Interplay between transcription regulators RUNX1 and FUBP1 activates an enhancer of the oncogene c-KIT and amplifies cell proliferation.

Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.

CD9, a key actor in the dissemination of lymphoblastic leukemia, modulating CXCR4-mediated migration via RAC1 signaling.

CD9, a member of the tetraspanin family, has been implicated in hematopoietic and leukemic stem cell homing. We investigated the role of CD9 in the dissemination of B acute lymphoblastic leukemia (B-ALL) cells, by stably downregulating CD9 in REH and NALM6 cells. CD9 expression was associated with higher levels of REH cell adhesion to fibronectin and C-X-C motif chemokine receptor 4 (CXCR4)-mediated migration. Death occurred later in NOD/SCID mice receiving REH cells depleted of CD9 for transplantation than in mice receiving control cells. After C-X-C motif chemokine ligand 12 (CXCL12) stimulation, CD9 promoted the formation of long cytoplasmic actin-rich protrusions. We demonstrated that CD9 enhanced RAC1 activation, in both REH cells and blasts from patients. Conversely, the overexpression of a competing CD9 C-terminal tail peptide in REH cytoplasm decreased RAC1 activation and cytoplasmic extension formation in response to CXCL12. Finally, the inhibition of RAC1 activation decreased migration in vitro, and the depletion of RAC1 protein from transplanted REH cells increased mouse survival. Furthermore, a testis-conditioned medium induced the migration of REH and NALM6 cells, and this migration was impeded by an anti-CD9 antibody. The level of CD9 expression also influenced the homing of these cells in mouse testes. These findings demonstrate, for the first time, that CD9 plays a key role in the CXCR4-mediated migration and engraftment of B-ALL cells in the bone marrow or testis, through RAC1 activation.

Hypoxia regulates CD9 expression and dissemination of B lymphoblasts.

Acute lymphoblastic leukemias (ALL) are the most frequent cancer in children and derive most often from B-cell precursors. Current survival rates roughly reach 90% at 10 years from diagnosis. However, 15-20% of children still relapse with a significant risk of death. Our previous work showed that the transmembrane protein CD9 plays a major role in lymphoblasts migration into sanctuary sites, especially in testis, through the activation of RAC1 signaling upon blasts stimulation with C-X-C chemokine ligand 12 (CXCL12). Here, we identified common factors shared by the bone marrow and extramedullary niches which could upregulate CD9 expression and function. We found that low oxygen levels enhance CD9 expression both at mRNA and protein levels. We further determined that Hypoxia Inducible Factor 1α (HIF1α), the master transcription factor involved in hypoxia response, binds directly CD9 promoter and induce CD9 transcription. We also showed that CD9 protein is crucial for leukemic cell adhesion and migration at low oxygen levels, possibly through its action on RAC1 signaling. Mouse xenograft experiments indicate that HIF1α signaling pathway promotes ALL cells engraftment in a CD9-dependent manner. The present work increments our understanding of CD9 implication in ALL pathogenesis.

Reduction of RUNX1 transcription factor activity by a CBFA2T3-mimicking peptide: application to B cell precursor acute lymphoblastic leukemia.

BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.

Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1-positive leukemia.

BACKGROUND: The t(12;21)(p13;q22) translocation is found in 20 to 25% of cases of childhood B-lineage acute lymphoblastic leukemia (B-ALL). This rearrangement results in the fusion of ETV6 (TEL) and RUNX1 (AML1) genes and defines a relatively uniform category, although only some patients suffer very late relapse. TEL/AML1-positive patients are thus an interesting subgroup to study, and such studies should elucidate the biological processes underlying TEL/AML1 pathogenesis. We report an analysis of gene expression in 60 children with B-lineage ALL using Agilent whole genome oligo-chips (44K-G4112A) and/or real time RT-PCR. RESULTS: We compared the leukemia cell gene expression profiles of 16 TEL/AML1-positive ALL patients to those of 44 TEL/AML1-negative patients, whose blast cells did not contain any additional recurrent translocation. Microarray analyses of 26 samples allowed the identification of genes differentially expressed between the TEL/AML1-positive and negative ALL groups. Gene enrichment analysis defined five enriched GO categories: cell differentiation, cell proliferation, apoptosis, cell motility and response to wounding, associated with 14 genes -RUNX1, TCFL5, TNFRSF7, CBFA2T3, CD9, SCARB1, TP53INP1, ACVR1C, PIK3C3, EGFL7, SEMA6A, CTGF, LSP1, TFPI - highlighting the biology of the TEL/AML1 sub-group. These results were first confirmed by the analysis of an additional microarray data-set (7 patient samples) and second by real-time RT-PCR quantification and clustering using an independent set (27 patient samples). Over-expression of RUNX1 (AML1) was further investigated and in one third of the patients correlated with cytogenetic findings. CONCLUSION: Gene expression analyses of leukemia cells from 60 children with TEL/AML1-positive and -negative B-lineage ALL led to the identification of five biological processes, associated with 14 validated genes characterizing and highlighting the biology of the TEL/AML1-positive ALL sub-group.

Optimization of proximity ligation assay (PLA) for detection of protein interactions and fusion proteins in non-adherent cells: application to pre-B lymphocytes.

BACKGROUND: Genetic abnormalities, including chromosomal translocations, are described for many hematological malignancies. From the clinical perspective, detection of chromosomal abnormalities is relevant not only for diagnostic and treatment purposes but also for prognostic risk assessment. From the translational research perspective, the identification of fusion proteins and protein interactions has allowed crucial breakthroughs in understanding the pathogenesis of malignancies and consequently major achievements in targeted therapy. METHODS: We describe the optimization of the Proximity Ligation Assay (PLA) to ascertain the presence of fusion proteins, and protein interactions in non-adherent pre-B cells. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. RESULTS: We propose an optimized PLA procedure. We overcome the issue of maintaining non-adherent hematological cells by traditional cytocentrifugation and optimized buffers, by changing incubation times, and modifying washing steps. Further, we provide convincing negative and positive controls, and demonstrate that optimized PLA procedure is sensitive to total protein level. The optimized PLA procedure allows the detection of fusion proteins and protein interactions on non-adherent cells. CONCLUSION: The optimized PLA procedure described here can be readily applied to various non-adherent hematological cells, from cell lines to patients' cells. The optimized PLA protocol enables detection of fusion proteins and their subcellular expression, and protein interactions in non-adherent cells. Therefore, the optimized PLA protocol provides a new tool that can be adopted in a wide range of applications in the biological field.

Pol32, a subunit of Saccharomyces cerevisiae DNA polymerase delta, suppresses genomic deletions and is involved in the mutagenic bypass pathway.

The Pol32 subunit of S. cerevisiae DNA polymerase (Pol) delta plays an important role in replication and mutagenesis. Here, by measuring the CAN1 forward mutation rate, we found that either POL32 or REV3 (which encodes the Pol zeta catalytic subunit) inactivation produces overlapping antimutator effects against rad mutators belonging to three epistasis groups. In contrast, the msh2Delta pol32Delta double mutant exhibits a synergistic mutator phenotype. Can(r) mutation spectrum analysis of pol32Delta strains revealed a substantial increase in the frequency of deletions and duplications (primarily deletions) of sequences flanked by short direct repeats, which appears to be RAD52 and RAD10 independent. To better understand the pol32Delta and rev3Delta antimutator effects in rad backgrounds and the pol32Delta mutator effect in a msh2Delta background, we determined Can(r) mutation spectra for rad5Delta, rad5Delta pol32Delta, rad5Delta rev3Delta, msh2Delta, msh2Delta pol32Delta, and msh2Delta rev3Delta strains. Both rad5Delta pol32Delta and rad5Delta rev3Delta mutants exhibit a reduction in frameshifts and base substitutions, attributable to antimutator effects conferred by the pol32Delta and rev3Delta mutations. In contrast, an increase in these two types of alterations is attributable to a synergistic mutator effect between the pol32Delta and msh2Delta mutations. Taken together, these observations indicate that Pol32 is important in ensuring genome stability and in mutagenesis.

CD9 expression can be used to predict childhood TEL/AML1-positive acute lymphoblastic leukemia: proposal for an accelerated diagnostic flowchart.

CD9 has been shown to be differentially expressed in childhood TEL/AML1-positive acute lymphoblastic leukemia (ALL). We confirmed this finding in large Affymetrix data sets and in 80 new cases at both RNA and protein levels. Moreover, we showed that mean fluorescence intensity of CD9 by flow cytometry can distinguish TEL/AML1-positive ALL from other BCP-ALL. Using ROC analysis, the most efficient model for predicting TEL/AML1-positive ALL combined CD9 (mean fluorescence intensity 40%). Finally, we propose a faster procedure for optimizing the diagnosis of childhood BCP-ALL subgroups.

A genomewide screen in Saccharomyces cerevisiae for genes that suppress the accumulation of mutations.

A genomewide screen of a collection of 4,847 yeast gene deletion mutants was carried out to identify the genes required for suppressing mutations in the CAN1 forward-mutation assay. The primary screens and subsequent analysis allowed (i) identification of 18 known mutator mutants, providing a solid means for checking the efficiency of the screen, and (ii) identification of a number of genes not known previously to be involved in suppressing mutations. Among the previously uncharacterized mutation-suppressing genes were six genes of unknown function including four (CSM2, SHU2, SHU1, and YLR376c) encoding proteins that interact with each other and promote resistance to killing by methyl methanesulfonate, one gene (EGL1) previously identified as suppressing Ty1 mobility and recombination between repeated sequences, and one gene (YLR154c) that was not associated with any known processes. In addition, five genes (TSA1, SOD1, LYS7, SKN7, and YAP1) implicated in the oxidative-stress responses were found to play a significant role in mutation suppression. Furthermore, TSA1, which encodes thioredoxin peroxidase, was found to strongly suppress gross chromosomal rearrangements. These results provide a global view of the nonessential genes involved in preventing mutagenesis. Study of such genes should provide useful clues in identification of human genes potentially involved in cancer predisposition and in understanding their mechanisms of action.