scholarly journals Development of a Notch pathway assay and quantification of functional Notch pathway activity in T-cell acute lymphoblastic leukemia

2020 ◽  
Author(s):  
Kirsten Canté-Barrett ◽  
Laurent Holtzer ◽  
Henk van Ooijen ◽  
Rico Hagelaar ◽  
Valentina Cordo ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Signal Transduction Pathway ◽  
Activity Score ◽  
Mrna Levels ◽  
Pathway Activity ◽  
Cell Acute Lymphoblastic Leukemia

AbstractThe Notch signal transduction pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis of many diseases including T-cell acute lymphoblastic leukemia. Various targeted drugs are available that inhibit Notch pathway signaling, but their effectiveness varies due to variable Notch pathway activity among individual patients. Quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. We here describe a new assay that infers a quantitative Notch pathway activity score from mRNA levels of conserved direct NOTCH target genes. Following biological validation, we assessed Notch pathway activity in a cohort of TALL patient samples and related it to biological and clinical parameters including outcome. High Notch pathway activity was not limited to T-ALL samples harbouring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate NOTCH signaling. The measured Notch pathway activity related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.

scholarly journals miR-22-3p Negatively Affects Tumor Progression in T-Cell Acute Lymphoblastic Leukemia

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1726
Author(s):  
Valentina Saccomani ◽  
Angela Grassi ◽  
Erich Piovan ◽  
Deborah Bongiovanni ◽  
Ludovica Di Martino ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Target Genes ◽  
Cell Transformation ◽  
Lymphoblastic Leukemia ◽  
T Cell Development ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Notch1 Signaling Pathway

T-cell acute lymphoblastic leukemia (T-ALL) is a rare, aggressive disease arising from T-cell precursors. NOTCH1 plays an important role both in T-cell development and leukemia progression, and more than 60% of human T-ALLs harbor mutations in components of the NOTCH1 signaling pathway, leading to deregulated cell growth and contributing to cell transformation. Besides multiple NOTCH1 target genes, microRNAs have also been shown to regulate T-ALL initiation and progression. Using an established mouse model of T-ALL induced by NOTCH1 activation, we identified several microRNAs downstream of NOTCH1 activation. In particular, we found that NOTCH1 inhibition can induce miR-22-3p in NOTCH1-dependent tumors and that this regulation is also conserved in human samples. Importantly, miR-22-3p overexpression in T-ALL cells can inhibit colony formation in vitro and leukemia progression in vivo. In addition, miR-22-3p was found to be downregulated in T-ALL specimens, both T-ALL cell lines and primary samples, relative to immature T-cells. Our results suggest that miR-22-3p is a functionally relevant microRNA in T-ALL whose modulation can be exploited for therapeutic purposes to inhibit T-ALL progression.

scholarly journals Identification of a New Targeted Therapy for T-Cell Acute Lymphoblastic Leukemia and Studies on Its Mechanism of Action

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2752-2752
Author(s):  
Kinjal Shah ◽  
Julhash U. Kazi
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Predictive Biomarkers ◽  
Protein Kinase Inhibitors ◽  
Maturation Stage ◽  
Mrna Levels ◽  
Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy, of which T- cell acute lymphoblastic leukemia (T-ALL) constitutes an aggressive subset. Due to the advent of new therapies, T-ALL now has a 5-year event-free survival (EFS) rate exceeding 85%. However, some patients still relapse and display resistance to therapy. Moreover, adverse side-effects of intensive chemotherapy worsen the duration of treatment. Therefore, we still need to improve our current treatment beyond that of the chemotherapeutic approaches. It has been shown that the maturation stage of T-ALL decides its dependency on Bcl-2/Bcl-xL. The immature early T cell progenitor ALL (ETP-ALL) rely on Bcl-2 for their survival while all the other stages of T-ALL and primary patient samples depend on Bcl-xL. Bcl-2 inhibitors have thus shown to display promising antitumor activity against ETP-ALL, a subgroup with a high risk of relapse, but with a variable response across these patients. Therefore, there is a need for predictive biomarkers and further investigation towards finding a combination of drugs for the treatment of these patients. Methodology & Aim: We screened 10 different T-ALL cell lines with a combination of Bcl-2 inhibitor and a panel of 378 protein kinase inhibitors and identified polo-like kinase inhibitor as a promising candidate. We thus aimed to study the combined effect of Bcl-2 and PLK1 inhibition in a panel of T-ALL cell lines and in a PDX model of chemo-resistant childhood T-ALL. We also investigated the underlying mechanism of drug synergy by various biochemical assays. Results: Cell viability of 14 T-ALL cell lines was determined after being subjected to Bcl-2 inhibitor (ABT-199) and PLK1 inhibitor (BI-6727). All cell lines responded well to BI6727 with an EC50 of less than 70nM. However, they showed differential response to ABT199 with only 3 cell lines being sensitive with an EC50 of less than 40nM. The mRNA levels of Bcl-2, Bcl-xL and PLK 1, 2, 3 and 4 were determined by qRT-PCR. PLK1 was found to be highly expressed in all the cell lines as compared to the rest of the 3 PLK family proteins. ABT-199-sensitive cell lines showed lower Bcl-xL mRNA levels irrespective of their Bcl-2 expression, and displayed synergy with BI-6727. A higher degree of apoptosis was also observed in the combination treatment as compared to a single drug. Immunoblot analysis revealed cleavage of PARP1 and lower levels of c-Myc and MCL1 expression in the presence of both ABT-199 and BI-6727. Conclusions: Upregulation of the anti-apoptotic BCL2 family members is one of the canonical ways for cancer cells to escape apoptosis. In the past years, several highly selective and potent BCL2 inhibitors have been developed and showed promising efficacy in various cancers. We found that the sensitivity of T-ALL cell lines to ABT-199 is largely determined by the lower levels of Bcl-xL expression. Furthermore, ABT-199 displays synergy with the PLK inhibitor. T-ALL cell lines predominantly express PLK1 and thus the combinatorial effect of ABT-199 and BI-6727 is mediated through the pharmacological inhibition of both BCL2 and PLK1. Currently, we are generating iRFP-expressing T-ALL cell lines which will be used to check drug efficacy in vivo. Furthermore, we have collected chemo-resistant PDX cell lines which will be used to verify the cell line data. Besides its role in cell cycle control, we still have very limited knowledge about the function of PLK1 in leukemia. Thus, studying its role in T-ALL cell lines by knocking down PLK1 with CRISPR/Cas9 technology will provide an important insight. Disclosures No relevant conflicts of interest to declare.

T-cell acute lymphoblastic leukemia--the associated gene SCL/tal codes for a 42-Kd nuclear phosphoprotein

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2858-2866 ◽  
Author(s):  
AN Goldfarb ◽  
S Goueli ◽  
D Mickelson ◽  
JM Greenberg
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Nuclear Localization ◽  
Dna Sequences ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Bhlh Protein ◽  
Helix Loop Helix ◽  
Cell Acute Lymphoblastic Leukemia

SCL/tal is a putative oncogene originally identified through its involvement in the translocation t(1;14)(p32;q11) present in the leukemic cell line DU.528. Subsequent studies have shown an upstream deletion activating expression of SCL/tal to be one of the most common genetic lesions in T-cell acute lymphoblastic leukemia (T-ALL). The cDNA sequence of SCL/tal encodes a basic helix-loop-helix (bHLH) protein with regions of marked homology to lyl-1 and tal-2, two other bHLH proteins involved in T-ALL chromosomal translocations. The bHLH motif suggests that the SCL/tal product localizes to the nucleus, binds to specific DNA sequences, and regulates transcription of a specific array of target genes. Our studies directly identify the SCL/tal product as a 42-Kd phosphoprotein that efficiently localizes to the nucleus. Deletion mutagenesis has allowed identification of a region critical for nuclear localization, a region that corresponds to the DNA- binding basic domain within the bHLH motif. Because this domain is shared by lyl-1 and tal-2, these latter putative T-cell oncoproteins probably use a nuclear localization mechanism identical to that of SCL/tal.

Collaboration Between RUNX and NOTCH Pathways in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1279-1279 ◽  
Author(s):  
Christopher R Jenkins ◽  
Hongfang Wang ◽  
Olena O Shevchuk ◽  
Sonya H Lam ◽  
Vincenzo Giambra ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Notch Signaling ◽  
Cell Lines ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Brdu Incorporation ◽  
Knock Down ◽  
Primary Mouse ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 1279 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy characterized by the clonal outgrowth of developmentally arrested T-lymphoid blasts. Notch signaling is activated by mutation of NOTCH1 and/or FBW7 in over half of cases, and ultimately results in increased expression of target genes via the NOTCH/CSL transcriptional complex. Enforced expression of activated NOTCH1 in mouse hematopoietic progenitors leads to the development of clonal T-cell leukemias, suggesting that collaborating mutations are required for establishment and/or propagation of malignant clones. To identify candidate collaborating loci, Beverly and Capobianco performed a retroviral insertional mutagenesis screen in mice expressing a relatively weak activated Notch1 transgene and found recurrent insertions into Ikaros (Ikzf1). These insertions resulted in expression of dominant negative isoforms of Ikaros and thus potentiated Notch signaling since Ikaros and Notch/CSL compete for occupancy at target gene regulatory elements. In an attempt to identify collaborating mutations outside of the Notch pathway, we performed a similar screen, but employed instead a very potent activated NOTCH1 allele (ΔE) in hopes of saturating the Notch signaling pathway. We thus cloned out the insertion sites from 88 primary mouse leukemias generated by transduction of bone marrow with ΔE retrovirus. While recurrent insertions into Ikzf1 were again identified, we also observed frequent insertions into other regions including the Runx3 locus. The Runx3 integrations were tightly clustered in a region 40–60kb upstream of the transcriptional start site, suggesting the retroviral LTR might be inducing an increase in Runx3 expression. A single integration upstream of Runx1 was also identified in a region frequently mutated in similar screens. Of note, analysis of publically available gene expression profile data revealed that RUNX1 and RUNX3 are ubiquitously expressed in patient T-ALL samples. In order to functionally characterize the roles of RUNX1 and RUNX3 in T-ALL, we utilized lentiviral shRNAs to knock down RUNX1 and/or RUNX3 across a broad panel of 26 human T-ALL cell lines. Despite recent studies suggesting RUNX1 may act as a tumor suppressor in T-ALL, we observed the overwhelming majority of cell lines to show substantial growth defects after knock-down of RUNX1/3 as measured by competitive growth assay. These results were confirmed in a subset of cell lines and also in xenograft-expanded primary T-ALL samples by BrdU incorporation/DNA content assays which showed reduced proliferation/G1 cell cycle arrest following RUNX1/3 knock-down. Conversely, overexpression of RUNX3 induced T-ALL cells to proliferate more rapidly and to resist ABT-263-induced apoptosis. To explore potential target genes responsible for these pro-growth/survival effects, we mined available ChIP-Seq data and found NOTCH1/CSL and RUNX1 binding sites to co-localize within IGF1R and IL7R loci at intronic enhancer-like regions with associated H3K4me1>H3K4me3 marks and reduced H3K27me3 marks. Importantly, we show that NOTCH1 and RUNX factors co-regulate surface protein expression of IGF1R and IL7R in a synergistic/additive manner. As we and others have previously demonstrated important roles for both IGF1R and IL7R in T-ALL cell growth and leukemia-initiating activity, these studies reveal a novel collaborative mechanism between NOTCH1 and RUNX proteins in supporting propagation of established T-ALL disease. Disclosures: No relevant conflicts of interest to declare.

RUNX1 promotes cell growth in human T-cell acute lymphoblastic leukemia by transcriptional regulation of key target genes

2018 ◽  
Vol 64 ◽  
pp. 84-96 ◽  
Author(s):  
Catherine E. Jenkins ◽  
Samuel Gusscott ◽  
Rachel J. Wong ◽  
Olena O. Shevchuk ◽  
Gurneet Rana ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Human T Cell ◽  
Cell Acute Lymphoblastic Leukemia

scholarly journals A Test to Quantify NOTCH Pathway Activity in T Cell Acute Lymphoblastic Leukemia Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4661-4661
Author(s):  
Kirsten Canté-Barrett ◽  
Laurent Holtzer ◽  
Henk van Ooijen ◽  
Rico Hagelaar ◽  
Valentina Cordo' ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Cell Types ◽  
Activity Score ◽  
Free Survival ◽  
Pathway Activity ◽  
Notch Activity ◽  
Protein Levels

Background The NOTCH signaling pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis in many diseases including T-cell acute lymphoblastic leukemia (T-ALL). Over 70% of T-ALL patient samples contain mutations in NOTCH1 and/or FBXW7 that result in the activation of the NOTCH pathway. Various targeted drugs are available that inhibit NOTCH signaling, but their effectiveness varies due to variable NOTCH pathway activities among individual patients. Moreover, patients' leukemic cells that lack these mutations may still require NOTCH signaling. A quick and robust quantification of NOTCH pathway activity in primary patient samples would identify patients who could benefit from NOTCH targeted treatment. Aims In primary human T-ALL samples, we aimed to determine the NOTCH pathway activity in relation to active, intracellular NOTCH1 (ICN1) levels and in relation to NOTCH1 and/or FBXW7 mutations. Additionally, we investigated whether the NOTCH pathway activity score is more accurate than a mutation-based activity prediction. Methods Our test to assess functional NOTCH pathway activity in various cell types was applied to primary human T-ALL samples. The NOTCH test infers a quantitative NOTCH pathway activity score from mRNA levels of conserved direct NOTCH target genes based on a Bayesian network model. This model describes the causal relation between up- or downregulation of NOTCH target genes and the presence of an active or inactive NOTCH transcription complex. The Bayesian model was calibrated on publically available Affymetrix U133 Plus2.0 microarray datasets of samples with an active or inactive NOTCH pathway. Following validation on multiple cell types and malignancies, we scored NOTCH pathway activation in our well-characterized cohort of 117 T-ALL patient samples and related it to clinical and biological parameters including outcome. Results The NOTCH pathway model was calibrated using a microarray dataset containing high-grade serous ovarian cancer-which has high NOTCH activity-and normal ovarian tissue samples that lack NOTCH activity. Validation of the test using datasets from primary cells and cell lines of various origins revealed that it measures the NOTCH activity status in different cellular contexts. In primary diagnostic T-ALL samples, we observed a significant relationship between NOTCH pathway activity scores and active, intracellular cleaved NOTCH1 (ICN1) protein levels and the presence of NOTCH1-activating mutations. We next scored NOTCH pathway activity over the four T-ALL subgroups ETP-ALL, TLX, Proliferative and TALLMO. The TLX subgroup had the highest NOTCH activity levels compared to the other subgroups, consistent with the high percentage of TLX cases with NOTCH1/FBXW7 mutations. Strikingly, the significance of the correlation between ICN1 levels and NOTCH pathway activity was mainly attributed to the strong NOTCH1-activating mutations that include NOTCH1 juxtamembrane domain mutations, or hetero-dimerization mutations combined with PEST domain or FBXW7 mutations. When assessing the event-free survival and relapse-free survival curves, we observed that patients with the lowest (lower than the 25th-percentile) NOTCH pathway activity scores had the shortest event-free survival compared to the others (p<0.05, log-rank test). Summary/Conclusion High NOTCH pathway activation was mostly present in-but not limited to-T-ALL samples harboring strong NOTCH1 mutations, indicating that additional mechanisms can activate NOTCH signaling. Combined with the observation that the measured NOTCH pathway activity relates to ICN1 protein levels, this indicates that the pathway activity score more accurately reflects NOTCH pathway activity than the predicted activity based on NOTCH1 mutations alone. Disclosures Holtzer: Philips Research: Employment. Verhaegh:Philips Research: Employment. van de Stolpe:The Netherlands: Employment; Eindhoven: Employment; Philips Research: Employment.

Direct Inhibition of the Notch Transactivation Complex with Synthetic Constrained Peptides in T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2819-2819 ◽  
Author(s):  
Raymond Moellering ◽  
Melanie Cornejo ◽  
Jennifer Rocknik ◽  
Michael Hancock ◽  
Christina DelBianco ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Notch Signaling ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Stem Cell Differentiation ◽  
Gamma Secretase ◽  
Clinical Resistance ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Notch signaling represents a central pathway regulating hematopoiesis, stem cell differentiation, and malignant transformation in human cancer. Activation of highly conserved Notch1 receptors results in cleavage and release of an intracellular domain (ICN1). Following translocation to the nucleus, ICN1 forms a ternary complex with the transcriptional repressor CSL (CBF-1, Suppressor of Hairless and Lag-1) bound to cognate DNA. This event triggers a repressor-to-activator switch, as an interfacial groove is formed which recruits the Mastermind-Like (MAML1) co-activator protein. Activating mutations in NOTCH1 are found in more than 50% of patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL), promoting protein stability and establishing a direct link to disease pathogenesis. Pharmacologic efforts to target the Notch pathway in T-ALL have been directed at gamma secretase, a regulatory enzyme in Notch activation. Recently, the observed clinical resistance to gamma secretase inhibitors has been explained, in part, by additional mutations in the Notch-targeting ubiquitin ligase, Fbxw7, which further increases oncoprotein stability. Therefore, direct inhibitors of ICN1 function are highly desirable. Drawing upon insights afforded by the resolved crystal structure of the DNA-bound ICN1:MAML1:CSL complex, we synthesized a series of hydrocarbon stapled alpha-helical peptides targeting Notch (SAHNs) based on minimal motifs of the MAML protein predicted to engage the composite ICN1:CSL interface. Direct, high-affinity binding to purified components of the Notch complex was confirmed using surface plasmon resonance (SPR). Nuclear access of SAHN1 was confirmed using quantitative epifluorescent and confocal microscopy. Intracellular association with ICN1 and CSL was established using bidirectional affinity chromatography. Using a novel CSL-responsive reporter construct, we observed inhibition of endogenous Notch transactivation by SAHN1 in T-ALL cell lines. Furthermore, SAHN1 induces a dose-dependent knockdown of endogenous Notch1 target genes including HES1, HEY1 and cMYC in T-ALL cell lines. Remarkably, inhibition of Notch signaling by SAHN1 confers selective cytotoxicity at 48 hours in a panel of T-ALL cell lines with known mutations in NOTCH, including those resistant to gamma secretase inhibitors. Supporting an on-target mechanism of action, we have prepared a damaged analogue of SAHN1 containing a two-residue rearrangement (SAHN1D). SAHN1D possesses reduced binding affinity for the Notch complex and despite comparable intracellular access, SAHN1D lacks both transcriptional and cytotoxic effects on cultured T-ALL cell lines in vitro. Efficacy studies have also been performed in vivo using a novel murine model of T-ALL. In summary, we report here the design, biochemical characterization and translational rationale supporting the first direct inhibitor of the Notch transactivation complex in T-ALL.

LEF1 Is a Tumor Suppressor in T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3802-3802
Author(s):  
Alejandro Gutierrez ◽  
Takaomi Sanda ◽  
Stuart Winter ◽  
Richard S. Larson ◽  
Lewis B. Silverman ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Suppressor Activity ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Expression Microarrays

Abstract In an effort to further unravel the molecular pathogenesis of T cell acute lymphoblastic leukemia (T-ALL), we performed array CGH on diagnostic specimens from 47 pediatric patients with T-ALL. The LEF1 transcription factor is best known as a positive mediator of oncogenic β-catenin signaling, and it is required for the expression of MYC, Cyclin D1 and Survivin in some contexts, including some normal hematopoietic progenitors. Additionally, Lef1 has been shown to be required for the survival of murine Notch-dependent T cell lymphoma cells. We were thus surprised to find highly focal deletions of LEF1 in 10.6 % (n = 5 of 47) of primary T-ALL patient samples, with 3 cases harboring homozygous deletions and 2 harboring heterozygous deletions of this locus. These deletions involved no other known genes. We then sequenced this gene in 45 of these cases and found heterozygous mutations in 3 additional T-ALL samples, including 2 cases with frameshift mutations predicted to truncate the protein prior to its context-dependent activation and HMG box domains, and one case with an Asp85Asn substitution. Seven of the eight samples harboring LEF1 abnormalities also had mutations in the heterodimerization or PEST domains of NOTCH1. In an effort to characterize the biologic consequences of LEF1 loss in T-ALL, we took advantage of expression microarrays that were previously performed on most of these samples. LEF1 loss defines a novel T-ALL subtype characterized by arrest at the CD1-positive early cortical stage, and whose gene expression profile resembles that of HOX11-positive cases, although expression of HOX11 was generally low in these samples. Loss of LEF1 was mutually exclusive to overexpression of TAL1 and of the HOXA/MEIS1 cluster. Interestingly, LEF1-negative T-ALL cases were associated with increased expression of MYC, and gene set enrichment analysis identified a significant association between LEF1 loss and upregulation of MYC target genes. In addition to its role as a transcriptional activator in the setting of active WNT/β-catenin signaling, LEF1 can also act as a transcriptional repressor in some cellular contexts. Experiments are currently underway to establish the mechanism mediating the tumor suppressor activity of LEF1 in T-ALL.

scholarly journals miR106a-363 Cluster Has Oncogenic Potential in Childhood T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5142-5142
Author(s):  
Monika Drobna ◽  
Bronislawa Szarzynska-Zawadzka ◽  
Maria Kosmalska ◽  
Roman Jaksik ◽  
Tomasz Szczepanski ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Luciferase Reporter ◽  
Pathway Enrichment Analysis ◽  
Thermo Fisher Scientific ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Fisher Scientific

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy originating from T-cell precursors and is characterized by high genetic, immunophenotypic, and clinical heterogeneity. MicroRNAs (miRNAs) belong to the class of small noncoding RNAs and are implicated in the regulation of hematopoiesis and in the development of leukemia. miRNAs control expression of their target genes at the post-transcriptional level by blocking translation of messenger RNAs (mRNAs) or promoting their degradation. Some miRNAs are encoded within clusters, giving rise to policistronic transcripts. Such miRNAs are co-expressed and may co-regulate the expression of genes involved in certain biological processes and pathways. In our recent study we performed miRNA profiling in pediatric T-ALL using Next-Generation Sequencing (Dawidowska M et al. Blood 2017; 130:1443) and identified miRNAs differentially expressed in T-ALL. The set of overexpressed miRNAs included, among others, miR-20b-5p, miR-363-3p and miR-92a-2-5p, belonging to a cluster of six miRNAs: miR-106a-363 (ChrXq26.2). miR-106a-363 cluster is a paralog of miR-17-92 cluster (Chr13q31.3), a prototypic oncogenic cluster of eminent importance in human hematopoietic cancers, with reported role in T-ALL pathogenesis (Mavrakis KJ et al., Nature Cell Biology 2010, 12:4). Despite the similarity of seed sequences between miRNAs from miR-17-92 and miR-106a-363 clusters, the significance of miR-106a-363 cluster in T-ALL remains to be elucidated. In this study we investigated the expression of the miR-20b-5p, miR-363-3p and miR-92a-2-5p in children with T-ALL, healthy donor thymocytes, normal bone marrow samples and 6 T-ALL cell lines. RT-qPCR analysis (TaqMan Advanced miRNA Assays; Thermo Fisher Scientific) confirmed overexpression of 2 miRNAs from cluster miR-106a-363 (miR-20b-5p and miR-363-3p) in children with T-ALL and in T-ALL cell lines, suggesting their oncogenic function. To predict potential target genes of overexpressed miRNAs belonging to miR106a-363 cluster, we applied 8 target prediction algorithms and pathway enrichment analysis. This revealed the enrichment of miR-20b-5p and miR-363-3p target genes in GO term: positive regulation of apoptosis. We further validated predicted miRNA-mRNA interactions (Dual Luciferase Reporter Assays; Promega) confirming the majority of them (e.g. PTEN, FBXW7, BCL2L11). Finally, we assessed the effect of mimicry/inhibition (miRVana, Thermo Fisher Scientific) of overexpressed miRNAs from miR-106a-363 cluster on proliferation, cell cycle distribution and apoptosis in 3 T-ALL cell lines. Overexpression of miR-20b-5p and miR-363-3p in CCRF-CEM, DND-41 and P12-Ichikawa cells resulted in increased proliferation and inhibited apoptosis. To summarize, in this study we showed that miRNAs belonging to miR-106a-363 cluster directly interact with mRNAs implicated in the regulation of apoptosis and that miR-20b-5p and miR-363-3p have pro-proliferative and anti-apoptotic effects in T-ALL cells in vitro. These results indicate that miR-106a-363 cluster may have an oncogenic role in the pathogenesis of T-ALL via suppression of pro-apoptotic genes. Research funded by National Science Centre, Poland grants: 2014/15/B/NZ2/03394, 2017/25/N/NZ2/01132 and National Centre of Research and Development (NCRD) grant STARTEGMED3/304586/5/NCBR/2017. Disclosures No relevant conflicts of interest to declare.

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