GATA2 regulates the erythropoietin receptor in t(12;21) ALL.
ABSTRACT: The t(12;21) (p13;q22) chromosomal translocation resulting in the ETV6/RUNX1 fusion gene is the most frequent structural cytogenetic abnormality in children with acute lymphoblastic leukemia (ALL). The erythropoietin receptor (EPOR), usually associated with erythroid progenitor cells, is highly expressed in ETV6/RUNX1 positive cases compared to other B-lineage ALL subtypes. Gene expression analysis of a microarray database and direct quantitative analysis of patient samples revealed strong correlation between EPOR and GATA2 expression in ALL, and higher expression of GATA2 in t(12;21) patients. The mechanism of EPOR regulation was mainly investigated using two B-ALL cell lines: REH, which harbor and express the ETV6/RUNX1 fusion gene; and NALM-6, which do not. Expression of EPOR was increased in REH cells compared to NALM-6 cells. Moreover, of the six GATA family members only GATA2 was differentially expressed with substantially higher levels present in REH cells. GATA2 was shown to bind to the EPOR 5'-UTR in REH, but did not bind in NALM-6 cells. Overexpression of GATA2 led to an increase in EPOR expression in REH cells only, indicating that GATA2 regulates EPOR but is dependent on the cellular context. Both EPOR and GATA2 are hypomethylated and associated with increased mRNA expression in REH compared to NALM-6 cells. Decitabine treatment effectively reduced methylation of CpG sites in the GATA2 promoter leading to increased GATA2 expression in both cell lines. Although Decitabine also reduced an already low level of methylation of the EPOR in NALM-6 cells there was no increase in EPOR expression. Furthermore, EPOR and GATA2 are regulated post-transcriptionally by miR-362 and miR-650, respectively. Overall our data show that EPOR expression in t(12;21) B-ALL cells, is regulated by GATA2 and is mediated through epigenetic, transcriptional and post-transcriptional mechanisms, contingent upon the genetic subtype of the disease.
Project description:The t(12;21) translocation that generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. Single-nucleotide polymorphism array analysis of ETV6-RUNX1 patient samples has identified multiple additional genetic alterations; however, the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that cooperate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to coexpress the Etv6-RUNX1 fusion and Sleeping Beauty transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a Sleeping Beauty transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insight into the cooperating genetic alterations in ETV6-RUNX1 leukemia.
Project description:Genome binding/occupancy profiling of ETS Variant Transcription Factor 6- Runt Related Transcription Factor 1 fusion protein (ETV6-RUNX1) in REH cells by high throughput sequencing. ETV6-RUNX1 is expressed in pediatric t(12;21) ETV6-RUNX1 B cell precursor acute lymphoblastic leukemia. Overall design: ETV6-RUNX1 genome-wide distribution is determined using ChIP-seq. Cells used in this study are pre-B lymphoblast REH cells. ChIP samples and libraries were done by Sérandour A.A and Debaize L. High-throughput sequencing was conducted on a Genome HiSeq1500/2500. The input, REH RUNX1 Chip-Seq and histone marks are deposited in GEO GSE117684.
Project description:The t(12;21) translocation generating the ETV6/RUNX1 fusion gene represents the most frequent chromosomal rearrangement in childhood leukemia. Presence of ETV6/RUNX1 alone is usually not sufficient for leukemia onset, and additional genetic alterations have to occur in ETV6/RUNX1-positive cells to cause transformation. We have previously generated an ETV6/RUNX1 transgenic mouse model where the expression of the fusion gene is restricted to CD19-positive B cells. Since BCL2 family members have been proposed to play a role in leukemogenesis, we investigated combined effects of ETV6/RUNX1 with exogenous expression of the antiapoptotic protein BCL2 by crossing ETV6/RUNX1 transgenic animals with Vav-BCL2 transgenic mice. Strikingly, co-expression of ETV6/RUNX1 and BCL2 resulted in significantly shorter disease latency in mice, indicating oncogene cooperativity. This was associated with faster development of follicular B cell lymphoma and exacerbated immune complex glomerulonephritis. ETV6/RUNX1-BCL2 double transgenic animals displayed increased B cell numbers and immunoglobulin titers compared to Vav-BCL2 transgenic mice. This led to pronounced deposition of immune complexes in glomeruli followed by accelerated development of immune complex glomerulonephritis. Thus, our study reveals a previously unrecognized synergism between ETV6/RUNX1 and BCL2 impacting on malignant disease and autoimmunity.
Project description:The t(12;21)(p13;q22) chromosomal translocation is the most frequent translocation in childhood B cell precursor-acute lymphoblastic leukemia and results in the expression of an ETV6/RUNX1 fusion protein. The frequency of ETV6/RUNX1 fusions in newborns clearly exceeds the leukemia rate revealing that additional events occur in ETV6/RUNX1-positive cells for leukemic transformation. Hitherto, the mechanisms triggering these second hits remain largely elusive. Thus, we generated a novel ETV6/RUNX1 transgenic mouse model where the expression of the fusion protein is restricted to CD19(+) B cells. These animals harbor regular B cell development and lack gross abnormalities. We established stable pro-B cell lines carrying the ETV6/RUNX1 transgene that allowed us to investigate whether ETV6/RUNX1 itself favors the acquisition of second hits. Remarkably, these pro-B cell lines as well as primary bone marrow cells derived from ETV6/RUNX1 transgenic animals display elevated levels of reactive oxygen species (ROS) as tested with ETV6/RUNX1 transgenic dihydroethidium staining. In line, intracellular phospho-histone H2AX flow cytometry and comet assay revealed increased DNA damage indicating that ETV6/RUNX1 expression enhances ROS. On the basis of our data, we propose the following model: the expression of ETV6/RUNX1 creates a preleukemic clone and leads to increased ROS levels. These elevated ROS favor the accumulation of secondary hits by increasing genetic instability and double-strand breaks, thus allowing preleukemic clones to develop into fully transformed leukemic cells.
Project description:Translocation t(12;21), resulting in the ETV6-RUNX1 (or TEL-AML1) fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia and is considered a first hit in leukemogenesis. A targeted therapy approach is not available for children with this subtype of leukemia. To identify the molecular mechanisms underlying ETV6-RUNX1-driven leukemia, we performed gene expression profiling of healthy hematopoietic progenitors in which we ectopically expressed ETV6-RUNX1. We reveal an ETV6-RUNX1-driven transcriptional network that induces proliferation, survival and cellular homeostasis. In addition, Vps34, an important regulator of autophagy, was found to be induced by ETV6-RUNX1 and up-regulated in ETV6-RUNX1-positive leukemic patient cells. We show that induction of Vps34 was transcriptionally regulated by ETV6-RUNX1 and correlated with high levels of autophagy. Knockdown of Vps34 in ETV6-RUNX1-positive cell lines severely reduced proliferation and survival. Inhibition of autophagy by hydroxychloroquine, a well-tolerated autophagy inhibitor, reduced cell viability in both ETV6-RUNX1-positive cell lines and primary acute lymphoblastic leukemia samples, and selectively sensitized primary ETV6-RUNX1-positive leukemia samples to L asparaginase. These findings reveal a causal relationship between ETV6-RUNX1 and autophagy, and provide pre-clinical evidence for the efficacy of autophagy inhibitors in ETV6-RUNX1-driven leukemia.
Project description:Childhood acute lymphoblastic leukemia (ALL) with t(12;21), which results in expression of the ETV6/RUNX1 fusion gene, is the most common chromosomal lesion in precursor-B (pre-B) ALL. We identified 17 microRNAs that were downregulated in ETV6/RUNX1+ compared with ETV6/RUNX1- clinical samples. Among these microRNAs, miR-181a-1 was the most significantly reduced (by ~75%; P < 0.001). Using chromatin immunoprecipitation, we demonstrated that ETV6/RUNX1 directly binds the regulatory region of MIR181A1, and knockdown of ETV6/RUNX1 increased miR-181a-1 level. We further showed that miR-181a (functional counterpart of miR-181a-1) could target ETV6/RUNX1 and cause a reduction in the level of the oncoprotein ETV6/RUNX1, cell growth arrest, an increase in apoptosis, and induction of cell differentiation in ETV6/RUNX1+ cell line. Moreover, ectopic expression of miR-181a also resulted in decreased CD10 hyperexpression in ETV6/RUNX1+ primary patient samples. Taken together, our results demonstrate that MIR181A1 and ETV6/RUNX1 regulate each other, and we propose that a double negative loop involving MIR181A1 and ETV6/RUNX1 may contribute to ETV6/RUNX1-driven arrest of differentiation in pre-B ALL.
Project description:BACKGROUND:The t(12;21)(p13;q22), which fuses ETV6 and RUNX1 genes, is the most common genetic abnormality in children with B-cell precursor acute lymphoblastic leukaemia. The implication of the fusion protein in leukemogenesis seems to be clear. However, its role in the maintenance of the disease continues to be controversial. METHODS:Generation of an in vitro ETV6/RUNX1 knock out model using the CRISPR/Cas9 gene editing system. Functional characterization by RNA sequencing, proliferation assays, apoptosis and pharmacologic studies, and generation of edited-cell xenograft model. RESULTS:The expression of ETV6/RUNX1 fusion gene was completely eliminated, thus generating a powerful model on which to study the role of the fusion gene in leukemic cells. The loss of fusion gene expression led to the deregulation of biological processes affecting survival such as apoptosis resistance and cell proliferation capacity. Tumour cells showed higher levels of apoptosis, lower proliferation rate and a greater sensitivity to PI3K inhibitors in vitro along as a decrease in tumour growth in xenografts models after ETV6/RUNX1 fusion gene abrogation. CONCLUSIONS:ETV6/RUNX1 fusion protein seems to play an important role in the maintenance of the leukemic phenotype and could thus become a potential therapeutic target.
Project description:MicroRNAs (miRNAs) regulate the expression of multiple proteins in a dose-dependent manner. We hypothesized that increased expression of miRNAs encoded on chromosome 21 (chr 21) contribute to the leukemogenic function of trisomy 21. The levels of chr 21 miRNAs were quantified by qRT-PCR in four types of childhood acute lymphoblastic leukemia (ALL) characterized by either numerical (trisomy or tetrasomy) or structural abnormalities of chr 21. Suprisingly, high expression of the hsa-mir-125b-2 cluster, consisting of three miRNAs, was identified in leukemias with the structural ETV6/RUNX1 abnormality and not in ALLs with trisomy 21. Manipulation of ETV6/RUNX1 expression and chromatin immunoprecipitation studies showed that the high expression of the miRNA cluster is an event independent of the ETV6/RUNX1 fusion protein. Overexpression of hsa-mir-125b-2 conferred a survival advantage to Ba/F3 cells after IL-3 withdrawal or a broad spectrum of apoptotic stimuli through inhibition of caspase 3 activation. Conversely, knockdown of the endogenous miR-125b in the ETV6/RUNX1 leukemia cell line REH increased apoptosis after Doxorubicin and Staurosporine treatments. P53 protein levels were not altered by miR-125b. Together, these results suggest that the expression of hsa-mir-125b-2 in ETV6/RUNX1 ALL provides survival advantage to growth inhibitory signals in a p53-independent manner.
Project description:The t(12;21)(p13;q22) ETV6-RUNX1 gene fusion is one of the most common chromosomal translocation in childhood acute lymphoblastic leukemia (ALL). It is associated with favorable prognosis. The identification of the genomic sequence of the breakpoint flanking regions of the ETV6-RUNX1 translocation should be the best strategy to monitor minimal residual disease (MRD) in patients with ETV6-RUNX1-positive ALL. In this study, the ETV6-RUNX1 translocation was sequenced by next-generation sequencing (NGS) in 26 patients with ETV6-RUNX1-positive ALL and re-sequenced by using the Sanger method. Interestingly, the three-way translocation, including ETV6-RUNX1, was detected in five patients. Four of them relapsed during or after therapy, while 21 patients without the three-way translocation were still in remission (P < 0.0001). The three-way translocation pattern was identical between the diagnosis and relapse samples in three patients, excluding one patient (SCMC-001245). The relapse samples retained the translocation of ETV6-RUNX1 relative to the three-way translocation t(8;12;21) at diagnosis, suggesting that the three-way translocation might be an important risk factor for relapse in patients with ETV6-RUNX1-positive ALL and should be further studied.
Project description:The presence of the chromosomal rearrangement t(12;21)(ETV6-RUNX1) in childhood B-acute lymphoblastic leukemia (B-ALL) is an independent predictor of favorable prognosis, however relapses still occur many years later after stopping therapy, and patients often display resistance to current treatments. Since spleen tyrosine kinase (SYK), a cytosolic nonreceptor tyrosine kinase interacting with immune receptors, has been previously associated with malignant transformation and cancer cell proliferation, we aimed to assess its role in ETV6-RUNX1 cell survival and prognosis. We evaluated the effects on cell survival of three SYK inhibitors and showed that all of them, in particular entospletinib, are able to induce cell death and enhance the efficacy of conventional chemotherapeutics. By using reverse phase protein arrays we next revealed that activated SYK is upregulated at diagnosis in pediatric ETV6-RUNX1 patients who will experience relapse, and, importantly, hyperactivation is maintained at a high level also at relapse occurrence. We thus treated primary cells from patients both at diagnosis and relapse with the combination entospletinib + chemotherapeutics and observed that SYK inhibition is able to sensitize resistant primary cells to conventional drugs. Entospletinib could thus represent a new therapeutic option supporting conventional chemotherapy for relapsed ETV6-RUNX1 patients, and these evidences encourage further studies on SYK for treatment of other relapsed resistant acute lymphoblastic leukemia (ALL) subgroups.