Role of Ts-RNAs in CLL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2016-2016
Author(s):  
Veronica Balatti ◽  
Alexey Palamarchuk ◽  
Lara Rizzotto ◽  
Dario Veneziano ◽  
Giovanni Nigita ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Research Funding ◽  
Prognostic Markers ◽  
Clinical Course ◽  
Differentially Expressed ◽  
Expression Level ◽  
Transcriptional Level ◽  
Indolent Disease ◽  
Aggressive Clinical Course

Abstract Chronic lymphocytic leukemia (CLL) is the most common human leukemia. This disease occurs in two forms, aggressive and indolent, both characterized by clonal expansion of CD5+ B-lymphocytes. Hallmarks of aggressive CLL are the high expression level of ZAP-70, the unmutated sequence of IgVH genes, and activation of the TCL1 oncogene. While investigating whether the expression of miR-3676/4521 cluster in CLL is correlated with TCL1 regulation and ZAP-70 methylation in aggressive CLLs, we found that these two microRNAs are associated with tRNA sequences and that this region produces two small RNAs, members of a novel class of small non-coding RNAs (tsRNAs). Ts-3676/4521 cluster is located on the short arm of chromosome 17 and is co-deleted with p53 in 17p deleted CLL patients. We previously demonstrated that ts-3676 and ts-4521 are down-regulated and mutated in all CLL types. Furthermore, ts-3676 targets TCL1 3'UTR, indicating that tsRNAs can interfere with gene expression at a post-transcriptional level in a micoRNA-like manner. However, tsRNAs do not share the biogenesis mechanism of microRNAs but are cleaved from the 3' ends of pre-tRNAs by the endonuclease RNase Z and represent unique sequences starting at the 3' ends of the tRNAs and ending at a sequence of 4 consecutive T nucleotides. Thus tsRNAs are single stranded RNAs, more similar to piRNAs than miRNAs. Since piRNAs interact with Piwi proteins to affect gene methylation, we hypothesized that tsRNAs could also interact with Piwi proteins. By performing a series of RNA immunoprecipitation experiments, we verified the interaction of ts-3676 and ts-4521 with Piwi proteins, suggesting that these molecules could be involved in gene promoter methylation, hence affecting gene expression at a pre-transcriptional level in a piRNA-like manner. Since ts-3676 and ts-4521 are deregulated in CLLs, we studied if other tsRNAs are differentially regulated CLL. We retrieved the tsRNAs sequences from 3' ends of tRNA genes, obtaining a total of 120 tsRNA sequences, and designed a tsRNA microarray chip to investigate signatures of tsRNAs that can distinguish different classes of CLLs. We hybridized total RNAs extracted from 23 CLL samples (11 indolent, 12 aggressive) and 8 CD19+ B cells of healthy donors and found 17 tsRNA differentially expressed in these cohorts of samples. 9 tsRNAs are differentially expressed in aggressive CLL vs. normal B-cells, 10 tsRNAs are differentially expressed in indolent CLL vs. normal B-cells, 15 tsRNAs are differentially expressed in indolent vs. aggressive CLL. In these signatures, we identified ts-46 and ts-47 strongly downregulated in aggressive CLL, suggesting these ts-RNAs as potential tumor-suppressors. Finally, we found that ts-3676 and ts-4521 have a prognostic value for particular subgroups of CLLs. Patients with low ZAP-70 expression and mutated IgVH generally show indolent disease, while patients with high ZAP-70 expression and un-mutated IgVH show aggressive disease. However, a relatively small cohort of patients shows aggressive prognostic markers but clinically indolent disease. Our analysis revealed that expression of ts-3676 is lower in aggressive CLLs vs indolent CLLs, although, we did not test these tsRNAs in cohort of patients with aggressive prognostic markers but indolent disease. Currently there is no good marker to discriminate these patients from aggressive CLLs. Thus, we selected 8 patients with unfavorable prognostic markers but indolent disease and 12 patients with both aggressive clinical course and unfavorable prognosis markers, to test for differences in the expression level of ts-3676 and ts-4521. We found that in patients with unfavorable prognostic markers but indolent disease, expression of ts-3676 and ts-4521 was almost double of that in patients displaying both aggressive clinical course and markers. In conclusion, we provided an evidence that tsRNAs deregulation can affect gene expression by interfering with the epigenetic machinery and/or with the post-transcriptional stability of target genes. We also identified the first tsRNAs important in CLL as new markers and/or targets for therapy: ts-3676, ts-4521, ts-46 and ts-47. Disclosures Kipps: Celgene: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Speakers Bureau.

scholarly journals Gene expression profiling reveals light regulation on adventitious root formation in lotus (Nelumbo nucifera Gaertn.)

2020 ◽  
Author(s):  
Cheng libao ◽  
Han Yuyan ◽  
Zhao Minrong ◽  
Xu xiaoyong ◽  
Shen Zhiguang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Light Intensity ◽  
Gene Expression Profiling ◽  
Differentially Expressed Genes ◽  
Expression Profiling ◽  
Sugar Metabolism ◽  
High Light ◽  
Differentially Expressed ◽  
Expression Level ◽  
Transcriptional Level

Abstract Background: Lotus is an aquatic horticultural crop, and widely cultivated in most regions of China. Lotus is often used as a kind of an important off-season vegetable with various nutrients. Principle root of lotus is degenerated, and the role of adventitious roots (ARs) is Irreplaceable for plant growth. We found that no ARs could be formed under darkness condition, and high light significantly promote the development of root primordium. Therefore, four libraries with three light intensities were constructed to monitor metabolism changes, especially in IAA and sugar metabolism. Results: We found that ARs formation was significantly affected by light, high light intensity accelerated ARs development. The change of metabolism during ARs formation under different light intensity was evaluated according to gene expression profiling by high-throughput tag-sequencing. It was shown that more than 2.2× 10 4 genes was obtained in each library, and the expression level of most genes were distributed between 1e-01 and 1e+03 (FPKF value). Among these identified genes, 1739, 1683 and 1462 genes were up-regulated, and 1533, 995 and 834 genes were down-regulated in D/CK, E/CK and F/CK libraries respectively. In addition, we also found that 1454 and 478 genes changed expression in D/CK and F/CK libraries when compared D/CK with F/CK libraries. In F/D libraries, the transcriptional level of most differentially expressed genes was between -5~5 fold, and twenty differentially expressed genes were involved in signal transduction pathway. Twenty-eight genes related with IAA response and thirty-five genes involved in sugar metabolism were found to change expression. It was elucidated that IAA content was enhanced after seed germinated, even in darkness condition, which was responsible for ARs formation. Conclusion: The process of ARs formation was very complex, and regulated by multiple factors. The ARs formation was regulated by IAA, even in the dark, induction and developmental process could also be completed. In addition, the genes (36 genes) changed expression level in carbohydrate metabolism showed the third number, so sucrose metabolism was involved in the ARs development (expressed stage) according to genes expression and content change characteristic.

scholarly journals A shared gene expression signature in mouse models of EBV-associated and non–EBV-associated Burkitt lymphoma

Blood ◽  
2011 ◽  
Vol 118 (26) ◽  
pp. 6849-6859 ◽  
Author(s):  
Kathryn T. Bieging ◽  
Kamonwan Fish ◽  
Subbarao Bondada ◽  
Richard Longnecker
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Mouse Model ◽  
Tumor Cells ◽  
Burkitt Lymphoma ◽  
Gene Expression Signature ◽  
Differentially Expressed ◽  
Cell Phenotype ◽  
Phenotypic Differences ◽  
Tumor Onset

AbstractThe link between EBV infection and Burkitt lymphoma (BL) is strong, but the mechanism underlying that link has been elusive. We have developed a mouse model for EBV-associated BL in which LMP2A, an EBV latency protein, and MYC are expressed in B cells. Our model has demonstrated the ability of LMP2A to accelerate tumor onset, increase spleen size, and bypass p53 inactivation. Here we describe the results of total gene expression analysis of tumor and pretumor B cells from our transgenic mouse model. Although we see many phenotypic differences and changes in gene expression in pretumor B cells, the transcriptional profiles of tumor cells from LMP2A/λ-MYC and λ-MYC mice are strikingly similar, with fewer than 20 genes differentially expressed. We evaluated the functional significance of one of the most interesting differentially expressed genes, Egr1, and found that it was not required for acceleration of tumor onset by LMP2A. Our studies demonstrate the remarkable ability of LMP2A to affect the pretumor B-cell phenotype and tumorigenesis without substantially altering gene expression in tumor cells.

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

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 950-950
Author(s):  
Xu Zhang ◽  
Jihyun Song ◽  
Binal N. Shah ◽  
Jin Han ◽  
Taif Hassan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Research Funding ◽  
Clinical Manifestations ◽  
Hemoglobin Concentration ◽  
Differentially Expressed ◽  
Erythroid Progenitors ◽  
Advisory Committees ◽  
Deconvolution Analysis ◽  
Stress Erythropoiesis

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.

scholarly journals DYRK1A Is Regulated By Oncogenic KMT2A and Required for Survival of KMT2A-Rearranged Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2742-2742
Author(s):  
Christian Hurtz ◽  
Gerald Wertheim ◽  
Rahul S. Bhansali ◽  
Anne Lehman ◽  
Grace Jeschke ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Negative Regulator ◽  
Advisory Committees ◽  
Normal Tissues ◽  
Pharmacologic Inhibition

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

scholarly journals Differential long non-coding RNA expression profile and function analysis in primary Sjogren’s syndrome

2021 ◽  
Author(s):  
Xiaochan Chen ◽  
Qi Cheng ◽  
Yan Du ◽  
Lei Liu ◽  
Huaxiang Wu
Keyword(s):  
B Cells ◽  
Primary Sjögren’S Syndrome ◽  
Differentially Expressed ◽  
Expression Level ◽  
Primary Sjogren’S Syndrome ◽  
Primary Sjögren's Syndrome ◽  
Cerna Network ◽  
Non Coding Rna ◽  
Long Non Coding Rna ◽  
Healthy Persons

Abstract Background: Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by abnormal immune cell activation. This study aimed to investigate differentially expressed long non-coding RNA (lncRNA) in peripheral blood mononuclear cells (PBMCs) in patients with pSS to identify lncRNAs that affect pSS pathogenesis. Methods: Total RNA was extrated from PBMCs of 30 patients with pSS and 15 healthy persons. Transcriptome sequencing was used to screen differentially expressed lncRNAs and mRNAs in 8 RNA samples from the discovery cohort. The differentially expressed mRNAs underwent functional enrichment analysis. A protein interaction relationship (PPI) and ceRNA network was constructed. Real-time PCR was used to validate screened lncRNAs in all 45 RNA samples. Results: 1180 lncRNAs and 640 mRNAs were differentially expressed in pSS patients (fold change > 2 in healthy persons). The PPI network was constructed with 640 mRNAs and a ceRNA network with four key lncRNAs (GABPB1-AS1, PSMA3-AS1, LINC00847 and SNHG1). RT-PCR revealed that GABPB1-AS1 and PSMA3-AS1 were significantly upregulated 3.0-and 1.4-fold in the pSS group, respectively. The GABPB1-AS1 expression level was positively correlated with the percentage of B cells and IgG levels. Conclusions: GABPB1-AS1 was significently upregulated in pSS patients, and its expression level is positively correlated with the percentage of B cells and IgG levels. GABPB1-AS1 may be involved in the pathogenesis of pSS.

Sensitivity to Wnt Pathway Inhibition in CLL Is Associated with Specific Gene Expression Signatures

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 801-801
Author(s):  
Lili Wang ◽  
Alex K Shalek ◽  
Jellert Gaublomme ◽  
Nir Yosef ◽  
Jennifer R Brown ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Viability ◽  
B Cell ◽  
Cell Survival ◽  
Wnt Pathway ◽  
Differentially Expressed ◽  
Differential Sensitivity ◽  
Specific Gene ◽  
Disease Heterogeneity

Abstract Abstract 801 We have recently found that the Wnt/b-catenin signaling pathway plays a key role in chronic lymphocytic leukemia (CLL). We were, however, intrigued by the question of whether this aberrant pathway may function differently in independent leukemias, and contribute to disease heterogeneity. To assess differential activity of the Wnt pathway across patients, we tested the effects of blocking Wnt activation on CLL cell survival. We knocked down a key downstream gene, LEF1, which is the most differentially expressed gene in CLL compared to normal B cells (based on gene expression microarrays). Addressing this question requires genetic manipulation of primary normal and malignant human B cells, and yet these cells are notoriously difficult to transfect. We therefore focused on developing a method for introducing siRNAs into normal and malignant B cells. We adapted a novel delivery system consisting of vertical silicon nanowires (SiNWs, Shalek et al PNAS 2010) that penetrate the plasma membrane in a minimally invasive fashion and deliver biomolecular cargo directly into the cytoplasm. We achieved consistent and reliable delivery of fluorescently labeled siRNAs (at 50–200 pmol) into normal and CLL B cells. siRNA was delivered to >90% of cells with >85% cell viability remaining after 48 hours. We used this platform to knockdown LEF1 in 20 CLL-B and 5 normal CD19+ B cell samples, and examined cell survival 48 hours after siRNA delivery using an ATP-based CellTiter-Glo assay. Indeed, our studies revealed a heterogeneous response among CLL-B cells to LEF1 inhibition. As a group, CLL-B cells were significantly more sensitive to LEF1 knockdown with a survival rate of 77% (12% s.e.m) compared to 97% (13% s.e.m) in normal B cells. CLL B cells from different patients showed differential sensitivity to LEF1 knockdown, with 8 non-responders, 8 intermediate responders and 4 strong responders (i.e. significant death). Sensitivity to LEF1 inhibition did not correlate with known CLL cytogenetic prognostic factors. To determine if the differential response to LEF1 knockdown was associated with specific gene signatures, we examined gene expression data generated from CLL-B cells from 12 (4 strong, 3 intermediate, and 5 non-responders) of the 20 CLLs tested (using the Affymetrix U133 Plus 2 Array). To increase statistical power, we used each CLL's expression profile (using only genes that showed variability across samples) to create clusters of ∼19 CLLs that showed similar expression profiles (using microarray data from our compendium of 177 additional CLLs). We further reduced the number of genes to ∼4000 genes by retaining only those whose expression levels were significantly different in at least one associated cluster relative to normal CD19+ B cell controls (T-test, FDR<10−4; p-values converted using the Benjamini-Hochberg method). These analyses led to the identification of several hundred genes whose expression correlated significantly with LEF1 knockdown's effect on cell viability. Analysis of these differentially expressed genes identified several potentially important pathways. Ongoing analyses include the identification and validation of a molecular signature for this effect. This signature could enable rapid identification of patients who would be most responsive to therapy with LEF1 inhibitors, which are under development along with other Wnt pathway inhibitors. Disclosures: No relevant conflicts of interest to declare.

ANKL Is Strongly EBV-Related, Frequent In Chromosome Aberrations and Gene Mutations, LMP2A/LMP1-CDK6-MDM2-CD44-Positive and Might Be Sensitive To L-Asparaginase-Containing Regimen

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3012-3012
Author(s):  
Hongsheng Zhou ◽  
Yongjian Deng ◽  
Changxin Yin ◽  
Xiaolei Xue ◽  
Xiangzhao Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Chromosome Aberrations ◽  
Research Funding ◽  
Clinical Course ◽  
Molecular Cytogenetics ◽  
Sh2 Domain ◽  
Clinical Feature ◽  
Hematopoietic Stem ◽  
Aggressive Clinical Course

Abstract Background Aggressive natural killer leukemia (ANKL), previously being classified as large granular lymphocyte (LGL) leukemia or malignant histiocytosis, is a rare subset neoplasm with high aggressive clinical course. Until now, apart from clinical feature, the frequent chromosome aberrations, somatic genes mutations, signal pathway and the optimal treatment regimen are still poor understood. Patient and Methods A retrospective analysis was performed upon review of clinical database, including additional immunohistochemistry (IHC) staining and integrated mutation analysis on reserved samples. Twelve samples of extranodal NK/T-cell lymphoma-nasal type (ENKL-NT) were used in IHC analysis for control. Results A total of 26 cases were collected during 2001 to 2013, including 17 males and 9 females, median age of 28 years old (range 4-76 years old). Most patients presented with acute-onset high swinging fever (n=26), deteriorating jaundice (n=19) and pancytopenia (n=22) at diagnosis. The organ most frequently involved organs were bone marrow (n=25), liver (n=23), spleen (n=22) and gastrointestinal (n=6). Disseminated intravascular coagulopathy (DIC) was present in 15 out of 26 patients, significantly associated with liver involvement and jaundice (p<0.01). Epstein-Barr virus (EBV) viremia was present in 7 of 8 tested cases (5.12x10*,2-5.41x10*,6 copies/mL). The characteristic morphological appearance of ANKL in bone marrow smears were deep purple-red staining granules in cytoplasm and prominent vacuoles located in cytoplasm and/or nucleus. FACS demonstrated ANKL cells were CD2+cCD3+CD7+CD11b+/-CD11c+/-CD29+CD38+/-CD45+CD56+CD86+BCL-2+, CD3-TCR-cCD79a-cMPO-CD5-CD10-CD19-CD25-CD33-CD83-CD123-. Surprisingly, ANKL cells were negative for CD21, an established EBV receptor. Additional IHC on 14 reserved bone marrow samples revealed ANKL cells were strongly positive for LMP2A (n=14), LMP1 (n=11) and EBER (n=10). In ENKL-NT, LMP2A and LMP1 were moderately positive in 5 and 4 out of 12 cases, respectively. EBERs were detected in all ENKT-NT cases. Moreover, IHC analysis showed ANKL cells were strongly stained by CDK6 (n=13), MDM2 (n=14), CD44 (n=13), IFN-γ (n=14), slightly stained by CDK4 (n=2), negative for SH2 domain-containing inositol 5’-phosphatase-1 (SHIP-1), which is responsible for 2B4-mediated inhibition in NK cells. In ENKL-NT samples, CDK6 (n=11), MDM2 (n=9) and CD44 (n=8) were positive; furthermore, SHIP-1 was slightly positive in 4 samples. Chromosome aberrations were present in 7 patients, including dup1(q22q25), inv(3)(p21p25), del(3)(p13), t(3;11)(q21;q23), i(7)(q10), del(7)(q32), del(14)(q24), der(15)t(7;15)(q10;q10), -8, -12,+13,-18,+19,-22. Multiple somatic mutations were detected in 7 cases, including TET2 (D1938E, S69R, V292L, E1207D, G1391C, T1393K, Y1998X), FBXW7 (S427L,Q428K, D431E, R505C, Q508K, G517V, D775Y), CEBPA (E57D, N292K, S85I), FLT3 (M837I, E604X), KRAS (G77R, Q22H, G13D), PAX5 (P93T, A111S), PHF6 (R15S, S155X, V5F), DNMT3A (P602H, H613D), EGFR (E736X, S155X), IDH2 (K166M),SH2B3 (E190X), c-Kit (G812C), JAK1 (D775Y), NOTCH1 (A1701S). Only 1 out of 10 patients obtained partial remission after anthracycline-based or platinum-based regimens (CHOP, CHOP-Bleomycin, CDOP, EPOCH, HyperCVAD and ESHAP). Notably, two cases rapidly achieved durable complete remission after L-asparaginase-containing regimen (CHOP/PEG-L-asp and VDLP). Two patients received allogeneic hematopoietic stem cell transplantation, one maintained durable remission and another died of invasive pulmonary infection before engraftment. The median overall survival of 26 cases was only 7.5 days (range 2-330 days). Conclusions ANKL is Trojan story about EBV and NK cells, being presented with strong evidence of EBV infection/transformation, highly aggressive clinical course and prominent chromosome/genomic instability, which deserves more research efforts to dissect the role of EBV, molecular cytogenetics make-up, signaling pathway of LMP1/LMP2A-PI3K/AKT-CDK6-MDM2 and optimal regimen such as L-asp-contained protocol for this rare leukemia subset. Disclosures: Zhou: Guangzhou Pearl River of Science & Technology New Star (No. 2011J2200069 to HS.Zhou): Research Funding. Liu:863 Program (No. 2011AA020105) and National Public Health Grand Research Foundation ( No. 201202017): Research Funding; National Natural Science Foundation of China (Grant No.81000231, No.81270647) and Science and Technology Program of Guangzhou of China (11A72121174): Research Funding.

GATA2 Expression Level in Chronic Myeloid Leukemia (CML) Patients Correlates with Their Prognostic Scores and Is Associated with Disease Stage at Diagnosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2768-2768
Author(s):  
Philippe Guardiola ◽  
Corentin Orvain ◽  
Nick C.P. Cross ◽  
Martine Gardembas ◽  
Marc Spentchian ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Platelet Count ◽  
Peripheral Blood ◽  
Research Funding ◽  
Scoring Systems ◽  
Gene Expression Dataset ◽  
Expression Level ◽  
Prognostic Scores ◽  
Eutos Score

Abstract The Sokal, Hasford and EUTOS scoring systems have been successively proposed to predict the outcome of CML patients in chronic phase (CP-CML), both in terms of disease response and survival. The EUTOS score, which is the only one developed for patients treated with tyrosine kinase inhibitors, considers 2 risk groups based on spleen size and peripheral blood basophil percentage. No clear link has been identified between the covariates used to calculate those scores and CML molecular pathophysiology. To better delineate the molecular mechanisms underlying those scoring systems, a gene expression profiling study was performed on 48 CP-CML patients included in the Novartis ENEST1st study, for whom good quality peripheral blood total RNA was available at diagnosis. First, patients with low-risk (n=21) and high-risk (n=12) Sokal scores were compared, leading to identify 13 genes differentially expressed between those groups (q<.05). GATA2, which encodes a critical hematopoietic transcription factor, was among the 10 genes significantly overexpressed in high Sokal score CP-CMLs. Robust regression analyses confirmed that GATA2 expression level was correlated to the 3 prognostic scores, when considering those as continuous covariates (R2=.39, p<10-4 [Sokal]; R2=.16, p=.007 [Hasford]; R2=.16, p=.007 [EUTOS]). Among the covariates used to calculate those scores, platelet count (R2=.35, p<10-4), basophil (R2=.22, p=.0012), eosinophil (R2=.14, p=.01) and peripheral blood blast (R2=.13, p=.017) percentages were correlated to GATA2 expression level. Using a quantitative RT-PCR assay, GATA2 expression was subsequently assessed on an independent set of 80 CP-CML patients followed at Angers University Hospital from 2005 to 2014 (Sokal low, n=32; int, n=30; high, n=18 of which 7 EUTOS high). This validation set confirmed that GATA2 expression level was significantly correlated to the 3 prognostic scores (R2=.31, p<10-4 [Sokal]; R2=.28, p<10-4 [EUTOS]; R2=.11, p=.003 [Hasford]). Among the covariates used in those scores, basophil percentage (R2=.44, p<10-4), platelet count (R2=.31, p<10-4) and peripheral blood blast percentage (R2=.22, p<10-4) were significantly correlated to GATA2 expression level in this second cohort. The ENEST1st gene expression dataset and a linear regression approach were then used to better define the role of GATA2 in CP-CMLs. This analysis identified 12 genes significantly correlated to GATA2 in terms of expression levels (q<10-5). Half of those - CPA3, FCER1A, ENPP3, HDC, IL1RL1, SLC45A3 - were basophil-related genes. Interestingly, in normal umbilical cord blood-derived myelopoiesis, the expressions of GATA2, CPA3, FCER1A, and HDC were significantly correlated and coexpressed in common myeloid progenitors (CMP), granulocyte/monocyte progenitors (GMP) and megakaryocyte/erythroid progenitors (MEP) (GEO Microarray, GSE24759). Transcription factor ChIP-Seq data from the ENCODE project confirmed that, in K562 cell line, among the 12 genes identified above, GCSAML, EPAS1, FCER1A, HDC, IL1RL1, MGAT3, RDH11 and SLC45A3 were direct GATA2 targets. To extend this analysis, the gene expression dataset from Radich et al study (Proc Natl Acad Sci USA 2006;103:2794-9 - GEO Microarray, GSE4170), based on 108 peripheral blood samples from patients with CP-CML (n=57), accelerated phase (AP)-CML (n=17) or blastic crisis (BC)-CML (n=22 myeloid BC, n=12 B-cell lymphoblastic BC) were reanalyzed, and confirmed that GATA2 expression level was significantly correlated to the one of the basophil-related genes FCER1A, HDC and SLC45A3 in CP-CMLs. It also showed that GATA2 expression level significantly increased from CP-CMLs to myeloid BC-CMLs, whereas it was expressed at the lowest levels in lymphoblastic BC-CMLs. Similar results were observed when analyzing the dataset from Cramer-Morales et al study (Blood 2013;122:1293-304 - GEO Microarray, GSE47927), in which GATA2 was expressed at significantly higher levels in the hematopoietic stem cell, multipotent progenitors, CMP, GMP, and MEP compartments in myeloid BC-CMLs, when compared to the same compartment in AP-CMLs, CP-CMLs or in healthy volunteers. Therefore, GATA2 is a key gene affecting the outcome of CP-CMLs and the evolution into myeloid BC-CMLs. GATA2 is associated with a basophil-related gene expression signature that might explain the prognostic influence of the basophil percentage within the EUTOS score. Disclosures Gardembas: Novartis: Speakers Bureau. Spentchian:Novartis: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Radich:Gilliad: Consultancy; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Rousselot:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Speakers Bureau.

scholarly journals Repressed Chromatin Drives Leukaemogenesis in Mutant IDH2 Acute Myeloid Leukaemia Via Inhibition of Granulocyte Differentiation and Cell Cycle Progression

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3467-3467
Author(s):  
Douglas RA Silveira ◽  
Prodromos Chatzikyriakou ◽  
Olena Yavorska ◽  
Sarah Mackie ◽  
Roan Hulks ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Research Funding ◽  
Differentially Expressed ◽  
Rna Seq ◽  
Induced Differentiation ◽  
Effector Genes ◽  
Acute Myeloid

Abstract Differentiation arrest in acute myeloid leukaemia (AML) results in accumulation of leukaemic progenitors (L-Prog) and bone marrow failure. Mutant isocitrate dehydrogenase enzyme produces d-2-hydroxyglutarate (2HG), which inhibits α-ketoglutarate-dependent dioxygenases, including Jumonji histone demethylases (JKDM) and TET2, but how this causes AML is unclear. Inhibitors of mutant IDH enzyme (mIDHi) restore differentiation in IDH-mutant (mIDH) AML (Amatangelo et al., 2018). Here, we studied transcriptional networks involved using single-cell (SC) gene expression (GEX) and transcription factor (TF) motif accessibility in primary AML treated with the mIDH2 inhibitor enasidenib (ENA) and found that ENA activates cell cycle (CC) and pro-differentiation programmes through increased promoter accessibility of granulocyte-monocyte (GM)-TF targets. We treated patient L-Prog in vitro with ENA or vehicle, and performed SC RNA-seq (Chromium 10x) in 4 responsive (R), and one non-responsive (NR) patient samples in early, mid and late timepoints. GEX signatures were used to annotate cells according to function (undifferentiated [U], early and late GM [EGM and LGM]) and CC states. In R samples, ENA yielded more dividing late-GM at mid-late timepoints than DMSO (18% vs 6.5%), and more terminally differentiated neutrophils at late timepoints (46% vs 16%). Using SCENIC (Aibar et al., 2017) to assign highly differentially-expressed genes to TF motifs, we computed regulatory networks (regulons, 'R'). Expression of the SP1 R was strongly correlated with active proliferation and ENA conditions led to generation of more cells that co-expressed CEBPA R or CEBPE R with SP1 R, emphasising simultaneous engagement of CC and GM programmes. SP1 function is associated with CC and GM differentiation, and silencing of its binding to its targets contributes to AML pathogenesis (Maiques-Diaz et al., 2012). Control and NR samples failed to produce neutrophils, had reduced co-expression of CEBPE/SP1 R and yielded more poorly differentiated cells expressing GATA2 R. At the individual gene level, ENA stimulated downregulation of GATA2, GFI1B, IKZF1/2, and RUNX3 together with upregulation of immediate early genes which respond to cytokine and mitogenic stimuli (EGR1, IER2, AP-1) in early-mid phase. Later there is upregulation of CEBP TFs and effector genes FUT4, ELANE, AZU1 and PRTN3. Interestingly, expression of some GM-TFs (RUNX1, SPI1/PU.1, GFI1) was similar between ENA and DMSO, indicating that gene expression alone was insufficient for GM differentiation. Given the effects of 2-HG on JKDM, we assessed chromatin accessibility and TF binding using SC ATAC-seq. Overall, we had 25% of differentially accessible (DA) peaks, from which 75% were more accessible in ENA than in DMSO. ENA DA peaks were highly enriched in promoters. Using ArchR (Granja et al., 2021), we clustered cells and used ELANE expression levels to compute trajectories in parallel with SC RNA-seq data. ENA peaks were sequentially enriched for CBF/RUNX and GATA families, followed by AP-1 (JUN/FOS) and EGR/CEBP/KLF motifs. Footprinting analysis showed sequential decrease and increase of TF binding for GATA2 and CEBPA/E respectively during ENA-induced differentiation. Although it did not cause higher expression of SPI1/PU.1, ENA induced increased accessibility of its target binding sites at promoters, which included CEBPA/E and GM effectors (MPO, FUT4, PRTN3). This provides a novel mechanism by which ENA induces differentiation of L-prog. Regulatory network analysis around active, differentially expressed TFs at different phases of ENA-induced differentiation showed a switch from a repressive transcriptional landscape driven by stem-progenitor TFs, to one where AP-1 and GM-TFs activate expression of GM-effector genes. We postulate a model where MYC, E2F8 and EGR1 upregulate the CEBP family in early-mid differentiation. In addition to stimulation of promoter accessibility of TFBS, we find that ENA increases accessibility of cis-regulatory elements of CEBP TFs, adding another mechanism by which differentiation of L-Prog occurs. Our data on the mechanism of action of ENA suggest that differentiation arrest in IDHm AML involves suppression of CC and GM differentiation programs in a repressive chromatin landscape, likely via inhibition of KDM6A and demethylation of repressive H3K27me3 marks. Disclosures Silveira: Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Servier/Agios: Research Funding; BMS/Celgene: Research Funding. Hasan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Vyas: Gilead: Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding.

scholarly journals Single-Cell and Spatial Analyses Characterize Distinct Subsets of Malignant T Cells in Angioimmunoblastic T Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2393-2393
Author(s):  
Francois Lemonnier ◽  
Chuang Dong ◽  
Bruno Tesson ◽  
Laurine Gil ◽  
Noudjoud Attaf ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
T Cell Subsets ◽  
Patient Specific ◽  
Malignant T Cells

Abstract Introduction Angioimmunoblastic T-cell lymphoma (AITL) is the most frequent of nodal peripheral T-cell lymphomas. AITL results from the transformation of T follicular helper (T FH) cells and is characterized by chemo-resistance and poor survival (5-year OS around 30%). Recent data from prospective clinical trials suggest that disease outcome may be impacted by factors other than genomic features, such as the tumor microenvironment (TME) and overall intra-tumoral heterogeneity. Our understanding of AITL intra-tumoral genetic, transcriptional and functional heterogeneity is limited because most molecular data generated so far have come from bulk analyses. Single-cell RNA sequencing (scRNA-seq) enables fine characterization of cell types and functional cell states. When focused on T or B cells, 5'-end scRNA-seq also yields the TCR or BCR sequences that allow tracking clonally related cells. Here we studied the intra-tumor heterogeneity of AITL tumors using integrative scRNA-seq. Methods We analyzed lymph node live cell suspensions from AITL patients (n=10) using droplet-based 10x Genomics 5'-end scRNA-seq. Malignant T cells from 4 AITL samples were also analyzed by FACS index sorting and plate-based 5'-end scRNA-seq to link cell surface phenotype and gene expression profile. We identified malignant T cell clones by intersecting the gene expression and TCR sequencing data, and performed separate focused analyses of TME subsets and malignant T cells. We compared subsets of malignant T cells from all patients using marker gene-based metaclustering to identify AITL T cell states conserved across patients. We explored the genetic heterogeneity of malignant T cells by mapping RHOA G17V mutations and inferring copy number variation (CNV) subclones from scRNA-seq data. In select cases, we performed in situ analysis by immunohistochemistry (IHC) or spatial transcriptomics to characterize the spatial distribution of malignant T cell subsets identified by scRNA-seq. Results Based on gene expression, malignant T cells grouped in patient-specific clusters, while non-malignant T, B and myeloid TME cells from all patients clustered by cell type or cell state. Among TME cells, we identified 7 subsets of B cells (including activated B cells, plasma cells, and one patient-specific monoclonal B cell proliferation), 6 subsets of myeloid cells (including macrophages, conventional and plasmacytoid dendritic cells), and 8 subsets of non-malignant T cells (including activated cytotoxic T lymphocytes (CTL) with clonal expansions). Patient-specific malignant T cells were heterogeneous and divided into several gene-expression based clusters. Metaclustering of malignant T cell subsets identified T central memory (T CM)-like and T FH-like states in 10/10 samples. We also identified in 3/10 samples clusters of CTL-like malignant T cells expressing characteristic marker genes (including NKG7, GNLY, GZMK, PRF1). We observed an intra-sample continuum of gene expression states from quiescent T CM-like to proliferating T FH-like states. T FH-like cells were larger in size and expressed higher levels of surface PD1 and ICOS than T CM-like and CTL-like subsets. We detected the RHOA G17V mutation in malignant T cells of 4/4 mutated cases, with no evidence of subclonal heterogeneity for that mutation. We detected clonal and subclonal CNV in most AITL malignant T cells. CTL-like states were enriched in specific CNV subclones, but the T CM-like to T FH-like continuum was observed in all CNV subclones, suggesting that functional plasticity and subclonal genetic evolution may occur independently. In situ staining of markers for T FH-like (PD1, ICOS, CD200) and CTL-like (GZMK, GZMA) cells showed that T FH-like and CTL-like cells occupied distinct tissue niches within the tumor. In spatial transcriptomics analysis, T FH-like cells mapped to follicular dendritic cell (FDC)-rich areas, while T CM-like cells were associated with T-zone reticular cells. Conclusions Our analyses recapitulate known characteristics of AITL TME, and uncover previously unrecognized heterogeneity among malignant T cells across multiple patients. The distinct gene expression programs, phenotypes, genetics, and locations of T FH-like, T CM-like and CTL-like states suggest that AITL malignant T cells undergo significant functional plasticity and genetic divergence, which could influence response to therapy and overall clinical course. Figure 1 Figure 1. Disclosures Lemonnier: Institut Roche: Research Funding; Gilead: Other: travel grant. Gaulard: Gilead: Consultancy; Innate Pharma: Research Funding; Sanofi: Research Funding; Alderaan: Research Funding; Takeda: Consultancy, Honoraria. Milpied: Institut Roche: Research Funding; Innate Pharma: Research Funding; Bristol Myers Squibb: Research Funding.

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