miR-130b and miR-128a are essential lineage-specific codrivers of t(4;11) MLL-AF4 acute leukemia.

t(4;11) MLL-AF4 acute leukemia is one of the most aggressive malignancies in the infant and pediatric population, yet we have little information on the molecular mechanisms responsible for disease progression. This impairs the development of therapeutic regimens that can address the aggressive phenotype and lineage plasticity of MLL-AF4-driven leukemogenesis. This study highlights novel mechanisms of disease development by focusing on 2 microRNAs (miRNAs) upregulated in leukemic blasts from primary patient samples: miR-130b and miR-128a. We show that miR-130b and miR-128a are downstream targets of MLL-AF4 and can individually drive the transition from a pre-leukemic stage to an acute leukemia in an entirely murine Mll-AF4 in vivo model. They are also required to maintain the disease phenotype. Interestingly, miR-130b overexpression led to a mixed/B-cell precursor (BCP)/myeloid leukemia, propagated by the lymphoid-primed multipotent progenitor (LMPP) population, whereas miR-128a overexpression resulted in a pro-B acute lymphoblastic leukemia (ALL), maintained by a highly expanded Il7r+c-Kit+ blast population. Molecular and phenotypic changes induced by these two miRNAs fully recapitulate the human disease, including central nervous system infiltration and activation of an MLL-AF4 expression signature. Furthermore, we identified 2 downstream targets of these miRNAs, NR2F6 and SGMS1, which in extensive validation studies are confirmed as novel tumor suppressors of MLL-AF4+ leukemia. Our integrative approach thus provides a platform for the identification of essential co-drivers of MLL-rearranged leukemias, in which the preleukemia to leukemia transition and lineage plasticity can be dissected and new therapeutic approaches can be tested.

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.

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.

Defining the fetal origin of MLL-AF4 infant leukemia highlights specific fatty acid requirements.

Infant MLL-AF4-driven acute lymphoblastic leukemia (ALL) is a devastating disease with dismal prognosis. A lack of understanding of the unique biology of this disease, particularly its prenatal origin, has hindered improvement of survival. We perform multiple RNA sequencing experiments on fetal, neonatal, and adult hematopoietic stem and progenitor cells from human and mouse. This allows definition of a conserved fetal transcriptional signature characterized by a prominent proliferative and oncogenic nature that persists in infant ALL blasts. From this signature, we identify a number of genes in functional validation studies that are critical for survival of MLL-AF4+ ALL cells. Of particular interest are PLK1 because of the readily available inhibitor and ELOVL1, which highlights altered fatty acid metabolism as a feature of infant ALL. We identify which aspects of the disease are residues of its fetal origin and potential disease vulnerabilities.

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.

The Ouzo effect: A tool to elaborate high-payload nanocapsules.

We investigate the encapsulation in hybridosomes®, a type of capsules unique regarding their structure and method of elaboration. Hybridosomes® are made of a single shell of inorganic nanoparticles (~5 nm) crosslinked with a polymer and are easily obtained via spontaneous emulsification in a ternary mixture THF/water/butylated hydroxytoluene (BHT). Our main finding is that an exceptionally high concentration of a hydrophobic model dye can be loaded in the hybridosomes®, up to 0.35 mol.L-1 or equivalently 170 g.L-1 or 450,000 molecules/capsule. The detailed investigation of the encapsulation mechanism shows that the dye concentrates in the droplets during the emulsification step simultaneously with capsule formation. Then it precipitates inside the capsules during the course of solvent evaporation. In vitro fluorescence measurements show that the nano-precipitated cargo can be transferred from the core of the hybridosomes® to the membrane of liposomes. In vivo studies suggest that the dye diffuses through the body during several days. The released dye tends to accumulate in body-fat, while the inorganic nanoparticles remain trapped into the liver and the spleen macrophages.

Mechanisms of extramedullary relapse in acute lymphoblastic leukemia: Reconciling biological concepts and clinical issues.

Long-term survival rates in childhood acute lymphoblastic leukemia (ALL) are currently above 85% due to huge improvements in treatment. However, 15-20% of children still experience relapses. Relapses can either occur in the bone marrow or at extramedullary sites, such as gonads or the central nervous system (CNS), formerly referred to as ALL-blast sanctuaries. The reason why ALL cells migrate to and stay in these sites is still unclear. In this review, we have attempted to assemble the evidence concerning the microenvironmental factors that could explain why ALL cells reside in such sites. We present criteria that make extramedullary leukemia niches and solid tumor metastatic niches comparable. Indeed, considering extramedullary leukemias as metastases could be a useful approach for proposing more effective treatments. In this context, we conclude with several examples of potential niche-based therapies which could be successfully added to current treatments of ALL.

Promises and limitations of nanoparticles in the era of cell therapy: Example with CD19-targeting chimeric antigen receptor (CAR)-modified T cells.

A number of nanoparticles has been developed by chemists for biomedical applications to meet imaging and targeting needs. In parallel, adoptive T therapy with chimeric antigen receptor engineered T cells (CART cells) has recently held great promise in B-cell malignancy treatments thanks to the development of anti-CD19 CAR T cells. Indeed, CD19 is a reliable B cell marker and a validated target protein for therapy. In this perspective article, we propose to discuss the advantages, limits and challenges of nanoparticles and CAR T cells, focusing on CD19 targeting objects: anti-CD19 nanoparticles and anti-CD19 CAR T cells, because those genetically-modified cells are the most widely developed in clinical setting. In the first part, we will introduce B cell malignancies and the CD19 surface marker. Then we will present the positioning of nanomedicine in the topic of B cell malignancy, before exposing CAR T technology. Finally, we will discuss the complementary approaches between nanoparticles and CAR T cells.

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.

Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene.

Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO(®) expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD. Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS).