Kpm/Lats2 is linked to chemosensitivity of leukemic cells through the stabilization of p73

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3856-3866 ◽  
Author(s):  
Masahiro Kawahara ◽  
Toshiyuki Hori ◽  
Kazuhisa Chonabayashi ◽  
Tsutomu Oka ◽  
Marius Sudol ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Immunoprecipitation ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Gene Promoter ◽  
Leukemic Cells ◽  
Nuclear Accumulation ◽  
Conventional Chemotherapy ◽  
Puma Gene ◽  
The Stability

Down-regulation of the Kpm/Lats2 tumor suppressor is observed in various malignancies and associated with poor prognosis in acute lymphoblastic leukemia. We documented that Kpm/Lats2 was markedly decreased in several leukemias that were highly resistant to conventional chemotherapy. Silencing of Kpm/Lats2 expression in leukemic cells did not change the rate of cell growth but rendered the cells more resistant to DNA damage–inducing agents. Expression of p21 and PUMA was strongly induced by these agents in control cells, despite defective p53, but was only slightly induced in Kpm/Lats2-knockdown cells. DNA damage–induced nuclear accumulation of p73 was clearly observed in control cells but hardly detected in Kpm/Lats2-knockdown cells. Chromatin immunoprecipitation (ChIP) assay showed that p73 was recruited to the PUMA gene promoter in control cells but not in Kpm/Lats2-knockdown cells after DNA damage. The analyses with transient coexpression of Kpm/Lats2, YAP2, and p73 showed that Kpm/Lats2 contributed the stability of YAP2 and p73, which was dependent on the kinase function of Kpm/Lats2 and YAP2 phosphorylation at serine 127. Our results suggest that Kpm/Lats2 is involved in the fate of p73 through the phosphorylation of YAP2 by Kpm/Lats2 and the induction of p73 target genes that underlie chemosensitivity of leukemic cells.

Kpm/Lats2 of the Hippo Pathway Is Linked to Chemo-Sensitivity of Leukemic Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1797-1797 ◽  
Author(s):  
Masahiro Kawahara ◽  
Toshiyuki Hori ◽  
Kazuhisa Chonabayashi ◽  
Tsutomu Oka ◽  
Marius Sudol ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Line ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Hippo Pathway ◽  
Leukemic Cells ◽  
Nuclear Accumulation ◽  
Cell Leukemia ◽  
Down Regulation ◽  
The Hippo Pathway

Abstract The Hippo pathway is now recognized as a major signaling network for development and organ size control in mammals as well as in Drosophila melanogaster. We previously identified human kpm/Lats2, the key serine/threonine kinase in the Hippo pathway, and reported that its overexpression induced cell cycle arrest and apoptosis. Down-regulation of kpm/Lats2 expression has been described in various malignancies. It is of particular importance in hematology that low expression of kpm/Lats2 has been reported to be associated with poor prognosis in acute lymphoblastic leukemia. In the present study, we first measured the expression level of kpm/Lats2 mRNA in various hematological malignancies and found that it was markedly decreased in adult T cell leukemia (ATL) and NK cell leukemia/lymphoma, both of which are known to be highly resistant to conventional chemotherapy. In order to investigate the relationship between down-regulation of kpm/Lats2 expression and chemo-resistance, we made kpm/Lats2-knockdown sublines from KG-1a, an AML-derived cell line, and ED-40515+, an ATL-derived cell line, by using shRNA-expression retrovirus vector targeting kpm/Lats2. Silencing of kpm/Lats2 expression in both leukemic cell lines did not change the rate of cell growth but rendered them resistant to DNA damage-inducing agents such as DOX and ETP. Expression of p21 and PUMA was strongly induced by these agents in control cells, despite defective p53, but was only slightly induced in kpm/Lats2-knockdown cells. DNA damage-induced nuclear accumulation of p73, a member of p53 family, was clearly observed in control cells but hardly detected in kpm/Lats2-knockdown cells. ChIP assay showed that p73 was recruited to the puma gene promoter in control cells but not in kpm/Lats2-knockdown cells after DNA damage stress. The analyses with transient co-expression of Kpm/Lats2, YAP2, and p73 showed that Kpm/Lats2 contributed to the stability of YAP2 and p73, which was dependent on the kinase function of Kpm/Lats2 and YAP2 phosphorylation at serine 127. These results strongly suggest that Kpm/Lats2 is involved in the fate of p73 and the induction of p73-target genes that underlie chemo-sensitivity of leukemic cells.

A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3417-3423 ◽  
Author(s):  
Marina Bousquet ◽  
Cyril Broccardo ◽  
Cathy Quelen ◽  
Fabienne Meggetto ◽  
Emilienne Kuhlein ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Cell Acute Lymphoblastic Leukemia

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

E2a-Pbx1 Direct Targets and Secondary Mutations in Leukemogenesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3343-3343
Author(s):  
Christofer Diakos ◽  
Yuanyuan Xiao ◽  
Jerry Hofmann ◽  
Shichun Zheng ◽  
Michelle Kang ◽  
...  
Keyword(s):  
Target Genes ◽  
Cell Transformation ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Translocation ◽  
Gene Promoter ◽  
Transactivation Domain ◽  
Oncogenic Potential ◽  
Expression Arrays ◽  
Functional Regulation ◽  
Copy Number Changes

Abstract E2a-Pbx1 is expressed as a result of the t(1;19) chromosomal translocation in nearly 5% of cases of acute lymphoblastic leukemia. The E2a-Pbx1 chimeric transcription factor contains the N-terminal transactivation domain of E2A fused to the C-terminal DNAbinding homeodomain of Pbx1. Previews studies indicate that additional genetic events may be required for E2A-Pbx1-leukemic transformation, based on long incubation times and monoclonal nature of tumors observed in mouse models. While there is no doubt of its oncogenic potential, the mechanisms of E2a-Pbx1-mediated pre-B cell transformation and the nature of direct E2a-Pbx1 target genes and additional events that complement the fusion oncogene to create full-blown leukemia are still unclear. Herein we used chromatin immunoprecipitation (ChIP-chip) assays to identify direct targets of E2a-Pbx1, and we used gene and miRNA expression arrays of siRNA E2a-Pbx1-silenced cells to evaluate changes in expression induced by the fusion protein. To identify complementary genetic rearrangements, analyses of primary E2a-Pbx1 leukemias were performed to copy number changes and loss of heterozygosity which might identify mutations that synergize with the direct/functional E2a-Pbx1 targets to produce the leukemic phenotype. These arrays were analyzed in comparison to high-density gene promoter methylation arrays. Our data identified members of the WNT pathway as direct targets of E2a-Pbx1. Expression data from E2a-Pbx1 silenced cells support this finding as they demonstrate a functional regulation of this pathway. We further show a differential impact of E2a-Pbx1 silencing on the miRNA profile and identify E2a-Pbx1 dependent miRNAs. Using CGH arrays on primary E2a-Pbx1 samples we were able to pinpoint candidate secondary mutations as well as broad genetic categories: cases with 1q+, 1q+ combined with 9p-, and, separately, cases with +8. In summary we present direct and functional E2a-Pbx1 targets as well as candidate secondary mutations. We propose a model were direct and functional E2a- Pbx1 driven pathways that might include both genes and miRNAs might collaborate with identified auxiliary events to produce the E2a-Pbx1 leukemia.

scholarly journals NKX3.1 is a direct TAL1 target gene that mediates proliferation of TAL1-expressing human T cell acute lymphoblastic leukemia

2010 ◽  
Vol 207 (10) ◽  
pp. 2141-2156 ◽  
Author(s):  
Sophie Kusy ◽  
Bastien Gerby ◽  
Nicolas Goardon ◽  
Nathalie Gault ◽  
Federica Ferri ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Notch Pathway ◽  
Gene Promoter ◽  
Leukemic Cells ◽  
Regulatory Sequence ◽  
Human T Cell ◽  
T Cell Leukemogenesis

TAL1 (also known as SCL) is expressed in >40% of human T cell acute lymphoblastic leukemias (T-ALLs). TAL1 encodes a basic helix-loop-helix transcription factor that can interfere with the transcriptional activity of E2A and HEB during T cell leukemogenesis; however, the oncogenic pathways directly activated by TAL1 are not characterized. In this study, we show that, in human TAL1–expressing T-ALL cell lines, TAL1 directly activates NKX3.1, a tumor suppressor gene required for prostate stem cell maintenance. In human T-ALL cell lines, NKX3.1 gene activation is mediated by a TAL1–LMO–Ldb1 complex that is recruited by GATA-3 bound to an NKX3.1 gene promoter regulatory sequence. TAL1-induced NKX3.1 activation is associated with suppression of HP1-α (heterochromatin protein 1 α) binding and opening of chromatin on the NKX3.1 gene promoter. NKX3.1 is necessary for T-ALL proliferation, can partially restore proliferation in TAL1 knockdown cells, and directly regulates miR-17-92. In primary human TAL1-expressing leukemic cells, the NKX3.1 gene is expressed independently of the Notch pathway, and its inactivation impairs proliferation. Finally, TAL1 or NKX3.1 knockdown abrogates the ability of human T-ALL cells to efficiently induce leukemia development in mice. These results suggest that tumor suppressor or oncogenic activity of NKX3.1 depends on tissue expression.

scholarly journals Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dalila Naci ◽  
Sofiane Berrazouane ◽  
Frédéric Barabé ◽  
Fawzi Aoudjit
Keyword(s):  
Dna Damage ◽  
Cell Adhesion ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Major Pathway ◽  
Histone H2ax ◽  
Anti Cancer ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Α2β1 Integrin

AbstractChemoresistance is a major hurdle in anti-cancer therapy. Growing evidence indicates that integrin-mediated cell adhesion to extracellular matrix plays a major role in chemoresistance. However, the underlying mechanisms are not fully understood. We have previously shown that the collagen-binding integrin α2β1 promoted doxorubicin resistance in acute T cell lymphoblastic leukemia (T-ALL). In this study, we found that acute myeloid leukemia (AML) cell lines also express α2β1 integrin and collagen promoted their chemoresistance as well. Furthermore, we found that high levels of α2 integrin correlate with worse overall survival in AML. Our results showed that doxorubicin-induced apoptosis in leukemic cells is associated with activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and that collagen inhibited this pathway. The protective effect of collagen is associated with the inhibition of Rac1-induced DNA damage as evaluated by the comet assay and the phosphorylated levels of histone H2AX (γ-H2AX). Together these results show that by inhibiting pro-apoptotic Rac1, α2β1 integrin can be a major pathway protecting leukemic cells from genotoxic agents and may thus represent an important therapeutic target in anti-cancer treatment.

scholarly journals The eukaryotic initiation factor 2 kinase GCN2 protects against hepatotoxicity during asparaginase treatment

2013 ◽  
Vol 305 (9) ◽  
pp. E1124-E1133 ◽  
Author(s):  
Gabriel J. Wilson ◽  
Piyawan Bunpo ◽  
Judy K. Cundiff ◽  
Ronald C. Wek ◽  
Tracy G. Anthony
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Target Genes ◽  
Binding Protein ◽  
Lymphoblastic Leukemia ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Triglyceride Accumulation ◽  
Eukaryotic Initiation Factor 2 ◽  
Initiation Factor 2

Asparaginase is an important drug in the treatment regimen for acute lymphoblastic leukemia. Asparaginase depletes circulating asparagine and glutamine, activating an amino acid stress response (AAR) involving phosphorylation of eukaryotic initiation factor 2 (eIF2) by general control nonderepressible kinase 2 (GCN2). We hypothesized that GCN2 functions to mitigate hepatic stress during asparaginase therapy by activating the AAR. To test this idea, C57BL/6J wild-type mice ( Gcn2+/+) and those deleted for Gcn2 ( Gcn2−/−) were injected with asparaginase or saline excipient one time daily for 1 or 6 days. In liver, increased phosphorylation of eIF2 and mRNA expression of AAR target genes activating transcription factor 4, asparagine synthetase, eIF4E-binding protein 1, and CAAT enhancer-binding protein homologous protein were significantly blunted or blocked in the liver of G cn2−/− mice. Loss of AAR during asparaginase coincided with increases in mammalian target of rapamycin signaling, hepatic triglyceride accumulation, and DNA damage in association with genetic markers of oxidative stress ( glutathione peroxidase) and inflammation ( tumor necrosis factor alpha-α). Although asparaginase depleted circulating asparagine in both Gcn2+/+ and Gcn2−/− mice, all other amino acids, including plasma glutamine, were elevated in the plasma of Gcn2−/− mice. This study shows that loss of GCN2 promotes oxidative stress and inflammatory-mediated DNA damage during asparaginase therapy, suggesting that patients with reduced or dysfunctional AAR may be at risk of developing hepatic complications during asparaginase treatment.

scholarly journals Ebf1 or Pax5 haploinsufficiency synergizes with STAT5 activation to initiate acute lymphoblastic leukemia

2011 ◽  
Vol 208 (6) ◽  
pp. 1135-1149 ◽  
Author(s):  
Lynn M. Heltemes-Harris ◽  
Mark J.L. Willette ◽  
Laura B. Ramsey ◽  
Yi Hua Qiu ◽  
E. Shannon Neeley ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Active Form ◽  
Cell Development ◽  
B Cell Development ◽  
Leukemic Cells ◽  
Small Perturbations ◽  
Potential Tumor Suppressor

As STAT5 is critical for the differentiation, proliferation, and survival of progenitor B cells, this transcription factor may play a role in acute lymphoblastic leukemia (ALL). Here, we show increased expression of activated signal transducer and activator of transcription 5 (STAT5), which is correlated with poor prognosis, in ALL patient cells. Mutations in EBF1 and PAX5, genes critical for B cell development have also been identified in human ALL. To determine whether mutations in Ebf1 or Pax5 synergize with STAT5 activation to induce ALL, we crossed mice expressing a constitutively active form of STAT5 (Stat5b-CA) with mice heterozygous for Ebf1 or Pax5. Haploinsufficiency of either Pax5 or Ebf1 synergized with Stat5b-CA to rapidly induce ALL in 100% of the mice. The leukemic cells displayed reduced expression of both Pax5 and Ebf1, but this had little effect on most EBF1 or PAX5 target genes. Only a subset of target genes was deregulated; this subset included a large percentage of potential tumor suppressor genes and oncogenes. Further, most of these genes appear to be jointly regulated by both EBF1 and PAX5. Our findings suggest a model whereby small perturbations in a self-reinforcing network of transcription factors critical for B cell development, specifically PAX5 and EBF1, cooperate with STAT5 activation to initiate ALL.

scholarly journals A Novel Functional Polymorphism in the CCAAT/Enhancer Binding Protein (C/EBP), Epsilon (CEBPE) Gene Promoter Influences Acute Lymphoblastic Leukemia Risk Via Interaction with IKZF1

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 489-489
Author(s):  
Kyle M. Walsh ◽  
Adam J. de Smith ◽  
Jianqiao Xiao ◽  
Marcus O. Muench ◽  
Marina Fomin ◽  
...  
Keyword(s):  
B Cell ◽  
Chromatin Immunoprecipitation ◽  
Protein C ◽  
Risk Allele ◽  
Lymphoblastic Leukemia ◽  
Gene Promoter ◽  
Expression Vectors ◽  
Genome Wide ◽  
Enhancer Binding Protein ◽  
Increased Risk

Abstract Purpose: We sought to identify and characterize the functional polymorphism in the CCAAT/enhancer binding protein (C/EBP), epsilon (CEBPE) gene, which is known to harbor a genetic risk modifier for childhood acute lymphoblastic leukemia (ALL). Background: Association between ALL risk and polymorphic variation at the CEBPE gene, a zinc-finger hematopoietic and apoptotic regulator expressed exclusively in myeloid cells, was established in a genome-wide association study and replicated in other populations. The actual functional genetic variation that actually contributes the leukemia risk is not known. The discovery and functional characterization of the functional variant may lead to risk stratification, prevention, and/or therapeutic modalities. Methods: Utilizing California Childhood Leukemia Study genome-wide SNP data from Hispanic subjects, we refine the genetic signal to a specific single nucleotide polymorphism (SNP) within the CEBPE gene promoter (rs2239635). This SNP was identified first using genetic imputation using 1000 Genomes data as a framework, and further validated in additional subjects using Taqman genotyping. Functional work to characterize the impact of the polymorphism was performed using prediction methods (database searches), chromatin immunoprecipitation assays from normal subjects heterozygous for rs2239635, and luciferase expression vectors carrying the SNP. Results: The minor allele of rs2239635 conferred a 1.87-fold increased risk of B-cell ALL (95% CI = 1.51-2.30; P = 6.2x10-9) and a 2.94-fold increased risk of high hyperdiploid B-cell ALL (95% CI = 2.12-4.06; P = 8.2x10-11), but only a 1.51-fold increased risk of non-hyperdiploid B-cell ALL (95% CI = 1.11-2.04; P = 8.8x10-3). Adjusting for rs2239635 attenuated all SNP associations in the region, including the original GWAS allele, strongly suggest that the GWAS signal in CCLS Hispanics is attributable to rs2239635. We also demonstrate, using published functional data and our own chromatin immunoprecipitation assays, that this SNP disrupts a canonical transcription factor binding site for Ikaros (IKZF1), an essential hematopoietic transcription factor. Using expression vectors, we found the polymorphic locus to have transcriptional repressive properties that are diminished in the presence of the ALL risk allele. Additionally, a heritable risk allele within IKZF1 interacts with rs2239635 in a multiplicative fashion (P = 0.02) in case-control association analyses. Discussion: In sum, evidence for a functional CEBPE polymorphism links two hematopoietic developmental transcriptional factors and highlights feedback control between them. The minor, risk-associated variant of CEBPE may alter the ability of IKZF1 to repress CEBPE expression in precursor B-cells, causing lineage confusion and enhanced pre-B cell leukemia risk. This risk primarily impacts risk of pediatric ALL that exhibits the high hyperdiploid phenotype. Disclosures No relevant conflicts of interest to declare.

scholarly journals HMGA2 As a Potential Molecular Target in MLL-AF4 Positive Infant Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2244-2244
Author(s):  
Minenori Eguchi-Ishimae ◽  
Mariko Eguchi ◽  
Zhouying Wu ◽  
Wen Ming ◽  
Hidehiko Iwabuki ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Growth ◽  
Target Genes ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Demethylating Agent ◽  
Mll Gene ◽  
Hmga2 Protein

Abstract Leukemic cells of acute lymphoblastic leukemia (ALL) in infants are frequently characterised by chromosome translocations involving 11q23, resulting in the rearrangement of the mixed-lineage leukemia (MLL) gene and subsequent generation of MLL fusion gene. Among more than 50 genes which have been identified as the fusion partner of the MLL gene, fusion with AF4 is characteristically observed in infant ALL representing a hallmark of poor prognosis. Although recent progress of intensive chemotherapy with or without stem cell transplantation has improved its treatment outcome, the treatment is often accompanied by long-term side effects. Less toxic molecular targeting therapies are therefore necessary for infant ALL. We have previously reported that in infant ALL with MLL fusion gene, microRNA let-7b is significantly downregulated by DNA hypermethylation of its promoter region. The downregulation of let-7b is one of the consequences of oncogenic MLL fusion proteins contributing to leukemogenesis possibly through upregulation of let-7b-regulated target genes with oncogenic potential such as high mobility group AT-hook 2 (HMGA2). HMGA2 is a chromatin-remodelling factor, which alters chromatin architecture by binding to AT-rich regions in the DNA, either promoting or inhibiting the expression of its target genes. One of the targets of HMGA2 is CDKN2A gene which encodes 2 cell cycle regulators p16INK4A and p14ARF. This let-7b-HMGA2-CDKN2A axis regulates cellular growth and senescence of stem cells both in normal and pathological state such as cancer. We initially examined the expression of HMGA2 in leukemic cells obtained from 35 MLL-rearranged infant ALL patients (MLL-AF4, n = 26; MLL-AF9, n = 4; MLL-ENL, n = 5) using quantitative RT-PCR. As results, HMGA2 was highly expressed in most of the patients with MLL fusion gene, especially in MLL-AF4-positive cases, compared to those without the fusion. These results indicate that deregulation of let-7b-HMGA2 axis by MLL fusion may contribute to leukemogenesis and could be a possible target of molecular therapy against MLL-rearranged ALL. As let-7b is downregulated by promoter hypermethylation, demethylating agents such as 5-azacytidine could be applied to recover the expression of the gene in leukemic cells with MLL fusion gene. To test this possibility, leukemic cell lines with MLL-AF4 fusion gene were used. The administration of 5-azacytidine alone was able to restore the expression of suppressed let-7b as well as p16INK4A gene in the leukemic cells, but the effects was incomplete, showing persistent partial promoter methylation. In addition, the recovered expression disappeared when 5-azacytidine was removed. On the other hand, when HMGA2 inhibitor was combined with 5-azacytidine, the expression of let-7b was upregulated and sustained resulting in suppression of HMGA2 protein itself. This upregulation of let-7b and suppression of HMGA2 protein persisted even after the removal of 5-azacytidine, possibly through maintaining of the demethylating status by HMGA2 inhibitor. Inhibition of HMGA2 by either siRNA or HMGA2 inhibitor suppressed the growth of MLL-AF4-positive leukemic cells when analysed by MTT assay. The effects of HMGA2 inhibitor on cell growth inhibition became more prominent in combination with demethylating agent 5-azacytidine. Our results revealed the functional significance of let-7b and HMGA2 in controlling MLL-AF4-positive leukemic cell growth and the therapeutic potential of combining demethylating agent and the HMGA2 inhibitor in the treatment of MLL-AF4-positive ALL. Disclosures No relevant conflicts of interest to declare.

Collaboration Between MiR-125b, MiR-100 and MiR-99a Induces Vincristine Resistance in Childhood TEL-AML1-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1475-1475
Author(s):  
Farhad Akbari Moqadam ◽  
Ellen Lange-Turenhout ◽  
Rob Pieters ◽  
Monique L. Den Boer
Keyword(s):  
Target Genes ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Protein Coding ◽  
Expression Levels ◽  
Over Expression ◽  
Viable Cells ◽  
Reh Cells

Abstract Abstract 1475 MicroRNAs (miRNAs) are involved in the pathobiology of leukemia. We previously observed that miR-125b, miR-99a and miR-100 were highly expressed in ex vivo vincristine (VCR) resistant leukemic cells of children with precursor B-cell acute lymphoblastic leukemia (ALL) (Schotte et al. Haematologica 2011). VCR is a microtubulin-interfering drug applied in the treatment of children with newly diagnosed ALL. Leukemic cells of TEL-AML1-positive precursor B-cell ALL were found to be resistant to this drug (Ramakers-van Woerden et al. Blood 2000). The expression levels of miR-125b, miR-99a and miR-100 were highly correlated in childhood precursor B-ALL cells. MiR-125b, as the most well-known miRNA among these three, was shown to contribute to drug resistance in different types of solid tumors. Down-regulation of miR-125b was also shown to sensitize the TEL-AML1-positive Reh cell line to doxorubicin and staurosporine (Gefen et al. Leukemia 2010). However, recent studies suggest that miRNAs co-operate and the combination of multiple distinct miRNAs co-regulates expression of associated target genes. The current study addressed whether miR-125b alone or in combination with miR-99a and/or miR-100 contributed to VCR resistance in childhood TEL-AML1-positive ALL cells. MiRNAs were over-expressed in TEL-AML1-positive Reh cells using lentiviral particles and/or miRNA precursors. Expression level of mature (and hence processed precursor) miRNAs was analyzed by qRT-PCR. The cell cycle distribution and the amount of leukemic cells in apoptosis were determined by flow cytometry of propidium iodide stained nuclei and Annexin V-propidium iodide stained cells, respectively. Leukemic cells were incubated with 9 ng/mL VCR and after 3 days of exposure the cellular response to VCR was measured by an MTT-based cell survival assay. The expression level of mature miR-125b, miR-99a and miR-100 was raised >100-fold upon lentiviral and precursor miRNA transduction compared to basal expression levels of TEL-AML1-positive Reh cells. Over-expression of miR-125b, miR-99a or miR-100 as single miRNAs did not significantly affect cellular survival after 3 days of exposure to VCR (20±5%, 29±17% and 29±17% viable cells, respectively) compared to the cells transduced with a scrambled miR-control (10±4% viable cells, p>0.05 each) when cell viability in absence of VCR was set to 100%. Over-expression of the combination of miR-100 and miR-99a only had a limited protective effect on cell viability (30±7% viable cells compared to 10±4% viable cells for the scrambled miR-control, p<0.05). In contrast, co-expression of miR-99a or miR-100 together with miR-125b or the combination of miR-99a and miR-100 together with miR-125b strongly and significantly induced resistance to VCR in TEL-AML- positive Reh cells; 91±4% of miR-99a/miR-125b-transduced, 93±5% of miR-100/miR-125b-transduced and 82±17% of miR-99a/miR-100/miR-125b-transduced cells remained alive upon VCR exposure compared to only 38±13% of miR-125b-transduced cells (p<0.05). The combination of these miRNAs did not change the cell cycle distribution or the amount of apoptotic cells in the absence of VCR. This suggests that these 3 miRNAs synergize in the development of resistance to VCR. Computational miRNA target prediction algorithms (TargetScan and EIMMo3) predicted 14 possible protein-coding target genes for the combination of miR-125b, miR-99a and miR-100. Gene expression profiling of ex vivo VCR-resistant (n=20) and VCR-sensitive (n=10) leukemic cells of TEL-AML1-positive ALL patients revealed that the expression levels of these 14 predicted target genes did not differ between resistant and sensitive cases (p>0.05). Ongoing studies currently explore which other protein-coding genes can be directly targeted by the miR125b, miR-99a and/or miR100 combination using functional gene expression studies. In conclusion, miR-125b, miR-99a or miR-100 as single factors were not effective to induce VCR resistance. In contrast, miR-125b in combination with miR-99a and/or miR-100 strongly increased VCR resistance in leukemic cells of TEL-AML1-positive precursor B-ALL. Discovery of directly regulated protein-coding target genes of these 3 miRNAs may point to ways to modulate resistance to VCR in children with ALL. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document