scholarly journals Low-Dose Ionizing Radiation Modulates Expression of Gpam in Immortalized EBV Infected B Cells through miRNA Network

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4829-4829
Author(s):  
Yoko Tabe ◽  
Yasuhito Hatanaka ◽  
Mayumi Nakashiro ◽  
Kazumasa Sekihara ◽  
Shinichi Yamamoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Ionizing Radiation ◽  
Mirna Expression ◽  
Stromal Cells ◽  
Research Funding ◽  
Low Dose ◽  
Binding Protein ◽  
B Cell Lymphoma ◽  
Stromal Microenvironment

Abstract Low-dose ionizing radiation (LDIR, ≤ 0.1 Gy) which is typically associated with therapeutic and diagnostic radiological modalities, is known to induce remodeling of the stromal microenvironment. Whereas the biologic responses to LDIR are commonly described as a stress response, the carcinogenic potential of this environmental stressor remains unknown. Recently, ionizing radiation (IR)-induced alterations of miRNA expression that play a fundamental role in cell signaling events, have been demonstrated in various cell types (Marsit, Cancer Res. 2006). To assess a potential determinant influencing of LDIR induced miRNA alterations in pre-malignant cells in a microenvironment, we utilized immortalized pre-malignant Epstein-Barr virus infected-B (EBV-B) cells which are continuously proliferating in circulation or in lymph nodes. The LDIR system (0.1 Gy, 4MV X ray from a LINAC) was utilized in the in vitro co-culture of EBV-B and mesenchymal stromal cells (MSC) to mimic the lymph node stromal microenvironment. We confirmed that MSC protected co-cultured EBV-B cells from spontaneous apoptosis and caused accumulation of EBV-B cells in the G0/G1 phase (EBV-B; monoculture vs coculture with MSC; Sub G1% 42.0±2.4 vs 34.1±0.9 p< 0.01, G0/G1 % 42.6±2.1 vs 47.6±2.0, p<0.05). To identify LDIR-induced modulation of specific miRNAs in EBV-B cells, a high-confidence list of 44 known LDIR-associated miRNAs from three separate screening studies of lymphoblastic cells was assembled (Cha et al., Oncol Rep.2009, Chaudhry et al., J Biomed Sci. 2010, Lhakhang et al., Comp Funct Genomics. 2012). From these miRNA, we focused on four miRNAs let-7a, miR-16, miR-19b and miR-21 that have been listed in more than two of the screens. Our studies showed that LDIR upregulated let-7a and miR-16 levels in irradiated mono-cultured EBV-B, but on the contrary downregulated all tested miRNAs in MSC co-cultured EBV-B cells (at 24-hour). We next investigated alterations of EBV-B gene expression by LDIR using the DNA microarray (Affymetrix). In mono-cultured EBV-B cells, cDNA microarray analysis detected upregulation of TGFB1 mRNA, and downregulation of the genes encoding a lipid biosynthesis enzyme glycerol-3-phosphate acyltransferase (GPAM), a growth factor amphiregulin (AREG), cell survival-/growth-related factors Rho-GTPase effector gene folmin-1 (FMN1), chemotaxis-inducing chemokine gene IL-8 and B-cell lymphoma 2 gene BCL2, all of which were confirmed by qRT-PCR. Downregulation of the GPAM protein was further shown on the protein level by western blot, and the ontology analysis demonstrated that LDIR caused the TGFbeta-dependent induction of the epithelial-mesenchymal transition (EMT) pathway in mono-cultured EBV-B cells. In contrast to the mono-culture condition, the GPAM mRNA and protein expression were upregulated in MSC co-cultured EBV-B cells. To determine the potential targets of LDIR-altered miRNAs, we next identified 79 genes that are commonly targeted by let-7a, miR-16, miR-19b, or miR-21 from a microrna.org database, and found that the expression changes of GPAM mRNA and protein were strikingly matched with miRNA profiling. Partial concordant changes of DNAJ(HSP40)A2 coding heat shock protein (HSP) 40 homolog, a cochaperone of HSP70s, CPEB3 coding RNA binding protein cytoplasmic polyadenylation element binding protein 3 (CEBP3) and its transcriptional target GLUR2 (glutamate receptor subunit), a receptor of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), were also observed in the microarray results. Since GPAM is a lipid-related metabolism gene which is known to be regulated by miR-27b and to negatively impact survival of breast cancer cells we next investigated the expression changes of miR-27b after LDIR in EBV-B cells. As expected, LDIR upregulated miR-27b in mono-cultured EBV-B cells, and downregulated in co-cultured EBV-B cells with MSC. Intriguingly, no significant changes of GPAM mRNA as well as tested miRNA expression was observed by 1Gy irradiation. In summary, we demonstrated that LDIR directly modulates EBV-B cells gene expression. Importantly, LDIR additionally impacts EBV-B cells indirectly through miRNA modulation by the neighboring stromal cells. Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Integrative Genomic and Proteomic Analysis Of Low-Dose ionizing Irradiation Effects On Bone Marrow Stromal Microenvironment and On Survival Of Pre-Leukemic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3775-3775
Author(s):  
Yoko Tabe ◽  
Linhua Jin ◽  
Yasuhito Hatanaka ◽  
Hiromichi Matsushita ◽  
Saiko Kazuno ◽  
...  
Keyword(s):  
B Cells ◽  
Ionizing Radiation ◽  
Stromal Cells ◽  
Low Dose ◽  
Culture Conditions ◽  
Leukemic Cells ◽  
Cell Growth Inhibition ◽  
Stromal Microenvironment ◽  
Viable Cells ◽  
Related Proteins

Abstract Ionizing radiation is known to induce remodeling of stromal microenvironment and enhance cancer progression. In this study, we investigated the molecular alterations of low-dose ionizing radiation (LDIR) induced non-targeted/bystander responses which affect a complex interplay of stromal cells and pre-leukemic cells in the bone marrow (BM) microenvironment. As a model of BM stromal cells and pre-leukemic cells, we utilized primary BM-derived stromal cells (MSCs) and the Epstein- Barr virus (EBV) infected and immortalized pre-leukemic B-lymphocyte cell line (EBV-B). LDIR (100 mGy, 4MV X ray from a LINAC) caused cell growth inhibition and moderate apoptosis induction in MSCs (viable cells % of control; 75.8 ± 2.4, specific SubG1 % 7.1 ± 0.8 at 24 h) but not in EBV-B cells. We further observed persistent upregulation of p21 mRNA (p< 0.001, RQ-PCR) after acute low-dose irradiation in MSCs but not in EBV-B cells, suggesting radiation induced senescence-like changes in MSCs. In EBV-B cells co-cultured with MSCs, low-dose irradiation induced moderate cell growth inhibition (viable cells % of control; 81.3 ± 6.5) without significant apoptosis induction. To gain insights into the molecular changes induced by LDIR in both, MSC and EBV-B cells we utilized genomic and proteomic analyses. To exclude possible contamination of MSCs, we confirmed negative expression of CD90 mRNA in the tested EBV-B samples. We first screened up to 28,869 genes by cDNA microarray (Affymetrix) and performed functional network analysis by MetaCore (GeneGo). LDIR induced upregulation of 48 genes and the downregulation of 45 (i.e., > 1.3-fold regulation) with prominent stimulation of cell adhesion pathways in MSCs. Of note, 31 of 48 up-regulated genes were small nucleolar RNAs. In EBV-B cells, LDIR upregulated 69 genes/downregulated 130 genes with significant stimulation of the TGFbeta dependent induction of epithelial-mesenchymal transition (EMT) pathways. In EBV-B cells co-cultured with MSCs, LDIR induced immune response signaling along with integrin-mediated cell-matrix adhesion pathway with 42 genes upregulation / 34 genes downregulation. Up-regulation of inflammatory IL8 mRNA (2.0±0.03 fold, p<0.001) by LDIR were further detected in EBV-B cells co-cultured with MSCs. Since ionizing radiation is known to change levels of specific microRNA depending on cell type, we investigated the changes of four microRNA, let-7a, miR-16, miR-19b and miR-21 by RT-PCR. After acute LDIR, all of the tested miRNAs were upegulated in EBV-B cells cultured alone, but downregulated in EBV-B cells co-cultured with MSCs. These changes were accompanied by the coordinate modulation of their common target GPAM mRNA, a key enzyme of phospholipids, and ribozyme CPEB3 mRNA. Indeed, proteomic analysis by isobaric tags for relative and absolute quantitation (iTRAQ, Applied Biosystems) detected the upregulation of Protein kinase C delta (p = 0.04) which is strictly dependent on the presence of phospholipids. iTRAQ detected more than 1500 proteins in MSCs and EBV-B cells. In MSCs LDIR resulted in 32 upregulated proteins and 1 downregulated protein (p < 0.05) with the activation of focal adhesion (6 proteins, p < 0.001) and apoptosis/senescence pathways (three proteins, p = 0.02), concordant with cDNA microarray data. In EBV-B cells, LDIR induced upregulation of 47 proteins including 19 ribosome related proteins (40%) and downregulation of 19 proteins including 6 apoptosis/senescence related proteins, suggesting an activation of molecular repair mechanisms from LDIR-triggeredstress. In contrast, under MSC co-culture conditions LDIR downregulated 25 of ribosomal proteins (74% of the 34 downregulated proteins) in EBV-B cells, indicating suppression of the ribosomal biogenesis and translational activity, which might cause the observed cell quiescence with cell cycle arrest of EBV-B cells mediated by the low-dose irradiated neighboring MSCs. In summary, we have demonstrated the LDIR effects on BM-derived stromal cells and pre-leukemic cells under MSC co-culture conditions mimicking the BM microenvironment. We conclude that LDIR may support pre-malignant cells survival via direct activation of TGFbeta dependent EMT pathways, which contribute to antiapoptosis, and via interactions with irradiated neighboring BM stromal cells which promote quiescence and survival of pre-leukemic cells. Disclosures: No relevant conflicts of interest to declare.

The Actin Binding Protein Plastin-3 Is Involved in the Pathogenesis of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1662-1662
Author(s):  
Arne Velthaus ◽  
Kerstin Cornils ◽  
Saskia Grüb ◽  
Hauke Stamm ◽  
Daniel Wicklein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Binding Protein ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Mutational Status ◽  
Acute Myeloid

Abstract Leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where they undergo complex interactions with their surrounding stromal cells. In order to identify genes being implicated in the interaction of acute myeloid leukemia (AML) cells and stromal cells, we performed co-cultures of primary AML cells with primary endothelial cells and osteoblasts. The gene expression of co-cultured AML blasts was compared to AML cells grown without adherent cells using microarray analysis. Amongst those genes being dysregulated upon co-culture was the actin binding protein plastin-3 (PLS3). Further RT-qPCR analysis revealed an endogenous PLS3 expression in about 50% of BM samples from AML patients (n=25). In contrast, expression of PLS3 was only detected in 2 of 12 analyzed AML cell lines with Kasumi-1 showing strong and THP-1 showing only weak expression. Therefore, functional analysis of PLS3 in AML was studied using shRNA knockdown and overexpression of PLS3 in Kasumi-1 cells. We could show that PLS3 has an impact on the colony formation capacity of AML cells in vitro as the knockdown resulted in significantly reduced colony numbers while increased colony growth was observed in the Kasumi-1 cells overexpressing PLS3 (p<0.001 and p<0.001, respectively). To investigate the role of PLS3 in vivo, NSG mice were transplanted with the PLS3 knockdown Kasumi-1 cells. Compared to mice transplanted with Kasumi-1 cells transduced with a vector carrying a scrambled shRNA, the PLS3 knockdown mice survived significantly longer (median survival time 64 vs. 110 days, respectively; p<0.001; n=9 mice per group). Finally, we investigated whether the expression of PLS3 was associated with AML patients' outcome using published microarray-based gene expression data (Verhaak et al, Haematologica 2009;94). Clinical data of 290 AML patients were available. Based on the mean gene expression value, the patient cohort was divided into high vs low PLS3 expressors. The overall survival was analyzed in a multivariate Cox proportional hazards model including PLS3 gene expression and the baseline parameters age, karyotype and FLT3 mutational status. After a stepwise removal of insignificant terms, the patient's age and a high PLS3 expression remained as independent prognostic survival markers (for PLS3: HR 1.58 (CI 1.05 - 2.37) and for age: HR 1.01 (CI 1.00 - 1.03)). In conclusion, our results identify the actin binding protein PLS3 as potential novel therapeutic target in AML. Disclosures Stamm: Astellas: Other: Travel, Accommodation, Expenses. Heuser:BerGenBio: Research Funding; Tetralogic: Research Funding; Novartis: Consultancy, Research Funding; Celgene: Honoraria; Bayer Pharma AG: Research Funding; Pfizer: Research Funding; Karyopharm Therapeutics Inc: Research Funding. Fiedler:Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; Teva: Other: Travel; GSO: Other: Travel; Pfizer: Research Funding; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding. Wellbrock:Astellas: Other: Travel, Accommodation, Expenses.

scholarly journals CG'806, a First-in-Class Pan-FLT3/Pan-BTK Inhibitor, Exhibits Broader and Greater Potency Than Ibrutinib Against Primary and Cultured Malignant B Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3503-3503
Author(s):  
Hongying Zhang ◽  
Andrea Local ◽  
Khalid Benbatoul ◽  
Peter Folger ◽  
Susan Sheng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
B Cell Malignancies ◽  
Apoptotic Effect ◽  
Btk Inhibitor ◽  
Induced Apoptosis

Abstract Mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) account for >70% of cases of B cell lymphoma. Targeting Bruton tyrosine kinase (BTK) with ibrutinib in B cell malignancies led to a paradigm shift in therapy. However, primary resistance to ibrutinib has been observed in about 30% MCL patients; more than 50% patients with CLL and MCL treated with ibrutinib discontinue treatment due to intolerance or emergence of resistant disease (Woyach et al., 2017; Shpilberg et al., 2018). CG'806 is an oral small molecule non-covalent pan-FLT3/pan-BTK inhibitor designed to address the shortcomings of ibrutinib. It is in development for acute myeloid leukemia (AML) and B cell lymphoma. CG'806 inhibited cell proliferation and induced apoptosis with a potency that was 50-6,000 times greater than that of ibrutinib when tested against 14 established malignant B-cell lines in vitro. When tested against 124 samples freshly isolated from the marrow of CLL patients the median IC50 for CG'806 was 0.11 µM and the median for ibrutinib was 4.09 µM, respectively, p<0.001). Since stromal-mediated signaling plays important roles in malignant B cell survival and chemoresistance, the apoptotic effect of CG'806 was further analyzed on cultured and primary malignant B cells in the presence of stromal cells. CG'806 produced similar dose-dependent apoptotic effect on Mino cells, an MCL cell line, in the absence or presence of human stromal HS5 cells indicating that its potency was not impaired by factors released by these stroma cells. Most importantly, CG'806 dose-dependently induced apoptosis in ibrutinib-refractory primary MCL samples in the presence of CD40L-expressing stromal cells (N=4). Whereas 0.1 µM and 1 µM CG'806 caused about 25% and 45% apoptotic cell death, respectively, 1 µM ibrutinib induced less than 10% cell death under the same culture conditions. CG'806 inhibited malignant B cell colony formation and migration towards SDF1α about 2-fold more effectively than ibrutinib. Given the role of activated B cell receptor (BCR) and NFκB pathways in lymphoma, CG'806 was tested for its ability to impair signaling in these pathways. CG'806 produced cell line dependent and dose/time dependent decreases in the phosphorylation of BTK, PLCγ2, PI3K, AKT, mTOR, PKC, and ERK, and reduced. These effects were correlated with induction of PARP cleavage and cell cycle arrest. We conclude that CG'806 inhibits driver and rescue pathways to directly and potently kill a broad range of malignant B cells, including both establish cell lines and freshly isolated patient samples, thereby distinguishing CG'806 from ibrutinib and supporting clinical development of CG'806 in patients with CLL and other B-cell malignancies intolerant, resistant, or refractory to ibrutinib. Disclosures Zhang: Aptose Biosciences, Inc: Employment. Local:Aptose Biosciences, Inc: Employment. Benbatoul:Aptose Biosciences, Inc: Employment. Folger:Aptose Biosciences, Inc: Employment. Sheng:Aptose Biosciences, Inc: Employment. McLaughlin:Aptose Biosciences, Inc: Other: internship. Danilov:Astra Zeneca: Consultancy; Genentech: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Takeda Oncology: Research Funding. Tyner:Constellation: Research Funding; Aptose: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding. Howell:Aptose Biosciences, Inc: Research Funding. Rice:Aptose Biosciences, Inc: Equity Ownership.

scholarly journals Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein

2005 ◽  
Vol 203 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Chang Hoon Lee ◽  
Mark Melchers ◽  
Hongsheng Wang ◽  
Ted A. Torrey ◽  
Rebecca Slota ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Transcriptional Activation ◽  
Germinal Center ◽  
Target Genes ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Consensus Sequence ◽  
B Cell Lymphoma ◽  
Regulatory Factor

Interferon (IFN) consensus sequence-binding protein/IFN regulatory factor 8 (IRF8) is a transcription factor that regulates the differentiation and function of macrophages, granulocytes, and dendritic cells through activation or repression of target genes. Although IRF8 is also expressed in lymphocytes, its roles in B cell and T cell maturation or function are ill defined, and few transcriptional targets are known. Gene expression profiling of human tonsillar B cells and mouse B cell lymphomas showed that IRF8 transcripts were expressed at highest levels in centroblasts, either from secondary lymphoid tissue or transformed cells. In addition, staining for IRF8 was most intense in tonsillar germinal center (GC) dark-zone centroblasts. To discover B cell genes regulated by IRF8, we transfected purified primary tonsillar B cells with enhanced green fluorescent protein–tagged IRF8, generated small interfering RNA knockdowns of IRF8 expression in a mouse B cell lymphoma cell line, and examined the effects of a null mutation of IRF8 on B cells. Each approach identified activation-induced cytidine deaminase (AICDA) and BCL6 as targets of transcriptional activation. Chromatin immunoprecipitation studies demonstrated in vivo occupancy of 5′ sequences of both genes by IRF8 protein. These results suggest previously unappreciated roles for IRF8 in the transcriptional regulation of B cell GC reactions that include direct regulation of AICDA and BCL6.

scholarly journals Bone Marrow Lymphoid Niche Adaptation to Mature B Cell Neoplasms

2021 ◽  
Vol 12 ◽  
Author(s):  
Erwan Dumontet ◽  
Stéphane J. C. Mancini ◽  
Karin Tarte
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Tumor Cells ◽  
Stromal Cells ◽  
Therapeutic Option ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Niche Adaptation ◽  
Functional Features

B-cell non-Hodgkin lymphoma (B-NHL) evolution and treatment are complicated by a high prevalence of relapses primarily due to the ability of malignant B cells to interact with tumor-supportive lymph node (LN) and bone marrow (BM) microenvironments. In particular, progressive alterations of BM stromal cells sustain the survival, proliferation, and drug resistance of tumor B cells during diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). The current review describes how the crosstalk between BM stromal cells and lymphoma tumor cells triggers the establishment of the tumor supportive niche. DLBCL, FL, and CLL display distinct patterns of BM involvement, but in each case tumor-infiltrating stromal cells, corresponding to cancer-associated fibroblasts, exhibit specific phenotypic and functional features promoting the recruitment, adhesion, and survival of tumor cells. Tumor cell-derived extracellular vesicles have been recently proposed as playing a central role in triggering initial induction of tumor-supportive niches, notably within the BM. Finally, the disruption of the BM stroma reprogramming emerges as a promising therapeutic option in B-cell lymphomas. Targeting the crosstalk between BM stromal cells and malignant B cells, either through the inhibition of stroma-derived B-cell growth factors or through the mobilization of clonal B cells outside their supportive BM niche, should in particular be further evaluated as a way to avoid relapses by abrogating resistance niches.

scholarly journals Changes in the Multipotent Stromal Mesenchymal Cells from the Bone Marrow of the Patients with Hematological Diseases in Debut and after the Treatment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5014-5014
Author(s):  
Irina N. Shipounova ◽  
Nataliya A. Petinati ◽  
Nina J. Drize ◽  
Aminat A. Magomedova ◽  
Ekaterina A. Fastova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Marker Gene ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Stromal Microenvironment ◽  
Expression Of Genes

Introduction. Stromal microenvironment of the bone marrow (BM) is essential for normal hematopoiesis; the very same cells are involved in the interaction with the leukemic stem cells. The aim of the study was to reveal the alterations in stromal microenvironment of patients in debut and after the therapy using multipotent mesenchymal stromal cells (MSC) as a model. Methods. MSC of patients with acute myeloid leukemia (AML, N=32), acute lymphoblastic leukemia (ALL, N=20), chronic myeloid leukemia (CML, N=19), and diffuse large B-cell lymphoma without BM involvement (DLBCL, N=17) were isolated by standard method from the patients' BM. Each BM sample was acquired during diagnostic aspiration after the informed signed consent was obtained from the patient. Groups of BM donors comparable by age and gender were used as controls for each nosology. Gene expression was analyzed with real-time RT-PCR. The significance of differences was evaluated with Mann-Whitney U-test. Results. The results of gene expression analysis are summarized in Table. The expression of genes regulating hematopoietic stem and precursor cells (JAG1, LIF, IL6) was significantly upregulated in MSC of the patients in debut, except for DLBCL. The latter was characterized with upregulation of osteogenic marker gene SPP1 and downregulation of FGFR1 gene. The upregulation of the expression of genes regulating proliferation of stromal cells (PDGFRA, FGFR1) and adipogenic marker gene (PPARG) was common for AML and CML. Both acute leukemias were characterized by the upregulation of genes associated with inflammation and regulation of hematopoietic precursors (CSF1, IL1B, IL1BR1) and by the downregulation of chondrogenic differentiation marker gene (SOX9). CML and DLBCL demonstrated the upregulation of FGFR2. BM of the DLBCL patients did not contain any malignant cells; nevertheless, stromal precursors from the BM were significantly affected. This indicates the distant effects of DLBCL malignant cells on the patients' BM. Myeloid malignancies seem to affect MSC more profoundly then lymphoid ones. Effect of leukemic cells on stromal microenvironment in case of myeloid leukemia was more pronounced. The treatment significantly affected gene expression in MSC of patients. In all studied nosologies the IL6 gene expression was upregulated, which may reflect the inflammation processes ongoing in the organism. The expression of LIF was upregulated and ICAM1, downregulated in MSCs of AML, ALL, and CML patients. In the MSC of patients with AML, who had received the highest doses of cytostatic drugs to achieve remission, a significant decrease in the expression of most studied genes was found. In patients with ALL with long-term continuing treatment in combination with lower doses of drugs, IL1B expression was increased, while the decrease in expression was detected for a number of genes regulating hematopoietic stem cells (SDF1, TGFB1), differentiation and proliferation (SOX9, FGFR1, FGFR2). Treatment of CML patients is based on tyrosine kinase inhibitors in doses designed for long-term use, and is less damaging for MSC. The upregulation of TGFB1, SOX9, PDGFRA genes and downregulation of IL1B gene was revealed. MCS of DLBCL patients, unlike the other samples, were analyzed after the end of treatment. Nevertheless, significant upregulation of IL8 and FGFR2 genes was found. Thus, both the malignant cells and chemotherapy affect stromal precursor cells. The changes are not transient; they are preserved for a few months at least. MSCs comprise only a minor subpopulation in the BM in vivo. When expanded in vitro, they demonstrate significant changes between groups of patients and healthy donors. Conclusions. Leukemia cells adapt the stromal microenvironment. With different leukemia, the same changes are observed in the expression of genes in MSC. MSC of patients with acute forms have a lot of changes which coincide among these two diseases. MSC of AML patients are most affected both in debut and after the therapy. Treatment depends on the nosology and in varying degrees changes the MSC. This work was supported by the Russian Foundation for Basic Research, project no. 17-00-00170. Disclosures Chelysheva: Novartis: Consultancy, Honoraria; Fusion Pharma: Consultancy. Shukhov:Novartis: Consultancy; Pfizer: Consultancy. Turkina:Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; fusion pharma: 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.

Gene expression changes in mouse brain after exposure to low‐dose ionizing radiation

2003 ◽  
Vol 79 (10) ◽  
pp. 759-775 ◽  
Author(s):  
E. Yin ◽  
D. O. Nelson ◽  
M. A. Coleman ◽  
L. E. Peterson ◽  
A. J. Wyrobek
Keyword(s):  
Gene Expression ◽  
Ionizing Radiation ◽  
Mouse Brain ◽  
Low Dose

New Pathogenetic Insights Into Classical Hodgkin Lymphoma Revealed by Gene Expression Profiling of Microdissected Hodgkin/Reed-Sternberg Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 266-266 ◽  
Author(s):  
Enrico Tiacci ◽  
Verena Brune ◽  
Susan Eckerle ◽  
Wolfram Klapper ◽  
Ines Pfeil ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Gene Expression Profiling ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Expression Profiles ◽  
B Cell Lymphoma ◽  
Cell Phenotype

Abstract Abstract 266 Background. Previous gene expression profiling studies on cHL have been performed on whole tissue sections (mainly reflecting the prominent reactive background in which the few HRS cells are embedded), or on cHL cell lines. However, cultured HRS cells do not likely reflect primary HRS cells in all aspects, being derived from end-stage patients and from sites (e.g. pleural effusions or bone marrow) which are not typically involved by cHL and where HRS cells lost their dependence on the inflammatory microenvironment of the lymph node. Methods. ∼1000–2000 neoplastic cells were laser-microdissected from hematoxylin/eosin-stained frozen sections of lymph nodes taken at disease onset from patients with cHL (n=16) or with various B-cell lymphomas (n=35), including primary mediastinal B-cell lymphoma (PMBL) and nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL). After two rounds of in vitro linear amplification, mRNA was hybridized to Affymetrix HG-U133 Plus 2.0 chips. Expression profiles were likewise generated from sorted cHL cell lines and several normal mature B-cell populations. Results. Primary and cultured HRS cells, although sharing hallmark cHL signatures such as high NF-kB transcriptional activity and lost B-cell identity, showed considerable transcriptional divergence in chemokine/chemokine receptor activity, extracellular matrix remodeling and cell adhesion (all enriched in primary HRS cells), as well as in proliferation (enriched in cultured HRS cells). Unsupervised and supervised analyses indicated that microdissected HRS cells of cHL represent a transcriptionally unique lymphoma entity, overall closer to nLPHL than to PMBL but with differential behavior of the cHL histological subtypes, being HRS cells of the lymphocyte-rich and mixed-cellularity subtypes close to nLPHL cells while HRS cells of NS and LD exhibited greater similarity to PMBL cells. HRS cells downregulated a large number of genes involved in cell cycle checkpoints and in the maintenance of genomic integrity and chromosomal stability, while upregulating gene and gene signatures involved in various oncogenic signaling pathways and in cell phenotype reprogramming. Comparisons with normal B cells highlighted the lack of consistent transcriptional similarity of HRS cells to bulk germinal center (GC) B cells or plasma cells and, interestingly, a more pronounced resemblance to CD30+ GC B cells and CD30+ extrafollicular B cells, two previously uncharacterized subsets that are transcriptionally distinct from the other mature B-cell types. Conclusions. Gene expression profiling of primary HRS cells provided several new insights into the biology and pathogenesis of cHL, its relatedness to other lymphomas and normal B cells, and its enigmatic phenotype. Disclosures: No relevant conflicts of interest to declare.

IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2431-2431
Author(s):  
Marta Coscia ◽  
Francesca Pantaleoni ◽  
Chiara Riganti ◽  
Candida Vitale ◽  
Micol Rigoni ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Board Of Directors ◽  
Stromal Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Spontaneous Apoptosis ◽  
Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

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