Julien Calvo

Expression of CD34 and CD7 on human T-cell acute lymphoblastic leukemia discriminates functionally heterogeneous cell populations.

Leukemia-initiating/repopulating cells (LICs), also named leukemic stem cells, are responsible for propagating human acute leukemia. Although they have been characterized in various leukemias, their role in T-cell acute lymphoblastic leukemia (T-ALL) is unclear. To identify and characterize LICs in T-ALL (T-LIC), we fractionated peripheral blood cell populations from patient samples by flow cytometry into three cell fractions by using two markers: CD34 (a marker of immature cells and LICs) and CD7 (a marker of early T-cell differentiation). We tested these populations in both in vitro culture assays and in vivo for growth and leukemia development in immune-deficient mice. We found LIC activity in CD7+ cells only as CD34+CD7− cells contained normal human progenitors and hematopoietic stem cells that differentiated into T, B lymphoid and myeloid cells. In contrast, CD34+CD7+ cells were enriched in LICs, when compared with CD34−CD7+ cells. These CD34+CD7+ cells also proliferated more upon NOTCH activation than CD34−CD7+ cells and were sensitive to dexamethasone and NOTCH inhibitors. These data show that CD34 and CD7 expression in human T-ALL samples help in discriminating heterogeneous cell populations endowed with different LIC activity, proliferation capacity and responses to drugs.

Interleukin-18 produced by bone marrow- derived stromal cells supports T-cell acute leukaemia progression

Development of novel therapies is critical for T‐cell acute leukaemia (T‐ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T‐ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T‐ALL cell samples cultured on stromal cells independently of NOTCH activation and maintained their ability to propagate in vivo. Similar results were obtained when T‐ALL cells were cultured with ERK1/2‐knockdown stromal cells or with conditioned medium from MEKi‐treated stromal cells. Microarray analysis identified interleukin 18 (IL‐18) as transcriptionally up‐regulated in MEKi‐treated MS5 cells. Recombinant IL‐18 promoted T‐ALL growth in vitro, whereas the loss of function of IL‐18 receptor in T‐ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL‐18R was activated by IL‐18 in blast cells. IL‐18 circulating levels were increased in T‐ALL‐xenografted mice and also in T‐ALL patients in comparison with controls. This study uncovers a novel role of the pro‐inflammatory cytokine IL‐18 and outlines the microenvironment involvement in human T‐ALL development.

The SCL/TAL1 Transcription Factor Represses the Stress Protein DDiT4/REDD1 in Human Hematopoietic Stem/Progenitor Cells.

Hematopoietic stem/progenitor cells (HSPCs) are regulated through numerous molecular mechanisms that have not been interconnected. The transcription factor stem cell leukemia/T‐cell acute leukemia 1 (TAL1) controls human HSPC but its mechanism of action is not clarified. In this study, we show that knockdown (KD) or short‐term conditional over‐expression (OE) of TAL1 in human HSPC ex vivo, respectively, blocks and maintains hematopoietic potentials, affecting proliferation of human HSPC. Comparative gene expression analyses of TAL1/KD and TAL1/OE human HSPC revealed modifications of cell cycle regulators as well as previously described TAL1 target genes. Interestingly an inverse correlation between TAL1 and DNA damage‐induced transcript 4 (DDiT4/REDD1), an inhibitor of the mammalian target of rapamycin (mTOR) pathway, is uncovered. Low phosphorylation levels of mTOR target proteins in TAL1/KD HSPC confirmed an interplay between mTOR pathway and TAL1 in correlation with TAL1‐mediated effects of HSPC proliferation. Finally chromatin immunoprecipitation experiments performed in human HSPC showed that DDiT4 is a direct TAL1 target gene. Functional analyses showed that TAL1 represses DDiT4 expression in HSPCs. These results pinpoint DDiT4/REDD1 as a novel target gene regulated by TAL1 in human HSPC and establish for the first time a link between TAL1 and the mTOR pathway in human early hematopoietic cells. Stem Cells 2015;33:2268–2279

Optimized gene transfer into human primary leukemic T cell with NOD-SCID/leukemia-initiating cell activity

Optimized gene transfer into human primary leukemic T cell with NOD-SCID/leukemia-initiating cell activity

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells.

The human hemopoietic progenitor hierarchy producing lymphoid and granulocytic-monocytic (myeloid) lineages is unclear. Multiple progenitor populations produce lymphoid and myeloid cells, but remain incompletely characterized. Here, we demonstrated cord blood lympho-myeloid containing progenitor populations - the lymphoid-primed multi-potential progenitor (LMPP), granulocyte-macrophage progenitor (GMP) and multi-lymphoid progenitor (MLP) - were functionally and transcriptionally distinct and heterogeneous at the clonal level, with progenitors of many different functional potentials present. Though most progenitors had uni-lineage myeloid or lymphoid potential, bi- and rarer multi-lineage progenitors occurred in LMPP, GMP and MLP. This, coupled with single cell expression analyses, suggested a continuum of progenitors execute lymphoid and myeloid differentiation rather than only uni-lineage progenitors being present downstream of stem cells.

Assessment of Human Multi-Potent Hematopoietic Stem/Progenitor Cell Potential Using a Single In Vitro Screening System

Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34+CD38−/low(CD45RA−CD90+) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.

Bone marrow sites differently imprint dormancy and chemoresistance to T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) expands in various bone marrow (BM) sites of the body. We investigated whether different BM sites could differently modulate T-ALL propagation using in vivo animal models. We observed that mouse and human T-ALL develop slowly in the BM of tail vertebrae compared with the BM from thorax vertebrae. T-ALL recovered from tail BM displays lower cell-surface marker expression and decreased metabolism and cell-cycle progression, demonstrating a dormancy phenotype. Functionally, tail-derived T-ALL exhibit a deficient short-term ex vivo growth and a delayed in vivo propagation. These features are noncell-autonomous because T-ALL from tail and thorax shares identical genomic abnormalities and functional disparities disappear in vivo and in prolonged in vitro assays. Importantly tail-derived T-ALL displays higher intrinsic resistance to cell-cycle-related drugs (ie, vincristine sulfate and cytarabine). Of note, T-ALL recovered from gonadal adipose tissues or from cocultures with adipocytes shares metabolic, cell-cycle, and phenotypic or chemoresistance features, with tail-derived T-ALL suggesting adipocytes may participate in the tail BM imprints on T-ALL. Altogether these results demonstrate that BM sites differentially orchestrate T-ALL propagation stamping specific features to leukemic cells such as quiescence and decreased response to cell-cycle-dependent chemotherapy.

Speed of leukemia development and genetic diversity in xenograft models of T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) develops through accumulation of multiple genomic alterations within T-cell progenitors resulting in clonal heterogeneity among leukemic cells. Human T-ALL xeno-transplantation in immunodeficient mice is a gold standard approach to study leukemia biology and we recently uncovered that the leukemia development is more or less rapid depending on T-ALL sample. The resulting human leukemia may arise through genetic selection and we previously showed that human T-ALL development in immune-deficient mice is significantly enhanced upon CD7+/CD34+ leukemic cell transplantations. Here we investigated the genetic characteristics of CD7+/CD34+ and CD7+/CD34− cells from newly diagnosed human T-ALL and correlated it to the speed of leukemia development. We observed that CD7+/CD34+ or CD7+/CD34− T-ALL cells that promote leukemia within a short-time period are genetically similar, as well as xenograft-derived leukemia resulting from both cell fractions. In the case of delayed T-ALL growth CD7+/CD34+ or CD7+/CD34− cells were either genetically diverse, the resulting xenograft leukemia arising from different but branched subclones present in the original sample, or similar, indicating decreased fitness to mouse micro-environment. Altogether, our work provides new information relating the speed of leukemia development in xenografts to the genetic diversity of T-ALL cell compartments.

Homeobox protein TLX3 activates miR-125b expression to promote T-cell acute lymphoblastic leukemia

The oncogenic mechanisms driven by aberrantly expressed transcription factors in T-cell acute leukemia (T-ALL) are still elusive. MicroRNAs (miRNAs) play an important role in normal development and pathologies. Here, we examined the expression of 738 miRNA species in 41 newly diagnosed pediatric T-ALLs and in human thymus-derived cells. We found that expression of 2 clustered miRNAs, miR-125b/99a, peaks in primitive T cells and is upregulated in the T leukemia homeobox 3 (TLX3)-positive subtype of T-ALL. Using loss- and gain-of-function approaches, we established functional relationships between TLX3 and miR-125b. Both TLX3 and miR-125b support in vitro cell growth and in vivo invasiveness of T-ALL. Besides, ectopic expression of TLX3 or miR-125b in human hematopoietic progenitor cells enhances production of T-cell progenitors and favors their accumulation at immature stages of T-cell development resembling the differentiation arrest observed in TLX3 T-ALL. Ectopic miR-125b also remarkably accelerated leukemia in a xenograft model, suggesting that miR125b is an important mediator of the TLX3-mediated transformation program that takes place in immature T-cell progenitors. Mechanistically, TLX3-mediated activation of miR-125b may impact T-cell differentiation in part via repression of Ets1 and CBFβ genes, 2 regulators of T-lineage. Finally, we established that TLX3 directly regulates miR-125b production through binding and transactivation of LINC00478, a long noncoding RNA gene, which is the host of miR-99a/Let-7c/miR-125b. Altogether, our results reveal an original functional link between TLX3 and oncogenic miR-125b in T-ALL development.