, in response to fasting. To examine the functional role of mitochondria-LD interaction on lipid handling, we overexpressed PLIN5 variants. We found that the phosphorylation state of PLIN5 impacts its capacity to form mitochondria-LD contact sites. PLIN5 S155A promoted extensive organelle interactions, triglyceride (TG) synthesis, and LD expansion in mice fed a control diet. Conversely, PLIN5 S155E expressing cells had fewer LDs and contact sites and contained less TG. Wild-type (WT) PLIN5 overexpression in WD-fed mice reduced steatosis and improved redox state despite continued WD consumption. These findings highlight the importance of organelle interactions in lipid metabolism, revealing a critical mechanism by which hepatocytes maintain homeostasis during metabolic stress. Our study underscores the potential utility of targeting mitochondria-LD interactions for therapeutic intervention.

cell biology↗

bioRxiv2024

TRAIL-induced cytokine production via NFKB2 pathway promotes neutrophil chemotaxis and immune suppression in triple negative breast cancers

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo, TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.

cancer biology↗

bioRxiv2024

KSHV promotes oncogenic FOS to inhibit nuclease AEN and transactivate RGS2 for AKT phosphorylation

Kaposis sarcoma-associated herpesvirus (KSHV) ORF57 is a lytic RNA-binding protein. We applied BCBL-1 cells in lytic KSHV infection and performed UV cross-linking immunoprecipitation (CLIP) followed by RNA-seq of the CLIPed RNA fragments (CLIP-seq). We identified ORF57-bound transcripts from 544 host protein-coding genes. By comparing with the RNA-seq profiles from BCBL-1 cells with latent and lytic KSHV infection and from HEK293T cells with and without ORF57 expression, we identified FOS and CITED2 RNAs being two common ORF57-specific RNA targets. FOS dimerizes with JUN as a transcription factor AP-1 involved in cell proliferation, differentiation, and transformation. Knockout of the ORF57 gene from the KSHV genome led BAC16-iSLK cells incapable of FOS expression in KSHV lytic infection. The dysfunctional KSHV genome in FOS expression could be rescued by Lenti-ORF57 virus infection. ORF57 protein does not regulate FOS translation but binds to the 13-nt RNA motif near the FOS RNA 5' end and prolongs FOS mRNA half-life 7.7 times longer than it is in the absence of ORF57. This binding of ORF57 to FOS RNA is competitive to the binding of a host nuclease AEN (also referred to as ISG20L1). KSHV infection inhibits the expression of AEN, but not exosomal RNA helicase MTR4. FOS expression mediated by ORF57 inhibits AEN transcription, but transactivates RGS2, a regulator of G-protein coupled receptors. FOS binds a conserved AP-1 site in the RGS2 promoter and enhances RGS2 expression to phosphorylate AKT. Altogether, we have discovered that KSHV ORF57 specifically binds and stabilizes FOS RNA to increase FOS expression, thereby disturbing host gene expression and inducing pathogenesis during KSHV lytic infection. SignificanceWe discovered that FOS, a heterodimer component of oncogenic transcription factor AP- 1, is highly elevated in KSHV infected cells by expression of a viral lytic RNA-binding protein, ORF57, which binds a 13-nt RNA motif near the FOS RNA 5' end to prolong FOS RNA half-life. This binding of ORF57 to FOS RNA is competitive to the binding of host RNA destabilizer(s). KSHV infection inhibits expression of host nuclease AEN (or ISG20L1), but not MTR4. FOS inhibits AEN transcription, but transactivates RGS2 by binding to a conserved AP-1 site in the RGS2 promoter, thereby enhancing RGS2 expression and phosphorylation of AKT. Our data conclude that viral RNA-binding protein ORF57 controls the expression of a subset of genes for signaling, cell cycle progression, and proliferation to contribute viral pathogenesis.

microbiology↗

bioRxiv2023

A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal (PP) and pericentral (PC) axis. How these mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combined intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We found that PP and PC mitochondria are morphologically and functionally distinct; beta-oxidation was elevated in PP regions, while lipid synthesis was predominant in the PC mitochondria. In addition, comparative phosphoproteomics revealed spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifted mitochondrial phenotypes in the PP and PC regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

cell biology↗

bioRxiv2022

Spatial structure may favor or disfavor microbial coexistence

Microbes often exist in spatially structured environments and many of their interactions are mediated through diffusible metabolites. How does such a context affect microbial coexistence? To address this question, we used a model in which the spatial distributions of species and diffusible interaction mediators are explicitly included. We simulated the enrichment process, examining how microbial species spatially reorganize and how eventually a subset of them coexist. In our model we find that slower motility of cells promotes coexistence by allowing species to co-localize with their facilitators and avoid their inhibitors. We additionally find that a spatially structured environment is more influential when species mostly facilitate each other, rather than when they are mostly competing. More coexistence is observed when species produce many mediators and consume some (not many or few) mediators, and when overall consumption and production rates are balanced. Interestingly, coexistence appears to be disfavored at low diffusion constants of diffusible mediators, because they lead to weaker interaction strengths. Overall, our results offer new insights into how production, consumption, motility, and diffusion intersect to determine microbial coexistence in a spatially structured environment.

microbiology↗

bioRxiv2022

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splicing junctions from RNA-seq identified hundreds of novel viral RNA splicing events, including the splicing events spanning large parts of the viral genome and production of vIRF4 circRNAs. We found that the 57KO alters the viral RNA splicing efficiency of affected RNA splicing events. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with multiple exons and introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF70-K3 RNA bears two introns, of which the first intron is positioned in the ORF70 coding region as an alternative intron and the second intron in the intergenic region as a constitutive intron. In WT cells expressing ORF57, most ORF70-K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out without affecting the splicing efficiency of the second intron. Using a minigene comprising of ORF70-K3 locus, we further confirmed ORF57 regulation of ORF70-K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70-K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection. Author summaryLike other herpesviruses, the expression of KSHV genes is in a tightly regulated manner during viral infection. The contribution of RNA splicing in the regulation of KSHV gene expression was extensively investigated in this report. We found that approximately one-third of all viral gene transcripts are spliced, with many undergoing RNA alternative splicing. We identified a subset of viral RNA splicing events susceptible to the regulation of viral RNA-binding protein ORF57. This led to the discovery of ORF57-mediated regulation of the bicistronic ORF70-K3 RNA splicing and production of viral thymidylate synthase ORF70 and viral E3-ubiquitin ligase K3. Such regulation allows timely but mutually exclusive expression of these two viral proteins to execute their unique roles in immune evasion and viral genome replication during the KSHV lytic cycle. Thus, these data provide new insight into KSHV biology. Finally, our study represents a blueprint for RNA splicing studies of other pathogens in infected cells.

microbiology↗

bioRxiv2021

H3K27 Acetylated Nucleosomes Facilitate HMGN Protein Localization at Regulatory Sites to Modulate The Interaction of Transcription Factors with Chromatin

Nucleosomes containing acetylated H3K27 are a major epigenetic mark of active chromatin and identify cell-type specific chromatin regulatory regions which serve as binding sites for transcription factors. Here we show that the ubiquitous nucleosome binding proteins HMGN1 and HMGN2 bind preferentially to H3K27ac nucleosomes at cell-type specific chromatin regulatory regions. HMGNs bind directly to the acetylated nucleosome; the H3K27ac residue and linker DNA facilitate the preferential binding of HMGNs to the modified nucleosomes. Loss of HMGNs increases the levels of H3K27me3 and the histone H1 occupancy at enhancers and promoters and alters the interaction of transcription factors with chromatin. These experiments indicate that the H3K27ac epigenetic mark affects the interaction of architectural protein with chromatin regulatory sites and provide insights into the molecular mechanism whereby ubiquitous chromatin binding proteins, which bind to chromatin without DNA sequence specificity, localize to regulatory chromatin and modulate cell-type specific gene expression.

molecular biology↗

bioRxiv2020

Iterative Epigenomic Analyses in the Same Single Cell

Gene expression in individual cells is epigenetically regulated by DNA modifications, histone modifications, transcription factors and other DNA-binding proteins. It has been shown that multiple histone modifications can predict gene expression and reflect future responses of bulk cells to extracellular cues. However, the predictive ability of epigenomic analysis is still limited for mechanistic research at a single cell level. To overcome this limitation, it is useful to acquire reliable signals from multiple epigenetic marks in the same single cell. Here, we propose a new approach for analysis of several components of the epigenome in the same single cell. The new method allows reanalysis of the same single cell. We found that reanalysis of the same single cell is feasible, and provides confirmation of the signals and allows application of statistical analysis to identify reliable signals using data sets generated only from the single cell. Reanalysis of the same single cell is also useful to acquire multiple-epigenetic marks from the same single cells. The method can acquire at least 4 epigenetic marks, H3K27ac, H3K27me3, mediator complex subunit 1 and a DNA modification. We predicted active signaling pathways in K562 single cells using the data. We confirmed that the prediction results showed a strong correlation with actual active signaling pathways shown by RNA-seq results. These results suggest that the new approach provides mechanistic insights for cellular phenotypes through multi-layered epigenome analysis in the same single cells.

genomics↗

bioRxiv2020

Expression of HPV oncogenes from a single integration site in cervical cancer cell lines

Integration of a virus genome into human chromosomal DNA is critical in viral carcinogenesis. In this report, we discovered that virus-host fusion transcripts are characteristically originated mainly from a single integration site in three cervical cancer cell lines, CaSki and SiHa cells with multiple HPV16 DNA integration sites and HeLa cells with multiple HPV18 DNA integration sites. The host genomic elements surrounding the integrated HPV genome are critical for efficient expression of the viral oncogenes. We found that HPV E6 and E7 are expressed by hijacking a host 3’ splice site and/or RNA polyadenylation signal for their production. The viral-host fusion transcripts may encode a chimeric viral-host fusion protein through alternative RNA splicing. One such E6* protein in SiHa cells was found to antagonize E6 function and knockdown of its expression further decreased p53 protein level and increased cell growth by promoting S phase entry. Together, our findings of the integrated virus genome expression only from a given integrated host genomic site will shed new light on possible application of precision medicine and the understanding of HPV carcinogenesis.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗