MS-5 Bone Marrow Stromal Cells Research Articles

FGF7 Supports Hematopoietic Stem and Progenitor Cells and Niche-Dependent Myeloblastoma Cells Via Autocrine Action On Bone Marrow Stromal Cells In Vitro

Some fibroblast growth factor (FGF)/FGF receptor (FGFR) systems, namely FGF1 and FGF2 which are produced mainly by megakaryocytes, and their cognate receptors expressed on either hematopoietic stem or progenitor cells (HSPCs) or bone marrow (BM) stromal cells, are reported to support HSPCs under stressed conditions. In this study, we show that mesenchymal cell-derived FGF7 (also called keratinocyte growth factor or KGF) may be a novel niche factor for HSPC support and niche-dependent leukemia-initiating cell growth. We previously reported that mouse embryonic fibroblasts (MEFs) have characteristics that mimic BM stromal cells and possess an ability of HSPCs support in vitro, and that this ability is profoundly attenuated in MEFs deficient for the MED1 subunit of the Mediator transcriptional master coregulator complex, subcomplex of RNA polymerase II holoenzyme. We found through the microarray analyses that the FGF7 expression (both at mRNA and protein levels) was specifically and profoundly attenuated in Med1-/- MEFs compared with Med1+/+ MEFs. When normal mouse BM cells were cocultured with mitomycin C-treated Med1+/+ MEFs or BM stromal cells (OP-9 or MS-5) in the presence of anti-FGF7 antibody, the DNA synthesis and resultant growth of BM cells, and the number of long-term culture-initiating cells (LTC-ICs), were significantly suppressed. However, the addition of anti-FGF7 antibody did not enhance cell death in BM cells. In contrast, the addition of recombinant FGF7 to the coculture of BM cells with mitomycin C-treated Med1-/- MEFs robustly upregulated the DNA synthesis and growth of BM cells, and the number of LTC-ICs. The Ph+ myeloblastoma cells MB-1, originally isolated from a patient with blast crisis chronic myeloid leukemia, is niche-dependent myeloblastic cells that require mesenchymal stromal cells for survival and growth. During coculture typically with OP-9 BM stromal cells, these MB-1 cells form cobblestone areas, which most probably reconstitute a stochastic model of leukemic stem or initiating cells in vitro. We next analyzed the role of FGF7 in support of MB-1 cells for growth and survival during coculture. Indeed, the addition of anti-FGF7 antibody to the coculture with mitomycin C-treated BM stromal cells (either OP-9 or MS-5) substantially attenuated DNA synthesis and resultant proliferation of stromal cell-dependent myeloblastoma cells MB1. Notably, cobblestone formation (i.e., signature of stem cell phenotype) of MB-1 cells was significantly attenuated when anti-FGF7 antibody was added to the coculture with OP-9 cells. However, the addition of anti-FGF7 antibody did not elicit cell death of MB-1 cells. Thus, FGF7 appears to promote both proliferation and maintenance of leukemia stem or initiating cell feature of MB-1 cells. While neither FGF7 nor its cognate receptor FGFR2IIIb mRNAs were detectable in BM cells, the mesenchymal cells including Med1+/+ MEFs and (OP-9 and MS-5) BM stromal cells expressed them. The expression levels of FGFR2IIIb were comparable between Med1+/+ and Med1-/- MEFs, and BM stromal cells. Therefore, we hypothesized that FGFR2IIIb on these mesenchymal stromal cells might be functional and possibly elicit secretion of niche signal(s) secondarily in response to the downstream target gene(s) of FGFR2IIIb, followed by subsequent support of HSPCs and leukemic cells. To prove that FGF7 is functional in these cells, we examined response of the downstream targets of FGFR2IIIb in these cells, including phosphorylation of FRS2 (the immediate downstream target of FGFR2IIIb tyrosine kinase), phosphorylation of MAP kinases (the intermediate hub of various intracellular signals), and mRNA expression of immediate early response genes including c-Jun, c-Fos and c-Myc. Indeed, exogenously added recombinant FGF7 elicited phosphorylation of both FRS2 and MAP kinases within 10 min, and full induction of these immediate early response genes within 30 min, similarly in both Med1+/+ and Med1-/- MEFs. The OP-9 and MS-5 BM stromal cells also exhibited similar responses to exogenously added FGF7. These results indicate that FGFR2IIIb expressed on mesenchymal stromal cells are functional, and that FGF7 and FGFR2IIIb produced by these cells constitute an autocrine (or paracrine) loop. Taken together, we propose that FGF7 produced by BM stromal cells supports HSPCs and leukemic progenitor cells indirectly via FGFR2IIIb expressed on the BM stromal cells. Disclosures:No relevant conflicts of interest to declare.

Putative Autophagy Protein VMP1 Amplifies Protective Autophagy Under Stress Conditions in AML Cells

Autophagy is a multi-step catabolic process orchestrated by a large set of specific proteins. We previously reported that autophagy is essential for normal and AML cells. However, how (basal) autophagy is regulated in these cells remains elusive. Vacuole membrane protein (VMP1) is a putative autophagy protein residing in the endoplasmic reticulum membrane. Upon autophagy induction, VMP1 can recruit and activate the PI3K complex, thereby facilitating and initiating the biogenesis of autophagy vesicles.Transcriptome analyses of autophagy-associated genes was performed on a previously established gene expression dataset of a cohort of patient AML CD34+ cells (n=62) in comparison with normal bone marrow CD34+ cells (n=22). VMP1 mRNA expression was upregulated in 66% of AML cases, with an average increase of 1.4 fold (p<0.001), which was confirmed by qPCR. Within the panel of tested AMLs, VMP1 was significantly higher expressed in AML cells belonging to the monocytic (M4-M5) compared to myeloid (M1-M2) lineage. Moreover, in publicly available expression data sets (R2: Genomics Analysis and Visualization Platform), VMP1 expression was significantly lower in AMLs harbouring C/EBPα mutations.Functional studies demonstrated that downregulation of VMP1 by shRNAs in cord blood (CB)-derived CD34+ HSPCs resulted in a decline of autophagic-flux, coinciding with reduced progenitor frequencies (2.9 fold reduction, p=<0.05). Similar, in vitro expansion was reduced under both myeloid (~2.5 fold reduction, p=<0.05) and erythroid (~7.6 fold reduction, p=<0.01) culture conditions. The reduced cell expansion was due to delayed differentiation and increased apoptosis. Notably, the strongest effects were observed in the erythroid lineage. Co-culturing of shVMP1-transduced CD34+ cells on MS5 bone marrow stromal cells did not rescue the observed negative phenotype. Next, shVMP1-GFP or shSCR-GFP-transduced CD34+ HSPCs were transplanted in immunodeficient NSG mice after sublethal irradiation. Chimerism levels were defined by the percentage of human CD45 in peripheral blood. While GFP levels for shSCR remained stable at around ~60%, the contribution of the shVMP1-transduced cells to the engrafted HSPCs was severely reduced (<2%, p<0.0001). Similar to normal HSPCs, knockdown of VMP1 in leukemic cell lines (n=6) or primary patient CD34+ AML cells (n=3) resulted also in a strong reduction in expansion compared to control. Moreover, knockdown of VMP1 resulted in accumulation of reactive oxygen species (ROS) and mitochondrial mass as determined by FACS, which might be due to impaired mitophagy.In order to model high VMP1 expressing AMLs, we constructed a lentiviral VMP1 overexpression vector. In contrast to VMP1 knockdown, overexpression of VMP1 did not affect cell expansion, differentiation or autophagy flux under normal culture conditions in CB CD34+ cells. However, under hypoxia or during glucose starvation normal CD34+ or AML cells overexpressing VMP1 showed an increased autophagic-flux. Of interest, the VMP1 expression in CB 34+ cells increased (~1.5 fold, p=<0.05) under hypoxia compared to normoxia. Finally to enhance autophagic-flux, leukemic TF-1 and NB4 cells were serum starved (1% FCS) or cultured overnight under hypoxic conditions (1% oxygen). Both serum starvation and hypoxia triggered cell death in TF-1 and NB4 cells, which could be in part rescued by ectopic overexpression of VMP1 (~2 fold increased survival, p=<0.05).In conclusion, our data demonstrate that in normal hematopoietic- and leukemic cells VMP1 is associated with mitochondrial quality control and increased VMP1 expression amplifies protective autophagy under adverse growth conditions such as hypoxia or starvation. DisclosuresNo relevant conflicts of interest to declare.

MGP Interacts with BMP-4 and BMP-2 and Supports Normal and Malignant Hematopoietic Stem/Progenitor Cells

The general transcriptional coregulatory complex Mediator, subcomplex of the RNA polymerase II holoenzyme, is the endpoint convergence of a variety of intracellular signals, and initiates transcription through recruitment of general initiation factors and formation of a functional preinitiation complex. Among the Mediator subunits, MED1 is crucial for hematopoiesis: it is known to be employed in transcription of genes involved in hematopoietic niche function, as well as in differentiation of hematopoietic precursor cells (e.g., RARα- and VDR-mediated myelomonopoiesis, GATA1-mediated erythromegakaryopoiesis, and iNKT cell development). Among the attenuated genes in Med1-/- mesenchymal stromal cells was matrix Gla protein (MGP), modulator of the BMP-SMAD signals. As MGP is abundantly expressed in bone tissues and reportedly interacts with niche factors BMP-4 and BMP-2, we hypothesized that MGP might modulate niche function in hematopoiesis through the interaction with these BMPs. We tested this possibility by an in vitro niche model composed of a coculture of bone marrow (BM) mesenchymal stromal cells and BM hematopoietic cells. When mouse BM hematopoietic cells were cocultured with OP-9 or MS-5 mouse BM stromal cells in the presence of the blocking antibody against MGP, the growth and DNA synthesis of BM hematopoietic cells were attenuated and the number of long-term culture-initiating cells (LTC-ICs), which corresponded to the supported hematopoietic stem/progenitor cells (HSPCs), decreased. We next asked if MGP also functioned as a niche factor for malignant HSPCs, To this end, we utilized MB-1 cells, blast crisis CML-derived myeloblastic leukemia cells. MB-1 cells are dependent on cocultured BM stromal cells and possess characteristics of leukemia stem cells (LSCs). When MB-1 cells were cocultured with OP-9 or MS-5 BM stromal cells in the presence of the anti-MGP antibody, the number of MB-1 cells, as well as the number of cobblestone formation, i.e., LSC nature, of MB-1 cells, was profoundly attenuated. Therefore, MGP appears to have a trophic effect on hematopoietic cells and to support normal and malignant HSPCs in our niche model. MGP was, both at mRNA and protein levels, expressed abundantly in BM stromal cells but scarcely in normal hematopoietic cells. However, MB-1 leukemia cells (presumably ectopically) expressed a meaningfully high level of MGP. During the coculture of normal BM hematopoietic cells with OP-9 or MS-5 BM stromal cells, MGP was induced prominently and transiently after one day. MGP, expressed in MS-5 or OP-9 BM stromal cells, was likewise transiently induced after 1 day of culture with BM hematopoietic cells that were physically dissociated from these BM stromal cells by the transwell apparatus, indicating that secreted humoral factor(s) induced the MGP expression in the BM stromal cells. As for a malignant situation, the expressions of MGP in both MB-1 leukemia cells and OP-9 or MS-5 BM stromal cells were fluctuated in an oscillated days period. Since the support of HSPCs required their association with BM stromal cells, the induced MGP expression was apparently insufficient for HSPCs support, and the action of induced MGP may be indirect. Therefore, we next asked if BMP-4 and BMP-2, which reportedly associate with MGP, were also induced by the coculture and were employed in HSPCs support jointly with MGP. Indeed, during the coculture, BMP-4 was rapidly and transiently induced within a few days, simultaneously with MGP, followed by a subsequent and sustained induction of BMP-2 that lasted for over a week. However, neither BMP-4 nor BMP-2 was produced by BM stromal cells when cultured with dissociated BM hematopoietic cells using the transwell apparatus. Therefore, the induction of BMP2/BMP4 was dependent on physical association of hematopoietic cells and stromal cells. GST-pulldown assays and mammalian two-hybrid assays confirmed that MGP specifically interacted with both BMP-4 and BMP-2. To elucidate the mechanism of action of the blocking antibody against MGP, we tested if the antibody inhibited the interaction between MGP and BMP-4. Indeed, serial GST pulldown assays disclosed that the addition of the antibody specifically abolished the interaction between GST-MGP and BMP-4. These results indicate that MGP might act as a niche factor for normal and malignant HSPCs, at least in part, through interacting with, and modulating the action of, BMP-4 and BMP-2. DisclosuresNo relevant conflicts of interest to declare.

Periostin Supports Hematopoietic Stem/Progenitor Cells and Niche-Dependent Myeloblastoma Cells In Vitro

Periostin (POSTN), the fasciclin family extracellular matrix protein also known as osteoblast-specific factor 2 (OSF-2), was previously reported to be required for optimal B lymphopoiesis in vitro. Now, our study first demonstrates the proof that POSTN might be a bona fide niche factor for both normal and malignant myelopoiesis, indicating that it is a niche molecule for hematopoietic stem cells and diverse hematopoietic precursor cells. The Mediator, composed of about 31 subunits, is a master transcriptional coregulator complex that is essential for global transcription governed by RNA polymerase II. Among the Mediator subunits, MED1 acts as a specific coactivator for activators that include nuclear receptors and GATA1. We previously reported that Med1−/− mouse embryonic fibroblasts (MEFs) have a decreased capability to support hematopoietic stem/progenitor cells (HSPCs) relative to wild-type MEFs in vitro, and that the attenuated expression of full-length osteopontin and FGF7 in Med1−/− MEFs is responsible for the observed phenotype. The microarray analyses, showing that the expression of POSTN was also suppressed in Med1−/− MEFs, prompted us to study the role for POSTN in support of both normal and malignant HSPCs in our in vitro niche model. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1+/+ MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN blocking antibody, the mitogenicity and growth of BM cells were attenuated. The number of long-term culture-initiating cells (LTC-ICs), i.e., number of both granulo-monocytic and erythroid colonies, was also decreased. When BM cells were cocultured with Med1-/- MEFs in the presence of recombinant POSTN, the mitogenicity and growth of BM cells and the number of LTC-ICs were restored. These results suggest that POSTN mediates mitogenicity of BM cells and HSPCs support. The MB-1 myeloblastoma cell line, originally established from a patient with myeloid crisis chronic myeloid leukemia, is a mesenchymal stromal cell-dependent cell line. These cells are unique in that they grow by forming cobblestone areas in the presence of niche cells but die of apoptosis when detached from stromal cells, thus faithfully conforming to a stochastic model of leukemic stem cells in vitro. Intriguingly, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the mitogenicity and growth, as well as the cobblestone formation, a leukemic stem cell feature, of MB-1 myeloblastoma cells. Therefore, it appears that niche cell-derived POSTN supports niche-dependent MB-1 myeloblastoma cells. While POSTN was expressed both in BM cells and variably in different BM stromal cells, expression in the latter cells was markedly increased by tactile interaction with hematopoietic cells. Specifically, POSTN was robustly induced 6 hours after BM stromal cells were cocultured with BM cells or MB-1 myeloblastoma cells, and the induction sustained for as long as 24 hours. However, POSTN expression was not enhanced when BM cells were cocultured but physically separated from MS-5 or OP-9 cells using transwell culture wells. Therefore, the major source of POSTN in the coculture appears to be the BM stromal cells associated with hematopoietic cells. The receptor for POSTN, integrin αvβ3, was expressed abundantly in BM stromal cells. Although β3 mRNA was especially prominent in both BM cells and MB-1 cells, in accordance with a previous report that integrin β3/CD61 marks HSPCs, western blot analysis showed that αv and β3 expression levels were below the detection level on BM cells. Hence, integrin αvβ3 is scarce on BM cells compared to BM stromal cells, although it does not exclude the possibility that functional integrin αvβ3 might be enriched on HSPCs as suggested previously. When an excess amount of exogenous POSTN was added to MS-5 or OP-9 BM stromal cells after 24-h serum starvation, FAK (the immediate target of integrin αvβ3) and MAP kinases ERK1/ERK2 (the intermediate hub of various intracellular signals) were robustly phosphorylated as early as 10 min, and the phosphorylation was sustained for over 60 min. Thus, POSTN effectively activates integrin αvβ3 and subsequent intracellular signaling in BM stromal cells. These results suggest that stromal cell POSTN supports both normal HSPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner. DisclosuresNo relevant conflicts of interest to declare.

Periostin supports hematopoietic progenitor cells and niche-dependent myeloblastoma cells in vitro

The expression of extracellular matrix protein periostin (POSTN) was attenuated in Med1−/− mouse embryonic fibroblasts (MEFs), which exhibited a decreased capability to support hematopoietic progenitor cells (HPCs) in vitro. When bone marrow (BM) cells were cocultured with mitomycin C-treated Med1+/+ MEFs, or OP-9 or MS-5 BM stromal cells, in the presence of anti-POSTN antibody, the growth of BM cells and number of long-term culture-initiating cells (LTC-ICs) were attenuated. When BM cells were cocultured with Med1−/− MEFs in the presence of recombinant POSTN, the growth of BM cells and the number of LTC-ICs were restored. Moreover, antibody-mediated blockage of stromal cells-derived POSTN markedly reduced the growth and cobblestone formation, a leukemic stem cell feature, of stromal cell-dependent MB-1 myeloblastoma cells. POSTN was expressed both in BM cells and variably in different BM stromal cells. Expression in the latter cells was increased by physical interaction with hematopoietic cells. The receptor for POSTN, integrin αvβ3, was expressed abundantly in BM stromal cells. The addition of recombinant POSTN to BM stromal cells induced intracellular signaling downstream of integrin αvβ3. These results suggest that stromal cell POSTN supports both normal HPCs and leukemia-initiating cells in vitro, at least in part, indirectly by acting on stromal cells in an autocrine or paracrine manner.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p&lt;0.01) and, to some extent, granulocytic and macrophage colony formation as well (p&lt;0.09, p&lt;0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p&lt;0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

Targeting Autophagy As a Potential Therapeutic Strategy for the Treatment of AML

Autophagy is a catabolic process whereby redundant organelles and proteins are digested and recycled to maintain cellular homeostasis. Malignant cells might activate autophagy in response to cellular stress, and/or to provide metabolites required for rapid cell proliferation. In addition, elevated autophagy is associated with increased chemoresistance. In normal hematopoiesis, autophagy is important for stem and progenitor cell (HSPC) maintenance. We previously observed that basal autophagy is higher in HSPCs compared more differentiated cells. Genetic interference with the autophagy machinery resulted in a strong reduction in HSPC frequencies, without interfering with the differentiation program.To study the role of autophagy in acute myeloid leukemia (AML), steady-state autophagy was measured in a panel of different myeloid leukemic cell lines (n=8). The autophagic flux was determined by the relative accumulation of cyto-ID (a marker for autophagic vesicles) measured by flow cytometry after hydroxychloroquine (HCQ) or Bafilomycin-1A treatment and LC3-II/p62 accumulation by Western blotting. Interestingly, in leukemic cell lines, a higher basal autophagy flux correlated with increased sensitivity to prolonged inhibition (6 days) of autophagy with different concentrations of HCQ (5 uM R2 =0.52 and 20 uM R2 =0.76), while a mild reduction in expansion was observed in cord blood (CB) derived normal CD34+ cells, after treatment with HCQ (5 uM). In line with the leukemic cell lines, primary sorted AML CD34+ cells (n=36) also showed decreased survival upon HCQ (20 uM) treatment compared to normal bone marrow CD34+ cells (N=6; NBM CD34+: 41.7% ± 7.1 vs. AML CD34+: 21.3% ± 3.2, p=<0.05).Gene expression analysis of autophagy genes by qPCR revealed increased expression of essential autophagy genes ATG5 and ATG7 in primary AML CD34+ compared to NBM CD34+ cells (AML vs. NBM: ATG5 10.3 fold increase, p=<0.05; ATG7 5.9 fold increase, p=<0.01). Therefore, to study the functional consequences in AML, ATG5 and ATG7 were downregulated in leukemic cell lines and primary AML CD34+ cells (n=6), using lentiviral shRNAs. Significant downregulation of ATG5 and ATG7 was confirmed by qPCR, which was also sufficient to inhibit the autophagy flux as demonstrated by reduced accumulation of LC3-GFP puncta. In line with results of HCQ treatment, downmodulation of ATG5 and ATG7 resulted in a strong reduction in expansion of leukemic cell lines. Also, transduced primary AML blasts cultured on MS5 bone marrow stromal cells for 4 weeks showed a distinct reduction in expansion (shSCR vs. shATG5 2.1 fold reduction, p=<0.05; shATG7 2.5 fold reduction p=<0.01).The genetic or pharmaceutical inhibition of autophagy triggered an apoptotic response in leukemic cellsas determined by Annexin V measurements. This coincided with increased expression of p53 and the downstream pro-apoptotic target genes BAX and PUMA and the putative AKT inhibitor PHLDA3. Moreover, HCQ induced apoptosis could be rescued by downregulation of p53 by RNAi in leukemic cell lines that express wild-type P53. Prolonged exposure to HCQ did also result in an apoptotic response in leukemic cell lines harboring p53 mutations, but without the upregulation of BAX, PUMA and PHLDA3.In conclusion, our results indicate that AML CD34+ cells are highly dependent on the autophagy machinery for growth and cell survival. Targeting autophagy might provide a new therapeutic option for treatment of this type of leukemia. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic targeting of Polo-like kinase-1 and Aurora kinases in T-cell acute lymphoblastic leukemia

Polo-like kinases (PLKs) and Aurora kinases (AKs) act as key cell cycle regulators in healthy human cells. In cancer, these protein kinases are often overexpressed and dysregulated, thus contributing to uncontrolled cell proliferation and growth. T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous malignancy arising in the thymus from T-cell progenitors. Primary chemoresistant and relapsed T-ALL patients have yet a poor outcome, therefore novel therapies, targeting signaling pathways important for leukemic cell proliferation, are required. Here, we demonstrate the potential therapeutic effects of BI6727, MK-5108, and GSK1070916, three selective inhibitors of PLK1, AK-A, and AK-B/C, respectively, in a panel of T-ALL cell lines and primary cells from T-ALL patients. The drugs were both cytostatic and cytotoxic to T-ALL cells by inducing G2/M-phase arrest and apoptosis. The drugs retained part of their pro-apoptotic activity in the presence of MS-5 bone marrow stromal cells. Moreover, we document for the first time that BI6727 perturbed both the PI3K/Akt/mTORC2 and the MEK/ERK/mTORC1 signaling pathways, and that a combination of BI6727 with specific inhibitors of the aforementioned pathways (MK-2206, CCI-779) displayed significantly synergistic cytotoxic effects. Taken together, our findings indicate that PLK1 and AK inhibitors display the potential for being employed in innovative therapeutic strategies for improving T-ALL patient outcome.

Induction therapy in a multiple myeloma mouse model using a combination of AS101 and melphalan, and the activity of AS101 in a tumor microenvironment model

We previously showed that the organotellurium compound, ammonium trichloro (dioxyethylene-0-0') tellurate (AS101), has antitumoral activity in multiple myeloma (MM) cell lines. Here, we evaluated the antimyeloma activity of AS101 combined with low-dose melphalan, and also examined the activity of AS101 in the myeloma tumor microenvironment. Isobologram analysis was performed to determine the interactions of AS101 and melphalan as a combination therapy. Growth arrest, apoptosis, and CD81 antigen were detected by flow cytometry. Using the 5T33MM mouse model, we evaluated mouse survival and serum levels of vascular endothelial growth factor (VEGF) and IgG(2b) paraprotein. We established cocultures of MS-5 bone marrow stromal cells and 5T33 MM cells in order to examine AS101 activity in a myeloma microenvironment model. Combined treatment of AS101 with melphalan in vitro resulted in a synergistic inhibitory effect on growth, G(2)/M phase growth arrest, reduced IgG(2b) secretion, apoptotic cell death, and reduced fibronectin-mediated adhesion of MM cells. AS101 reduced VEGF secretion and protein expression in myeloma and cocultured cells, downregulated production of the matrix metalloproteinases (MMPs), MMP-9 and MMP-2, and also inhibited growth of the treated myeloma coculture. Combined treatment using AS101 and low dose of melphalan in vivo resulted in modest survival improvement of myeloma-bearing mice and in reduced IgG(2b) and VEGF serum levels. AS101 in combination with a subtherapeutic dose of melphalan had increased beneficial effect relative to each agent alone in a mouse MM model. In addition, AS101 might be useful for targeting interactions between myeloma cells and the bone marrow microenvironment.

Perivascular/Mesenchymal Stem Cells from Multiple Human Tissues Support Hematopoiesis in Vitro.

Mesenchymal stem cells (MSC) can be derived selectively in culture from multiple organs, an omnipresence we have recently suggested to be explained by the perivascular location of native MSC ancestors within intact tissues (Crisan et al. 2008, in press). We have now analyzed the ability of MSC extracted pro- or retrospectively from different human tissues to support hematopoiesis. MSC were either classically derived in primary cultures of umbilical cord blood (UCB) lineage-depleted mononuclear cells (n=3) or enzymatically dissociated adult adipose tissue (n=3), or grown as CD146+ NG2+ CD34-CD56- CD45- pericytes (n=2) purified by flow cytometry from fetal skeletal muscle and cultured over the long term. In both settings, identical MSC were obtained that maintained a stable CD146+ CD90+ CD73+ CD105+ CD34- CD45- surface phenotype and could differentiate into skeletal muscle, fat, bone and cartilage. CD34+ hematopoietic progenitors (n=3) immunoselected from term UCB were seeded (5×10e3cells/cm2 in triplicate) onto confluent irradiated layers of MSC derived from UCB, adipose tissue or fetal muscle pericytes (MSCu, MSCa and MSCmp, respectively) or, as a control, MS5 bone marrow stromal cells that allow the proliferation of very primitive human progenitor cells. All studies were approved by the relevant institutional regulatory board. The cells were cocultured for 5 weeks in a classical long-term culture-initiating cell assay in a complete medium (MyeloCult H5100, Stem Cell Technologies) containing hydrocortisone but no added cytokine. Wells were scored daily for the presence of cobblestone areas (CA) and half of the medium was replaced every week. Eventually, trypsinized cells from each well were characterized by flow cytometry for the expression of hematopoietic cell markers and assayed for CFC potential. After 14 days of incubation, colonies grown in semi-solid medium were scored as derived from colony forming units (CFU)-granulocyte, erythroid, macrophage, megakaryocyte (GEMM) and as high-proliferative-potential colony precursors (HPPC), the most primitive hematopoietic cell so far identified in a clonogenic assay in vitro. Within the CD45+ gate, all trypsinized cultures contained comparable percentages of CD34+lin- cells (MSCu: 51±9%; MSCa: 58±14%; MSCmp: 61±19%; MS5: 59±18%), the most immature hematopoietic cell compartment maintained during the long-term coculture. MSCu and MSCmp supported a similar cell proliferation during the whole culture while on MSCa, CA formed very rapidly and consistently but eventually decreased over the long-term culture. Interestingly, MSCu and MSCmp supported the development of the highest numbers of HPPC and of CFU giving rise to the largest GEMM colonies, as compared to MSCa that gave the same results as the control MS5 cell line. In summary, all MSCs tested were able to support hematopoiesis and CA formation, albeit with differences in growth kinetics and morphology of the colonies. Herein we show for the first time that purified human perivascular cells exhibit robust hematopoiesis support in vitro, in addition to multilineage mesodermal developmental potential. In conclusion, we demonstrate that MSC from novel sources distinct from the bone marrow are able to support hematopoiesis. These results further sustain the identity, beyond acronyms, between marrow stromal cells, long known for their support of hematopoiesis, and mesenchymal stem cells that gained more recent credit in the field of regenerative medicine because of their multilineage differentiation potential.

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Establishing long-term cultures with self-renewing acute myeloid leukemia stem/progenitor cells

With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology. We have cultured acute myeloid leukemia CD34(+) cells on bone marrow stroma. Long-term expansion was monitored and self-renewal was addressed by replating of Leukemic-cobblestone area-forming cells (L-CAs). Also, lentiviral vectors were generated that could target L-CAs. A strong expansion was observed in about 75% of the acute myeloid leukemia cases (n = 30) and long-term cultures could be maintained for up to 24 weeks on MS5 bone marrow stromal cells. Cells that were able to initiate leukemic cobblestone areas resided in the CD34(+) population and were absent from the CD34(-) population. Self-renewal within these L-CAs was determined by sequential passaging of these L-CAs onto new MS5 stromal layers, which resulted in the generation of second, third, and fourth L-CAs, which were able to sustain long-term expansion and generated high numbers of immature undifferentiated suspension cells. CD34(+) cells that were able to initiate long-term cultures all coexpressed MEIS1 and HOXA9, and expressed elevated BMI1 levels. We present a novel long-term leukemic stem/progenitor assay in which new drugs can be tested and in which genes can be overexpressed or downmodulated using a lentiviral approach in order to obtain more insight into the process of leukemic transformation and self-renewal.