HIF-Mediated and Non-HIF-Mediated Differential Gene Expressions in Sickle Cell Reticulocyte and Their Impact on Clinical Manifestations

Abstract Reticulocytosis in sickle cell disease (SCD) is driven by tissue hypoxia from hemolytic anemia and vascular occlusion. Gene expression changes caused by hypoxia and other factors during reticulocytosis may impact SCD outcomes. We detected 1226 differentially expressed genes in SCD reticulocyte transcriptome compared to normal Black controls. To assess the role of hypoxia-mediating HIFs from other regulation of changes of the SCD reticulocyte transcriptome, we compared differential expression in SCD to that in Chuvash erythrocytosis (CE), a disorder characterized by constitutive upregulation of HIFs in normoxia. Of the SCD differentially expressed genes, 28% were shared between CE and SCD and thus classified as HIF-mediated. The HIF-mediated changes were generally in genes promoting erythroid maturation. We found that genes encoding the response to endoplasmic reticulum stress generally lacked HIF mediation. We then investigated the clinical correlation of erythroid gene expression for the 1226 differentially expressed genes detected in SCD reticulocytes, using clinical measures and gene expression data previously profiled in peripheral blood mononuclear cells (PBMCs) of 157 SCD patients at the University of Illinois at Chicago (UIC). Normal PBMCs contain only a small number of erythroid progenitors, but in SCD or CE PBMCs the erythroid transcriptome is enriched due to elevated circulating erythroid progenitors from heightened erythropoiesis (PMID: 32399971). We applied deconvolution analysis to assess the clinical correlation of erythroid gene expression, using a 16-gene expression signature of erythroid progenitors previously identified in SCD PBMCs. Deconvolution analysis uses the proportion of cell/tissue or specific marker genes (here the erythroid specific 16-gene signature) to dissect gene expression variation in biological samples with cell/tissue type heterogeneity. We correlated, in the 157 UIC patients, erythroid gene expression with i) degree of anemia as indicated by hemoglobin concentration, ii) vaso-occlusive severe pain episodes per year, and iii) degree of hemolysis measured by a hemolysis index. The analysis identified 231 genes associated with at least one of the complications. Increased expression of 40 erythroid specific genes, including 15 HIF-mediated genes, was associated with all three complications. These 40 genes are all upregulated in SCD reticulocytes and correlated with low hemoglobin concentration, frequent severe pain episodes, and high hemolysis index, suggesting that these manifestations may share a relationship to stress erythropoiesis-driven transcriptional activity. Expression quantitative trait loci (eQTL) contain genetic polymorphisms that associate with gene expression level, which can be viewed as a natural experiment to investigate the causal relations between gene expression change and phenotypic outcomes. To assess the causal effect of erythroid gene expression, we tested association between erythroid eQTL and the clinical manifestations in 906 SCD patients from the Walk-PHaSST and PUSH cohorts. We first mapped erythroid eQTL in the 157 UIC patients, who were previously genotyped by array, applying deconvolution algorithm on the same PBMC data for the 1226 differential genes in SCD reticulocytes, and detected 54 distinct eQTL for 30 genes at 5% false discovery rate. After adjusting for multiple comparisons, we found that the C allele of rs16911905, located in the β-globin cluster and associated with increased erythroid expression of HBD (encodes δ-globin of hemoglobin A 2), significantly correlated with lower hemoglobin concentration (β=-0.064, 95% CI -0.092 - -0.036, P=6.7×10 -6). The C allele was also associated with higher hemolytic rate (P=0.031), less frequent pain episodes (P=0.045), and increased erythroid expression of HBB here encoding sickle β-globin (P=5.1x10 -5). The association of the C allele with lower hemoglobin concentration was then validated in 242 patients from the UIC cohort (β=-0.071, 95% CI -0.13 - -0.011, P=0.023), as was the trend of association with higher hemolytic rate (P=0.0031) and less pain episodes (P=0.034). Our findings reveal HIF- and non-HIF-mediated genes in SCD stress erythropoiesis, and identify novel clinical associations for a HBD eQTL. Our study highlights the correlation of altered erythroid gene expression with SCD hemolytic and vaso-occlusive manifestations. Disclosures Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding. Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.

Download Full-text

P135 MAJOR GENE REGULATORS AFFECTED IN COLON AND BLOOD OF DEXTRAN SODIUM SULFATE ACUTE COLITIS MURINE MODEL

Inflammatory Bowel Diseases ◽

10.1093/ibd/zaa010.079 ◽

2020 ◽

Vol 26 (Supplement_1) ◽

pp. S32-S32

Author(s):

Reza Yarani ◽

Oana Palasca ◽

Nadezhda Tsankova Doncheva ◽

Christian Anthon ◽

Bartosz Pilecki ◽

...

Keyword(s):

Gene Expression ◽

Differentially Expressed Genes ◽

Intestinal Inflammation ◽

Major Gene ◽

Clinical Manifestations ◽

Ease Of Use ◽

Differentially Expressed ◽

In Vivo Studies ◽

P Value ◽

Sequencing Analysis

Abstract Background Dextran sulfate sodium (DSS) ulcerative colitis (UC) murine models have long been used for in vivo studies. DSS is a negatively charged polysaccharide with colitogenic properties. Although the mechanisms by which DSS induces intestinal inflammation are not fully understood, there are several good reasons why the DSS chemical colitis model for investigating the immunopathogenesis mechanism of UC is widely used. These include strong phenotypic clinical manifestations which emulate numerous clinical and histopathological features of human UC, ease of use, low mortality rate and high reproducibility. Here, by using high-throughput RNA sequencing analysis we set to investigate the major predicted gene regulators (GRs) affected by differentially expressed genes in the DSS treated UC model in order to obtain regulatory insights into the pathogenic mechanisms of UC development. Methods A DSS-induced mouse model of UC was established. Total RNA from colon tissue and blood of 3 healthy and 3 DSS-treated mice was extracted and sequenced by Illumina HiSeq 4000. Gene expression levels were obtained by mapping and quantification to the annotated mouse genome. Subsequently, differential gene expression analysis between DSS-treated and control mice both in colon and blood was performed. Ingenuity pathway analysis software (IPA®, Qiagen) was used to predict/identify major GRs affected by significantly differentially expressed genes (SDEGs, FC > |2|, p ≤0.05) in both colon and blood. Results Our analysis revealed how many and which major GRs are affected in DSS-treated mice and also the direction of change as compared to healthy (untreated) mice. In colon, 595 activated and 198 inhibited major GRs (p-value of overlap ≤0.05) in relation to ∼ 3180 SDEGs were identified, while in blood, we identified 205 activated and 62 inhibited GRs (in relation to ∼650 SDEGs). Colon and blood share 181 activated and 41 inhibited GRs. Identified GRs include transcription regulators, cytokines, transmembrane receptors and enzymes that mainly contribute to the development of inflammatory/immune responses. In colon and blood, the top 10 activated and inhibited regulators with the highest positive and negative activation z-score with target molecules as well as expression in the datasets are indicated in Figure 1a and 1b, respectively. Conclusion In this study, we analyzed linkage of GRs to SDEGs through coordinated expression and identified potential major regulators that have significant effect on UC pathogenic-related gene expression. These GRs seem to be the key regulators of transcriptomic changes induced by inflammation. These findings expand our molecular understanding of putative new targets that may be important in the pathophysiology of UC and provide biological insights into the observed expression changes between the UC and healthy controls.

Download Full-text

The Transcriptome of Immunomodulator-Resistant Multiple Myeloma

Blood ◽

10.1182/blood-2019-123407 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1772-1772

Author(s):

Moritz Binder ◽

S. Vincent Rajkumar ◽

Martha Q. Lacy ◽

Jessica L. Haug ◽

Angela Dispenzieri ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Overall Survival ◽

Differentially Expressed Genes ◽

Research Funding ◽

Differentially Expressed ◽

Resistant Disease ◽

Kaplan Meier ◽

Gene Coding ◽

Cellular Pathways

Introduction: While the molecular target of immunomodulators such as pomalidomide (POM) and lenalidomide (LEN) has been identified, the mechanisms underlying therapeutic resistance remain incompletely understood. The uniformly emerging resistance to therapy over time in the absence of identifiable cereblon pathway mutations in the majority of patients raises questions about alternative mechanisms including aberrant gene expression. Methods: We performed gene expression profiling using an Affymetrix GeneChip Human Genome U133 Plus 2.0 microarray on CD138+ bone marrow cells from patients with relapsed / refractory (RRMM) and newly diagnosed (NDMM) multiple myeloma prior to initiating treatment. Patients were treated on two phase II clinical trial protocols (MC0789: POM ± dexamethasone in RRMM; MC0884: LEN ± dexamethasone in NDMM) between 2007 and 2012. We categorized patients based on their IMWG response as non-responders (SD) and responders (VGPR+). We selected 15 responders and 15 non-responders from MC0789 (n = 30) and compared overall survival, gene expression patterns, and involved cellular pathways between the two groups. We selected 5 responders and 5 non-responders from MC0884 (n = 10) for targeted validation of differentially expressed candidate genes. After data quality control and normalization of gene expression values, differential gene expression was estimated using limma. Statistical significance was adjusted for multiple testing in the discovery set using a false discovery rate-based approach for genome-wide experiments (q-value). We used Gene Ontology and PANTHER pathway analysis for functional annotation of differentially expressed genes. Overall survival estimates were calculated using the Kaplan-Meier method. Computation and visualization were performed in R. Results: Median age at treatment initiation on MC0789 was 65 years (40 - 82), 65% of the patients were male. Pomalidomide resistance was associated with an increase in mortality (median overall survival 1.6 versus 6.4 years, p = 0.009, Kaplan-Meier plot). There were 1076 differentially regulated genes between responders and non-responders (521 up- and 555 down-regulated, q < 0.050 for all genes, volcano plot). Expression of CRBN was 1.5-fold down-regulated in non-responders (q = 0.005). Supervised hierarchical clustering of the top 500 differentially expressed genes demonstrated distinct patterns in pomalidomide-resistant disease (heatmap). Gene ontology analysis revealed protein synthesis as one of the most enriched biological processes (bar graph). Pathway analysis showed a 6-fold enrichment (FDR = 0.007) of the ubiquitin proteasome pathway in pomalidomide-resistant disease. Differentially expressed genes involved key protein degradation pathways, epigenetic modifiers, and transcription factors. Targeted validation in MC0884 revealed 13 common genes with at least 1.5-fold differential expression (5 up- and 8 down-regulated), 12 of which have previously been implicated in the regulation of apoptosis, tumor glucose metabolism, Rho and Wnt signaling, miRNA-driven resistance to chemotherapy, and ubiquitin-dependent protein degradation (Table and Sankey diagram). The most up-regulated gene in non-responders was MYRIP, a gene coding for a vesicle trafficking protein associated with platinum resistance and suppression of pro-apoptotic BCL-2 family members in solid malignancies. The most down-regulated gene was FRZB, a gene coding for a negative regulator of Wnt signaling, previously implicated in the progression of monoclonal gammopathy of undetermined significance to multiple myeloma. Conclusions: Overall survival of patients with pomalidomide-resistant RRMM remains poor. Pomalidomide resistance was associated with differential gene expression in several potentially targetable cellular pathways beyond the known drug target cereblon. Targeted validation of candidate genes revealed common cellular pathways in immunomodulator-resistant disease. Elucidating the exact molecular mechanisms underlying immunomodulator resistance is of considerable interest for biomarker development and the identification of novel therapeutic targets and warrants further exploration. Figure Disclosures Lacy: Celgene: Research Funding. Dispenzieri:Celgene: Research Funding; Alnylam: Research Funding; Intellia: Consultancy; Janssen: Consultancy; Pfizer: Research Funding; Akcea: Consultancy; Takeda: Research Funding. Kumar:Takeda: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.

Download Full-text

Transcriptomic Systems Biology Analyses with Buparlisib (BKM120) Pan PI3K Inhibition in Canine B Cell Lymphoma (BCL): Leveraging Comparative Oncology for Cancer Therapeutics and Biomarker Discovery

Blood ◽

10.1182/blood-2018-99-118548 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4114-4114

Author(s):

Ravi Dashnamoorthy ◽

Afshin Beheshti ◽

Sarah Cass ◽

Athena Kritharis ◽

Kristine Burgess ◽

...

Keyword(s):

Clinical Study ◽

Systems Biology ◽

Cancer Risk ◽

Board Of Directors ◽

Differentially Expressed Genes ◽

Research Funding ◽

Biological Pathways ◽

Differentially Expressed ◽

Advisory Committees ◽

Key Genes

Abstract Background: The canine is a highly appealing model for cancer research and discovery in part due to comparable histopathological features with humans, a fully intact immune system, similar clinicopathologic features, a more comparable body size and pharmacokinetic properties than the mouse, varied breed-specific incidence rates as well as a shared environment with humans. We and others have shown prominent transcriptomic overlap of human and canine NHL (cNHL) (McDonald T et al. Onctogarget, 2018). PI3K/Akt signaling plays an important role in lymphomagenesis, which is also a promising therapeutic target. However, identification of predictive genetic aberrations of therapeutic efficacy remains elusive. We evaluated the clinical activity of the pan-PI3K inhibitor, buparlisib, in a pilot clinical study in cNHL. Methods :We enrolled and treated 10 dogs with buparlisibwho were diagnosed with BCL in an IRB and IACUC approved clinical study. Cases included 2 treatment naïve and 8 dogs with relapsed disease that had relapsed s/p CHOP (6), L' asparaginase (1) and VELCAP (1) treatment. Pet owners were consented and the study subjects received buparlisib9mg/kg orally for 28 consecutive days. Analysis for tumor response were evaluated on weekly basis through direct tumor measurement or use of x-rays. Post-therapy fine needle aspirates (FNA) were collected on Days 0, 7 and 21 to examine predictors of response to BKM120. RNA from fine need aspirate cells were isolated and the transcriptomic changes were evaluated using Canine Genome 2.0 Affymetrix Array, followed by unbiased systems biology assessment for biological pathways using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). We performed unbiased assessment to determine pertinent biological pathways associated with treatment response. The overall impact was used to determine the global effect on tumor progression and cancer risk based on the specific regulation of each gene. A Carcinogenic Risk Score (CRS) was calculated based on these values to determine if there is a promoted risk for cancer (positive value) or inhibitory risk for cancer (negative value) by summing the log2 fold-change values of key genes and subtracting this from the sum of log2 fold-change values of the tumor suppressors when comparing pre-treated to BKM120 treated dogs. Results: Following four weeks of BKM120 treatment, the overall response rate was 30% with 1 complete response lasting 42 days; 2 partial responses lasting 55 and 72 days; 3 stable disease; and 4 progressive disease. Mild treatment related toxicities such elevated blood glucose, thrombocytopenia and anemia, fever, nausea and lethargic symptoms, with no treatment related toxicities in 2 cases were noted. Principal Component Analysis (PCA) and hierrachical clustering analysis of differentially expressed genes show that differentially expressed genes to cluster together in all dogs during post 2 week, indicating a consistent biological activity by BKM120 in all dogs regardless of breed, prior treatment or disease status. Pathway network analysis based on differentially expressed genes predicted activation of upstream regulators associated with tumor suppression including SOX1, SOX3 and GMNN (Week 1) and CEBPA (Week 2). Analysis of "key genes" involved in multiple biological processes appeared to be associated with response of PI3K inhibitortreatment. This included down regulation of CREBBP with a Cancer Risk Score (CRS) of -0.97 and downregulation of VIM, CDH3, WNT3, WNT5B and FGFR2 with a CRS of -2.98 (Fig 1). Conclusion: Results from our pilot study in cNHL showed encouraging clinical responses with a pan-PI3K inhibitor in 3 of 10 dogs. Furthermore, our unbiased characterization of biological pathways revealed that the observed GEP changes associated with tumor suppression and they reduced the risk for cancer progression. Overall, the canine model appears to be particularly attractive model that may be leveraged for the study of clinical and biological responses to novel therapeutic oncologic agents. Disclosures Evens: Bayer: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Novartis: Consultancy; Acerta: Consultancy; Seattle Genetics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics International DMC: Membership on an entity's Board of Directors or advisory committees; Tesaro: Research Funding; Janssen: Consultancy; Affimed: Consultancy.

Download Full-text

Functional Effects of Low-Dose IL-2 in Patients with Chronic Graft Versus Host Disease

Blood ◽

10.1182/blood.v128.22.667.667 ◽

2016 ◽

Vol 128 (22) ◽

pp. 667-667

Author(s):

Jennifer Whangbo ◽

Bryn Falahee ◽

John Koreth ◽

Haesook T. Kim ◽

Sarah Nikiforow ◽

...

Keyword(s):

T Cells ◽

Clinical Response ◽

Board Of Directors ◽

Differentially Expressed Genes ◽

Research Funding ◽

Low Dose ◽

Differentially Expressed ◽

Advisory Committees ◽

Response Groups ◽

Pre Treatment

Abstract Introduction: CD4+CD25+FoxP3+ regulatory T cells (Treg) have broad suppressive activity and play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). Interleukin-2 (IL-2) is a key growth factor for Treg and we previously reported that daily administration of low-dose IL-2 in patients with refractory cGVHD induces selective expansion of Treg and NK cells. No expansion of CD4 conventional T cells (Tcon) or CD8 T cells was noted. In a Phase 2 study, 64% of patients had clinical responses. Predictors of clinical response included younger age, early IL-2 initiation and a higher Treg:Tcon ratio at study baseline and at week 1 of IL-2 therapy. However, there were no significant differences in overall Treg expansion or plasma IL-2 levels between responders and non-responders during the initial 12 week treatment period. We therefore asked whether functional or gene expression differences in Treg or effector T cells could distinguish clinical response to IL-2. Methods: In vitro Treg suppression assays were performed using cryopreserved patient samples obtained at study baseline and week 6 of IL-2 therapy. CD4+CD25+CD127- Treg were cultured with CellTrace Violet-labeled CD4+CD25- Tcon in the presence of stimulating antibody-coated beads. Proliferation was measured as CellTrace Violet dilution by flow cytometry after 4 days of incubation. All patient samples were tested against the same healthy donor (HD) Treg and Tcon. Differential gene expression between clinical responders and non-responders was examined by RNA Seq analysis of sorted Treg, Tcon, CD8 and NK cells at study baseline and week 4 of IL-2 therapy. Results: To compare Treg cell function between response groups, we tested the ability of patient Treg to suppress the proliferation of HD Tcon. To determine whether there were qualitative differences in effector T cells between the response groups, we measured suppression of patient Tcon by HD Treg. At study baseline, clinical responders appear to have higher Treg suppressive function although the differences between the response groups were not significant due to small sample size (Figure 1A). Patient Tcon in responders and non-responders were similarly suppressed by HD Treg. At week 6 of IL-2 therapy, Treg from non-responders showed significant improvement in suppressive activity against HD Tcon, whereas there was no significant change in suppressive function of Treg from responders (Figure 1B). Thus, despite the lack of clinical improvement in cGVHD symptoms, non-responder Treg show both numeric increase and functional improvement in response to IL-2 therapy. To identify other cell-intrinsic differences that distinguish clinical response to IL-2, we performed RNA Seq on sorted Treg, Tcon, CD8 and NK cells in 3 responders and 3 non-responders. Differentially expressed genes were identified using the DEseq package. There were very few differentially expressed genes between response groups within the pre-treatment Treg, CD8 and NK cell samples (Figure 2). In contrast, 93 genes (92 upregulated, 1 downregulated) with greater than 4-fold difference in expression levels were identified in pre-treatment Tcon samples between responders and non-responders. The top 10 gene ontogeny terms associated with these differentially expressed genes include cell-cell adhesion and ion transport processes. By week 4 of IL-2 therapy, the gene expression profiles of responder and non-responder cells were very similar. There were no differentially expressed genes within Treg, and only 5 within Tcon. Conclusions: In addition to increasing absolute Treg numbers, daily low-dose IL-2 improves Treg suppressive function in patients who do not exhibit clinical cGVHD improvement. Interestingly, RNA Seq results suggest that the determinants of clinical response do not lie within the Treg. Instead, the greatest differences between response groups were detected in pre-treatment CD4 Tcon, implying that lack of clinical response reflects resistance of CD4 effector cells to suppression mediated by Treg in vivo. Further analysis of differentially expressed Tcon genes may help elucidate the mechanisms of resistance to IL-2 therapy in patients with cGVHD. Disclosures Koreth: LLS: Research Funding; prometheus labs inc: Research Funding; kadmon corp: Membership on an entity's Board of Directors or advisory committees; amgen inc: Consultancy; millennium pharmaceuticals: Research Funding; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Armand:Otsuka: Research Funding; Roche: Research Funding; BMS: Consultancy, Research Funding; Infinity: Consultancy; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta: Research Funding; Sigma Tau: Research Funding; Tensha: Research Funding. Soiffer:Kiadis: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Exposure to hypoxia causes stress erythropoiesis and downregulates immune response genes in spleen of mice

BMC Genomics ◽

10.1186/s12864-021-07731-x ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Haijing Wang ◽

Daoxin Liu ◽

Pengfei Song ◽

Feng Jiang ◽

Xiangwen Chi ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Expression Patterns ◽

Differentially Expressed ◽

Hypoxic Exposure ◽

Control Group ◽

Sequencing Analysis ◽

Erythroid Progenitors ◽

Stress Erythropoiesis ◽

Expression Of Genes

Abstract Background The spleen is the largest secondary lymphoid organ and the main site where stress erythropoiesis occurs. It is known that hypoxia triggers the expansion of erythroid progenitors; however, its effects on splenic gene expression are still unclear. Here, we examined splenic global gene expression patterns by time-series RNA-seq after exposing mice to hypoxia for 0, 1, 3, 5, 7 and 13 days. Results Morphological analysis showed that on the 3rd day there was a significant increase in the spleen index and in the proliferation of erythroid progenitors. RNA-sequencing analysis revealed that the overall expression of genes decreased with increased hypoxic exposure. Compared with the control group, 1380, 3430, 4396, 3026, and 1636 genes were differentially expressed on days 1, 3, 5, 7 and 13, respectively. Clustering analysis of the intersection of differentially expressed genes pointed to 739 genes, 628 of which were upregulated, and GO analysis revealed a significant enrichment for cell proliferation. Enriched GO terms of downregulated genes were associated with immune cell activation. Expression of Gata1, Tal1 and Klf1 was significantly altered during stress erythropoiesis. Furthermore, expression of genes involved in the immune response was inhibited, and NK cells decreased. Conclusions The spleen of mice conquer hypoxia exposure in two ways. Stress erythropoiesis regulated by three transcription factors and genes in immune response were downregulated. These findings expand our knowledge of splenic transcriptional changes during hypoxia.

Download Full-text

Gene Expression and Epigenetic Analysis in Relapsed/Refractory Diffuse Large B Cell Lymphoma Provides Insights into Evolution of Treatment Resistance to R-CHOP

Blood ◽

10.1182/blood-2020-138645 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 26-26

Author(s):

Manishkumar S. Patel ◽

Ellen K. Kendall ◽

Sarah Ondrejka ◽

Agrima Mian ◽

Yazeed Sawalha ◽

...

Keyword(s):

Gene Expression ◽

B Cell ◽

Differentially Expressed Genes ◽

Research Funding ◽

B Cell Lymphoma ◽

Cell Receptor ◽

B Cell Receptor ◽

Gene Set Enrichment Analysis ◽

Differentially Expressed ◽

P Value

Background Diffuse large B cell lymphoma (DLBCL) is curable in ~60-70% of patients using standard chemoimmunotherapy, but the prognosis is poor for relapsed/refractory (R/R) DLBCL. Therefore, understanding the underlying molecular mechanisms will facilitate early prediction and effective management of resistance to therapy. Recent studies of paired diagnostic-relapse biopsies from patients have relied on a single "omics" approach, examining either gene expression or epigenetic evolution. Here we present a combined analysis of gene expression and DNA methylation profiles of paired diagnostic-relapse DLBCL biopsies to identify changes responsible for relapse after R-CHOP. Methods Biopsies from 23 DLBCL patients were obtained at the time of diagnosis and relapse following frontline R-CHOP chemoimmunotherapy. The cohort had 18 (78.3%) male patients with median age of 62 (range, 35-86) years and median IPI of 2.5 (range, 1-5). The median time from diagnosis to relapse was 7 (range, 0-57) months. DNA and RNA were extracted simultaneously from formalin-fixed paraffin embedded (FFPE) biopsy samples. DNA methylation levels were measured through Illumina 850k Methylation Array for 22 pairs of diagnostic-relapse biopsies. RNA from diagnostic-relapse paired biopsies from 6 patients was sequenced using Illumina HiSeq4000. Differentially methylated probes were identified using the DMRcate package, and differentially expressed genes were identified using the DESeq2 package. Gene set enrichment analysis was performed using canonical pathway gene sets from MSigDB. Pearson's correlation with a Bonferroni correction to the p-value was used to calculate the correlation between regularized log transformed gene expression counts and methylation beta values. Results In a pairwise comparison of gene expression between diagnostic and R/R biopsy pairs, we found 14 differentially expressed genes (FDR<0.1 & Log2FC>|1|) consistent across all pairs. Compared to gene expression at diagnosis, five genes (CYP1B1, LGR4, ATXN1, CTSC, ZMAT3) were downregulated, and eight genes (ERBB3, CD19, CARD11, MT-RNR2, IGHG3, CCDC88C, ATP2A3, CENPE, and PCNT) were up-regulated in the R/R samples. Many of these genes have been previously implicated in oncogenesis, such as ERBB3, a member of the epidermal growth receptor family. Importantly, some of these genes have known roles in DLBCL biology, such as CD19, a member of the B-cell receptor complex, and CARD11, a gene in which several oncogenic mutations have been identified in DLBCL as a mediator of NF-KB activation. Gene set enrichment analysis revealed overexpression of immune signatures such as cytokine-cytokine receptor interaction, chemokine receptor-chemokine binding, and the IL-12-STAT4 pathway at diagnosis. At relapse, cell cycle, B-cell receptor, and NOTCH signaling pathways were overexpressed. Interestingly, in a pairwise comparison of methylation between diagnostic and R/R biopsy pairs, there were no differentially methylated probes (FDR<0.05), suggesting no coordinated epigenetic evolution between diagnostic and R/R pairs. For biopsy pairs that had both gene expression and methylation data (5 pairs), we correlated gene expression and methylation values. We found that none of the differentially expressed genes between the diagnostic and R/R biopsies were significantly correlated with methylation status (adjusted p-value<0.05). Conclusions By analyzing paired diagnostic and relapse DLBCL biopsies, we found that at the time of relapse, there are significant transcriptomic changes but no significant epigenetic changes when compared to diagnostic biopsies. Activation of B-cell receptor and NOTCH signaling, as well as the loss of immune signaling at relapse, cannot be attributed to coordinated epigenetic changes in methylation. As the epigenetic profile of the biopsies did not consistently evolve, these data emphasize the need for better understanding of the baseline methylation profiles at the time of diagnosis, as well as acquired somatic mutations that may contribute to the emergence of therapeutic resistance. Future studies are needed to focus on how activation of signaling pathways triggered by genomic alterations can be targeted in relapsed/refractory DLBCL. Disclosures Hsi: Seattle Genetics: Consultancy, Honoraria; Miltenyi: Consultancy, Honoraria; Abbvie: Research Funding; Eli Lilly: Research Funding; CytomX: Consultancy, Honoraria. Hill:Takeda: Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding.

Download Full-text

Gene Expression after Hemarthrosis Differs between FVIII-Deficient Mice Treated with Recombinant FVIII or FVIII-Fc Fusion Protein

Blood ◽

10.1182/blood-2021-149923 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3175-3175

Author(s):

Bilgimol Chumappumkal Joseph ◽

Esther Cooke ◽

Jenny Y Zhou ◽

Sonha Nguyen ◽

Samantha Ruiz ◽

...

Keyword(s):

Gene Expression ◽

Fusion Protein ◽

Board Of Directors ◽

Differential Expression ◽

Differentially Expressed Genes ◽

Research Funding ◽

Molecular Pathways ◽

Advisory Committees ◽

Fc Fusion Protein ◽

Deficient Mice

Abstract Introduction Local and systemic molecular responses to hemarthroses in hemophilia are not well understood. Emerging clinical evidence suggests that treatment with FVIII-Fc Fusion protein (FcFVIII), using the Fc-portion of immunoglobulin for half-life extension, may mitigate joint inflammation and modulate immune stimulation. We analyzed gene expression profiles in synovium and spleen (a major immune regulatory organ) in FVIII-deficient mice at baseline and after hemarthrosis treated with recombinant human FVIII (rhFVIII) or murine (m)FcFVIII to characterize the respective differences and molecular pathways for each FVIII preparation. Methods Hemarthrosis was induced by sub-patellar needle puncture in FVIII-deficient mice treated with saline (vehicle), 100 IU/kg mFcFVIII (Fc murine due to species specificity) or 120 IU/kg rhFVIII (to account for differences in t 1/2) administered intravenously 2 hours before and 6 hours after injury. Spleen and synovium were harvested on day 3 or day 14 post-injury (n=3-5 per group). Spleen and synovium from uninjured mice served as controls. RNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit and sequenced on an Illumina NextSeq500 platform (single-end; 75bp reads). The limma-voom method (R Bioconductor) was used for differential expression analyses. The criteria for differential expression were: i) a log fold-change (logFC) >1 or <-1, and ii) an adjusted p value <0.05. Functional enrichment was performed using Signaling Pathway Impact Analysis (SPIA). Molecular pathways were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome pathway databases. Results Knee injury in FVIII-deficient mice resulted in hemarthrosis and a drop in mean hematocrit from 44.7% to 27.5% (saline), which was prevented by rhFVIII and mFcFVIII prophylaxis (mean day 2 hematocrit ~45%). In the spleen, differential gene expression (DGE) in saline treated mice was pronounced but fleeting with 5295 and 9 differentially expressed genes (DEG) on day 3 and 14. DGE was abrogated by either rhFVIII or mFcFVIII, without significant expression differences between rhFVIII and mFcFVIII at the single gene level. However, differences were noted between rhFVIII and mFcFVIII during KEGG pathway analyses, where rhFVIII predominantly perturbed pathways related to inflammation, T-cell signaling and innate immune regulation, while mFcFVIII perturbed pathways related to B-cell signaling and cellular homeostasis. In the synovium, DGE in saline treated mice was also pronounced on day 3 (3388 DEG) but, in contrast to the spleen, continued on day 14 (1030 DEG). Unlike in the spleen, DGE was not fully corrected with rhFVIII or mFcFVIII; moreover, gene expression differed between rhFVIII and mFcFVIII. This was particularly evident on day 14, with a partial reduction to 603 and 294 differentially expressed genes with rhFVIII and mFcFVIII, respectively. Only 163 genes overlapped, whereas 440 were unique to rhFVIII and 131 to mFcFVIII. A comparison between gene expression with rhFVIII and mFcFVIII yielded that ≥ 2 log-fold expression difference was present for 74 genes, with 72 genes upregulated with rhFVIII relative to mFcFVIII. Many of these genes are involved in pathways related to innate and adaptive immune responses, cell cycle regulation and extracellular matrix organization in the Reactome database. This response was dampened by mFcFVIII. Conclusions Joint bleeding in hemophilic mice induced pronounced systemic and synovial gene expression, which was diminished by rhFVIII or mFcFVIII. However, while there were many similarities with regard to regulation of gene expression and/or molecular pathways, there were also notable differences, especially involving immune regulatory, inflammatory and tissue repair functions. These observations provide new molecular insights regarding Fc-driven effects of FVIII after hemarthrosis, suggesting that the type of FVIII preparation used for bleed control may have implications beyond hemostasis. Disclosures von Drygalski: Hematherix, Inc: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Super FVa; uniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Biomarin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Download Full-text

Integrated Transcriptomic and Proteomic Analyses of Inflammasome in Myelodysplastic Syndromes and Chronic Myelomonocytic Leukemia

Blood ◽

10.1182/blood-2019-128094 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2991-2991

Author(s):

María Zurdo ◽

Ana M Hurtado López ◽

Tzu Hua Chen-Liang ◽

Helios Martínez-Banaclocha ◽

Laura Palomo ◽

...

Keyword(s):

Board Of Directors ◽

Differentially Expressed Genes ◽

Myelodysplastic Syndromes ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Myelomonocytic Leukemia ◽

Differentially Expressed ◽

Advisory Committees ◽

Myelomonocytic Leukemia

Background and aim: Inflammasome and pyroptosis overactivation have recently been associated as fundamental mechanisms in the ineffective hematopoiesis of myelodysplastic syndromes (MDS). Chronic myelomonocytic leukemia (CMML) shares histological and clinical characteristics with MDS but, within clinical differences, It stands out a high association with inflammatory/autoimmune diseases in which a disproportionate activation of inflamasome has been implicated. Our hypothesis is that CMML cases show a higher inflammasome activation with respect to the MDS subset, a relevant difference both in terms of potential therapeutic targets and pathogenic clues. The main objective is to confirm, describe and quantify these differences using high-performance and multi-gene/protein methods. Methods: We performed enhanced RNA-seq in bone marrow mononucleated cells of 27 CMML at diagnosis, 10 MDS and 9 controls (103 million average readings). We selected 116 genes related to the inflammasome and reviewed the differential expression between cases and controls. We evaluated by multiplex immunoassay the profile of 28 cytokines in peripheral blood in 35 CMML patients, 37 MDS and 8 controls. Subsequently, we studied whether these differentially expressed genes / cytokines showed differences in CMML depending on the mutational state of TET2, SRSF2 and ASXL1. Finally, we compared in vitro the degree of activation of inflamasome in the monocytoid component of 8 CMML patients versus 7 controls. Results: In the transcriptomic analysis of the inflamasome genes in patients with CMML, we found 30 of 116 differentially expressed genes compared with healthy controls. Of those 30 genes, 26 showed a pro-inflammatory function and, of them, 18 were up-regulated. Of the 4 differentially expressed genes with an anti-inflammatory function, 3 were significantly under-expressed in CMML patients. We highlight, due to the quantitative difference, the overexpression of two genes coding for monocyte chemotactic proteins, CCL7 and CCL2 (FC = 269.21, p = 0.032; FC = 11.79, p = 0.03) That pro-inflammatory transcriptional profile was not so evident in the cases of MDS: of the 29 differentially expressed genes with pro-inflammatory function, 18 were down-regulated. Subsequently, we designed a customized panel for proteomic analysis including 9 of the 30 differentially expressed genes in CMML. We found that, in a relevant percentage of cases, also proinflammatory cytokines derived from these differentially expressed genes were elevated (62.5%) in peripheral blood of patients, compared to healthy donors; pointing towards the key role of gene transcription in the definition of the pro-inflammatory sense of the proteomic dimension of inflammasome in CMML. Next, we found that those patients with CMML and somatic mutations of TET2 had a higher expression of CCL7 and CCL2 compared to patients with CMML wild type, with a tendency to significance in the first case and significant in the second (FC 11.9, p = 0.15; FC 7.8, p = 0.03). Finally, we conducted in vitro stimulation studies at diagnosis in patients with CMML confirming that the canonical activation of the NLRP3 inflammasome (increased production of IL-1β) is significantly enhanced with respect to control individuals. Conclusion: We describe for the first time, a hyperactivation in CMML, compared with MDS, of the components of the inflammasome. Hyperactivation associated in CMML to a gene transcriptional mechanism and related, in the case of the two most over-expressed genes, CCL2 and CCL7, to the presence of mutations in TET2. Our findings point to new therapeutic targets whose modulation could restore inefficient hemopoiesis and potential diagnostic and prognostic biomarkers in CMML. Disclosures Díez-Campelo: Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jerez:Novartis: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Differential gene expression analysis of ankylosing spondylitis shows deregulation of the HLA-DRB, HLA-DQB, ITM2A, and CTLA4 genes

Egyptian Journal of Medical Human Genetics ◽

10.1186/s43042-021-00161-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Rowan AlEjielat ◽

Anas Khaleel ◽

Amneh H. Tarkhan

Keyword(s):

Gene Expression ◽

Ankylosing Spondylitis ◽

Differentially Expressed Genes ◽

Gene Network ◽

Disease Diagnosis ◽

Receptor Activation ◽

Functional Enrichment ◽

Differentially Expressed ◽

Inflammatory Disorder ◽

Data Set

Abstract Background Ankylosing spondylitis (AS) is a rare inflammatory disorder affecting the spinal joints. Although we know some of the genetic factors that are associated with the disease, the molecular basis of this illness has not yet been fully elucidated, and the genes involved in AS pathogenesis have not been entirely identified. The current study aimed at constructing a gene network that may serve as an AS gene signature and biomarker, both of which will help in disease diagnosis and the identification of therapeutic targets. Previously published gene expression profiles of 16 AS patients and 16 gender- and age-matched controls that were profiled on the Illumina HumanHT-12 V3.0 Expression BeadChip platform were mined. Patients were Portuguese, 21 to 64 years old, were diagnosed based on the modified New York criteria, and had Bath Ankylosing Spondylitis Disease Activity Index scores > 4 and Bath Ankylosing Spondylitis Functional Index scores > 4. All patients were receiving only NSAIDs and/or sulphasalazine. Functional enrichment and pathway analysis were performed to create an interaction network of differentially expressed genes. Results ITM2A, ICOS, VSIG10L, CD59, TRAC, and CTLA-4 were among the significantly differentially expressed genes in AS, but the most significantly downregulated genes were the HLA-DRB6, HLA-DRB5, HLA-DRB4, HLA-DRB3, HLA-DRB1, HLA-DQB1, ITM2A, and CTLA-4 genes. The genes in this study were mostly associated with the regulation of the immune system processes, parts of cell membrane, and signaling related to T cell receptor and antigen receptor, in addition to some overlaps related to the IL2 STAT signaling, as well as the androgen response. The most significantly over-represented pathways in the data set were associated with the “RUNX1 and FOXP3 which control the development of regulatory T lymphocytes (Tregs)” and the “GABA receptor activation” pathways. Conclusions Comprehensive gene analysis of differentially expressed genes in AS reveals a significant gene network that is involved in a multitude of important immune and inflammatory pathways. These pathways and networks might serve as biomarkers for AS and can potentially help in diagnosing the disease and identifying future targets for treatment.

Download Full-text

Behavioral sensitization induced by methamphetamine causes differential alterations in gene expression and histone acetylation of the prefrontal cortex in rats

BMC Neuroscience ◽

10.1186/s12868-021-00616-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Hui Li ◽

Jing-An Chen ◽

Qian-Zhi Ding ◽

Guan-Yi Lu ◽

Ning Wu ◽

...

Keyword(s):

Gene Expression ◽

Prefrontal Cortex ◽

Histone Acetylation ◽

Differentially Expressed Genes ◽

Behavioral Sensitization ◽

Functional Enrichment ◽

Differentially Expressed ◽

Adult Rats ◽

Mrna Microarray ◽

H3 Acetylation

Abstract Background Methamphetamine (METH) is one of the most widely abused illicit substances worldwide; unfortunately, its addiction mechanism remains unclear. Based on accumulating evidence, changes in gene expression and chromatin modifications might be related to the persistent effects of METH on the brain. In the present study, we took advantage of METH-induced behavioral sensitization as an animal model that reflects some aspects of drug addiction and examined the changes in gene expression and histone acetylation in the prefrontal cortex (PFC) of adult rats. Methods We conducted mRNA microarray and chromatin immunoprecipitation (ChIP) coupled to DNA microarray (ChIP-chip) analyses to screen and identify changes in transcript levels and histone acetylation patterns. Functional enrichment analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, were performed to analyze the differentially expressed genes. We then further identified alterations in ANP32A (acidic leucine-rich nuclear phosphoprotein-32A) and POU3F2 (POU domain, class 3, transcription factor 2) using qPCR and ChIP-PCR assays. Results In the rat model of METH-induced behavioral sensitization, METH challenge caused 275 differentially expressed genes and a number of hyperacetylated genes (821 genes with H3 acetylation and 10 genes with H4 acetylation). Based on mRNA microarray and GO and KEGG enrichment analyses, 24 genes may be involved in METH-induced behavioral sensitization, and 7 genes were confirmed using qPCR. We further examined the alterations in the levels of the ANP32A and POU3F2 transcripts and histone acetylation at different periods of METH-induced behavioral sensitization. H4 hyperacetylation contributed to the increased levels of ANP32A mRNA and H3/H4 hyperacetylation contributed to the increased levels of POU3F2 mRNA induced by METH challenge-induced behavioral sensitization, but not by acute METH exposure. Conclusions The present results revealed alterations in transcription and histone acetylation in the rat PFC by METH exposure and provided evidence that modifications of histone acetylation contributed to the alterations in gene expression caused by METH-induced behavioral sensitization.

Download Full-text