G2-M Checkpoint Research Articles

Comprehensive transcriptomic analyses identify the immunosuppressive effects of LLDT-8 in ART-treated SIV-infected rhesus macaques

BackgroundChronic immune activation plays a significant role in the pathogenesis and disease progression of human immunodeficiency virus (HIV), and the existing interventions to address this issue are limited. In a phase II clinical trial, (5R)-5-hydroxytriptolide (LLDT-8) demonstrated promising potential in enhancing CD4+ T cell recovery. However, the therapeutical effects of LLDT-8 remained to be systemic explored. MethodsTo assess the treatment effects of LLDT-8, we conducted flow cytometry and RNA-seq analyses on eight Chinese rhesus monkeys infected with simian immunodeficiency virus (SIV). Additionally, we performed comprehensive transcriptomic analyses, including cross-sectional and longitudinal differentially expressed gene (DEG) analysis, gene set enrichment analysis (GSEA), weighted gene co-expression network analysis (WGCNA), and deconvolution analysis using peripheral blood mononuclear cell (PBMC) samples from 14-time points. These findings were further validated with RNA-seq analysis on patients who received LLDT-8 treatment, along with in vitro cellular experiments using human PBMCs. ResultsFlow cytometry analysis revealed that LLDT-8 treatment significantly reduced the percentage of HLA-DR+CD38+CD8+ T cells in SIV-infected rhesus monkeys (P < 0.001). The cross-sectional and longitudinal analysis identified 2531 and 1809 DEGs, respectively. GSEA analysis indicated that LLDT-8 treatment led to significant downregulation of proliferation-related pathways, such as E2F targets, G2M checkpoint, and mitotic spindle pathways. WGCNA analysis identified two modules and 202 hub genes associated with CD8 activation levels. Deconvolution analysis showed a significant decrease in the proportion of CD8+ T cells and activated CD4+ T cells during LLDT-8 treatment. Gene ontology results demonstrated that the common DEGs between LLDT-8-treated patients and rhesus monkeys were primarily enriched in cell activation and cell cycle progression. Furthermore, in vitro cellular experiments validated the consistent impact of LLDT-8 in inhibiting proliferation, activation (HLA-DR and CD38 expression), exhaustion (PD-1 expression), and IFN-γ production in human CD4+ and CD8+ T cells. ConclusionLLDT-8 exhibited notable efficacy in alleviating immune activation in both an in vivo animal model and in vitro human cell experiments. These findings suggest that LLDT-8 may hold potential as a drug for managing systemic immune activation associated with SIV/HIV infection, warranting further prospective clinical exploration.

CSIG-10. SEX-DIFFERENCES IN NF-ΚB SIGNALING DRIVE SEX-BIASED RESPONSES TOWARDS SENESCENCE AND INFLAMMATION IN GLIOBLASTOMA

Abstract Male glioblastoma (GBM) patients have a higher incidence rate and worse median survival in comparison to females. The molecular mechanisms that drive these sex differences in incidence and survival are not well understood. Using a murine in vitro GBM model, we identified that male and female cells tend to segregate into two unique groups based on differential gene expression. Male-enriched processes included myc, E2F and G2-M checkpoint signaling. Female-enriched processes included epithelial-mesenchymal transition, interferon responses and TNF-α signaling by NF-κB. Reports from human GBM patients corroborate an increased enrichment for inflammatory and innate immune responses in females. Since the NF-κB pathway regulates senescence and its associated inflammatory response, the senescence-associated secretory phenotype, we examined if there are sex differences in NF-κB signaling as we have reported that female GBM cells undergo senescence more readily. We found that low levels of NF-κB activity were correlated with higher levels of senescence, and that cells from the female-enriched group correspondingly had lower levels of NF-κB activity. Interestingly, p-p65 S536 expression (a marker of increased NF-κB transcriptional activity) was negatively correlated with NF-κB activity in cells within the female-enriched group. Furthermore, female cells with low NF-κB activity responded more readily to TNF-α treatment. These results suggest that female cells can more readily modulate the NF-κB pathway in response to stimuli, thereby poising them towards a pro-inflammatory state. Since inflammation and senescence regulate therapeutic responses in GBM, and we have previously reported that female GBM cells undergo senescence more readily following radiation, these results suggest that further studies to understand the sex differences in NF-κB signaling may improve patient response to radiation and chemotherapy treatment.

Lysosomal Associated Membrane Protein Family Member 5 (LAMP5) As a Novel Mediator of, and Target in High Risk Multiple Myeloma (MM)

Background: Our recently published studies of single cell clonotypic and transcriptional evolution of multiple myeloma precursor disease (Dang et al. Cancer Cell, 2023), as well as related multi-omic studies of more advanced disease, identified a number of genes as potential contributors to progression. Among these is LAMP5, a gene on chromosome 20 that is markedly and specifically overexpressed in the malignant, monoclonal plasma cells in a high proportion of patients. As LAMP5 expression has also been associated with osteolytic bony disease in myeloma, we sought to delineate its potential contributions to the pathobiology of myeloma, which remain incompletely understood. Methods: The impact of LAMP5 knockdown and over-expression was studied using myeloma cell line models both in vitro and in vivo. Moreover, we generated a monoclonal antibody to LAMP5 after finding it was exposed on the myeloma cell plasma membrane, and converted this agent into an antibody drug conjugate (ADC) linked to monomethyl auristatin E (MMAE) or pyrrolobenzodiazepine ( PBD) dimers. Finally, we used the sequence of the single-chain variable fragment to generate LAMP5-targeted chimeric antigen receptor (CAR)-guided T-cells. Results: Analysis of the Multiple Myeloma Research Foundation's CoMMpass SM database indicated that high LAMP5 expression (defined as in the top quartile) in newly diagnosed myeloma patients was significantly associated with markers of increased disease burden, including a higher beta-2-microglobulin (p = 0.047), C-reactive protein (p = 0.0399), serum calcium (p &amp;lt; 0.0001), marrow plasmacytosis (p &amp;lt; 0.001), and a trend towards increased circulating plasma cells (p = 0.075). Moreover, high LAMP5 expression correlated with a shorter progression-free (p = 0.0006) as well as overall survival (p = 0.0006). Similarly, analysis of the University of Arkansas for Medical Sciences gene expression data indicated LAMP5 was increased in the hyperdiploid and proliferative groups that are associated with a poor prognosis. Inducible shRNA-mediated LAMP5 knockdown in myeloma cell lines conferred a survival disadvantage and activated apoptosis. RNA-sequencing analysis identified multiple dysregulated Hallmark pathways, including the G2M checkpoint, the mitotic spindle, apoptosis, mTORC1 signaling, and the unfolded protein response. Proteomic consequences of LAMP5 knockdown included Caspase-7, -8, and -3 cleavage, and increased abundance of Smac, Bim, and PUMA. By contrast, many G2M checkpoint proteins were down-regulated, including Aurora A, Checkpoint-1, Cell division cycle-6, and WEE1 kinases, and decreased levels of phospho-S807/S811 Rb protein were seen. Also, RNA-sequencing revealed mTORC1 pathway dysregulation, and proteomic studies showed decreased activity of AKT and AKT2, mTOR, and p70 S6 kinase. Notably, LAMP5 suppression was associated with reduced levels of expression of an RFP-LC3 fusion protein consistent with a role for LAMP5 in autophagy. In contrast, high LAMP5 expression was associated with improved colony formation in vitro ( A), and promoted disease progression in vivo. Since the LAMP5 promoter had consensus retinoic acid sites, we tested all-trans retinoic acid and found it suppressed LAMP5 protein expression, and reduced in vitro clonogenicity. Finally, since other LAMPs can under some circumstances be found at the plasma membrane, we performed flow and LAMP5 was indeed detected on the surface of unpermeabilized LAMP5-positive myeloma cells. We thus developed a panel of monoclonal antibodies that recognized the extracellular domains of LAMP5, and converted one of these into antibody drug conjugates (ADCs) linked to either MMAE or PBD. Both ADCs reduced the viability of LAMP5-positive myeloma cells in a time- and concentration-dependent manner ( B), activated immunogenic cell death in vitro, and also showed anti-tumor activity in vivo. Importantly, LAMP5-directed CAR T-cells similarly and specifically eliminated only LAMP5-positive myeloma cells with efficacy that was comparable to that of B-cell maturation antigen-targeted CAR T-cells. Conclusions: These pre-clinical in vitro and in vivo data support the possibility that LAMP5 represents a novel mediator of myeloma clonogenicity and high-risk behavior, and that it could serve as a rational therapeutic target leveraging immune therapies that recognize the extracellular domains of cell surface LAMP5.

Activity of Selinexor As a Single Agent and Synergistic Activity with Approved/Investigational Myelofibrosis Therapies in Vitro

Background: Myelofibrosis (MF) is a myeloproliferative neoplasm (MPN) that commonly harbors acquired somatic gene mutations in JAK2, CALR or MPL leading to constitutive JAK/STAT activation . Despite the current standard of care, the JAK1/2 inhibitor (JAKi) ruxolitinib (RUX), a significant unmet need remains for patients (pts) with MF, due to lack of response or JAKi resistance. Selinexor (SEL) is an investigational, oral XPO1 inhibitor that may inhibit multiple pathways relevant in MF including JAK/STAT, ERK, and AKT. Previously, we reported that 60 mg SEL in combination with RUX in pts with JAKi-naïve MF resulted in rapid, deep, and sustained spleen responses even in pts receiving suboptimal RUX doses, suggesting synergy between SEL and RUX while maintaining a generally manageable safety profile (Ali et al., ASCO 2023; Ali et al., AACR 2023). Here, we investigated the combinatorial effects of SEL with 5 approved/investigational MF therapeutic agents on human cell lines, then evaluated relevant downstream pathways impacted by SEL +/- RUX. Methods: JAK2 V617F mutant cell lines HEL ( TP53M113K), UKE-1 ( TP53WT), MUTZ-8 ( TP53WT), and JAK2WT ELF-153 ( TP53I251N) cells were treated with SEL alone or in combination with RUX, pacritinib (PAC), momelotinib (MMB), pelabresib (PELA), or navitoclax (NAV). Compounds were added to 1x10 4 cells in a 1:3 or 1:2 serial dilution, then cell viability was assessed 72 hours post-exposure. Single-agent non-linear regressions were used to determine inhibitory concentrations (IC), and drug synergy was evaluated using Synergy Finder 3.0 (Bliss). HEL ( JAK2V617F) and ELF-153 ( JAK2wt) cells were used for mechanistic studies including western blots and RNA sequencing. Results: Single agent SEL reduced cell viability in all cell lines tested, independent of JAK2 mutation status: HEL (IC 50 320nM), UKE-1 (320 nM), MUTZ-8 (12nM) and ELF-153 (570 nM) cells. SEL antiproliferative activity was more potent than RUX (IC 50 range 1-64.6 µM), MMB (1.9-27.8 µM), NAV (0.165-51.4 µM) and PELA (7.5-65.9 µM). When determined by Bliss scores, evidence of synergy was observed in the following co-treatments: SEL + RUX in HEL (10.8) and UKE-1 (11.9) cells; SEL + PELA in HEL (9.9) and UKE-1 (7.7); SEL + MMB (16.1) and NAV (12.4) in HEL ( Figure 1). Additive effects were observed: SEL + RUX in MUTZ-8 and ELF-153 ( JAK2wt) cells; SEL + MMB in UKE-1, MUTZ-8 ELF-153 cells; SEL + PAC in HEL and ELF-153 cells; SEL+ PELA in UKE-1, MUTZ-8 and ELF-153 cells; SEL+NAV in UKE-1 and ELF-153 cells. Mechanistically, SEL and the SEL + RUX combo both induced XPO1 protein degradation to a similar extent. The SEL + RUX combination in HEL cells resulted in a pro-apoptotic effect via decreased MCL-1 protein (P≤0.0001) and increased cleaved PARP vs RUX treatment. Preliminary analysis of mRNA transcript alterations after SEL + RUX treatment showed a modulation of expression of genes related to proliferation ( MYC), senescence ( CDKN1A/B) and cell cycle ( CYCLIND1/A2/B1). A RUX-resistant HEL cell clone was generated via culture with increasing RUX, resulting in a &amp;gt;10-fold increase in RUX IC 50. The SEL IC 50 of the resistant clone was not changed compared to parental cells. RUX-resistant HEL cells showed downregulation of several pathways including MYC, G2M checkpoint, ribosomal biogenesis, proteasome, spliceosome, and nuclear export when compared to parental HEL cells. SEL had synergistic or additive effects with MF agents in this resistant subclone. Conclusion: In a panel of MPN-derived cells with or without JAK2V617F, SEL showed single-agent antiproliferative activity, and synergism with other MF therapeutics at clinically achievable concentrations through regulation of XPO1, JAK/STAT signaling, and apoptosis/senescence regulators. In addition to positive Phase 1 clinical data in pts with JAKi-naïve MF who received SEL with RUX, these preclinical data support synergistic activity between SEL and other MF therapies, suggesting the potential for SEL as a backbone for novel treatment combinations for MF.

Clinical implications of RAB13 expression in pan-cancer based on multi-databases integrative analysis

Worldwide, cancer is a huge burden, and each year sees an increase in its incidence. RAB (Ras-related in brain) 13 is crucial for a number of tumor types. But more research on RAB13's tumor-related mechanism is still required. This study's goal was to investigate RAB13's function in human pan-cancer, and we have also preliminarily explored the relevant mechanisms. To investigate the differential expression, survival prognosis, immunological checkpoints, and pathological stage of RAB13 in human pan-cancer, respectively, databases of TIMER2.0, GEPIA 2, and UALCAN were employed. CBioPortal database was used to analyze the mutation level, meanwhile, PPI network was constructed based on STRING website. The putative functions of RAB13 in immunological infiltration were investigated using single sample gene set enrichment analysis (ssGSEA). The mechanism of RAB13 in hepatocellular cancer was also briefly investigated by us using gene set enrichment analysis (GSEA). RAB13 was differentially expressed in a number of different cancers, including liver hepatocellular carcinoma (LIHC), stomach adenocarcinoma (STAD), etc. Additionally, RAB13 overexpression in LGG and LIHC is associated with a worse prognosis, including overall survival (OS) and disease-free survival (DFS). Then, we observed that early in BLCA, BRAC, CHOL, ESCA, HNSC, KICH, KIRC, LIHC, LUAD, LUSC, and STAD, the level of RAB13 expression was raised. Next, we found that “amplification” was the most common mutation in RAB13. The expression of SLC39A1, JTB, SSR2, SNAPIN, and RHOC was strongly positively linked with RAB13, according to a correlation study. RAB13 favorably regulated B cell, CD8 + T cell, CD4 + T cell, macrophage, neutrophil, and dendritic cell in LIHC, according to immune infiltration analysis. Immune checkpoint study revealed a positive correlation between RAB13 expression and PD1, PDL1, and CTLA4 in LIHC. According to GSEA, RAB13 is involved in a number of processes in LIHC, including MTORC1 signaling, MYC targets v1, G2M checkpoint, MITOTIC spindle, DNA repair, P53 pathway, glycolysis, PI3K-AKT-MTOR signaling, etc. RAB13 is a possible therapeutic target in LIHC and can be used as a prognostic marker.

SAT632 The Functional Role And Regulatory Mechanism Of FTO M6A RNA Demethylase In Human Uterine Leiomyosarcoma

Abstract Disclosure: Q. Yang: None. A. Al-Hendy: None. Introduction: Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. However, the origin and molecular mechanism underlying driving its clinical and biological behavior remain to be determined. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications contributing to the pathogenesis of uLMS. However, the connectivity between reversible RNA m6A methylation and uLMS remains unclear. In this study, we assessed the role and mechanism of the m6A demethylase fat mass- and obesity-associated protein (FTO) in the pathogenesis of uLMS. Materials and Methods: We performed immunohistochemistry (IHC) and immunoblot analysis to measure the m6A erasers FTO and ALKBH5 protein levels in LMS and adjacent myometrium tissues (n=8) and cell lines. We performed LMS cell proliferation and cell cycle phase distribution analysis. RNA-seq was performed to determine the molecular mechanism underlying m6A eraser inhibition in LMS cells (n=4 for each group). The R package limma+voom was used for this analysis. Hallmark pathway analysis was used to identify enriched pathways in response to FTO inhibition. Real-time PCR was performed to validate the differentially expressed genes. Ingenuity pathway analysis (IPA) was performed to determine the upstream regulators targeting cell cycle-related genes. We used a cutoff −1.5 &amp;gt; fold-change &amp;gt; 1.5 and an adjusted p-value of 0.05. In addition, data were analyzed by a two-tailed unpaired Student’s t-test between any two groups. Results: IHC analysis revealed that m6A demethylases FTO and ALKBH5 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium (p&amp;lt;0.01, H-score). Furthermore, inhibition of FTO with its small, potent inhibitor (Dac51) decreased the uLMS proliferation dose-dependently via cell cycle arrest at G1. Notably, RNA-seq analysis revealed that inhibition of FTO with Dac51 exhibited a significant decrease in the expression of multiple CDK members and a significant increase in the expression of CDKN1A. Furthermore, IPA analysis revealed that transcription factors, including SP1 and E2F members are the upstream regulators targeting CDK members in response to Dac51 treatment. Moreover, Dac51 treatment altered the expression of several epigenetic regulators, including Sirt1 and 2, HDAC10 and SUV39H1, and rewiring of several critical pathways, including inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to suppression of uLMS phenotype. Conclusion: Our findings uncover the anti-uLMS function of a potent FTO inhibitor via reprogramming the oncogenic epitranscriptome. Therefore, targeted inhibition of FTO may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer. Presentation: Saturday, June 17, 2023

Targeting the fibroblast growth factor pathway in molecular subtypes of castration-resistant prostate cancer.

Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. However, castration-resistant prostate cancer (CRPC) tumors can resist AR signaling inhibitors through AR amplification and AR splice variants in AR-positive CRPC (ARPC), and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We have shown previously that DNPC can bypass AR-dependence through fibroblast growth factor receptor (FGFR) signaling. However, the role of the FGFR pathway in other CRPC phenotypes has not been elucidated. RNA-Seq analysis was conducted on patient metastases, LuCaP patient-derived xenograft (PDX) models, and CRPC cell lines. Cell lines (C4-2B, VCaP, and 22Rv1) and ex vivo LuCaP PDX tumor cells were treated with enzalutamide (ENZA) and FGFR inhibitors (FGFRi) alone or in combination and sensitivity was determined using cell viability assays. In vivo efficacy of FGFRi in ARPC, DNPC, and SCNPC were evaluated using PDX models. RNA-Seq analysis of FGFR signaling in metastatic specimens, LuCaP PDX models, and CRPC cell lines revealed significant FGF pathway activation in AR-low PC (ARLPC), DNPC, and SCNPC tumors. In vitro/ex vivo analysis of erdafitinib and CH5183284 demonstrated robust and moderate growth suppression of ARPC, respectively. In vivo studies using four ARPC PDX models showed that combination ENZA and CH5183284 significantly suppressed tumor growth. Additional in vivo studies using four ARPC PDX models revealed that erdafitinib monotherapy was as effective as ENZA in suppressing tumor growth, and there was limited combination benefit. Furthermore, two of three DNPC models and two of four SCNPC models responded to CH5183284 monotherapy, suggesting FGFRi responses were model dependent. RNA-Seq and gene set enrichment analysis of end-of-study ARPC tumors treated with FGFRi displayed decreased expression of E2F and MYC target genes and suppressed G2M checkpoint genes, whereas end-of-study SCNPC tumors had heterogeneous transcriptional responses. Although FGFRi treatments suppressed tumor growth across CRPC phenotypes, our analyses did not identify a single pathway or biomarker that would identify tumor response to FGFRi. This is very likely due to the array of FGFR1-4 expression and tumor phenotypes present in CRPC. Nevertheless, our data nominate the FGFR pathway as a clinically actionable target that promotes tumor growth in diverse phenotypes of treatment-refractory metastatic CRPC.

Comprehensive analysis of Cuproplasia and immune microenvironment in lung adenocarcinoma.

Background: Trace elements such as copper are essential for human health. Recently the journal Nat Rev Cancer has put forward the concept of Cuproplasia, a way of promoting tumor growth through reliance on copper. We attempted to conduct a comprehensive analysis of Cuproplasia-related genes in lung adenocarcinoma (LUAD) to explore the mechanism of action of Cuproplasia-related genes in LUAD. Method: Transcriptome data and clinical information of LUAD were obtained from TCGA-LUAD and GSE31210, and prognostic models of Cuproplasia-related genes were constructed and verified by regression analysis of GSVA, WGCNA, univariate COX and lasso. The signal pathways affected by Cuproplasia-related genes were analyzed by GO, KEGG and hallmarK pathway enrichment methods. Five immunocell infiltration algorithms and IMVIGOR210 data were used to analyze immune cell content and immunotherapy outcomes in the high-low risk group. Results: In the results of WGCNA, BROWN and TURQUOISE were identified as modules closely related to Cuproplasia score. In the end, lasso regression analysis established a Cuproplasia-related signature (CRS) based on 24 genes, and the prognosis of high-risk populations was worse in TCGA-LUAD and GSE31210 datasets. The enrichment analysis showed that copper proliferation was mainly through chromosome, cell cycle, dna replication, g2m checkpoint and other pathways. Immunoinfiltration analysis showed that there were differences in the content of macrophages among the four algorithms. And IMVIGOR210 found that the lower the score, the more effective the immunotherapy was. Conclusion: The Cuproplasia related gene can be used to predict the prognosis and immunotherapy outcome of LUAD patients, and may exert its effect by affecting chromosome-related pathways and macrophages.

P12.08.A CELL-EXTRINSIC D-2-HYDROXYGLUTARATE PROMOTES HYPERMETHYLATION AT LINEAGE-SPECIFIC REGULATORY REGIONS TO PREVENT MICROGLIAL ACTIVATION IN IDH-MUTANT GLIOMAS

Abstract BACKGROUND Tumor-associated macrophages and microglia (TAMs) are highly abundant myeloid cells in diffuse gliomas. Their composition and transcriptional programs differ according to the IDH mutation status of glioma cells. The underlying mechanisms remain until now little known. MATERIAL AND METHODS We compared bulk DNA methylome (Methylation EPIC array) and transcriptome of TAMs (CD11b+ purified by magnetic-activated cell sorting) from 25 IDH-mutant and 11 IDH-wildtype gliomas. The contamination of the CD11b+ fraction by tumor cells was determined by the detection of IDH and/or TERT mutations using droplet digital PCR. Only samples with a purity of at least 95% were used for analyses. To determine experimentally direct effects of D-2-hydroxyglutarate (D-2-HG), which is the oncometabolite produced and realeased into the microenvironment by IDH-mutated glioma cells, we used primary cultures of human microglial cells obtained from glioma surgery or epilepsy surgery. We exposed microglia or not to (D-2-HG) during 14 days, and analyzed their DNA methylome and transcriptome. RESULTS TAMs from IDH-mutant gliomas compared with TAMs from IDH-wildtype gliomas exhibit decreased expression of pro-inflammatory pathways (mainly IFN-γ, IFN-α, and TNF-α signaling) and genes related to G2-M checkpoint, glycolysis and Epithelial-Mesenchymal Transition (EMT). We uncovered that TAMs from IDH-mutant gliomas exhibit significant hypermethylation at promoters and enhancers linked to decreased expression of genes involved in inflammatory responses. Notably, these hypermethylated regions were enriched for binding motifs of transcription factors controlling an environment-dependent transcriptional network in microglial cells (such as PU.1, AP-1, C/EBP and RUNX). These findings were confirmed by comparing methylome data of TAMs from IDH-mutant gliomas and methylome of microglia from normal brains available in the literature. A 14 days exposition to D-2-HG of human microglial cells in vitro partially recapitulates the observed methylation changes. Transcriptome analyses of these cells show that this oncometabolite dampens the IFN-α/γ pathways and attenuates the response to LPS while inducing a metabolic shift toward oxidative phosphorylation. CONCLUSION Our findings establish a link between an extrinsic cell-effect of D-2-HG and the hypermethylation of DNA of microglial cells at regulatory regions in IDH-mutant gliomas. Our observations support the concept that oncometabolites may disrupt directly the function of immune cells of the tumor microenvironment.

Identification of m6A methylation-related genes in cerebral ischaemia‒reperfusion of Breviscapus therapy based on bioinformatics methods

BackgroundCerebral ischaemia‒reperfusion (I/R) frequently causes late-onset neuronal damage. Breviscapine promotes autophagy in microvascular endothelial cells in I/R and can inhibit oxidative damage and apoptosis. However, the mediation mechanism of breviscapine on neuronal cell death is unclear.MethodsFirst, transcriptome sequencing was performed on three groups of mice: the neuronal normal group (Control group), the oxygen-glucose deprivation/ reoxygenation group (OGD/R group) and the breviscapine administration group (Therapy group). Differentially expressed genes (DEGs) between the OGD/R and control groups and between the Therapy and OGD/R groups were obtained by the limma package. N6-methyladenosine (m6A) methylation-related DEGs were selected by Pearson correlation analysis. Then, prediction and confirmation of drug targets were performed by Swiss Target Prediction and UniProt Knowledgebase (UniProtKB) database, and key genes were obtained by Pearson correlation analysis between m6A-related DEGs and drug target genes. Next, gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA) were used to obtain the pathways of key genes. Finally, a circRNA-miRNA‒mRNA network was constructed based on the mRNAs, circRNAs and miRNAs.ResultsA total of 2250 DEGs between the OGD/R and control groups and 757 DEGs between the Therapy and OGD/R groups were selected by differential analysis. A total of 7 m6A-related DEGs, including Arl4d, Gm10653, Gm1113, Kcns3, Olfml2a, Stk26 and Tfcp2l1, were obtained by Pearson correlation analysis. Four key genes (Tfcp2l1, Kcns3, Olfml2a and Arl4d) were acquired, and GSEA showed that these key genes significantly participated in DNA repair, e2f targets and the g2m checkpoint. IPA revealed that Tfcp2l1 played a significant role in human embryonic stem cell pluripotency. The circRNA-miRNA‒mRNA network showed that mmu_circ_0001258 regulated Tfcp2l1 by mmu-miR-301b-3p.ConclusionsIn conclusion, four key genes, Tfcp2l1, Kcns3, Olfml2a and Arl4d, significantly associated with the treatment of OGD/R by breviscapine were identified, which provides a theoretical basis for clinical trials.

Identification of m5C-related molecular subtypes and prediction models in the prognosis and tumor microenvironment infiltration of soft tissue sarcoma

BackgroundThe epigenetic regulator in cancer progression and immune response has been demonstrated recently. However, the potential implications of 5-methylcytosine (m5C) in soft tissue sarcoma (STS) are unclear. MethodsThe RNA sequence profile of 911 normal and 259 primary STS tissues were obtained from GTEx and TCGA databases, respectively. We systematically analyzed the m5C modification patterns of STS samples based on 11 m5C regulators, and comprehensively correlated these modification patterns with clinical characteristics, prognosis, and tumor microenvironment (TME) cell-infiltrating. Furthermore, an m5C-related signature was generated using Cox proportional hazard model and validated by the GSE17118 cohort. ResultsTwo distinct m5C modification patterns (cluster1/2) were discovered. The cluster1 had favorable overall survival, higher immune score, higher expression of most immune checkpoints, and active immune cell infiltration. The GSVA analysis of the P53 pathway, Wnt signaling pathway, G2M checkpoint, mTORC1 signaling, Wnt/β catenin signaling, and PI3K/AKT/mTOR signaling were significantly enriched in the cluster2. Moreover, 1220 genes were differentially expressed between two clusters, and a m5C prognostic signature was constructed with five m5C-related genes. The signature represented an independent prognostic factor and showed the favorable performance in the GSE17118 cohort. Patients in the low-risk group showed higher immunoscore and higher expression of most immune checkpoints. Further GSVA analysis indicated that the levels of P53 pathway, Wnt signaling pathway, and TGF-β signaling pathway were different between low- and high-risk groups. Moreover, a nomogram incorporating m5C signature and clinical variables was established and showed well performance. ConclusionThis work showed that the m5C modification plays a significant role in the progression of STS and the formation of TME diversity. Evaluating the m5C modification pattern of tumor will enhance our cognition of TME infiltration characterization to guide more effective immunotherapy strategies.

Dealcoholized red wine inhibits occurrence and progression of hepatocellular carcinoma possibly by inducing cell cycle arrest and apoptosis

To investigate the inhibitory effect of dealcoholized red wine (DRW) on occurrence and progression of hepatocellular carcinoma (HCC) and explore its possible mechanisms. Three HCC cell lines (Huh7, HepG2 and SKHep-1) treated with 5, 10, 25, 50 and 100 μL/mL DRW were examined for changes in proliferation and colony formation ability using CCK-8 assay and colony formation assay. A nude mouse model bearing subcutaneous HCC xenograft was used to test the effect of 300 μL/day DRW for 4 weeks on tumor growth. The inhibitory effect of 300 μL/day DRW for 6 weeks on tumor growth was also observed in a mouse model of chemically induced HCC by examining the tumor number, largest tumor diameter and the liver/body ratio. RNA-seq technique was used for transcriptome sequencing of Huh7 cells treated with DRW (75 μL/mL) for 48 h, and gene-set enrichment analysis (GSEA) was performed to identify the changes in genes and pathways. Flow cytometry assay was used to analyze the changes in cell cycle and apoptosis of the cells. DRW inhibited the proliferation of the HCC cell lines in a concentration-and time-dependent manner, and concentration-dependently inhibited colony formation of the cells. Treatment with DRW significantly reduced the volume of subcutaneous tumor xenograft in the tumor-bearing nude mice (P < 0.05), and lowered the number of tumors (P < 0.001), the largest tumor diameter (P < 0.05) and the liver/body ratio (P < 0.01) in mice with chemically induced HCC. RNA-seq showed that 634 genes were significantly up-regulated and 478 were down-regulated in Huh7 cells after treatment with DRW. Gene-set enrichment analysis revealed that DRW significantly down-regulated cell cycle-related pathways (E2F Targets, G2M Checkpoint and MYC Targets) and up-regulated apoptosis pathways. Flow cytometry assay showed that DRW induced cell cycle arrest in G1 phase and apoptosis of Huh7 cells. DRW inhibits the occurrence and progression of HCC, and this effect is mediated possibly by inducing cell cycle arrest and apoptosis.

Characteristics and prognosis of adolescent and young adult breast cancer.

16 Background: Adolescent and young adult (AYA)-generation breast cancer has characteristics of high clinicopathological malignancy and poor prognosis compared to cancers of other age groups. In recent advances in cancer research, the tumor microenvironment is involved in cancer invasion. In this study, we investigated the biology of AYA-generation breast cancer and the prognostic effects of the cancer microenvironment in breast cancer patients. Methods: In the current study, we analyzed 2042 breast cancer patients with clinical and intratumoral gene expression data from the open public databases TCGA and METABRIC. Patients aged 15-39 were defined as AYA-generation breast cancer, and patients aged 40-65 as middle-aged breast cancer. Tumor biology and cancer microenvironment were assessed using gene set enrichment analysis (GSEA) and the xCell algorithm. Disease-specific survival (DSS) and disease-free survival (DFS) were used to evaluate the prognosis of AYA-generation breast cancer compared to that of middle-aged and elderly breast cancer. Results: In METABRIC, the proportion of advanced stages was higher in AYA generation breast cancer than in middle-aged and elderly breast cancer (p&lt;0.01). However, this was not the case with TCGA cohort. In the prognostic analysis, AYA-generation breast cancer showed shortened DSS and DFS compared with middle-aged breast cancer only in METABRIC. To clarify the association between prognosis and cancer biology, we performed GSEA. AYA-generation breast cancer enriched cell proliferation-related gene sets such as E2F Targets, G2M Checkpoints, and MYC Targets V1 in METABRIC. The analysis of TCGA also confirmed this result. Interestingly, the infiltration of macrophage M1 (p&lt;0.001) and helper T cell Th1 (p=0.007), which act as tumor suppressors, was significantly higher in AYA generation breast cancer in METABRIC cohort. In TCGA, there was only a tendency for Th1 infiltration to be high in AYA generation breast cancer. Conclusions: AYA-generation breast cancer enriched gene sets associated with cell proliferation which might explain the worse prognosis of AYA-generation breast cancer.

High Atlastin 2-2 (ATL2-2) Expression Associates with Worse Prognosis in Estrogen-Receptor-Positive Breast Cancer.

The disruption of endoplasmic reticulum (ER) homeostasis occurs in many human diseases. Atlastins (ATLs) maintain the branched network of the ER. The dysregulation of ATL2, located at ER network junctions, has been associated with cancer. ATL2 is necessary for lipid droplet formation in murine breast tissue. Thus, we analyzed whether ATL2 has a role in human breast cancer (BC) pathology. The expression of ATL2 variant ATL2-2 was analyzed in breast tumors from the BC cohorts of the TCGA, METABRIC, and two independent Icelandic cohorts, Cohort 1 and 2; its association with clinical, pathological, survival, and cellular pathways was explored. ATL2-2 mRNA and protein expression were higher in breast tumors than in normal tissue. ATL2-2 mRNA associated with tumor characteristics that indicate a worse prognosis. In METABRIC, high ATL2-2 mRNA levels were associated with shorter BC-specific survival (BCSS) in patients with estrogen-receptor-positive luminal breast tumors, which remained significant after correction for grade and tumor size (HR 1.334, CI 1.063-1.673). Tumors with high ATL2 mRNA showed an upregulation of hallmark pathways MYC targets v1, E2F targets, and G2M checkpoint genes. Taken together, the results suggest that high levels of ATL2-2 may support BC progression through key cancer driver pathways.

Non-SMC condensin I complex subunit D2 (NCAPD2) reveals its prognostic and immunologic features in human cancers.

Non-SMC condensin I complex subunit D2 (NCAPD2) is overexpressed in some malignant tumors. However, there are few studies on the function of NCAPD2 in pan-cancer. We used the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), Human Protein Atlas (HPA), and UALCAN to analyze NCAPD2 expression and promoter methylation levels in 33 tumors and normal samples. We performed immunohistochemistry (IHC) on liver cancer and corresponding normal tissues to examine NCAPD2 protein expression in LIHC. Kaplan-Meier survival and univariate regression analyses were performed to explore the pan-cancer clinical significance of NCAPD2. Moreover, correlative analysis between NCAPD2 expression and clinical characteristics, immune cell infiltration, immune checkpoints, immune regulators, tumor mutation burden (TMB), microsatellite instability (MSI), ribonucleic acid (RNA) methylation regulators, and drug sensitivity was conducted using data from TCGA. We also investigated the effects of NCAPD2 expression on immunotherapy efficacy and prognosis. Gene set enrichment analysis (GSEA) was conducted using NCAPD2. Bioinformatic analysis showed that NCAPD2 was overexpressed in most tumors and correlated with the clinical characteristics of some cancers. IHC results demonstrated that NCAPD2 protein expression was higher in LIHC than in normal liver. NCAPD2 expression was linked with T stage, clinical stage, and histologic grade in LIHC. Overexpression of NCAPD2 resulted in poor overall survival, and disease-specific survival in adrenocortical carcinoma, kidney renal papillary cell carcinoma, brain lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, mesothelioma, pancreatic adenocarcinoma, sarcoma, skin cutaneous melanoma, and uterine corpus endometrial carcinoma. NCAPD2 was considered an independent biomarker by Cox regression in LIHC. The time ROC curve demonstrated that the survival rate of 1-, 3-, and 5-year OS and DSS in LIHC was above 0.6. The expression of NCAPD2 was significantly correlated with immune cell infiltration, immune checkpoints, TMB, MSI, and RNA methylation regulators in several tumors. NCAPD2 had a high predictive value for immunotherapy efficiency in certain tumors. In our study, drugs sensitive to NCAPD2 protein were screened by sensitivity analysis. GSEA analysis showed that NCAPD2 mainly participated in the G2M checkpoint, mitotic spindle, and KRAS-signaling. NCAPD2 may act as a prognostic molecular marker in most cancers.

Glycolysis induces Th2 cell infiltration and significantly affects prognosis and immunotherapy response to lung adenocarcinoma.

Glycolysis has a major role in cancer progression and can affect the tumor immune microenvironment, while its specific role in lung adenocarcinoma (LUAD) remains poorly studied. We obtained publicly available data from The Cancer Genome Atlas and Gene Expression Omnibus databases and used R software to analyze the specific role of glycolysis in LUAD. The Single Sample Gene Set Enrichment Analysis (ssGSEA) indicated a correlation between glycolysis and unfavorable clinical outcome, as well as a repression effect on the immunotherapy response of LUAD patients. Pathway enrichment analysis revealed a significant enrichment of MYC targets, epithelial-mesenchymal transition (EMT), hypoxia, G2M checkpoint, and mTORC1 signaling pathways in patients with higher activity of glycolysis. Immune infiltration analysis showed a higher infiltration of M0 and M1 macrophages in patients with elevated activity of glycolysis. Moreover, we developed a prognosis model based on six glycolysis-related genes, including DLGAP5, TOP2A, KIF20A, OIP5, HJURP, and ANLN. Both the training and validation cohorts demonstrated the high efficiency of prognostic prediction in this model, which identified that patients with high risk may have a poorer prognosis and lower sensitivity to immunotherapy. Additionally, we also found that Th2 cell infiltration may predict poorer survival and resistance to immunotherapy. The study indicated that glycolysis is significantly associated with poor prognosis in patients with LUAD and immunotherapy resistance, which might be partly dependent on the Th2 cell infiltration. Additionally, the signature comprised of six genes related to glycolysis showed promising predictive value for LUAD prognosis.

Epigenetic alternations and targeted therapy in intrauterine adhesion: A comparative study

Although intrauterine adhesion (IUA) has been well recognized as a critical factor in infertility, little information is available regarding the molecular mechanisms. We performed a high-throughput RNA sequencing in the endometrium of three IUA patients and three normal controls. And another two gene expression profiles (PMID34968168 and GSE160365) were analyzed together. A total of 252 DEGs were identified. Cell cycle, E2F target, G2M checkpoint, integrin3 pathway and H1F1 signaling were aberrantly regulated in the IUA endometrium. 10 hub genes (CCL2, TFRC, THY1, IGF1, CTGF, SELL, SERPINE1, HBB, HBA1, LYZ) were exhibited in PPI analysis. FOXM1, IKBKB and MYC were three common transcription factors of DEGs. Five chemicals (MK-1775, PAC-1, TW-37, BIX-01294, 3-matida) were identified as putative therapeutic agents for IUA. Collectively, a series of DEGs associated with IUA were disclosed. Five chemicals and ten hub genes may be further explored as potential drugs and targets for IUA treatment.

Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Single cell transcriptome profiling reveals cutaneous immune microenvironment remodeling by photodynamic therapy in photoaged skin.

The immune microenvironment plays a critical role in maintaining skin homeostasis, which is closely related to the dysfunction in photoaged skin such as autoimmunity and tumorigenesis. Several recent studies have demonstrated the efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in alleviating photoaging and skin cancer. However, the underlying immune mechanisms and the immune microenvironment change by ALA-PDT remain largely unknown. To illustrate the effects of ALA-PDT on immune microenvironment in photoaged skin, single cell RNA sequencing (scRNA-seq) analysis of photoaged skin on the extensor side of the human forearm before and after ALA-PDT was performed. R-packages of Seurat, clusterProfiler, Monocle, CellChat were used for cell clustering, differentially expressed genes analysis, functional annotation, pseudotime analysis and cell-cell communication analysis. The gene sets related to specific functions were extracted from the MSigDB database, which were used to score the functions of immune cells in different states. We also compared our result with published scRNA-seq data of photoaged skin of the eyelids. The increase score of cellular senescence, hypoxia and reactive oxygen species pathway in immune cells and the decrease of immune receptor activity function and proportion of naive T cells were found in skin photoaging. Moreover, the function of T cell ribosomal synthesis was also impaired or down regulated and function of G2M checkpoint was up regulated. However, ALA-PDT showed promising results in reversing these effects, as it improved the above functions of T cells. The ratio of M1/M2 and percentage of Langerhans cells also decreased with photoaging and increased after ALA-PDT. Additionally, ALA-PDT restored the antigen presentation and migration function of dendritic cells and enhanced cell-cell communication among immune cells. These effects were observed to last for 6 months. ALA-PDT has potential to rejuvenate immune cells, partially reversed immunosenescence and improved the immunosuppressive state, ultimately remodelling the immune microenvironment in photoaged skin. These results provide an important immunological basis for further exploring strategies to reverse skin photoaging, chronological aging and potentially systemic aging.

Genomic landscape of multiple myeloma with extramedullary disease: Results from a large patient database.

8058 Background: Extramedullary disease (EMD) is associated with poor outcomes and the biological mechanisms driving this phenotype are poorly understood. We used the CoMMpass registry data to explore the genomic characteristics of EMD. Methods: The CoMMpass study is a prospective, international registry of newly diagnosed MM. 1143 patients had clinical data. Patients were classified as EMD if they had EMD at any point. Genomic data from bone marrow (BM) samples at diagnosis (whole genome sequencing, whole exome sequencing (WES), and RNA sequencing (RNAseq)) were compared between EMD and non-EMD. A multivariable model was constructed based on univariable results. Differential expression (DEA) and gene set enrichment analysis (GSEA) were performed after normalization using Hallmark and MM transcriptomic subgroups. The top 24 most mutated genes in MM were selected. Results: The median overall survival (mOS) and progression free survival (PFS) was shorter for EMD. The presence of EMD, anemia at diagnosis, age &gt;65 years at diagnosis, ISS stage, and use of ASCT at the first line were independently associated with worse mOS and PFS. EMD was associated with worse mOS (HR 1.7, 95% CI 1.3 - 2.2 p &lt;0.01) and PFS (HR 1.5, 95% CI 1.23 - 1.8, p &lt;0.01) in multivariable analysis and was not abrogated by ASCT, triplet induction or maintenance. Results did not vary when landmark analysis was used. MYC amplification, deletion 18q and 1q amplification were enriched in EMD cases. TP53 mutations and TP53 bi-allelic inactivation (mutation and deletion) were enriched in EMD (Table). DEA identified 1552 genes with increased and 3344 with decreased expression (2.7%, 5.4% of total genes respectively) in EMD when compared to non-EMD. On GSEA, EMD cases were enriched for the CD-1, PR, and CTA myeloma gene expression signatures, in addition to the Hallmark pathways MYC target, E2F targets, G2M checkpoint and unfolded protein response, while the adhesion molecules pathway and MM gene signatures LB, MS, and MF were downregulated in EMD. Conclusions: EMD cases were enriched for TP53 mutations and bi-allelic TP53 inactivation, both of which are prognostic. In addition, MYC amplification, 18q deletion and 1q gain were enriched in EMD which are novel CNA associated with EMD. EMD is also enriched for distinct molecular subgroup, including enrichment for CD-1, PR, and CTA signatures, the latter two are associated with poor outcomes, and the Hallmark pathways MYC, E2F and G2M checkpoints consistent with MYC amplification and PR molecular subgroup. WES and RNAseq of matching BM and EMD tissue samples from our institution is currently being performed. [Table: see text]