Sophie Amsellem

HOXC4 homeoprotein efficiently expands human hematopoietic stem cells and triggers similar molecular alterations as HOXB4

Background Expansion of hematopoietic stem cells represents an important objective for improving cell and gene therapy protocols. Retroviral transduction of the HoxB4 homeogene in mouse and human hematopoietic stem cells and hematopoietic progenitors is known to promote the cells’ expansion. A safer approach consists in transferring homeobox proteins into hematopoietic stem cells taking advantage of the natural ability of homeoproteins to cross cell membranes. Thus, HOXB4 protein transfer is operative for expanding human hematopoietic cells, but such expansion needs to be improved. Design and Methods To that aim, we evaluated the effects of HOXC4, a protein encoded by a HOXB4 paralog gene, by co-culturing HOXC4-producing stromal cells with human CD34+ hematopoietic cells. Numbers of progenitors and stem cells were assessed by in vitro cloning assays and injection into immuno-deficient mice, respectively. We also looked for activation or inhibition of target downstream gene expression. Results We show that the HOXC4 homeoprotein expands human hematopoietic immature cells by 3 to 6 times ex vivo and significantly improves the level of in vivo engraftment. Comparative transcriptome analysis of CD34+ cells subjected or not to HOXB4 or HOXC4 demonstrated that both homeoproteins regulate the same set of genes, some of which encode key hematopoietic factors and signaling molecules. Certain molecules identified herein are factors reported to be involved in stem cell fate or expansion in other models, such as MEF2C, EZH2, DBF4, DHX9, YPEL5 and Pumilio. Conclusions The present study may help to identify new HOX downstream key factors potentially involved in hematopoietic stem cell expansion or in leukemogenesis.

Improving the Outcome of Leukemia by Natural Killer Cell-Based Immunotherapeutic Strategies

Blurring the boundary between innate and adaptive immune system, natural killer (NK) cells are widely recognized as potent anti-leukemia mediators. Alloreactive donor NK cells have been shown to improve the outcome of allogeneic stem-cell transplantation for leukemia. In addition, in vivo transfer of NK cells may soon reveal an important therapeutic tool for leukemia, if tolerance to NK-mediated anti-leukemia effects is overcome. This will require, at a minimum, the ex vivo generation of a clinically safe NK cell product containing adequate numbers of NK cells with robust anti-leukemia potential. Ideally, ex vivo generated NK cells should also have similar anti-leukemia potential in different patients, and be easy to obtain for convenient clinical scale-up. Moreover, optimal clinical protocols for NK therapy in leukemia and other cancers are still lacking. These and other issues are being currently addressed by multiple research groups. This review will first describe current laboratory NK cell expansion and differentiation techniques by separately addressing different NK cell sources. Subsequently, it will address the mechanisms known to be responsible for NK cell alloreactivity, as well as their clinical impact in the hematopoietic stem cells transplantation setting. Finally, it will briefly provide insight on past NK-based clinical trials.

c-Myc regulates expression of NKG2D ligands ULBP1/2/3 in AML and modulates their susceptibility to NK-mediated lysis

Cytarabine (cytosine arabinoside) is one of the most effective drugs for the treatment of patients diagnosed with acute myeloid leukemia (AML). Despite its efficiency against AML cells, the emergence of drug resistance due to prolonged chemotherapy in most patients is still a major obstacle. Several studies have shown that drug resistance mechanisms alter the sensitivity of leukemia cells to immune system effector cells. To investigate this phenomenon, parental acute myeloid cell lines, HL-60 and KG-1, were continuously exposed to increasing doses of cytarabine in order to establish equivalent resistant cell lines, HL-60(R) and KG-1(R). Our data indicate that cytarabine-resistant cells are more susceptible to natural killer (NK)-mediated cell lysis as compared with parental cytarabine-sensitive cells. The increased susceptibility correlates with the induction of UL-16 binding proteins (ULBP) 1/2/3 and NK group 2, member D (NKG2D) ligands on target cells by a mechanism involving c-Myc induction. More importantly, chromatin immunoprecipitation assay revealed that ULBP1/3 are direct targets of c-Myc. Using drug-resistant primary AML blasts as target cells, inhibition of c-Myc resulted in decreased expression of NKG2D ligands and the subsequent impairment of NK cell lysis. This study provides for the first time, the c-Myc dependent regulation of NKG2D ligands in AML.

A GEIL flow cytometry consensus proposal for quantification of plasma cells: application to differential diagnosis between MGUS and myeloma.

Flow cytometry is the sole available technique for quantification of tumor plasma‐cells in plasma‐cell disorders, but so far, no consensus technique has been proposed. Here, we report on a standardized, simple, robust five color flow cytometry protocol developed to characterize and quantify bone marrow tumor plasma‐cells, validated in a multicenter manner.

The HOXB4 Homeoprotein Differentially Promotes Ex Vivo Expansion of Early Human Lymphoid Progenitors

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long‐term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34+ hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B‐cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.

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

Tetraspanin CD9 participates in dysmegakaryopoiesis and stromal interactions in primary myelofibrosis

Primary myelofibrosis is characterized by clonal myeloproliferation, dysmegakaryopoiesis, extramedullary hematopoiesis associated with myelofibrosis and altered stroma in the bone marrow and spleen. The expression of CD9, a tetraspanin known to participate in megakaryopoiesis, platelet formation, cell migration and interaction with stroma, is deregulated in patients with primary myelofibrosis and is correlated with stage of myelofibrosis. We investigated whether CD9 participates in the dysmegakaryopoiesis observed in patients and whether it is involved in the altered interplay between megakaryocytes and stromal cells. We found that CD9 expression was modulated during megakaryocyte differentiation in primary myelofibrosis and that cell surface CD9 engagement by antibody ligation improved the dysmegakaryopoiesis by restoring the balance of MAPK and PI3K signaling. When co-cultured on bone marrow mesenchymal stromal cells from patients, megakaryocytes from patients with primary myelofibrosis displayed modified behaviors in terms of adhesion, cell survival and proliferation as compared to megakaryocytes from healthy donors. These modifications were reversed after antibody ligation of cell surface CD9, suggesting the participation of CD9 in the abnormal interplay between primary myelofibrosis megakaryocytes and stroma. Furthermore, silencing of CD9 reduced CXCL12 and CXCR4 expression in primary myelofibrosis megakaryocytes as well as their CXCL12-dependent migration. Collectively, our results indicate that CD9 plays a role in the dysmegakaryopoiesis that occurs in primary myelofibrosis and affects interactions between megakaryocytes and bone marrow stromal cells. These results strengthen the “bad seed in bad soil” hypothesis that we have previously proposed, in which alterations of reciprocal interactions between hematopoietic and stromal cells participate in the pathogenesis of primary myelofibrosis.

Enhanced cytotoxic activity of ex vivo-differentiated human natural killer cells in the presence of HOXB4.

We have previously shown that human umbilical cord blood CD34+ progenitor cells undergo in vitro differentiation into functional natural killer (NK) cells and that their coculture in the presence of HOXB4-transduced stromal MS-5 cells resulted in an increase in differentiated NK number. The present study was conducted to compare the stromal effect on NK lytic potential in the presence and absence of HOXB4. Our results provide evidence that HOXB4-transduced MS-5 cells as compared with transduced GFP (+) MS-5 cells induced highly differentiated cytotoxic NK cells. Importantly, this difference was not because of the expression of activating NK receptors but was associated with an increased induction of granzyme B degranulation in response to stimulation with NK cell susceptible targets. DNA microarray-based global transcriptional profiling confirmed the upregulation of granzyme B. These findings provide further evidence that HOXB4 is a crucial regulator of NK function and that its use in generating functional NK cells with increased lytic potential may be significant for cancer immunotherapy.

Abstract 199: Increased killing potential of ex vivo differentiated human natural killer cells in the presence of HOXB4

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have previously shown that human umbilical cord blood CD34+ progenitor cells undergo in vitro differentiation into functional NK cells and that their co-culture in the presence of HOXB4 transduced stromal MS5 cells resulted in an increase in differentiated NK number. The present study was conducted to compare the stromal effect on NK lytic potential in the presence and absence of HOXB4. Our results provide evidence that HOXB4 transduced MS5 cells as compared to transduced, GFP (+) MS5 cells induced highly differentiated cytotoxic NK cells. Importantly, this difference was not due to the expression of activating NK receptors but was associated with an increased induction of granzyme B transcription and degranulation in response to stimulation with NK cell susceptible targets. DNA microarray-based global transcriptional profiling confirmed the up-regulation of Granzyme B. We provide evidence that HOXB4 is a crucial regulator of NK lytic function and that its use in generating functional NK cells with increased lytic potential may be significant for hematologic malignancies. Our findings provide further evidence that HOXB4 is a crucial regulator of NK function and that its use in generating functional NK cells with increased lytic potential may be significant for cancer immunotherapy. Citation Format: Salem Chouaib, Arash Nanbakhsh, Cecile Pochon, Sophie Amsellem, Gianfranco Pittari, Jean-Henri Bourhis. Increased killing potential of ex vivo differentiated human natural killer cells in the presence of HOXB4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 199. doi:10.1158/1538-7445.AM2014-199