Gene Expression Patterns In Cells Research Articles

Hijacking and rewiring of host CircRNA/miRNA/mRNA competitive endogenous RNA (ceRNA) regulatory networks by oncoviruses during development of viral cancers.

A significant portion of human cancers are caused by oncoviruses (12%-25%). Oncoviruses employ various strategies to promote their replication and induce tumourigenesis in host cells, one of which involves modifying the gene expression patterns of the host cells, leading to the rewiring of genes and resulting in significant changes in cellular processes and signalling pathways. In recent studies, a specific mode of gene regulation known as circular RNA (circRNA)-mediated competing endogenous RNA (ceRNA) networks has emerged as a key player in this context. CircRNAs, a class of non-coding RNA molecules, can interact with other RNA molecules, such as mRNAs and microRNAs (miRNAs), through a process known as ceRNA crosstalk. This interaction occurs when circRNAs, acting as sponges, sequester miRNAs, thereby preventing them from binding to their target mRNAs and modulating their expression. By rewiring the host cell genome, oncoviruses have the ability to manipulate the expression and activity of circRNAs, thereby influencing the ceRNA networks that can profoundly impact cellular processes such as cell proliferation, differentiation, apoptosis, and immune responses. This review focuses on a comprehensive evaluation of the latest findings on the involvement of virus-induced reprogramming of host circRNA-mediated ceRNA networks in the development and pathophysiology of human viral cancers, including cervical cancer, gastric cancer, nasopharyngeal carcinoma, Kaposi's sarcoma, hepatocellular carcinoma, and diffuse large B cell lymphoma. Understanding these mechanisms can improve our knowledge of how oncoviruses contribute to human tumourigenesis and identify potential targets for developing optimised therapies and diagnostic tools for viral cancers.

Dietary Intervention with Whey Protein Concentrate Does Not Affect Toll-like Receptor Responses and Gene Expression Patterns in Peripheral Blood Mononuclear Cells of Healthy Volunteers.

Bovine milk contains bioactive proteins, carbohydrates, and phospholipids with immunomodulatory properties impacting human immunity, potentially contributing to resistance to infections and allergies through diverse mechanisms. One such mechanism is the enhancing of the innate immune response to secondary pathogen-related stimuli, termed innate immune training. Although in vitro studies demonstrate that milk immunoglobulin G (IgG) can train human monocytes, evidence for in vivo immune training is limited. To explore the potential of bovine IgG for inducing innate immune training in vivo, this human study utilized an IgG-rich whey protein concentrate (WPC). Healthy male volunteers were assigned to a high dose WPC, low dose WPC, or placebo group. Blood was collected pre- and post-two weeks of WPC consumption. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with TLR ligands, evaluating IL-6 and TNF-α production by monocytes, myeloid DCs, and plasmacytoid DCs. Additionally, RNA was isolated for differential gene expression (DGE) analysis. Results indicated that the two-week WPC intervention did not influence the ex vivo response of studied cells to TLR agonists. Furthermore, PBMC gene expression patterns showed no significant differences between the placebo and high dose WPC groups. The data suggests that oral WPC ingestion did not enhance immune responses in young, healthy male participants.

Tissue-of-origin for cancers determines HIF-1 activation induced phenotypic heterogeneity.

Hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular response to hypoxia, and is activated in many cancers contributing to many steps in the metastatic cascade by acting as a key transcription co-regulator for a large number of downstream genes. Presence of hypoxia within a tumor is spatially nonuniform, and can also by dynamic. Further, although HIF-1 is primarily stabilized and activated by lack of molecular O2, its stability is also affected by other factors present in the tumor microenvironment. HIF-1 also crosstalks with other transcription factors in co-regulating gene expression. Consequently, it is nontrivial to predict the gene expression patterns in cells in response to hypoxia, or HIF-1 activation. Additionally, cancers originating from tissue origins with different basal level of partial oxygen tension may activate HIF-1 at different threshold of hypoxia. We analyzed large published single cell RNAseq data for colorectal, lung, and pancreatic cancers to investigate the phenotypic outcome of HIF-1 activation in cancer cells. We found that cancers from tissues with different partial O2 tension levels exhibit HIF-1 activation at different stages of metastasis, and phenotypically respond differently to HIF-1 activation, likely by contextual co-option of different transcription factors. We experimentally confirmed these predictions by using cell lines representative of colorectal, lung, and pancreatic cancers, finding that while hypoxia enhances growth of colorectal cancer, it induces increased invasion of lung, and pancreatic cancers. Our analysis suggest that HIF-1 activation may act as a rheostat regulating downstream gene expression towards phenotypic outcomes differently in various cancers.

The Impact of 3D Nichoids and Matrix Stiffness on Primary Malignant Mesothelioma Cells.

Malignant mesothelioma is a type of cancer that affects the mesothelium. It is an aggressive and deadly form of cancer that is often caused by exposure to asbestos. At the molecular level, it is characterized by a low number of genetic mutations and high heterogeneity among patients. In this work, we analyzed the plasticity of gene expression of primary mesothelial cancer cells by comparing their properties on 2D versus 3D surfaces. First, we derived from primary human samples four independent primary cancer cells. Then, we used Nichoids, which are micro-engineered 3D substrates, as three-dimensional structures. Nichoids limit the dimension of adhering cells during expansion by counteracting cell migration between adjacent units of a substrate with their microarchitecture. Tumor cells grow effectively on Nichoids, where they show enhanced proliferation. We performed RNAseq analyses on all the samples and compared the gene expression pattern of Nichoid-grown tumor cells to that of cells grown in a 2D culture. The PCA analysis showed that 3D samples were more transcriptionally similar compared to the 2D ones. The 3D Nichoids induced a transcriptional remodeling that affected mainly genes involved in extracellular matrix assembly. Among these genes responsible for collagen formation, COL1A1 and COL5A1 exhibited elevated expression, suggesting changes in matrix stiffness. Overall, our data show that primary mesothelioma cells can be effectively expanded in Nichoids and that 3D growth affects the cells' tensegrity or the mechanical stability of their structure.

The association of RBX1 and BAMBI gene expression with oocyte maturation in PCOS women

BackgroundPolycystic ovarian syndrome (PCOS) is a common endocrine disorder that affects 6–20% of women of reproductive age. One of the symptoms of PCOS is hyperandrogenism, which can impair follicular development. This disruption can cause issues with the development of oocytes and the growth of embryos. Although the exact cause of PCOS is not yet fully understood, studying the gene expression pattern of cumulus cells, which play a crucial role in the maturation and quality of oocytes, could help identify the genes associated with oocyte maturation in PCOS women. Through indirect activation of APC/Cdc20, RBX1 enables oocytes to bypass the GV (germinal vesicles) stage and advance to the MII (metaphase II) stage. our other gene is the BAMBI gene which stimulates WNT signaling, that is a crucial pathway for healthy ovarian function. This study aims to explore the expression level of the RBX1 and BAMBI genes between GV and MII oocytes of PCOS and non-PCOS groups.MethodsIn this experiment, we gathered the cumulus cells of MII (38 cases and 33 control) and GV (38 cases and 33 control) oocytes from women with/without PCOS. Besides, quantitative RT-PCR was used to assess the semi-quantitative expression of BAMBI and RBX1.ResultsAccording to our research, the expression level of RBX1 and BAMBI in MII and GV cumulus cells of PCOS patients was significantly lower than that in non-PCOS ones.ConclusionThis research raises the possibility of RBX1 and BAMBI involvement in oocyte quality in PCOS women.

Alteration of functionality and differentiation directed by changing gene expression patterns in myeloid-derived suppressor cells (MDSCs) in tumor microenvironment and bone marrow through early to terminal phase of tumor progression.

Myeloid-derived suppressor cells are heterogenous immature myeloid lineage cells that can differentiate into neutrophils, monocytes, and dendritic cells as well. These cells have been characterized to have potent immunosuppressive capacity in neoplasia and a neoplastic chronic inflammatory microenvironment. Increased accumulation of myeloid-derived suppressor cells was reported with poor clinical outcomes in patients. They support neoplastic progression by abrogating antitumor immunity through inhibition of lymphocyte functions and directly by facilitating tumor development. Yet the shifting genetic signatures of this myeloid lineage cell toward immunosuppressive functionality in progressive tumor development remain elusive. We have attempted to identify the gene expression profile using lineage-specific markers of these unique myeloid lineage cells in a tumor microenvironment and bone marrow using a liquid transplantable mice tumor model to trace the changing influence of the tumor microenvironment on myeloid-derived suppressor cells. We analyzed the phenotype, functional shift, suppressive activity, differentiation status, and microarray-based gene expression profile of CD11b+Gr1+ lineage-specific cells isolated from the tumor microenvironment and bone marrow of 4 stages of tumor-bearing mice and compared them with control counterparts. Our analysis of differentially expressed genes of myeloid-derived suppressor cells isolated from bone marrow and the tumor microenvironment reveals unique gene expression patterns in the bone marrow and tumor microenvironment-derived myeloid-derived suppressor cells. It also suggests T-cell suppressive activity of myeloid-derived suppressor cells progressively increases toward the mid-to-late phase of the tumor and a significant differentiation bias of tumor site myeloid-derived suppressor cells toward macrophages, even in the presence of differentiating agents, indicating potential molecular characteristics of myeloid-derived suppressor cells in different stages of the tumor that can emerge as an intervention target.

Single-cell multi-omics reveal stage of differentiation and trajectory-dependent immunity-related gene expression patterns in human erythroid cells.

The role of Erythroid cells in immune regulation and immunosuppression is one of the emerging topics in modern immunology that still requires further clarification as Erythroid cells from different tissues and different species express different immunoregulatory molecules. In this study, we performed a thorough investigation of human bone marrow Erythroid cells from adult healthy donors and adult acute lymphoblastic leukemia patients using the state-of-the-art single-cell targeted proteomics and transcriptomics via BD Rhapsody and cancer-related gene copy number variation analysis via NanoString Sprint Profiler. We found that human bone marrow Erythroid cells express the ARG1, LGALS1, LGALS3, LGALS9, and C10orf54 (VISTA) immunosuppressive genes, CXCL5, CXCL8, and VEGFA cytokine genes, as well as the genes involved in antimicrobial immunity and MHC Class II antigen presentation. We also found that ARG1 gene expression was restricted to the single erythroid cell cluster that we termed ARG1-positive Orthochromatic erythroblasts and that late Erythroid cells lose S100A9 and gain MZB1 gene expression in case of acute lymphoblastic leukemia. These findings show that steady-state erythropoiesis bone marrow Erythroid cells express myeloid signature genes even without any transdifferentiating stimulus like cancer.

Molecular and cellular basis of life cycle transition provides new insights into ecological adaptation in jellyfish

<p>Jellyfish are renowned for their complex life cycles and important ecological and evolutionary position. The unique transition from the sessile polyp to motile medusa stages is a key process determining a switch in jellyfish behaviour and regulating the formation of jellyfish blooms. Here, we presented a comprehensive cell atlas spanning four successive life cycle stages during the polyp-to-medusa transition in the scyphozoan jellyfish <i>Aurelia</i> <i>coerulea</i>. Moreover, we characterised the variation in cell composition and gene expression patterns during the phase transition, especially in the neuromuscular system. We found several previously unreported cell types that potentially underpin the complex swimming behaviour of jellyfish. Furthermore, we discovered the pivotal role of <i>HOX1</i> in modulating the genesis of striated muscles in <i>A</i>. <i>coerulea</i>. Collectively, this study provides valuable insights into the cellular and molecular mechanisms underlying the complex life cycle transition and helps to advance our understanding of ecological adaptation in jellyfish.</p>

Uncovering a unique pathogenic mechanism of SARS-CoV-2 omicron variant: selective induction of cellular senescence.

SARS-CoV-2 variants are constantly emerging with a variety of changes in the conformation of the spike protein, resulting in alterations of virus entry mechanisms. Solely omicron variants use the endosomal clathrin-mediated entry. Here, we investigate the influence of defined altered spike formations to study their impact on premature cellular senescence. In our study, in vitro infections of SARS-CoV-2 variants delta (B.1.617.2) and omicron (B.1.1.529) were analyzed by using human primary small alveolar epithelial cells and human ex vivo lung slices. We confirmed cellular senescence in human lungs of COVID-19 patients. Hence, global gene expression patterns of infected human primary alveolar epithelial cells were identified via mRNA sequencing. Solely omicron variants of SARS-CoV-2 influenced the expression of cell cycle genes, highlighted by an increased p21 expression in human primary lung cells and human ex vivo lungs. Additionally, an upregulated senescence-associated secretory phenotype (SASP) was detected. Transcriptomic data indicate an increased gene expression of p16, and p38 in omicron-infected lung cells. Significant changes due to different SARS-CoV-2 infections in human primary alveolar epithelial cells with an overall impact on premature aging could be identified. A substantially different cellular response with an upregulation of cell cycle, inflammation- and integrin-associated pathways in omicron infected cells indicates premature cellular senescence.

Immunologic barriers in liver transplantation: a single-cell analysis of the role of mesenchymal stem cells.

This study aimed to analyze the biomarkers that may reliably indicate rejection or tolerance and the mechanism that underlie the induction and maintenance of liver transplantation (LT) tolerance related to immunosuppressant or mesenchymal stem cells (MSCs). LT models of Lewis-Lewis and F344-Lewis rats were established. Lewis-Lewis rats model served as a control (Syn). F344-Lewis rats were treated with immunosuppressant alone (Allo+IS) or in combination with MSCs (Allo+IS+MSCs). Intrahepatic cell composition particularly immune cells was compared between the groups by single-cell sequencing. Analysis of subclusters, KEGG pathway analysis, and pseudotime trajectory analysis were performed to explore the potential immunoregulatory mechanisms of immunosuppressant alone or combined with MSCs. Immunosuppressants alone or combined with MSCs increases the liver tolerance, to a certain extent. Single-cell sequencing identified intrahepatic cell composition signature, including cell subpopulations of B cells, cholangiocytes, endothelial cells, erythrocytes, hepatic stellate cells, hepatocytes, mononuclear phagocytes, neutrophils, T cells, and plasmacytoid dendritic cells. Immunosuppressant particularly its combination with MSCs altered the landscape of intrahepatic cells in transplanted livers, as well as gene expression patterns in immune cells. MSCs may be included in the differentiation of T cells, classical monocytes, and non-classical monocytes. These findings provided novel insights for better understanding the heterogeneity and biological functions of intrahepatic immune cells after LT treated by IS alone or in combination with MSCs. The identified markers of immune cells may serve as the immunotherapeutic targets for MSC treatment of liver transplant rejection.

Comprehensive characterization of adipogenesis-related genes in colorectal cancer for clinical significance and immunogenomic landscape analyses

ObjectiveColorectal cancer (CRC) is a major global health concern, necessitating the identification of biomarkers and molecular subtypes for improved clinical management. This study aims to evaluate the clinical value of adipogenesis-related genes and molecular subtypes in CRC.MethodsA comprehensive analysis of adipogenesis-related genes in CRC was performed using publicly available datasets (TCGA and GEO database) and bioinformatics tools. Unsupervised cluster analysis was employed to identify the molecular subtypes of CRC, while LASSO regression analysis was utilized to develop a risk prognostic model. The immunogenomic patterns and immunotherapy analysis were used to predict patient response to immunotherapy. Furthermore, qPCR analysis was conducted to confirm the expression of the identified key genes in vitro.ResultsThrough the analysis of RNAseq data from normal and tumor tissues, we identified 50 differentially expressed genes. Unsupervised cluster analysis identified two subtypes (Cluster A and Cluster B) with significantly different survival outcomes. Cluster A and B displayed differential immune cell compositions and enrichment in specific biological pathways, providing insights into potential therapeutic targets. A risk-scoring model was developed using five ARGs, which successfully classified patients into high and low-risk groups, showing distinct survival outcomes. The model was validated and showed robust predictive performance. High-risk patients exhibited altered immune cell proportions and gene expression patterns compared to low-risk patients. In qPCR validation, four out of the five key genes were consistent with the results of bioinformatics analysis.ConclusionOverall, the findings of our investigation offer valuable understanding regarding the clinical relevance of ARGs and molecular subtypes in CRC, laying the groundwork for improved precision medicine applications and personalized treatment modalities.

SPI1 Is the Master Regulator of the Small Cell Variant of Anaplastic Large Cell Lymphoma Controlled By Methylation of SPI1 Gene Promoter Region

Introduction The small cell variant of anaplastic large cell lymphoma (SC-ALCL) is a subtype of anaplastic lymphoma kinase (ALK)-positive ALCL characterized by chemoresistance and poor prognosis, necessitating novel treatment strategies. Recent advances in targeted therapies have resulted in significant responses in various chemoresistant hematological malignancies. Therefore, understanding the underlying oncogenic mechanisms and identifying potential therapeutic targets are critical for overcoming SC-ALCL. Immunohistochemically, SC-ALCL comprises two distinct tumor cell populations: small tumor cells demonstrating negative to weak ALK and CD30 protein expression, and large tumor cells with robust expression of these proteins. We previously reported that small tumor cells display particular resistance to chemotherapy; however, the mechanisms underlying chemoresistance and the molecular characteristics of small tumor cells remain elusive. This study aimed to elucidate the molecular attributes of small tumor cells and pave the way for the development of innovative treatment approaches. Methods We investigated the characteristics of small tumor cells using patient blood samples diagnosed with SC-ALCL leukemic presentation and publicly available RNA-seq data from eight ALK-positive ALCL cell lines. Small and large tumor cells were separated from blood samples based on CD30 expression using CD30 - a phycoerythrin antibody and a magnetic bead separation kit. The presence of the NPM-ALK fusion gene was confirmed in both populations by PCR analysis. Bulk RNA-seq analysis was performed on four samples: small and large tumor cells at the onset and at the first (mainly occupied by small tumor cells) and second (mainly occupied by large tumor cells) relapses. Gene set enrichment analysis was used to identify gene expression patterns in small tumor cells. Signature genes of small tumor cells were identified by filtering with the following thresholds: false discovery rate-adjusted P-value <0.05, and fold-change >2 compared to large tumor cells. The transcription factors regulating the signature genes of small tumor cells were predicted using the g:Profiler software. Result Bulk RNA sequencing analysis of the four blood samples revealed that the SPI1 gene was significantly upregulated in small tumor cells compared to that in large tumor cells. SPI1 has emerged as the highest-scoring transcription factor, regulating the expression of 570 genes that characterize small tumor cells. Approximately 70% of these signature genes overlapped with the SPI1 target genes. Furthermore, gene set enrichment analysis revealed that small tumor cells display increased anti-apoptotic gene activity and decreased cell proliferation gene activity. These observations were validated by integrated gene expression analysis using publicly available RNA-seq data from eight ALK-positive ALCL cell lines (Figure 1). The study found high expression of SPI1 and SPI1 targeted genes in small tumor cells, accompanied by low expression of anti-apoptotic genes in ALK-positive ALCL cell lines, which were more analogous to large tumor cells than small tumor cells. Single-cell RNA-seq analysis validated the high expression of the SPI1 gene in small tumor cells. To explore the mechanism regulating SPI1 gene expression, we examined DNA methylation patterns in the SPI1 promoter region using conventional bisulfite sequencing. Conventional bisulfite sequencing revealed a hypomethylated SPI1 promoter region in small tumor cells and a hypermethylated region in large tumor cells, potentially accounting for the differential SPI1 expression. Conclusion This study highlights SPI1gene as the master regulator determining the unique characteristics of small tumor cells in SC-ALCL. Furthermore, the regulation of SPI1 gene expression appeared to be mediated by DNA methylation patterns within the SPI1 promoter region. These findings pave the way for innovative treatment strategies for SC-ALCL such as SPI1 targeting therapy and methylation-based therapies, offering promising avenues for treating SC-ALCL.

Melatonin alleviates the toxic effect of di(2-ethylhexyl) phthalate on oocyte quality resulting from CEBPB suppression during primordial follicle formation

Presently, the exposure of plasticizers to humans and animals occurs daily, which pose a potential threat to reproductive health. In the present study, a pregnant mouse model exposed to di(2-ethylhexyl) phthalate (DEHP, one of the most common plasticizers) and melatonin was established, and the single-cell transcriptome technology was applied to investigate the effects of melatonin in ovarian cells against DEHP. Results showed that DEHP markedly altered the gene expression pattern of ovarian cells, and severely weakened the histone methylation modification of oocytes. The administration of melatonin recovered the expression of LHX8 and SOHLH1 proteins that essential for primordial follicle formation, and increased the expression of CEBPB, as well as key genes of histone methylation modification (such as Smyd3 and Kdm5a). In addition, the ovarian damage caused by DEHP was also relieved after the overexpression of CEBPB, which suggested melatonin could improve primordial follicle formation progress via enhancing CEBPB expression in mice. Besides, the apoptosis of ovarian cells induced by DEHP also was diminished by melatonin. The study provides evidence of melatonin preventing the damage mediated by plasticizers on the reproductive system in females and CEBPB may serve as a downstream target factor of melatonin in the process.

Multidimensional progressive single-cell sequencing reveals cell microenvironment composition and cancer heterogeneity in lung cancer.

Despite substantial advances in cancer biology and treatment, the clinical outcomes of patients with lung cancer remain unsatisfactory. The tumor microenvironment (TME) is a potential target. Using single-cell RNA sequencing, we could distinguish eight distinct cell types in the lung cancer microenvironment, demonstrating substantial intratumoral heterogeneity in 19 different lung cancer tumor samples. Through the re-dimensional grouping of cancer-associated fibroblasts (CAFs), myeloid cells, epithelial cells, natural killer (NK) cells, and T cells, the difference in the TME of lung cancer was revealed. We discovered SFTPB, SFN, and KRT8 as possible predictive biomarkers for lung cancer by assessing the gene expression patterns in epithelial cells. Examining cell-to-cell communications showed a robust association between the quantity of matrix CAFs, epithelial cells, and macrophages in the thrombospondin signaling pathway. Additionally, we found that the amyloid precursor protein signaling pathway primarily originated from the matrix, and inflammatory cancer-associated endothelial and fibroblast cells showed a co-expression relationship with myeloid cells and B cells. Through cell-to-cell correlation analysis, we found positive regulation between NK cells, regulatory T cells, GZMB-CD8 T cells, and GZMK-CD8 T cells, which could play a role in developing immune TMEs. These findings support studies on cancer heterogeneity and add to our understanding of lung cancer's cellular microenvironment.

Defective Necroptosis Mediates Chemotherapy Resistance in AML

Acute myeloid leukemia (AML) is a hematopoietic malignancy that can affect individuals of any age and, if left untreated, is always fatal. Standard induction chemotherapy, known as “7+3,” using drugs like cytarabine (Ara-C) and an anthracycline (daunorubicin or idarubicin), is typically given to young and fit older patients. While this treatment regimen has shown positive outcomes in patients who respond to it, relapse and refractory disease remain the primary causes of death. Prognostic classification, which combines cytogenetic and mutational status, can estimate overall and event-free survival but is not effective in predicting induction failure. Despite decades of research and treatment with 7+3 chemotherapy, the reasons behind variable treatment responses in AML patients remain unclear, especially for those who experience induction failure. Understanding these mechanisms could aid in risk-adapted treatment decisions and the identification of new therapeutic targets. Recent research has investigated gene expression and signaling patterns in leukemic cells at the time of diagnosis to categorize patients according to their response to chemotherapy. Notably, increased NF-kB signaling at diagnosis has been linked to chemotherapy resistance. NF-kB is a family of transcription factors involved in regulating genes linked to cancer cell survival, proliferation, and therapy resistance. However, the exact mechanism by which NF-kB activation contributes to chemotherapy failure and resistance in AML is yet to be determined. Using publicly available datasets, we found that AML patients undergoing induction failure exhibited elevated expression of TNFAIP3/A20, which is driven by NF-kB signaling and leads to a worse prognosis for patients undergoing 7+3 chemotherapy. In experiments with primary AML samples, it was observed that A20-High AML are resistant to Doxorubicin treatment, whereas A20-Low AML samples are sensitive to Doxorubicin. Knockdown of A20 in A20-High AML samples resulted in increased cell sensitivity to Doxorubicin, suggesting that elevated A20 expression reduces AML cells' response to the Doxorubicin both in vitro and in vivo. To understand why A20 is overexpressed in AML, we investigated its requirement on AML cell function in various models, including human AML cell lines, patient-derived samples, and mouse models of AML. Deletion or knockdown of A20 led to reduced leukemic progenitor function, cell viability, and AML development in vitro and in vivo. A20 is a dual ubiquitin-editing protein that regulates signaling pathways by acting either as a deubiquitinase to modulate NF-kB signaling or as an E3 ligase to control necroptosis. Further analyses revealed that A20 prevents necroptosis by targeting the necroptosis effector, RIPK1, for degradation through the proteasome in AML. Preventing necroptosis, either pharmacologically or genetically, restored the viability and clonogenicity of A20-deficient AML cells in vitro and in vivo. Notably, Doxorubicin, but not AraC, induced necroptosis in AML cells, which is suppressed in leukemic clones expressing A20. Examination of single-cell RNA-sequencing data from AML patients before and after Doxorubicin treatment confirmed the persistence of A20-expressing clones during treatment. These findings demonstrate that NF-kB-driven overexpression of A20 contributes to failed induction chemotherapy in AML, highlighting the therapeutic potential of targeting an alternative cell death pathway in AML.

Cerebral Spinal Fluid Attenuates the Efficacy of Methotrexate Against Acute Lymphoblastic Leukemia Cells

The anti-folate methotrexate is a critical component of acute lymphoblastic leukemia (ALL) therapy. In addition to systemic administration, methotrexate is given intrathecally, or directly into the cerebral spinal fluid (CSF) via lumbar puncture, for central nervous system (CNS) leukemia treatment and prophylaxis. While IT methotrexate played a critical role in improving outcomes for ALL, CNS ALL relapse remains a major cause of treatment failure and therapy related morbidity. However, attempts to develop more efficacious and less toxic CNS ALL therapies have been largely unsuccessful and IT methotrexate has remained the standard of care for 60+ years. Based on our prior work showing that ALL cells are more poised to undergo apoptosis in nutrient poor CSF, we initially hypothesized that ALL chemosensitivity will be increased in CSF (Basile et al., 2020). While this was confirmed for several ALL drugs including cytarabine and anthracyclines, we strikingly found that methotrexate was significantly less efficacious and potent against ALL cells in CSF relative to standard tissue culture media or the more physiologically relevant human plasma-like media (HPLM). CSF also attenuated the sensitivity of ALL cells to other anti-folates including raltitrexed, trimetrexate, and pralatrexate. To define the mechanism(s) underlying this methotrexate resistance induced by CSF, we explored previously well-described mechanisms of methotrexate resistance in leukemia and other cancers. However, to date this work suggests that the molecular etiology of this CSF-induced relative methotrexate resistance is not entirely explained by diminished leukemia proliferation in CSF, altered methotrexate cellular influx/efflux, methotrexate metabolism, folate analog rescue, dysregulated expression of methotrexate target proteins, or compensation by nucleotide salvage pathways. To complement our work examining known mechanisms of methotrexate resistance, we also used a more discovery-based approach and compared the gene expression patterns of leukemia cells in CSF versus tissue culture media. Preliminary bioinformatic analyses identified dysregulation of multiple genes involved in the integrated stress response (ISR) and unfolded protein response (UPR) pathways in leukemia cells in CSF. The ISR/UPR are mechanisms by which cells adapt to diverse stress stimuli in the microenvironment, including potentially nutrient poor CSF which is low in glucose, proteins, and lipids relative to plasma. The hallmark event in this pathway is the phosphorylation of the translation initiation factor eIF2α, which inhibits global protein synthesis and enables the translation of selected genes that together enable cell survival. Supporting our gene expression data, ALL cells in CSF showed increased phosphorylation of eIF2α and a decrease in global protein synthesis. Intriguingly, it has been shown that activation of the UPR can divert metabolites from glycolysis to mitochondrial one-carbon metabolism with resulting resistance to anti-folates such as methotrexate (Reich et al., 2020). Accordingly, we next tested the effect of UPR/ISR activation on leukemia cell sensitivity to methotrexate in tissue culture media. Activation of the UPR with thapsigargin, a sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor that triggers ER stress, increased methotrexate resistance in leukemia cells. Similarly, BtdCPU a small-molecule activator of heme regulated inhibitor (HRI) kinase that phosphorylates eIF2α and activates the ISR, also enhanced leukemia resistance to methotrexate. Building upon these results, current investigations are focused on testing the role of UPR/IST activation in methotrexate resistance in leukemia cells in CSF and modulating this pathway to overcome resistance. While methotrexate is clearly an efficacious and critical component of CNS leukemia treatment and prophylaxis, we have shown that CSF attenuates the overall efficacy of methotrexate in targeting leukemia cells. Moreover, we anticipate that our current work defining the mechanisms driving this resistance may identify novel approaches for maximizing methotrexate efficacy and more completely eradicating leukemia cells in CSF and the CNS. Finally, this work highlights the importance of critically evaluating even long-established standards of care.

A Phase Ib/II Study Evaluating Navitoclax after Failure of Hypomethylating Agent and Venetoclax for Treatment of Relapsed or Refractory High-Risk Myelodysplastic Syndrome

BACKGROUND AND SIGNIFICANCE Myelodysplastic syndrome (MDS) is a heterogeneous group of blood cancers of myeloid progenitor cells. They vary widely in genetic landscape, risk of progression to acute myeloid leukemia, and risk of death, as predicted by the revised international prognostic score (IPSS-R). The hypomethylating agents (HMAs) azacitidine (AZA) and decitabine (DEC) are the standard of care for treatment of high risk (HR)-MDS, however complete response rates are <15% and most patients (pts) eventually progress. Outcomes after HMA failure are dismal. Defective apoptosis is a major feature of MDS. Increased expression of anti-apoptotic members of the BCL-2 family (BCL-2, BCL-X L and BCL-W) correlates with disease progression and poor outcomes. The addition of venetoclax (VEN), a selective BCL-2 inhibitor, has shown benefit when added to AZA in pts with relapsed or refractory (R/R) MDS (Zeidan, et al., AJH, 2023). However, patterns of BCL-2 family proteins are heterogeneous and response to inhibition of BCL-2 family proteins depends on the balance of pro- and anti-apoptotic proteins. Navitoclax (NAV) is a broad oral inhibitor of BCL-2, BCL-X L, and BCL-W. Increased expression of BCL-X L has been shown to make MDS cells insensitive to VEN. Further, VEN-resistant myeloid cell lines derived by prolonged VEN exposure showed up-regulation of BCL-X L, driving acquired drug resistance in these cell lines. Thus, the addition of NAV to the combination of VEN and HMA is a logical strategy to induce response in pts with R/R after HMA with or without prior VEN exposure. STUDY DESIGN AND METHODS We will perform a phase Ib/II, dose escalation and expansion study to evaluate escalating doses of NAV in combination with standard dose VEN and DEC in pts with R/R HR-MDS after failure of HMA (NCT05564650). The primary objective of the phase Ib portion of the study will be to evaluate the safety profile and determine the recommended phase II dose (RP2D) of NAV in the combination. The primary objective of the phase II portion of the study will be to evaluate the efficacy of combination therapy NAV/VEN/DEC in pts with R/R HR-MDS after failure of HMA/VEN. During cycle 1, to limit the risk of tumor lysis, pts will be treated with a short dose ramp-up with VEN 200 mg daily on day 1 and 400 mg daily on days 2-16, DEC 20 mg/m2 on days 3-7 and NAV 25 mg, 50 mg or 100 mg daily on days 3-16 during cycle 1. During cycles 2 and beyond, dosing will be similar to cycle 1 without dose ramp-up ( Figure 1). We will employ the Bayesian optimal interval (BOIN) design to identify the maximum tolerated dose and RP2D of NAV. Subsequently, the study will proceed to phase II to further evaluate the safety and efficacy of the combination. The study will enroll pts with IPSS-R ≥3 who have been previously treated with HMA/VEN. Pts in the phase I portion of the trial may be enrolled if they have not received VEN. Pts must have adequate performance status and organ function. Pts with a WBC >25,000 cells/µL and those taking prohibited medications will be excluded. Correlative studies will be performed to determine the impact of treatment on molecular targets and will include profiling of BCL-2 family members and single cell gene expression patterns pre- and post-treatment and at relapse. Pts who achieve a complete response (CR) or modified CR (mCR) will continue on the study until disease progression, dose limiting toxicity, availability of an alternative therapy or withdrawal of consent. Pts who have hematologic improvement but who do not attain CR or mCR can continue the study at the discretion of their treating physician in conjunction with the primary investigator.

Global m6A methylation and gene expression patterns in human microglial HMC3 cells infected with HIV-1

N6-methyladenosine (m6A) methylation of human immunodeficiency virus type 1 (HIV-1) RNA regulates viral replication, and the m6A of host RNA is affected by HIV-1 infection, but its global pattern and function are still unclear. In this study, we report that the number and position of m6A peaks in huge genes of human microglial HMC3 cells were modulated by a single cycle HIV-1 pseudotyped with VSV-G envelope glycoprotein infection using methylated RNA immunoprecipitation sequencing (MeRIP-seq). A conjoint analysis of MeRIP-seq and high-throughput sequencing for mRNA (RNA-seq) explored four groups of clearly classified genes, including 45 hyper-up (m6A-mRNA), 45 hyper-down, 120 hypo-up, and 54 hypo-down genes, in HIV-1 infected cells compared to uninfected ones. KEGG pathway analysis showed that these genes were mainly enriched in the Wnt and TNF signaling pathway, and cytokine–cytokine receptor interaction, which might be related to the immune response in HMC3 cells. And some of these genes might be associated with the pathway of axon guidance and neuroactive ligan-receptor interaction, which affect the neuronal state. However, the cognitive disorders caused by HIV-1 is associated with inflammatory changes that have not yet been well clarified. Furthermore, we confirmed the expression and m6A levels of four genes using RT-PCR and MeRIP-qPCR. Similar to the sequencing results, the expressions of these genes were significantly upregulated by HIV-1 infection. And the m6A level of IL-6 was downregulated, and those of HLA-B, CFB, and OLR1 were upregulated. These results suggest that HIV-1-induced changes in gene expression may be achieved through the regulation of methylation. Our study revealed the global m6A methylation and gene expression patterns under HIV-1 infection in human microglia, which might provide clues for understanding the interaction between HIV-1 and host cells and the cognitive disorders caused by HIV-1.

AP-1/c-Fos supports SIV and HIV-1 latency in CD4 T cells infected in vivo

Persistent HIV-1 reservoirs of infected CD4 Tcells are a major barrier to HIV-1 cure, although the mechanisms by which they are established and maintained invivo remain poorly characterized. To elucidate host cell gene expression patterns that govern virus gene expression, we analyzed viral RNA+ (vRNA) CD4 Tcells of untreated simian immunodeficiency virus (SIV)-infected macaques by single-cell RNA sequencing. A subset of vRNA+ cells distinguished by spliced and high total vRNA (7-10% of reads) expressed diminished FOS, a component of the Activator protein 1 (AP-1) transcription factor, relative to vRNA-low and -negative cells. Conversely, FOS and JUN, another AP-1 component, were upregulated in HIV DNA+ infected cells compared to uninfected cells from people with HIV-1 on suppressive therapy. Inhibiting c-Fos in latently infected primary cells augmented reactivatable HIV-1 infection. These findings implicate AP-1 in latency establishment and maintenance and as a potential therapeutic target to limit HIV-1 reservoirs.

Fibrin Stiffness Regulates Phenotypic Plasticity of Metastatic Breast Cancer Cells.

The extracellular matrix (ECM)-regulated phenotypic plasticity is crucial for metastatic progression of triple negative breast cancer (TNBC). While ECM faithful cell-based models are available for in situ and invasive tumors, such as cell aggregate cultures in reconstituted basement membrane and in collagenous gels, there are no ECM faithful models for metastatic circulating tumor cells (CTCs). Such models are essential to represent the stage of metastasis where clinical relevance and therapeutic opportunities are significant. Here, CTC-like DU4475 TNBC cells are cultured in mechanically tunable 3D fibrin hydrogels. This is motivated, as in circulation fibrin aids CTC survival by forming a protective coating reducing shear stress and immune cell-mediated cytotoxicity and promotes several stages of late metastatic processes at the interface between circulation and tissue. This work shows that fibrin hydrogels support DU4475 cell growth, resulting in spheroid formation. Furthermore, increasing fibrin stiffness from 57 to 175Pa leads to highly motile, actin and tubulin containing cellular protrusions, which are associated with specific cell morphology and gene expression patterns that markedly differ from basement membrane or suspension cultures. Thus, mechanically tunable fibrin gels reveal specific matrix-based regulation of TNBC cell phenotype and offer scaffolds for CTC-like cells with better mechano-biological properties than liquid.