Compared Gene Expression Research Articles

Single-Cell Analysis of Human Clonal Hematopoiesis Identifies Distinct Impact of DNMT3A and TET2 mutations on Hematopoietic Differentiation

Clonal hematopoiesis (CH) increases the risk of hematological malignancies and other adverse outcomes. The most common driver mutations occur in DNMT3A and TET2. However, it is still unclear how mutant DNMT3A and TET2 perturb human stem/progenitor cell homeostasis allowing mutant clones to expand. Studies of human CH have been limited by availability of samples from subjects without co-existing malignancy and high-precision methods for comparing mutant and wild-type (WT) cells in the same individual. To address this gap, we screened 195 bone marrow (BM) samples collected from subjects undergoing hip replacement surgery for CH by targeted DNA sequencing. As DNMT3A and TET2 regulate DNA methylation and gene expression, we employed multiomic single-cell analysis with TARGET-seq to combine single-cell genotyping with transcriptome sequencing on FACS index sorted cells. We developed an improved method, TARGET-seq+, which provides greater sensitivity transcript detection, resulting in a median of 4500 genes/cell with high-sensitivity genotyping (95% of cells genotyped; 7-11% allele dropout rate). We performed TARGET-seq+ on 12,942 BM stem, progenitor and mature cells from 9 CH samples with predicted loss-of-function mutations (4 single mutation cases & 5 multiple mutation cases) and 3 normal controls (Fig. 1). Of these, 87% of cells passed quality controls and were successfully genotyped. Integrated transcriptomic and genotyping analysis showed DNMT3Amut and TET2mut cells occupy a common differentiation trajectory with WT cells. Within DNMT3A-mutant samples, the mutant clone constituted 5-70% of transcriptomic and immunophenotypic long-term hematopoietic stem cells (LT-HSC). There was a further 1.5 to 1.7-fold expansion of DNMT3Amut cells in early erythro-megakaryocytic multipotent progenitors and lympho-myeloid multipotent progenitors (LMPP) compared to LT-HSC. Across most samples, clone size was maintained throughout differentiation with no evidence of differentiation delay or lineage bias. Differential expression analysis revealed subtle cell type-specific differences between WT and mutant cells, with upregulation of megakaryocytic lineage markers in mutant LMPP and downregulation of erythroid lineage markers in mutant erythroid progenitors. TET2mut clones were also identified in LT-HSCs at a clone size of 1-21% but expanded further in multiple downstream progenitors. Maximal expansion (2-12 fold) was seen in the myeloid lineage, but clones also expanded in the erythroid, megakaryocytic and lymphoid lineages (mean clone size 2 to 5-fold larger than in LT-HSC). Comparison of mutant and WT granulocyte-monocyte progenitors (GMP) identified upregulation of myeloid lineage-affiliated transcription factors, (e.g. CEBPD and IRF8) in TET2mut cells. Increased expression of myeloid gene signatures was also seen within TET2mut HSC and LMPP, suggesting myeloid lineage bias arises early in differentiation. Concordantly, TET2mut HSC were shifted towards a more differentiated state in pseudotime and downregulated LT-HSC signatures. In one sample with biallelic TET2 mutations, the TET2 single and double mutant clones were each detected in only 1 out of 109 (0.9%) CD90+CD49f+ LT-HSCs. However, the double mutant clone expanded dramatically in the myeloid lineage, contributing to >50% of cells within GMP, dendritic cell progenitor and neutrophil/monocyte precursor compartments, consistent with frequent detection of biallelic TET2 mutations in myeloid leukemias. Comparison of gene expression between differentiation stage-matched WT cells in CH samples and in age-matched normal controls allowed us to identify the impact of CH clones on WT cells, co-existing within the same BM micro-environment. WT HSCs in DNMT3A and TET2 mutant samples showed enhanced expression of inflammatory, quiescence and chemokine gene signatures compared to WT HSCs in non-CH samples. In summary, we present the first detailed analysis of the impact of DNMT3A and TET2 mutations on hematopoiesis in humans with unperturbed CH. The selective advantage of DNMT3A-mutant clones occurs primarily at the level of HSC and early progenitors, whereas TET2-mutant clones expand not only in HSC but also further throughout differentiation (Fig. 2). There is a non-cell autonomous impact on WT HSCs in CH. Our work provides a foundation for understanding the mechanistic basis of pre-leukemic clonal expansion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ZNF683 (Hobit) Marks a CD8+ T Cell Population Associated with Anti-Tumor Immunity Following Anti-PD-1 Therapy for Richter Syndrome

Richter syndrome (RS), an aggressive B cell lymphoma originating from transformation of indolent chronic lymphocytic leukemia (CLL), has been shown to be responsive to PD-1 checkpoint blockade (CPB) across clinical trials. To systematically discover the determinants of this response, we analyzed single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 RS patients enrolled in a phase I study of the anti-PD1 drug nivolumab with concurrent ibrutinib (NCT 02420912). At pre-treatment sampling, RS responders (RS-R) had a larger CD8 effector/effector memory (E/EM) population than RS non-responders (RS-NR) (p=0.04, 2-sample t-test). This population was rare in normal bone marrow (N=30, p=0.001, 2-sample t-test), displayed intermediate exhaustion, preserved cytotoxicity and was marked by expression of the transcription factor and PRDM1-homolog, ZNF683. Further implicating ZNF683-expressing CD8 T cells in CPB response, comparison of gene expression between RS-Rs and RS-NRs within transcriptionally defined CD8 T cell clusters identified higher ZNF683 expression in RS-Rs. Lastly, the kinetics of ZNF683 expression correlated with response, with relative expression per cell decreasing with RS progression. Trajectory analysis supported a T cell differentiation path from GZMK+ memory to ZNF683intermediate followed by branching towards either ZNF683high or terminal exhaustion. To establish the extent to which ZNF683high T cell signatures are detectable in a broader population of RS patients, we examined bulk RNA-seq data generated from 35 independent RS biopsy specimens and found that 7 of 35 (20%) displayed a ZNF683high signature. A similar distribution of samples with ZNF683high expression, when corrected for T cell fraction, was observed in 18 of 81 (20%) CLL samples for which non-CD19 selected transcriptomic data was available and was associated with a trend towards improved overall survival (p=0.11). We also detected a similar ZNF683high signature in a large external compendium of single cell data from tumor infiltrating lymphocytes across 18 solid and 3 hematologic tumor types and in the peripheral blood of melanoma CPB responders (p<0.001 hypergeometric test). Upon evaluation of TCGA data, we detected an association with survival in melanoma cases with high T cell expression of ZNF683 (P<0.0001). To probe the cellular impact of ZNF683, a FLAG-tagged ZNF683 construct was overexpressed in Jurkat cells. ZNF683 overexpression revealed upregulation of PRF1, ITGA1, CD244, CD226 and IL2RB and downregulation of PRDM1 and IL2. CUT&RUN was performed to identify the binding sites of ZNF683, yielding twenty-four differentially regulated peaks, among which included ENCODE-identified regulatory regions adjacent to key immune genes (TNFAIP8, BTN3A2, CD69/KLRF1, ARHGAP2, CAMK4, LMO2, IL2, TCF7). Evaluation of external ATAC-seq data generated from exhausted T cell subsets revealed PD-1high tumor-infiltrating lymphocytes to have reduced accessible chromatin across identified putative ZNF683 binding sites, including TCF7. Thus, terminal exhausted cells not only lose ZNF683 expression but also display chromatin remodeling that likely abrogates its effects. By applying the tool CISTROME-GO to integrate the CUT&RUN and RNA-seq data, we observed enrichment in pathways corresponding to antigen binding and presentation, transcription factor activity, and T cell mediated cytotoxicity and cell killing upon ZNF683 expression. To determine if the association between ZNF683 expression and response to PD-1 blockade in RS could be confirmed in peripheral blood, we analyzed RNA-seq data generated from T cells isolated from pre-treatment blood samples from 7 independent RS patients treated with PD-1 therapy. We again identified ZNF683 among the top upregulated genes in RS-R (Log2 fold change = 2.13, p=0.037), along with other genes enriched in our ZNF683high CD8 E/EM signature (BATF, CORO1A, CD38, ITGB2, GZMM), while RS-NR were enriched in NK-like genes (KLRC1, FCGR3A, KLRG1, KLRF1 and FCER1G). In conclusion, we identify ZNF683 as marking a CD8 T cell population associated with CPB response in RS blood and bone marrow and provide evidence that this transcription factor regulates key cellular pathways involved in immune anti-tumor response, with implications for understanding CPB response in RS and other malignancies.

Modeling Progression of CML Using Mutagenized Patient-Derived Induced Pluripotent Stem Cells (iPSCs): Potential Tool for Discovering Novel Therapeutic Targets

Despite significant progress obtained in CML therapy by using tyrosine kinase inhibitors (TKIs), progression towards accelerated phase and blast crisis (AP/BC) occurs, especially in patients developing resistance towards several lines of TKIs. AP/BC is characterized by an increased genetic instability, which is one of the major characteristics of CML and facilitated by the expression of the BCR-ABL fusion protein in leukemic cells. Although the major genomic events leading to BC have been described over several years of research (such as TP53 alterations, abnormalities of DNA repair genes) there is no model of reproducing these events in the context of stem cells in vitro. To this purpose we have designed a CML patient-derived, iPSC-based in vitro mutagenesis strategy to uncover novel genomic events associated with progression of CML. To mimic AP/BC in CML patients and to generate a "blast crisis in a dish" model, we have designed a strategy based on patient-derived iPSCs. For this purpose, we have generated iPSC from leukemic cells of three CML patients. At the time of cell reprogramming, two patients (UPN PB27 and UPN PB32) were in CP and the other (UPN PB34) in AP. The latter patient also had JAK2V617F mutation in the same Ph1+ stem cell clone. iPSC were treated using the mutagenic agent N- ethyl-N-nitrosourea (ENU) by daily addition of the drug in cultures. They were characterized for their pluripotency (expression of pluripotency genes, teratoma assays in NSG mice) as well as for their hematopoietic potential using StemDiff technology, before and after mutagenesis. Using this technology, iPSCs gave rise to hematopoietic cells from day +5 up to day 13, with a major expansion of hematopoietic cells and progenitors. As expected, cytological patterns differed between 3 pts. Amongst the three pts, UPN-PB32-ENU as compared to non-mutagenized cells, generated myeloid blasts exhibiting monoblastic patterns (AML5) and expression of MPO, CD34, CD33, CD11b, and CD43. To analyze the molecular events occurring during CML iPSC-based mutagenesis, we have performed single cell RNAseq analyses in hematopoiesis-induction cultures, in mutagenized versus non-mutagenized iPSC. These experiments have been performed at D+5, D+9 and D+13 of differentiation using 104 cells in each condition. They revealed 13 clusters, with evidence of delay of hematopoietic differentiation in ENU-treated iPSC and accumulation of Msx1-expressing cells at the mesoderm stage. Hematopoietic cells generated from the three mutagenized iPSCs (PB27, 32, and 34) and collected at day +13 were then compared to their non-mutagenized counterparts using transcriptomic analyses. As can be seen in Figure 1, several up-and down-regulated genes were identified in all three ENU-mutagenized hematopoietic cells. In UPN PB32-ENU (harboring the most potent BC phenotype), 1439 up- and 1641-down-regulated genes were identified. These results were analyzed along with the GSE4170 GEO dataset, in which Radich et al (PNAS 2006) have compared gene expression between CP, AP and BC by analyzing 91 cases of CML using normal immature CD34+ cells as a reference. A significant overlap was found between primary BC cells (GSE4170) and the signature induced in UPN PB32-ENU compared to its non-mutagenized counterparts. Among the overexpressed genes, the IL2RA (CD25) was found to be of interest as it has been shown to be overexpressed in AML. We have therefore analyzed its relevance using a cohort of CML patients in CP (n=22) AP/BP (n=14) and compared the CD25 mRNA expression to that observed in a cohort of AML (n=15), ALL (n=17), and normal controls (n=15). The mean percentage of CD25/ABL1 was highly increased in AP/BP CML as compared to other groups and, in particular, to CP-CML (Mean % 75 vs. 19.3). In addition, CD25 mRNA expression was evaluated in seven CML patients who have progressed to BC, in both CP and BP samples. CD25 up-regulation was clearly observed in all patients during AP/BC, with a 5- to 10-fold increase in BP of 4 of these 7 patients as compared to CP. Overall, our results provide a novel iPSC-based experimental tool to study CML progression, allowing to reveal genetic abnormalities that correlate with those observed in primary BC. They also suggest that iPSC technology could pave the way for discovering new therapeutic targets, not only for CML progression, but also for several other hematological malignancies progressing towards aggressive phases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LC3-Associated Phagocytosis Is Impaired in Bone Marrow Macrophages during Ageing Leading to Defective Processing of Apoptotic Cells and Suppression of Sting Activation

Introduction Macrophage phagocytic function declines with ageing, with potential deleterious consequences on inflammation, immune response, maintenance of tissue health, and cancer. Paradoxically, we and others have found that with increasing age, macrophage number increases within the bone marrow (BM) microenvironment, supporting the hypothesis that the ageing macrophages are functionally impaired. Phagocytosis by mononuclear cells occurs through either an LC3-dependent or -independent mechanism. LC3-associated phagocytosis (LAP) is physiologically triggered by pathogens and cellular debris via interaction with phagocyte surface receptors, including T cell membrane protein 4 (TIM-4) and FcR. Here, we investigate whether age-related macrophage functional impairment is associated with LC3. Results BM was isolated from young (8-12 weeks) and aged (18-24 months) female C57Bl/6 mice and macrophages were analysed using flow cytometry and isolated via FACS. We show increased BM macrophage numbers in old BM (2 fold), but no difference in total phagocytic potential as measured by uptake of E.coli bioparticles or apoptotic debris, when compared to young. Furthermore, using immunofluorescent microscopy to visualise macrophage phagocytosis, we observed that the phagosomes generated in BM macrophages from aged animals did not recruit LC3, whereas in young animals LC3 was recruited. Consequently, macrophages from older animals accumulated large amounts of E.coli bioparticles and apoptotic bodies when compared to young macrophages. This suggests defective processing of the E.coli bioparticles by the phagosome once the phagosome has formed. To understand if this is due to lack of LC3 protein, we next compared gene expression and protein levels of LC3 in young and old animals. There was no loss in LC3 RNA or protein expression in macrophages from old and young. As we have recently reported that BM macrophage LAP mediates the stimulator of interferon genes (STING) pathway, we examined STING activation in old and young BM macrophages by measuring Gbp2, Irf7, and Ifit3 gene expression. Results showed a significant reduction in STING mediated gene expression in BM macrophages from aged mice compared to young. Summary These data suggest the decline in macrophage phagocytic function associated with age occurs because of a failure of LC3-mediated phagosome clearance. Furthermore, we hypothesise that the observed increase in BM macrophage numbers in older animals is, at least in part, a compensatory mechanism for the defective individual macrophage phagocytic processing seen, helping preserve overall BM phagocytic function.

Mesenchymal Stromal Cells Protect Acute Myeloid Leukemia Cells from Venetoclax-Induced Apoptosis through Contact-Dependent Mechanisms

Acute myeloid leukemia (AML) patients have a poor prognosis with standard treatments due to a high relapse rate. Recently, Venetoclax (VEN), the first B-cell lymphoma 2 (BCL-2) selective inhibitor to enter the clinic, has shown a breaking-through effect in combination with demethylating agents in older patients with treatment-naïve AML. However, resistance mechanisms were described, hampering the complete remission of VEN-treated patients. It has been proposed that the bone marrow microenvironment in general and Mesenchymal stromal cells (MSCs), in particular, favor a protective niche that sustains leukemic cell viability and plays a crucial role indetermining drug sensitivity in several hematological disorders, including AML. A previous study demonstrated that VEN, as well as other drugs commonly used in AML therapy, showed a reduced effect in the presence of an MSC-conditioned medium, suggesting that MSC-dependent release of inflammatory signals and other soluble factors could be involved in drug resistance. Our study aims to further investigate the mechanisms of MSC-mediated VEN resistance in AML. We found that primary AML cells isolated from patients at diagnosis and treated with VEN showed different apoptotic rates, indicating an AML cell-specific resistance/sensitivity to the drug. In co-culture experiments, primary MSCs, isolated from healthy donors or AML patients, protected VEN-sensitive AML cells from apoptosis, confirming the critical role of MSCs in drug resistance. To deeply investigate this issue, we performed co-culture experiments using the AML cell line MV4-11, which we demonstrated as sensitive to VEN in a dose-dependent manner. We found that MSCs significantly reduced MV4-11 apoptosis after VEN exposure. The addiction of transwells to the co-culture system abrogated the MSC-protective effect on VEN-induced apoptosis, suggesting the establishment in our setting of a complex interplay between AML cells and MSCs, not only mediated by soluble factors as already demonstrated but also involving direct cell-contact. Furthermore, when we cultured MV4-11 cells in direct contact with MSCs, we were able to distinguish, among leukemic cells, a cell population that remained floating in the supernatant and a cell population that kept adherent to MSCs and could be collected by trypsin detachment only. Interestingly, MSCs protected the adherent but not the floating cells from VEN-induced apoptosis, suggesting a central role of a close adhesion in the MSC-driven resistance to VEN. To gain insight into this mechanism, we sorted adherent vs. floating cells and analyzed and compared Gene Expression Profiling (GEP). Interestingly, the principal component analysis suggested that the floating cell GEP was more similar to the GEP of MV4-11 cells cultured without MSCs compared to adherent cell GEP. We found 159 differential expressed genes (DEGs) in adherent vs. floating MV4-11 cells (FC| ≥ 2 and P ≤ 0.05, according to Transcriptome Analysis Console software guidelines). Enrichment analysis revealed that DEGs were involved in cell adhesion migration and proliferation pathways. Among DEGs, we found that CD90/Thy-1, an adhesion molecule whose expression correlated with unfavorable karyotypes and shorter survival in AML, was significantly up-regulated in adherent MV4-11 cells. Flow cytometry analysis confirmed that the adherent compared to the floating MV4-11 cells, showed a higher expression of CD90 as well as CXCR4 as expected. Our study demonstrated that MSCs protect AML cells from VEN-induced apoptosis through a complex mechanism involving tight adhesion-mediated pathways. Further clarification of these interactions can shed light on putative targets to counteract MSC-mediated drug resistance to VEN treatment.

Single-Cell Profiling of Acute Myeloid Leukemia Identified ARMH1, a Novel Protein Associated with Proliferation, Migration, and Drug Resistance

Despite advancements in treatments, half of pediatric acute myeloid leukemia (AML) patients relapse (Chen, J. & Glasser, C.L., 2020), making it pertinent to identify novel biomarkers that can be used for better stratification of patients post end of induction (EOI) therapy as well as to develop targeted therapies that could lead to higher clinical remission (CR) rates. Single-cell RNA sequencing (scRNAseq) analysis has revolutionized cancer research by identifying novel cell types, genes, and pathways. Our group used scRNAseq analysis of pediatric AML bone marrow (BM) samples collected at diagnosis (Dx) and EOI to identify the upregulation of multiple genes including a novel gene, Armadillo like helical domain containing 1 (ARMH1)/C1orf228, in cancerous blast cells (Thomas, B.E. et al., 2020). ARMH1 has not been previously associated with AML. Also, ARMH1 expression was observed to be significantly higher at Dx in samples from patients with relapse compared to samples from patients with CR (P<2.2e-16). Independent validation for ARMH1 blast-association using single cell and bulk transcriptome profiling revealed that ARMH1 expression was significantly higher in scRNAseq data of AML Dx blast cells compared to EOI non-blast cells (fold change, FC=1.8, P<2.2e-31) as well as in bulk RNAseq data of Dx samples with high blast% (>90%) compared to EOI samples (FC=1.8, P=.003) (Ulukaya, G. et al., 2021). Further analysis on other leukemias like T cell-acute lymphocytic leukemia (T-ALL) and mixed phenotypic acute leukemia (MPAL) also revealed higher expression of ARMH1 in the blast cells as compared to non-blast cells. Survival analysis on bulk RNAseq data using the survival genie platform (Dwivedi, B. et al., 2022) revealed that higher expression of ARMH1 was significantly associated with poor overall survival (OS) in both AML (P=.003) (Ulukaya, G. et al., 2021) and T-ALL (P=.014) datasets. Comparative gene expression analysis using the the Pediatric Single Cell Cancer Atlas - PedSCAtlas - tool (Mumme, H.L. et al., 2021), revealed negligible expression of ARMH1 in normal lymphoid, myeloid, and erythroid cell lineages, further confirming the leukemic blasts-specific over-expression of ARMH1. ARMH1 encodes a cytosolic protein of yet unknown function. The malignant blast specificity of ARMH1 suggested a role in leukemic cells' survival/maintenance. To ascertain its functional involvement in cancer cells, ARMH1 was silenced using siRNAs in both normal and AML cell lines. Through in vitro assays, we demonstrate that ARMH1 silencing significantly reduced leukemic cells' capacity to proliferate (P=.0041) and migrate (P=.0001). We also observed slightly increased sensitivity to chemotherapy drug, Cytarabine in the ARMH1 silenced cancer cells compared to control cancer cells in which the gene is fully functional. However, suppression of ARMH1 expression had no effect on cell growth in the normal HEK293t cell line (P=0.913). On the other hand, overexpression of ARMH1 via transient transfection of ARMH1 gene into the AML cell line, made the cells more resistant to Cytarabine and significantly increased both migration (P=.041) and cell proliferation (P=.032). In conclusion, we demonstrate for the first time that ARMH1, a still poorly understood protein, contributes to the increased proliferation and migration of malignant AML cells. With specific overexpression in blast cells of several pediatric leukemias and the newly discovered roles in proliferation, migration, and chemotherapy resistance, ARMH1 may be a good candidate for targeted treatment of pediatric leukemias.

Targeting PLZF Enhances Glucocorticoid Sensitivity in Acute Myeloid Leukemia

Glucocorticoids (GCs) are Glucocorticoid Receptor (GR) agonists that are extensively used in clinic, from management of autoimmune and inflammatory disorders to being the corner stone in treatment of adult and childhood lymphoid cancers. Unlike lymphoblastic leukemias, acute myeloid leukemia (AML) cells are generally resistant to glucocorticoids and the mechanisms underlying this resistance are yet to be elucidated. Recent efforts by our group and others suggest that certain genetic alterations, such as RUNX1 loss of function mutations, or therapy-induced stress by chemo-agents such as cytarabine or small molecules like FLT3-inhibitors could sensitize AML cells to GCs, most probably via up-regulation of glucocorticoid receptor (GR) expression. To better characterize GC-response in AML, we compared gene expression landscape of GC-resistant versus GC-sensitive AML cell lines exposed to dexamethasone (Dex) for different time points. Interestingly, we found that the number of down-regulated genes upon GC treatment is similar in both cell lines. However, GC-sensitive cell line showed 3 to 6 times more up-regulated genes following GC treatment compared to the resistant cell line. A significant portion of up-regulated genes in the sensitive cells were targets of GR transcription factor. Gene set enrichment analysis revealed up-regulation of genes associated with macrophage differentiation, inflammation, and apoptosis pathways only in the sensitive cells. To identify modulators of the GC response in AML, we conducted a genome-wide CRISPR/Cas9-based genetic screen on resistant cell lines in the presence or absence of Dex. We identified the promyelocytic leukemia zinc factor (PLZF) as a critical transcription factor that determines GC-response in AML cells. In line with this, we showed that PLZF knockdown conferred sensitivity to GC-resistant AML cells, whereas PLZF over-expression conferred resistance to the sensitive cells. Transcriptome analysis showed an overlap of up-regulated pathways between PLZF-depleted cells and GC-sensitive cells in presence of Dex including inflammation and apoptosis pathways, suggesting that PLZF depletion primes AML cells to GC-response. Furthermore, Dex treatment induced expression of a subset of GR target genes in PLZF depleted cells. Importantly, this effect was not associated with significant changes in GR global protein levels or sub-cellular distribution. PLZF has been recently shown to be a neosubstrate for Cereblon-mediated protein degradation. To define potential therapeutic strategies that target PLZF in AML, we selected two cereblon modulating molecules including Lenalidomide, which is commonly used in the clinic for treatment of hematological malignancies, and CC-3060 compound that is more potent in PLZF degradation. Combination of Dex with PLZF-degrading compounds increased cell death in a significant portion of 95 primary AML samples tested, and as expected, a broader synergistic effect was obtained with CC-3060 compound. Combination of Dex and Lenalidomide appeared more effective in samples harboring NPM1 mutation, independently of recurrent additional mutations of FLT3 or DNMT3A. Importantly, these combinations had no cytotoxicity effect on normal CD34+ cordblood cells in vitro. Current efforts are ongoing to assess efficacy of this newly identified combinatorial therapy in vivo. All together our study shows that GR trans-activating capacity is diminished in resistant AML cells leading to a failure in activating pro-differentiation and anti-leukemic genes upon Dex treatment. PLZF interferes with GC-induced cell death in AML cells at least in part via suppressing 100 of genes involved in GC-response. PLZF suppression induces inflammatory response and sensitizes AML cells to anti-inflammatory effects of glucocorticoids. Based on our results, potent PLZF degrading compounds hold promises to potentiate GC-response in AML.

Transcriptome analysis of AAV-induced retinopathy models expressing human VEGF, TNF-α, and IL-6 in murine eyes

Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-α, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-α and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-α-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-α expression increased the expression T-cell markers. Both TNF-α and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-α, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology.

Subsequent AS01-adjuvanted vaccinations induce similar transcriptional responses in populations with different disease statuses.

Transcriptional responses to adjuvanted vaccines can vary substantially among populations. Interindividual diversity in levels of pathogen exposure, and thus of cell-mediated immunological memory at baseline, may be an important determinant of population differences in vaccine responses. Adjuvant System AS01 is used in licensed or candidate vaccines for several diseases and populations, yet the impact of pre-existing immunity on its adjuvanticity remains to be elucidated. In this exploratory post-hoc analysis of clinical trial samples (clinicalTrials.gov: NCT01424501), we compared gene expression patterns elicited by two immunizations with the candidate tuberculosis (TB) vaccine M72/AS01, between three groups of individuals with different levels of memory responses to TB antigens before vaccination. Analyzed were one group of TB-disease-treated individuals, and two groups of TB-disease-naïve individuals who were (based on purified protein derivative [PPD] skin-test results) stratified into PPD-positive and PPD-negative groups. Although TB-disease-treated individuals displayed slightly stronger transcriptional responses after each vaccine dose, functional gene signatures were overall not distinctly different between groups. Considering the similarities with the signatures found previously for other AS01-adjuvanted vaccines, many features of the response appeared to be adjuvant-driven. Across groups, cell proliferation-related signals at 7 days post-dose 1 were associated with increased anti-M72 antibody response magnitudes. These early signals were stronger in the TB-disease-treated group as compared to both TB-disease-naïve groups. Interindividual homogeneity in gene expression levels was also higher for TB-disease-treated individuals post-dose 1, but increased in all groups post-dose 2 to attain similar levels between the three groups. Altogether, strong cell-mediated memory responses at baseline accelerated and amplified transcriptional responses to a single dose of this AS01-adjuvanted vaccine, resulting in more homogenous gene expression levels among the highly-primed individuals as compared to the disease-naïve individuals. However, after a second vaccination, response heterogeneity decreased and was similar across groups, irrespective of the degree of immune memory acquired at baseline. This information can support the design and analysis of future clinical trials evaluating AS01-adjuvanted vaccines.

SmDXS5, acting as a molecular valve, plays a key regulatory role in the primary and secondary metabolism of tanshinones in Salvia miltiorrhiza.

Red sage, the dry root and rhizome of the herbaceous plant Salvia miltiorrhiza Bunge, is widely used for treating various diseases. The low content of tanshinones (terpenoids) has always restricted development of the S. miltiorrhiza industry. Here, we found that SmDXS5, a rate-limiting enzyme-coding gene located at the intersection of primary and secondary metabolism, can effectively change the transcription level and secondary metabolome profile of hairy roots of S. miltiorrhiza, and significantly increase the content of tanshinones. Agrobacterium rhizogenes was used to infuse S. miltiorrhiza explants, and hairy roots of S. miltiorrhiza expressing the SmDXS5 gene were obtained successfully. We identified 39 differentially accumulated metabolites (DAMs) by metabolomics based on ultra-high performance liquid chromatography quadrupole exactive mass spectrometry and multivariate statistics. These DAMs might be key metabolites of SmDXS5 gene regulation. RNA sequencing was used to compare gene expression between the hairy roots of the SmDXS5 overexpressing group and the blank control (BC) group. Compared with the BC group, 18,646 differentially expressed genes were obtained: 8994 were upregulated and 9,652 downregulated. The combined transcriptome and metabolome analyses revealed that the mevalonate and methylerythritol phosphate pathways and synthase gene expression levels in the SmDXS5 overexpressing group were upregulated significantly, and the accumulation of tanshinone components was increased significantly, which promoted the process of glycolysis and promoted the transformation of carbohydrates to secondary metabolism. Moreover, the expression of SmPAL, the first rate-limiting enzyme gene of the phenylpropane pathway, decreased, reducing the accumulation of phenolic acid, another secondary metabolite. Therefore, SmDXS5 can be defined as a 'valve' gene, mainly responsible for regulating the distribution of primary and secondary metabolic flow of tanshinones in S. miltiorrhiza, and for other secondary metabolic pathways. The discovery of SmDXS5 and its molecular valve function in regulating primary and secondary metabolism will provide a basis for the industrial production of tanshinone components, and cultivation of high quality S. miltiorrhiza.

Construction of lncRNA TYMSOS/hsa-miR-101-3p/CEP55 and TYMSOS/hsa-miR-195-5p/CHEK1 Axis in Non-small Cell Lung Cancer.

As novel master regulators of initiation and progression, long non-coding RNAs (lncRNAs) can response to therapy in a wide variety of malignances. This study aimed to explore the regulatory mechanisms in non-small cell lung cancer (NSCLC) by constructing lncRNA-miRNA-mRNA networks. The whole transcriptome sequence was performed in five NSCLC tissues and their corresponding paired adjacent normal tissues. MicroRNAs (miRNAs) and mRNAs expression profiles were obtained from TCGA database. "EdgeR" R package was used for gene expression comparisons and the genes with | log2FC |> 2 and false discovery rate (FDR) adjusted P<0.05 were considered significant. Totally, 559 differentially expressed lncRNAs (DELs) (235 up-regulated, 324 down-regulated) were identified via whole transcriptome sequence analysis. Subsequently, 14 up-regulated lncRNAs were obtained in the intersection of our RNA-seq data and TCGA dataset. The dysregulations of the selected lncRNAs were evaluated through GEPIA. Three candidate lncRNAs (LINC01426, TYMSOS, VPS9D1-AS1) were finally selected for further study. Through bioinformatics prediction, with the three lnRNAs and their associated miRNAs (n=14) and mRNAs (n=218), a lncRNA-miRNA-mRNA network was constructed. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway associated with the targeted genes were proceeded. In addition, the protein-protein interaction (PPI) network construction was performed by STRING and lncRNA-miRNA-mRNA regulatory network was visualized via Cytoscape. The top ten significant hub-genes in the PPI network are identified based on their node degrees. Moreover, the lncRNA-miRNA and miRNA-mRNA interactions were predicted using the StarBase and survival analyses were performed in Kaplan-Meier plotter website. We concluded that TYMSOS/hsa-miR-101-3p/CEP55 and TYMSOS/hsa-miR-195-5p/CHEK1 ceRNA regulatory aixs may play vital roles in the occurrence and development of NSCLC, and may provide novel therapeutic targets in the future.

Axin1 Protects Colon Carcinogenesis by an Immune-Mediated Effect

Axin1 is a negative regulator of wingless-type MMTV integration site family, member 1 (Wnt)/β-catenin signaling with tumor-suppressor function. The Wnt pathway has a critical role in the intestine, both during homeostasis and cancer, but the role of Axin1 remains elusive. We assessed the role of Axin1 in normal intestinal homeostasis, with control, epithelial-specific, Axin1-knockout mice (Axin1ΔIEC) and Axin2-knockout mice. We evaluated the tumor-suppressor function of Axin1 during chemically induced colorectal tumorigenesis and dextran sulfate sodium-induced colitis, and performed comparative gene expression profiling by whole-genome RNA sequencing. The clinical relevance of the Axin1-dependent gene expression signature then was tested in a database of 2239 clinical colorectal cancer (CRC) samples. We found that Axin1 was dispensable for normal intestinal homeostasis and redundant with Axin2 for Wnt pathway down-regulation. Axin1 deficiency in intestinal epithelial cells rendered mice more susceptible to chemically induced colon carcinogenesis, but reduced dextran sulfate sodium-induced colitis by attenuating the induction of a proinflammatory program. RNA-seq analyses identified an interferon γ/T-helper1 immune program controlled by Axin1 that enhances the inflammatory response and protects against CRC. The Axin1-dependent gene expression signature was applied to human CRC samples and identified a group of patients with potential vulnerability to immune checkpoint blockade therapies. Our study establishes, invivo, that Axin1 has redundant function with Axin2 for Wnt down-regulation and infers a new role for Axin1. Physiologically, Axin1 stimulates gut inflammation via an interferon γ/Th1 program that prevents tumor growth. Linked to its T-cell-mediated effect, the colonic Axin1 signature offers therapeutic perspectives for CRC.

Abstract 9555: Human Valve Atlas in Health and Disease

Background: Semilunar and atrioventricular valves have distinct structures and are often affected differently in congenital valve diseases (CVD). Here we aim to define and compare the cellular composition and transcriptomic signatures of four different human fetal valves, which provide a reference map to engineer valves in vitro , and to study valve-specific impairment in CVD. Methods: Aortic, pulmonary, mitral, and tricuspid valves were barcoded respectively from a healthy human fetal heart at gestational day 105. Single-cell (sc)RNA-seq was performed. Spatial localization of new subpopulations was determined by immunostaining and in situ hybridization. Cell-cell interaction was revealed by cell-chat. Results: scRNA-seq analysis revealed major cell types including endothelial (VECs), interstitial (VICs), and immune cells. In addition to reported collagen &amp; glycosaminoglycan (GAG) VIC in mice, VIC sub-clustering identified two human-specific APOE+ and CLDN11+ subpopulations. APOE+ VICs specifically located underneath VECs facing unidirectional flow, indicating cell-cell interaction of APOE+ VICs &amp; VECs. VEC sub-clustering identified lymphatic VECs under turbulent flow, while CD55+ &amp; PTGDS+ VECs under unidirectional flow. Cell-chat uncovered NOTCH3-JAG1 pairs in facilitating the co-development of spatially adjacent APOE+ VICs &amp; PTGDS+ VECs. To further uncover valve-specific signatures, we compared gene expression profiles of four valves. In lymphatic VECs, genes regulating BMP pathway were enriched in semilunar valves. In GAG VICs, MAPK pathway was specifically enriched in pulmonary valve, indicating development of different valves may need activation of different pathways. Interestingly, when comparing valve with pulmonary atresia versus healthy pulmonary valve using scRNA-seq, both MAPK related genes and GAG VICs were significantly reduced, suggesting impaired MAPK may contribute to pulmonary-specific loss of GAG VICs leading to its abnormal development. Conclusion: Our high-resolution atlas of human fetal valves revealed subtypes of VIC &amp; VEC with unique spatial locations. Comparison of four valves identified pulmonary valve-specific MAPK pathway that may explain loss of GAG VICs in patients with pulmonary atresia.

Single cell profiling of CD45+ spinal cord cells reveals microglial and B cell heterogeneity and crosstalk following spinal cord injury

BackgroundImmune cells play crucial roles after spinal cord injury (SCI). However, incomplete knowledge of immune contributions to injury and repair hinders development of SCI therapies. We leveraged single-cell observations to describe key populations of immune cells present in the spinal cord and changes in their transcriptional profiles from uninjured to subacute and chronic stages of SCI.MethodsDeep-read single-cell sequencing was performed on CD45+ cells from spinal cords of uninjured and injured Swiss-webster mice. After T9 thoracic contusion, cells were collected 3-, 7-, and 60-day post-injury (dpi). Subpopulations of CD45+ immune cells were identified informatically, and their transcriptional responses characterized with time. We compared gene expression in spinal cord microglia and B cell subpopulations with those in published models of disease and injury. Microglia were compared with Disease Associated Microglia (DAM) and Injury Responsive Microglia (IRM). B cells were compared to developmental lineage states and to an Amyotrophic Lateral Sclerosis (ALS) model.ResultsIn uninjured and 7 dpi spinal cord, most CD45+ cells isolated were microglia while chronically B cells predominated. B cells accumulating in the spinal cord following injury included immature B to mature stages and were predominantly found in the injury zone. We defined diverse subtypes of microglia and B cells with altered gene expression with time after SCI. Spinal cord microglia gene expression indicates differences from brain microglia at rest and in inflammatory states. Expression analysis of signaling ligand–receptor partners identified microglia–B cell interactions at acute and chronic stages that may be involved in B cell recruitment, retention, and formation of ectopic lymphoid follicles.ConclusionsImmune cell responses to SCI have region-specific aspects and evolve with time. Developmentally diverse populations of B cells accumulate in the spinal cord following injury. Microglia at subacute stages express B cell recruitment factors, while chronically, they express factors predicted to reduce B cell inflammatory state. In the injured spinal cord, B cells create ectopic lymphoid structures, and express secreted factors potentially acting on microglia. Our study predicts previously unidentified crosstalk between microglia and B cells post-injury at acute and chronic stages, revealing new potential targets of inflammatory responses for SCI repair warranting future functional analyses.

Tmem45b is essential for inflammation- and tissue injury–induced mechanical pain hypersensitivity

Persistent mechanical pain hypersensitivity associated with peripheral inflammation, surgery, trauma, and nerve injury impairs patients' quality of life and daily activity. However, the molecular mechanism and treatment are not yet fully understood. Herein, we show that chemical ablation of isolectin B4-binding (IB4+) afferents by IB4-saporin injection into sciatic nerves completely and selectively inhibited inflammation- and tissue injury-induced mechanical pain hypersensitivity while thermal and mechanical pain hypersensitivities were normal following nerve injury. To determine the molecular mechanism involving the specific types of mechanical pain hypersensitivity, we compared gene expression profiles between IB4+ neuron-ablated and control dorsal root ganglion (DRG) neurons. We identified Tmem45b as one of 12 candidate genes that were specific to somatosensory ganglia and down-regulated by IB4+ neuronal ablation. Indeed, Tmem45b was expressed predominantly in IB4+ DRG neurons, where it was selectively localized in the trans Golgi apparatus of DRG neurons but not detectable in the peripheral and central branches of DRG axons. Tmem45b expression was barely detected in the spinal cord and brain. Although Tmem45b-knockout mice showed normal responses to noxious heat and noxious mechanical stimuli under normal conditions, mechanical pain hypersensitivity was selectively impaired after inflammation and tissue incision, reproducing the pain phenotype of IB4+ sensory neuron-ablated mice. Furthermore, acute knockdown by intrathecal injection of Tmem45b small interfering RNA, either before or after inflammation induction, successfully reduced mechanical pain hypersensitivity. Thus, our study demonstrates that Tmem45b is essential for inflammation- and tissue injury-induced mechanical pain hypersensitivity and highlights Tmem45b as a therapeutic target for future treatment.

Geobacter-associated prophages confer beneficial effect on dissimilatory reduction of Fe(III) oxides

The dissimilatory reduction of Fe(III) oxides driven by Fe(III)-reducing bacteria (FRB) is an important biogeochemical process that influences not only iron cycling but also the biogeochemical cycles of carbon, trace metals, nutrients and contaminants. Phages have central roles in modulating the population and activity of FRB, but the mechanism for phage-involved Fe(III) oxide reduction is still unclear. This work used a common FRB, Geobacter soli, to explore the roles and underlying mechanisms of FRB-harboring prophages in the dissimilatory reduction of Fe(III) oxides. Bioinformatic analysis predicted 185 phage-related genes in the G. soli genome, comprising functional prophages that were verified to be induced to form tailed phage particles. Ferrihydrite reduction was facilitated as prophage induction was stimulated and declined as prophage induction was inhibited, which indicated a positive role of G. soli-harboring prophages in Fe(III) oxide reduction. A comparison of gene expression and released phage particles in the cells grown with Fe(III)-citrate and ferrihydrite suggested that microbial ferrihydrite reduction would activate the SOS response and consequently induce the prophages to enter lytic cycles. The prophage-mediated lysis of the subpopulation resulted in an increased release of extracellular DNA and membrane vesicles that were conducive to Fe(III) oxide reduction, which might explain the positive role of prophages in ferrihydrite reduction. In summary, our results revealed the functional roles and underlying mechanisms of FRB-associated prophages in facilitating the dissimilatory reduction of Fe(III) oxides, which will advance our understanding of iron cycling in natural ecosystems.

Comparison of EMT-Related and Multi-Drug Resistant Gene Expression, Extracellular Matrix Production, and Drug Sensitivity in NSCLC Spheroids Generated by Scaffold-Free and Scaffold-Based Methods.

Multicellular 3D tumor models are becoming a powerful tool for testing of novel drug products and personalized anticancer therapy. Tumor spheroids, a commonly used 3D multicellular tumor model, more closely reproduce the tumor microenvironment than conventional 2D cell cultures. It should be noted that spheroids can be produced using different techniques, which can be subdivided into scaffold-free (SF) and scaffold-based (SB) methods. However, it remains unclear, to what extent spheroid properties depend on the method of their generation. In this study, we aimed to carry out a head-to-head comparison of drug sensitivity and molecular expression profile in SF and SB spheroids along with a monolayer (2D) cell culture. Here, we produced non-small cell lung cancer (NSCLC) spheroids based on human lung adenocarcinoma cell line A549. Drug sensitivity analysis of the tested cell cultures to five different chemotherapeutics resulted in IC50 (A549-SB) > IC50 (A549-SF) > IC50 (A549-2D) trend. It was found that SF and SB A549 spheroids displayed elevated expression levels of epithelial-to-mesenchymal transition (EMT) markers and proteins associated with drug resistance compared with the monolayer A549 cell culture. Enhanced drug resistance of A549-SB spheroids can be a result of larger diameters and elevated deposition of extracellular matrix (ECM) that impairs drug penetration into spheroids. Thus, the choice of the spheroid production method can influence the properties of the generated 3D cell culture and their drug resistance. This fact should be considered for correct interpretation of drug testing results.

ODP305 Differentiation Potential of Marginal Layer-Derived Adult Pituitary Stem/Progenitor Cells into Vascular Endothelial Cells and Pericytes in Mice

Abstract The anterior lobe (AL) of the pituitary gland consists five types of hormone-producing cells and fenestrated sinusoids (i. e., vascular endothelial cells and pericytes) that cooperate to maintain the physiological function of the gland. In addition, the population of cells located in pituitary stem/ progenitor cell niche, the marginal layer, which faces the residual lumen of the Rathke's pouch, serve to renew pituitary cells throughout life. We recently established a protocol for isolating marginal layer-derived adult pituitary stem/progenitor cells (MPSCs) by using differences in cell adhesion (1), and then showed differentiation potential into hormone-producing cells by subjecting MPSCs to three-dimensional culture. However, it is unclear whether MPSCs are capable of differentiating into all types of cells that constitute the pituitary gland. In this study, we performed DNA microarray analysis in MPSCs, which were proliferated by two-dimensional (2D) culture, for studying comprehensively gene expression profiles. Next, we investigated what kind of cells MPSCs would differentiate into. Comparing gene expression profiles of the AL and MPSCs, in MPSCs, 1776 genes were increased more than 4-fold and 1715 genes were decreased 4-fold compared to the AL (p&amp;lt;0. 05, respectively). The upregulated genes included pituitary stem/progenitor cell markers (ex. Sox2, Sox9, Klf4, Prrx1), niche markers (ex. Cdh1, Krt8), and cell surface antigens (ex. Cd9, Cd34, Cd44). On the other hand, most of the downregulated genes were differentiation markers (ex. Pou1f1, Gata2, Tbx19, Kdr). Next, immunofluorescent analysis revealed that most of the MPSCs were positive for CD34, the hematopoietic stem cell marker. After further 2D culture of MPSCs for 5-7days, vascular endothelial marker PECAM1-positive cells were detected by immunofluorescent analysis, but pericyte marker CSPG4-positive cell not. When TGFβ was applied to 2D culture of MPSCs, CSPG4-positive cells were detected instead of PECAM1-positive cells. Expression levels of Pecam1and Cspg4 were also examined by qPCR analysis, which corresponded to the results of immunofluorescent analysis. On the other hand, expression levels of anterior hormone genes were almost unchanged. These results have demonstrated that MPSCs could differentiate into vascular endothelial cells and pericytes. The present study suggests that pituitary stem/progenitor cells, which derived from the marginal layer, are a source of not only hormone-producing cells but also cells that constitute blood vessels. Reference: (1) Shintani and Higuchi, Stem Cell Res. 2021 Feb 2;52: 102223. Presentation: No date and time listed

Analyses of adult transcriptomes from four different populations of the spongy moth, Lymantria dispar L., from China and the USA

The spongy moth Lymantria dispar, formerly known as the gypsy moth, is a forest pest that occurs as two different biotypes: the European spongy moth (ESM), Lymantria dispar dispar, which is distributed in Europe and North America; and the Asian spongy moth (ASM), which consists of subspecies Lymantria dispar asiatica and Lymantria dispar japonica and is distributed in China, Russia, Korea, and Japan. The Asian biotype is classified as a quarantine pest by the U.S. Department of Agriculture because of the superior flight ability of adult females compared to females of the European biotype. To identify genes that potentially account for differences in female flight capability between the two biotypes, we assembled and compared transcriptional profiles of two North American populations of ESM and two Chinese populations of ASM, including samples of unmated female adults and females after mating and oviposition. Of 129,286 unigenes identified, 306 were up-regulated in ASM samples relative to ESM, including genes involved in egg production. In contrast, 2309 unigenes were down-regulated in ASM samples, including genes involved in energy production. Although a previous study found that ASM female flight was reduced after oviposition, a comparison of gene expression before and after mating and oviposition did not reveal any genes which were consistently up- or down-regulated in the two ASM populations.

Comparison of clinical severity, genotype and toxin gene expression of binary toxin-producing Clostridioides difficile clinical isolates in Japan.

The emerging Clostridioides difficile strain BI/NAP1/027 has been reported to be associated with more severe clinical symptoms and higher mortality rates, thought in part due to production of a novel binary toxin alongside conventional A and B toxins. However, recent studies suggest that this may not always be the case. Therefore, the purpose of this report was to investigate the correlation between clinical severity and microbiological characteristics of CDT-producing C. difficile isolates in Japan. Eight Japanese isolates of CDT producing C. difficile were investigated using genotyping, cytotoxic activity assays and toxin gene expression. Correlation with clinical severity was performed retrospectively using the patient record. Three of eight patients were assessed as having severe C. difficile infection (CDI). PCR ribotyping resolved six ribotypes including ribotype 027. No specific genes were identified determining severe compared with non-severe cases. Positive correlation of expression levels of tcdA, tcdB and cdtB were observed although these expression levels were not correlated with cytotoxicity. CDI severity index neither correlated with toxin gene expression level nor cytotoxicity. These data indicate that the possession of the CDT gene and toxin gene expression levels may not relate to C. difficile cytotoxicity or clinical severity.