Sebastien Morisot

CD34+ hematopoietic stem-progenitor cell microRNA expression and function: A circuit diagram of differentiation control

MicroRNAs (miRNAs) are a recently identified class of epigenetic elements consisting of small noncoding RNAs that bind to the 3′ untranslated region of mRNAs and down-regulate their translation to protein. miRNAs play critical roles in many different cellular processes including metabolism, apoptosis, differentiation, and development. We found 33 miRNAs expressed in CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human bone marrow and mobilized human peripheral blood stem cell harvests. We then combined these data with human HSPC mRNA expression data and with miRNA-mRNA target predictions, into a previously undescribed miRNA:mRNA interaction database called the Transcriptome Interaction Database. The in silico predictions from the Transcriptome Interaction Database pointed to miRNA control of hematopoietic differentiation through translational control of mRNAs critical to hematopoiesis. From these predictions, we formulated a model for miRNA control of stages of hematopoiesis in which many of the genes specifying hematopoietic differentiation are expressed by HSPCs, but are held in check by miRNAs until differentiation occurs. We validated miRNA control of several of these target mRNAs by demonstrating that their translation in fact is decreased by miRNAs. Finally, we chose miRNA-155 for functional characterization in hematopoiesis, because we predicted that it would control both myelopoiesis and erythropoiesis. As predicted, miRNA-155 transduction greatly reduced both myeloid and erythroid colony formation of normal human HSPCs.

Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4−/− fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4+/+ cells in competitive repopulation assays. However, hematopoietic “KLF4−/− chimeras” generated by transplantation of KLF4−/− fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115+Gr1+) monocytes, and had reduced numbers of resident (CD115+Gr1−) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4−/− chimeras, bone marrow monocytic cells from KLF4−/− chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

High frequencies of leukemia stem cells in poor-outcome childhood precursor-B acute lymphoblastic leukemias

In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-PrkdcscidIL2rgtmlWjl/SzJ (NOD-severe combined immune deficient (scid) IL2rg−/−) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1–7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg−/− mice. In this highly sensitive NOD-scid-IL2Rg−/−-based assay, 1–100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.

Activating Signals Dominate Inhibitory Signals in CD137L/IL-15 Activated Natural Killer Cells

Natural killer (NK) cells can mediate potent antitumor effects, but factors regulating the efficiency of tumor lysis remain unclear. Studies in allogeneic stem cell transplantation highlight an important role for killer cell immunoglobulin-like receptor (KIR) mismatch in overcoming human leukocyte antigen-mediated inhibitory signals. However, other activating and inhibitory signals also modulate tumor lysis by NK cells. We used rhIL15 and artificial antigen presenting cells expressing CD137L and IL15R&agr; to activate and expand peripheral blood NK cells (CD137L/IL15 NK) up to 1000-fold in 3 weeks. Compared with resting NK cells, CD137L/IL15 NK cells show modest increases in KIR expression and substantial increases in NKG2D, tumor necrosis factor-related apoptosis-inducing ligand, and natural cytotoxicity receptors (NCRs: NKp30, NKp44, NKp46). Compared with resting NK cells, CD137L/IL15 NK cells mediate enhanced cytotoxicity against allogeneic and autologous tumors and KIR signaling did not substantially inhibit cytotoxicity. Rather, tumor lysis by CD137L/IL15 activated NK cells was predominantly driven by NCR signaling as blockade of NCRs dramatically diminished the lysis of a wide array of tumor targets. Furthermore, tumor lysis by CD137L/IL15 NK cells was tightly linked to NCR expression levels that peaked on day 8 to 10 after NK activation, and cytotoxicity diminished on subsequent days as NCR expression declined. We conclude that KIR mismatch is not a prerequisite for tumor killing by CD137L/IL15 NK cells and that NCR expression provides a biomarker for predicting potency of CD137L/IL15 NK cells in studies of NK cell-based immunotherapy.

Selective Reduction of Graft-versus-Host Disease-Mediating Human T Cells by Ex Vivo Treatment with Soluble Fas Ligand

Previous work done in our laboratory, using mouse models, showed that soluble Fas ligand (sFasL) can efficiently delete donor anti-host T cells during their activation against irradiated host cells in MLCs. In the mouse models, this ex vivo sFasL treatment abrogated graft-vs-host disease (GVHD) while sparing donor T cells with antitumor reactivity. The present work was performed with human cells, to extend our work toward reduction of clinical GVHD. PBMC responders from a given individual (first party) were stimulated in vitro with irradiated PBMC stimulators from a second person (second party), in the presence of sFasL. In control MLCs without sFasL, alloreacting T cells began to up-regulate Fas (CD95) detectably and became sensitive to Fas-mediated apoptosis by as early as day 1–2. In MLCs containing sFasL, there were greatly reduced numbers of alloreacting CD3+CFSElo cells, activation Ag-expressing CD4hi and CD8hi cells, IFN-γ-producing CD4+ and CD8+ cells, and CD8+CD107a+ CTLs. Furthermore, mice transplanted with the ex vivo sFasL/MLR-treated cells had prolonged time to fatal GVHD in an in vivo xenogeneic GVHD model. Responder cells harvested from primary MLCs containing sFasL had reduced proliferation in response to second party cells, but proliferated in response to CMV Ags, PHA, and third party cells. In addition, sFasL/MLR-treated cell populations contained influenza-specific T cells, CD4+FOXP3+ T cells, and CD4+CD25+ T cells. These data indicate that this ex vivo sFasL/MLR depletion of alloreacting human donor anti-host T cells was efficient and selective.

Deletion of Tristetraprolin Caused Spontaneous Reactive Granulopoiesis by a Non–Cell-Autonomous Mechanism Without Disturbing Long-Term Hematopoietic Stem Cell Quiescence

Tristetraprolin (TTP, Zfp36, Nup475, Tis11) dramatically reduces the stability of target mRNAs by binding to AU-rich elements in their 3′ untranslated regions. Through this mechanism, TTP functions as a rheostatic, temporal regulator of gene expression. TTP knockout (KO) mice exhibit completely penetrant granulocytic hyperplasia. We have shown that the hematopoietic stem-progenitor cell compartment in TTP KO mice is also altered. Although no change was detected in long-term hematopoietic stem cell (HSC) frequency or function, as assayed by immunophenotypic markers or limiting dilution transplants, we observed increases in the frequencies and numbers of short-term HSCs, multipotent progenitors, and granulocyte–monocyte progenitors. This pattern is consistent with “reactive granulopoiesis,” in which committed myeloid progenitors and more primitive progenitors cycle more actively to increase production of mature granulocytes in response to infection or adjuvant. We created reverse chimeras by transplanting wild-type bone marrow into TTP KO mice and found the “reactive granulopoiesis” phenocopied, indicating a non–hematopoietic stem-progenitor cell–autonomous mechanism. Correspondingly, we found elevated levels of the granulopoietic TTP targets IL-1β, TNF-α, and IL-6 in the plasma of TTP KO mice. Consistent with the non–cell-autonomous nature of the phenotype, we found elevated levels of IL-1β, TNF-α, and IL-6 transcripts in the livers of TTP KO mice and no detectable difference in the bone marrows. These findings demonstrate the importance of TTP in inflammatory homeostasis and highlight the ability of the hematopoietic system to respond to stress without significant numbers of quiescent HSCs entering the cell cycle.

Correlated miR-mRNA Expression Signatures of Mouse Hematopoietic Stem and Progenitor Cell Subsets Predict “Stemness” and “Myeloid” Interaction Networks

Several individual miRNAs (miRs) have been implicated as potent regulators of important processes during normal and malignant hematopoiesis. In addition, many miRs have been shown to fine-tune intricate molecular networks, in concert with other regulatory elements. In order to study hematopoietic networks as a whole, we first created a map of global miR expression during early murine hematopoiesis. Next, we determined the copy number per cell for each miR in each of the examined stem and progenitor cell types. As data is emerging indicating that miRs function robustly mainly when they are expressed above a certain threshold (∼100 copies per cell), our database provides a resource for determining which miRs are expressed at a potentially functional level in each cell type. Finally, we combine our miR expression map with matched mRNA expression data and external prediction algorithms, using a Bayesian modeling approach to create a global landscape of predicted miR-mRNA interactions within each of these hematopoietic stem and progenitor cell subsets. This approach implicates several interaction networks comprising a “stemness” signature in the most primitive hematopoietic stem cell (HSC) populations, as well as “myeloid” patterns associated with two branches of myeloid development.

Abstract 3385: Expression of CD22, a late B lymphoid antigen, does not distinguish leukemia stem cells from the bulk population in acute lymphoblastic leukemia cases

Introduction: Pediatric ALL is still fatal in ∼20% of cases, motivating development and clinical trials of novel antineoplastic agents in recurrent ALL such as CD22 monoclonal antibody and immunotoxins. CD22 is expressed on most ALL cases, but not every cell in every ALL case is detectably CD22+. In normal B lymphocyte development, CD22 is first expressed on the cell membrane at the early B to proB cell stage, at the time of D-J gene rearrangement. Taken together, these 2 facts beg the question of whether most or all of the presumed earliest cells in ALL cases, the leukemia stem cells, might be CD22-; if so, CD22 monoclonal antibody treatment might miss ALL stem cells. We therefore investigated CD22 expression on pediatric ALL stem cells. Methods: Studies were performed using secondary xenotransplants of five cases of pediatric precursor-B ALL. Cells (>90% human leukemia cells from spleens of immunodeficient mice) were stained with monoclonal antibodies and fluorescence-activated cell sorted (FACS). Titered doses of human ALL cells (100 -100000 cells/mouse; n=5 mice/group) from (1) unsorted, (2) mock sorted, (3) CD22high, and (4) CD22low populations, excluding non-viable and mouse cells, were separately transplanted by intravenous injection into NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. NSG mice lack functional T, B cells, and NK cells, and are also deficient in innate immunity. Time to leukemia was determined from injection to onset of clinical signs of leukemia. Leukemia was confirmed by autopsy (splenomegaly) and FACS-analysis for human ALL. Pre- and post-sorted and pre- and post-transplanted cells were evaluated via multicolor FACS-analysis, using CD22, a viability dye, lymphoid (CD10, CD19), myeloid (CD13, CD33), stem-progenitor (CD34), and mouse cell (mouse CD45) markers. Results: Time to leukemia decreased in an inverse transplanted cell dose dependent manner in all 4 experimental groups (CD22high, CD22low, unsorted and mock sorted populations of each case), but was not statistically different among the four groups in any of the tested ALL cases. LSC quantitation is ongoing, based on frequency of mice with leukemias at the lowest transplanted cell doses. The heterogeneity of antigen expression in the leukemia cell populations was not different in cells analyzed before versus after NSG transplantation. Discussion: In these five cases of pediatric precursor-B ALL, the level of expression of CD22 did not, in evidence obtained to date, distinguish leukemia stem cells (as defined by NSG mouse-engrafting capacity) from the bulk population of ALL cells. Specifically, there is no suggestion from our data that leukemia stem cells were enriched in the CD22low populations, which provides further evidence against a hierarchical model of ALL stemness. Furthermore, targeting CD22 would not be predicted to differentially spare leukemia stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3385. doi:1538-7445.AM2012-3385

Abstract 4356: Cytotoxicity of the anti-CD22 immunotoxin HA22 against pediatric acute lymphoblastic leukemia (ALL)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Cure rates for children with acute lymphoblastic leukemia (ALL) have steadily improved, but a proportion of patients still relapse despite intensive and prolonged chemotherapy. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. We have developed recombinant anti-CD22 immunotoxins composed of the Fv portion of the anti-CD22 mAb RFB4 linked to a 38kDa truncated protein derived from Pseudomonas exotoxin A (PE38). These produce cell death through a unique and highly specific mechanism of action, entailing binding to cell surface CD22, rapid internalization, intracellular processing, and inhibition of protein synthesis through ADP-ribosylation of elongation factor-2. In phase I and II trials of patients with hairy cell leukemia the first generation agent, BL22, produced complete responses (CR) of 61% and 47% respectively. However, activity was modest in children with ALL (Blood 2007;110:262a). A modified agent, HA22, was derived by complementarity-determining region mutagenesis resulting in a significantly higher binding affinity for the CD22 antigen. We cultured pre-B ALL cells from newly diagnosed (n = 13) and relapsed children (n= 22), as well as human ALL cells derived from xenografts in immunodeficient mice (n = 4) and the cell line KOPN-8 on telomerase-immortalized human bone marrow mesenchymal cells. On day 2 of culture, HA22 was added at concentrations ranging from 0 to 500ng/ml and the cells were incubated for an additional 72 hours. Dexamethasone 10umol/L was used as a positive control. The number of viable CD19+ cells at the end of the cultures was determined by flow cytometry. Viability was assessed using gates set for light-scatter properties and Annexin-PE/7-AAD staining on Days 2 and 5. The percentage of CD19+ viable cells relative to the control was determined and the 50% inhibitory concentration (IC50) calculated. Mesenchymal-supported cell cultures maintained ALL cell viability (mean 77%, median 52%, range 6% - 280% viable cells at 96 hours). ALL samples varied in their sensitivity to HA22. IC50s ranged from 1-80 ng/mL in 29 of 40 samples. In 11 samples 50% killing was not achieved. Resistance to cytotoxicity from dexamethasone was also observed, which correlated with resistance to HA22 for samples from newly diagnosed patients (r = 0.55, p = 0.053). Peripheral blood blasts appeared to be more sensitive to HA22 in comparison to bone marrow derived blasts (p = 0.008). Blasts with higher CD22 site densities showed increased cytotoxicity to HA22 (r = −0.34, p = 0.16). These results indicate that most cases of childhood B-lineage ALL are sensitive to HA22 at concentrations well below clinically achievable blood levels. Clinical testing of HA22 in children with drug-resistant ALL has been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4356.