Activity of Targeted Molecular Therapeutics Against Primary AML Cells: Putative Role of the Bone Marrow Microenvironment.

Abstract In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow (BM) microenvironment such as mesenchymal stem cells (MSC). It is speculated that specific BM niches may provide a sanctuary for subpopulations of leukemic cells to evade chemotherapy-induced death and allow acquisition of a drug-resistant phenotype. In this study, we compared anti-leukemia effects of Ara-C and various signal transduction and apoptosis inhibitors in a co-culture system of primary AML and human bone marrow-derived MSC. AML blasts from 11 primary AML samples with high (>70%) blast count were co-cultured with MSC for 24 hours, after which they were exposed to the indicated concentrations of inhibitors for 48–96 hrs. Concentrations were selected on the basis of preliminary cell line studies which determined efficient inhibition of drug targets. Induction of apoptosis was analyzed by Annexin V flow cytometry after gating on the CD90 APC(−) (non-MSC) population. MSC protected leukemic blasts from spontaneous apoptosis in all 11 samples studied (mean annexinV positivity, 49.5±7.2% vs 25.3±4.8%, p<0.001) and from Ara-C-induced cytotoxicity in 6 out of 11 samples (p=0.02). No difference in the degree of protection was noted when MSC from older vs. younger donors were used (not shown). Co-culture of leukemic cells with MSC resulted in significant (p<0.03) suppression of inhibitor-induced apoptosis for all agents tested (Table 1), however PI3K/AKT inhibitors seemed to overcome MSC-mediated resistance. In addition, specific inhibitors of Bcl-2 and MDM2 induced apoptosis not only in suspension, but also in the MSC co-culture system, while Raf-1/MEK inhibitors were less effective. The AKT inhibitor A443654 caused apoptosis induction not only in leukemic cells, but also in MSC, which likely accounted for its high efficacy in stromal co-cultures (53±6% annexin V+). In a different study (Tabe et al, ASH 2005), we report that interactions of leukemic and BM stromal cells result in the activation of PI3K/ILK/AKT signaling in both, leukemic and stromal cells. We therefore propose that disruption of these interactions by specific PI3K/AKT inhibitors represents a novel therapeutic approach to eradicate leukemia in the BM microenvironment via direct effects on leukemic cells and by targeting activated BM stromal cells. Furthermore, Bcl-2 and MDM2 inhibitors appear to retain their efficacy in stroma-cocultured AML cells, while the efficacy of chemotherapy and Raf/MEK inhibitors in these conditions may be reduced. Further studies are aimed at the elucidation of the role of the BM microenvironment and its ability to activate specific signaling pathways in the pathogenesis of leukemias and on efforts to disrupt the MSC/leukemia interaction (Zeng et al, ASH 2005). Focus on this stroma-leukemia-stroma crosstalk may result in the development of strategies that enhance the efficacy of therapies in hematological malignancies and prevent the acquisition of a chemoresistant phenotype. Table 1. Leukemia Cell Apoptosis in a MSC/AML Co-Culture System Target Bcl-2/XL MDM2 PI3K AKT Raf-1 MEK Apoptosis was determined as percentage of Annexin V(+)CD90(−) cells, and calculated by the formula: % specific apoptosis = (test − control) x 100 / (100 − control). Compound, concentration Ara-C, 1 μM ABT-737, 0.1 μM Nutlin-3A, 2.5 μM LY294002, 10 μM A443654, 1 μM BAY43-9006, 2.5 μM CI1040, 3 μM AML 28±7 69±7 45±7 53.8±13.3 75±7 35±11 27±11 AML + MSC 16±4 38±6 28±6 31.2±6.9 53±6 18±8 15±5

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Inhibition of PI3K or Integrin-Linked Kinase (ILK) Target Primary AML Cells within the Bone Marrow Microenvironment in the In Vitro Co-Culture System.

Blood ◽

10.1182/blood.v108.11.1903.1903 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1903-1903

Author(s):

Teresa McQueen ◽

Yoko Tabe ◽

Marina Konopleva ◽

Michael Andreeff

Keyword(s):

Bone Marrow ◽

Culture System ◽

Hematological Malignancies ◽

Annexin V ◽

Bone Marrow Microenvironment ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Integrin Linked Kinase ◽

Induction Of Apoptosis

Abstract In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow microenvironment such as marrow stromal cells (MSC). It is proposed that specific niches within the bone marrow microenvironment provide a sanctuary for subpopulations of leukemic cells to evade chemotherapy-induced death, and we indeed demonstrated that MSC protect primary AML cells from Ara-C induced apoptosis in vitro (Konopleva, Leukemia 2002). Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate AKT in a PI3-kinase(PI3K)-dependent manner to promote cell survival and proliferation. In this study, we tested the hypothesis that selective inhibition of ILK signaling will provide a novel approach for targeting both leukemic cells and cells in their surrounding microenvironment. Direct co-culture of human MSC and leukemic NB4 cells results in activation of PI3K/ILK/AKT signaling as evidenced by enhanced ILK kinase activity, elevated phospho(p)-Akt, p-GSK3β and nuclear translocation of β-catenin. Both, PI3K inhibitor LY294002 (10μM) and specific ILK inhibitor QLT0267 (10μM) inhibited stroma-induced activation of AKT and suppressed GSK phosphorylation. This resulted in massive induction of apoptosis which was not abrogated by stromal co-culture (AnnexinV positivity %, MSC(−) vs MSC(+); 51.4+2.5 vs 55.8+3.5 p=0.26, LY 47.0+8.1 vs 47.9+6.1 p=0.85, 48hrs). In contrast, MSC co-culture effectively blocked apoptosis induced by MEK inhibitor PD98059 despite activation of pERK (62.5+3.2% vs 45.6+2.3%, p=0.02). We next examined anti-leukemia effects of PI3K and ILK inhibitors in the co-culture system of primary AML and human MSC. AML blasts from 7 primary AML samples with high (>54%) blast count were co-cultured with MSC for 24 hours, after which they were exposed to 10μM LY294002 or QLT0267 for 4–8 days. After this period, induction of apoptosis was analyzed in non-adherent AML cells by Annexin V flow cytometry after gating on the CD90-negative (non-MSC) population. To control for differences in spontaneous apoptosis, we calculated % specific apoptosis as (test - control) x 100 / (100 - control). MSCs protected leukemic blasts from spontaneous apoptosis in all 7 samples studied (mean annexin V positivity, 49.5±7.2% vs 25.3±4.8%, p<0.001). In contrast, inhibition of PI3K/ILK signaling induced unopposed apoptosis even in MSC co-cultures (% specific apoptosis, LY294002, 30.3±4.8%; LY+MSC, 28.3±7.7%; QLT0267, 26.9±9.8%; QLT+MSC, 33.1±9.3%, p>0.3 comparing cell death in the presence or absence of MSC). This resulted in corresponding loss of viability (% of control, LY294002, 66.0±11.0%; LY+MSC, 57.6±11.2%; QLT0267, 66.4±7.28%; QLT+MSC, 50.4±11.3%, p>0.1 comparing viability in the presence or absence of MSC). These observations indicate that disruption of leukemia/stroma interactions by specific PI3K/ILK inhibitors represents a novel therapeutic approach to eradicate leukemia in the bone marrow microenvironment. Further studies are aimed at the elucidation of the role of the BM microenvironment and its ability to activate specific signaling pathways in the pathogenesis of leukemias. Focus on this stroma-leukemia crosstalk may result in the development of strategies that alleviate the acquisition of a chemoresistant phenotype and enhance the efficacy of therapies in hematological malignancies.

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Novel Role of HDAC Inhibitors in Bone Marrow Microenvironment: Activation of Leukemia Cell Phagocytosis through Annexin A1.

Blood ◽

10.1182/blood.v106.11.2219.2219 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2219-2219

Author(s):

Yoko Tabe ◽

Rooha Contractor ◽

Susanne Radke ◽

Michael Andreeff ◽

Marina Konopleva

Keyword(s):

Bone Marrow ◽

Membrane Binding ◽

Hdac Inhibitors ◽

Leukemia Cell ◽

Cdna Array ◽

Bone Marrow Microenvironment ◽

Leukemic Cells ◽

Annexin A1 ◽

Induced Apoptosis

Abstract Annexin A1 (ANX-A1) is a calcium-dependent membrane-binding protein involved in the modulation of apoptosis and phagocytosis (FASEB J.2003;17:1544). We have previously reported that HDAC inhibitor depsipeptide (FK228) caused marked growth inhibition and apoptosis in t(8;21) Kasumi-1 AML cells with up-regulation of 123 genes (by cDNA array) including ANX-A1 (3.5 fold; Tabe, Blood 2004). By chromatin immunoprecipitation (ChIP) assay, FK228 induced H4 and H3-K9 acetylation in the ANX-A1 promoter with corresponding induction of ANX-A1 mRNA (7.2±1.7 fold, TaqMan RT-PCR) and protein (western blot analysis). The markedly increased ANX-A1 protein localized on the cell membrane of Kasumi-1 cells exposed to FK228 was confirmed by immunofluorecence analysis using confocal microscopy. ANX-A1 membrane localization was diminished by treatment with anti-ANX-A1 mAb. To investigate the contribution of ANX-A1 to FK228-induced apoptosis, we neutralized ANX-A1 by anti-ANX-A1 mAb. This moderately decreased FK228 induced apoptosis (36.0±4.1 vs 26.5±3.7% AnnexinV(+)/PI(+) cells, p=0.01). Similarly, Kasumi-1 cells transfected with siRNA/ANX-A1 were less sensitive to FK228-induced cell death compared with nonsense (N) siRNA transfected cells (siRNA 31.2±3.1% vs NsiRNA 39.5±2.9% annexin(+) cells, p=0.03). These data indicate that the upregulation of endogeneous ANX-A1 (either membrane-binding or secreted form) promotes cell apoptosis in an autocrine fashion. Next, we investigated the functional role of ANX-A1 on leukemia cell phagocytosis. The engulfment of Kasumi-1 cells by cocultured human THP-1 monocyte-derived macrophages was evaluated by cell adherence assay. Compared with untreated cells, the exposure to FK228 induced a dramatic increase in Kasumi-1 cells attachment to macrophages (untreated vs FK228 treated; 57 ± 9 cells vs 196 ± 33 cells/ microscopic fields (0.08 mm2/field), n = 5; p=0.01). FK228-induced cell attachment was completely abrogated in the siRNA/ANX-A1 transfected Kasumi-1 cells (60.5% ± 10.5% decrease; n = 5; p<0.001). Consistently, co-treatment with FK228 and anti-ANX-A1 mAb followed by washout of both compounds resulted in significantl repression of FK228-stimulated engulfment of leukemic cells by macrophages (54.1% ± 3.0% decrease; n = 5; p=0.02). This effect was not further enhanced by adding anti-ANX-A1 mAb to the co-culture medium, suggesting that membrane-associated but not soluble ANX-A1 contributes to leukemia cell engulfment by macrophages. Results presented here demonstrate a novel mechanism of action of HDAC inhibitors in the context of bone marrow microenvironment via histone acetylation, increased expression and externalization of ANX-A1, which provides an “eat-me” signal and mediates phagocytic clearance of apoptotic leukemic cells by macrophages. Our data further suggest that ANX-A1 is silenced via histone deacetylation in leukemic cells, and its re-expression by HDAC inhibitors may stimulate apoptosis in an autocrine fashion while diminishing the inflammatory response through activating phagocytosis in the bone marrow microenvironment.

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Protection of ALL Cells by Bone Marrow Stromal Cells and the Underlying Molecular Mechanism.

Blood ◽

10.1182/blood.v116.21.2587.2587 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2587-2587

Author(s):

Yang Yang ◽

Baohua Sun ◽

Saradhi Mallampati ◽

Zhen Cai ◽

Xiaoping Sun

Keyword(s):

Bone Marrow ◽

Signaling Pathways ◽

Cell Lines ◽

Stromal Cells ◽

Cell Apoptosis ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Leukemic Cells ◽

New Therapies ◽

Induced Apoptosis

Abstract Abstract 2587 Acute lymphoblastic leukemia (ALL) is one of the fastest-growing hematological malignancies affecting patients with all ages, particularly children. Significant advances have been made in recent years in our understanding of the disease and the development of new therapies, which have led to a greatly improved outcome. Nevertheless, in a significant number of patients with ALL, the disease relapse and become resistant to treatment, causing death of the patients. Increasing evidence suggests that relapse of the disease and resistant to treatment are largely attributed to the protection of the leukemic cells by various components in the microenvironment, such as bone marrow stromal cells. However, the cross-talk between leukemic cells and their microenvironment remains poorly understood. Therefore, better understanding the mechanisms underlying the protection of ALL cells by the microenvironment is of ultimate importance in developing new therapies targeting such protection and eventually eradicating all the leukemic cells to cure the disease. In this study, we used a coculture system with leukemic cells and bone marrow stromal cells (MSC) to mimic the in vivo interaction between the two cell types to explore the molecular events that might be responsible for the protection of ALL cells from Ara-C induced apoptosis. We cocultured human primary ALL cells with hTERT-immortalized normal human MSC and evaluated ALL cell apoptosis by FACS after staining with Annexin V and propidium iodide. In all 8 cases, the MSC provided significant protection of ALL cells from both spontaneous and Ara-C induced apoptosis. For example, the mean Ara-C induced apoptosis of ALL cells cultured without MCS was 42.7% (range, 27–54%), whereas it was 19.1% (range, 8–27%) with MSC. Similar results were obtained with human leukemia cell lines Reh, SEMK2 and RS4.11. We also found that the murine MSC line M210B4 could provide similar protection to ALL cells, whether the ALL cells are primary or cell lines. The reduced apoptosis in the coculture were confirmed by Western blot which showed that MSC could protect ALL cells from Caspase-3 and PARP cleavage. Furthermore, our results showed no significant Ara-C induced reduction in S phase when cocultured with MSC. This phenomenon was associated with decreased cyclinA and CDK2 expression. In addition, we found that cocultured with MSC resulted in phosphorylation of AKT in ALL cells and PI3K inhibitor LY294002 specifically inhibited MSC-induced activation of AKT and promoted ALL cell apoptosis. In addition, beta-catenin and c-myc had increased expression in ALL cells cocultured with MSC, suggesting that Wnt pathway could play a role in MSC-mediated protection. To identify candidate molecules potentially involved in the protection of ALL cells by MSC, we performed gene expression microarray analyses with ALL cells exposed to Ara-C in presence or absence of MSC. Our data indicated that several signaling pathways might be involved in this process, including apoptosis signaling and cell cycle checkpoint control, which confirmed above findings. The top expressed genes identified in the microarray studies were confirmed by RT-PCR. Collectively, our results demonstrated that MSC can protect ALL cells from Ara-C induced apoptosis by multiple signaling pathways, such as those involving PI3K/AKT and Wnt signaling. Hence, targeting these pathways may become potential novel therapeutic strategies to disrupt the support of the microenvironment to ALL cells and to eventually eradicate leukemic cells. Disclosures: No relevant conflicts of interest to declare.

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In the presence of bone marrow stromal cells human multiple myeloma cells become independent of the IL-6/gp130/STAT3 pathway

Blood ◽

10.1182/blood-2002-01-0102 ◽

2002 ◽

Vol 100 (9) ◽

pp. 3311-3318 ◽

Cited By ~ 65

Author(s):

Manik Chatterjee ◽

Dirk Hönemann ◽

Suzanne Lentzsch ◽

Kurt Bommert ◽

Christine Sers ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Marrow Stromal Cells ◽

Myeloma Cells ◽

Farnesyl Transferase ◽

Stat3 Pathway ◽

Induced Apoptosis

AbstractThe interleukin 6/glycoprotein 130/signal transducer and activator of transcription 3 (IL-6/gp130/STAT3) pathway has been reported to play an important role in the pathogenesis of multiple myeloma (MM) and for survival of MM cells. However, most data concerning the role of IL-6 and IL-6–triggered signaling pathways were obtained from experiments performed with MM cell lines and without considering the bone marrow microenvironment. Thus, the precise role of IL-6 and its intracellular signaling pathways for survival of human MM cells is still unclear. Here we show that treatment of human MM cells (IL-6–dependent MM cell line INA-6 and primary MM cells) with the IL-6 receptor antagonist Sant7 or with an anti-gp130 monoclonal antibody (mAb) induced apoptosis if the cells were cultured in the absence of bone marrow stromal cells (BMSCs). In contrast, apoptosis could not be observed if the MM cells were cocultured with BMSCs. The analysis of intracellular pathways revealed that Sant7 and anti-gp130 mAb were effectively inhibiting the phosphorylation of gp130 and STAT3 in the absence and presence of BMSCs, whereas ERK1 and ERK2 (ERK1,2) phosphorylation was only slightly affected. In contrast, treatment with the farnesyl transferase inhibitor, FPT III, induced apoptosis in MM cells in the absence or presence of BMSCs and led to a complete inhibition of the Ras/mitogen-activated protein kinase pathway. These observations indicate that the IL-6/gp130/STAT3 pathway is not essential for survival of human myeloma cells if they are grown in the presence of cells from the bone marrow microenvironment. Furthermore, we provide evidence that farnesyl transferase inhibitors might be useful for the development of novel therapeutic strategies for the treatment of MM.

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CXCR4 Inhibition as a Therapeutic Strategy in Leukemia.

Blood ◽

10.1182/blood.v104.11.456.456 ◽

2004 ◽

Vol 104 (11) ◽

pp. 456-456

Author(s):

Zhihong Zeng ◽

Randall L. Evans ◽

Ziwei Huang ◽

Michael Andreeff ◽

Marina Konopleva

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stromal Cells ◽

Surface Expression ◽

Bone Marrow Microenvironment ◽

Jurkat Cells ◽

Leukemic Cells ◽

Dependent Manner ◽

Viable Cells ◽

Induced Apoptosis

Abstract The chemokine receptor CXCR4 is critically involved in the migration of hematopoietic cells to the stroma derived factor (SDF-1α)-producing bone marrow microenvironment. We and others have previously demonstrated that stromal-leukemic interactions mediate protection of leukemic cells from chemotherapy-induced apoptosis. (Konopleva et al, Leukemia 2002; Tabe, Konopleva, et al, Blood 2004; Burger JA et al., Blood 2000). Using peptide based CXCR4 inhibitors, derived from the chemokine viral macrophage inflammatory protein II (vMIP II), we tested the hypothesis that CXCR4 inhibition interferes with stromal/leukemia cell interactions resulting in increased sensitivity to chemotherapy. CXCR4 was highly expressed on the cell surface of CML myeloid blood crisis cells (KBM5), KBM5/STI-resistant cells, lymphoid CEM and Jurkat cells, myeloid leukemic OCI-AML3 and U937 cells. In contrast, NB4 and TF-1 cells expressed low-levels surface CXCR4, and no surface expression was detected on KG-1 and HL-60 leukemic cells. Among CXCR4(+) cell lines, Jurkat cells demonstrated the highest chemoattractive response to SDF-1α(23 +/− 0.03% migration at SDF-1α50ng/ml, and 54 +/− 0.01% at 100ng/ml). The ability of three CXCR4 inhibitors to inhibit chemotaxis of Jurkat cells was examined in a standard migration assay. Results indicate that D10-vMIP-II, a polypeptide with the first 10 amino acids substituted by the D isoform, exhibits the strongest antagonistic effect on SDF-1α or stromal cell induced chemoattraction. D10-vMIP-II also decreases CXCR4 surface expression in a concentration-dependent manner: flow cytometry and live cell confocal microscopy revealed that within 30min of exposure D10-vMIP-II causes CXCR4 internalization that persisted for at least 4 hrs at 0.01μM and for 24 hrs at 0.1μM. Analysis of SDF-1α-mediated signaling demonstrated that D10-vMIP-II inhibits AKT and ERK phosphorylation. Finally, we examined the effects of D10-vMIP-II on the response to chemotherapy of leukemic cells co-cultured with MS5 stromal cells. Pre-treatment of Jurkat cells enhanced doxorubicin-induced apoptosis: Doxorubicin alone (10μM) 75 +/− 0.07% viable cells compared to control; Doxorubicin and D10-vMIP-II: 53 +/− 0.04% viable cells. Furthermore, D10-vMIP-II enhanced the sensitivity of primary CLL cells to Fludarabine in the in vitro stromal co-culture system. CLL samples with high surface expression of CXCR4 (n=3) co-cultured with stromal MS-5 cells were pre-treated with 0.1μM D10-vMIP II followed by 10μM Fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine). Stromal cells prevented Fludarabine-induced killing (64%±16.2 viable cells in stromal co-culture compared to 31% viable cells in medium only). Inhibition of CXCR4 signaling abrogated this protective effect and diminished CLL cell survival (26.9±7.1% viable cells, p=0.03 compared to Fludarabine-treated CLL cells co-cultured with MS-5). This growth inhibition was mediated by apoptosis induction as determined by CD45/annexinV flow cytometry (DMSO, 14.49±5.3% annexinV(+) leukemic cells; Fludarabine, 47.2±24.9%; D10-vMIP II followed by Fludarabine, 61.3±18.9%). Taken together, our data suggest that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to chemotherapy-induced apoptosis. Disruption of these interactions by the potent CXCR4 inhibitor D10-vMIP-II represents a novel strategy for the targeting leukemic cells within their bone marrow microenvironment.

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The Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Regulates the Function and Migration of Leukemic Blasts through CD63/PI3K/AKT/P21 Axis

Blood ◽

10.1182/blood.v126.23.2394.2394 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2394-2394

Author(s):

Dorian Forte ◽

Valentina Salvestrini ◽

Lucia Catani ◽

Roberto M Lemoli ◽

Michele Cavo ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Culture System ◽

Hematological Malignancies ◽

Leukemic Cells ◽

Tissue Inhibitor Of Metalloproteinases ◽

Tissue Inhibitor ◽

Hematopoietic Stem ◽

And Migration

Abstract Introduction. The Tissue Inhibitor of MetalloProteinases-1 (TIMP-1) is a member of the inflammatory network that dramatically increases in response to inflammation. First described as an inhibitor of MMPs, TIMP-1 exerts pleiotropic effects in the hematopoietic microenviroment. Along with the finding that hematopoietic stem progenitor cells (HSPCs) directly respond to inflammation, a persistent inflammatory stimulation may hinder HSPC functions and eventually lead to hematological malignancies. Specifically, the inflammatory pathways activated by the bone marrow (BM) microenvironment influences the transformation and progression of leukemia. In hematological malignancies, TIMP-1 promotes proliferation in lymphoma cells and increased TIMP-1 serum levels are associated with poor prognosis in a variety of cancers. However, recent studies have revealed the dichotomy of TIMP-1 in cancer progression. Here, in the attempt to provide further evidence for the critical role of inflammation in leukemic microenvironment, we investigated the role of TIMP-1 in leukemic blasts from patients with Acute Myeloid Leukemia (AML) and elucidated the downstream pathway ignited by rhTIMP-1. Methods. Human CD34+ HSPCs were isolated from cord blood (CB) units as control samples, while leukemic cells were collected from AML patients at diagnosis (percentage of blasts >90%; n=36). Cell proliferation was assessed by cell cycle analysis, and CFSE staining. In addition, we performed colony-forming unit assays. TIMP-1's contribution to cell survival was evaluated by AnnexinV/PI staining. The expression of the tetraspannin receptor CD63 (TIMP-1's putative receptor) was assessed by flow cytometry. Leukemic blasts were assayed towards a CXCL12 gradient after exposure to rhTIMP-1 or pre-treated with LY294002 (PI3K inhibitor); migrated cells were counted and characterized for CD63 expression. Leukemic blasts were sorted in two separate fractions, CD63- and CD63+, and their capability to respond to rhTIMP-1 was assessed. Downstream molecular targets of TIMP-1 (such as PI3K, pAkt, p21) were also confirmed by flow cytometry. RT-PCR was employed to detect the expression of hypoxia-inducible factor-1 (HIF-1α). Finally, co-culture system was performed with CFSE-positive leukemic blasts and mesenchymal stromal cells (MSCs) from normal or AML patients in the presence of rhTIMP-1. Results. We firstly found that TIMP-1 levels are increased in the plasma of BM of AML patients at diagnosis. Similarly to CD34+ HSPCs from CB, the clonogenic potential of primary human AML blasts is increased upon exposure to rhTIMP-1. Moreover, rhTIMP-1 promotes in vitro AML blast survival, possibly due to anti-apoptotic effects and a slight but significant increase in the proportion of leukemic cells entering S-phase of the cell cycle. Such effect is related to the downregulation of cyclin-dependent kinase inhibitor p21. As for the dissection of TIMP-1 signalling pathway, our data revealed that the tetraspannin CD63 receptor is required for TIMP-1's cytokine functions. Indeed, about half of leukemic blasts expresses CD63, whose activation leads to PI3K recruitment and Akt phopshorylation, key modulators of survival/proliferation pathways. Exposure of AML blasts to rhTIMP-1 resulted in the induction of HIF-1α, which is known to play an important role in survival of leukemic stem cells. Moreover, rhTIMP-1 increased the SDF1-driven migration of leukemic blasts through the activation of PI3K. Interestingly, migrating AML blasts are highly enriched in CD63+ cells, whereas the CD63-negative pre-sorted fraction of leukemic cells showed reduced response to migration and pAkt expression after exposure to rhTIMP-1.Finally, in the co-culture system between AML cells and normal versus leukemic MSCs, we defined the particular role of TIMP-1 in normal and leukemic microenviroment. In particular, our results suggest that a defective capacity of AML-MSCs in sustaining the proliferation and migration of AML blasts may be restored by rhTIMP-1. Conclusions. In our study TIMP-1 emerges as an important factor in the leukemic BM microenvironment, modulating leukemic blasts survival, migration and function. Specifically, our work suggests that TIMP-1 promotes survival and migration via CD63/PI3K/AKT/p21 signalling. Taken together, our findings indicates TIMP-1 pathway as a potential novel therapeutic target in AML. Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

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TPCK Induces Apoptosis in Human Acute Myeloid Leukemia U-937 Cells.

Blood ◽

10.1182/blood.v104.11.4478.4478 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4478-4478

Author(s):

Julia Mazar ◽

Alexandra Lichtenstein ◽

Leora Katz ◽

Ofer Shpilberg ◽

Itai Levi ◽

...

Keyword(s):

Chromatin Condensation ◽

Annexin V ◽

Leukemic Cells ◽

Dependent Manner ◽

Cytochrome C Release ◽

Apoptotic Pathway ◽

Concentration Dependent Manner ◽

Flip Flop ◽

Induced Apoptosis

Abstract Many types of antitumor therapy in general and AML in particular exert their effect by activating apoptosis. Apoptosis of AML cells can be induced by cytostatic drugs, corticosteroids, and radiation. Recently, the role of different proteases as possible targets for chemotherapy was described. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like protease (CLP) inhibitor was shown to exert a dual effect on leukemic cells: proapoptotic and antiapoptotic. In the present study the mechanism of its proapoptotic effect was addressed. It was found that the CLP inhibitors, TPCK and 3,4 dichloroisicoumarine induced apoptosis in a time- and concentration-dependent manner. Apoptosis was accompanied by a decrease in mitochondrial membrane potential, cytochrome c release, caspase-3 (but not caspase-8) activation, PS flip-flop (measured by Annexin-V staining followed by flow cytometry analysis) and chromatin condensation, but not fragmentation (detected by acridine orange/ethidium bromide staining). All apoptotic processes induced by TPCK were completely inhibited by cycloheximide. The ability of cycloheximide to inhibit TPCK-induced cell death suggests that protein synthesis plays a role in TPCK-induced apoptosis. Additionaly, the proapoptotic effect of TPCK was abolished by elimination of glucose from the medium. The data supports the role of mitochondria in this process. In the present study the apoptotic pathway driven by inhibition of CLP was demonstrated. Moreover, these findings suggest possible ways of preventing the proapoptotic activity of TPCK and thereby enhancimg its antiapoptotic action.

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Rooting into the Soil, a Model for the Role of Sox4 in Leukemia

Blood ◽

10.1182/blood.v112.11.3794.3794 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3794-3794

Author(s):

Saradhi Mallampati ◽

Baohua Sun ◽

Yun Gong ◽

Enze Wang ◽

M. James You ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Stromal Cells ◽

Bone Marrow Stromal Cells ◽

Leukemic Cell ◽

Marrow Stromal Cells ◽

Leukemic Cells ◽

Cell Stage

Abstract Development and progression of leukemia requires interaction of leukemia-initiating cells with their bone marrow niches. The niches serve as the nursery and shelter for the leukemic cells, which can result in drug resistance, disease recurrence, and minimal residual disease, the most important causes for the death of patients with leukemia. Therefore, obliteration of the interaction between the leukemic cells and their niches is of utmost importance in eradicating leukemic cells during therapy to cure the disease. However, little is currently known of the molecular mechanisms underlying the interaction of the two types of cells. Sox4, a SRY-related HMG-box containing transcription factor that is vital during development, plays an important role in leukemia. Published mouse studies demonstrated that increased expression of Sox4 was associated with leukemogenesis. We determined the expression levels of Sox4 by real-time RT-PCR in 100 human leukemic samples and found high levels of expression in B- and T-ALL, but not in AML, CML, CLL, Sezary syndrome, or T cell prolymphocytic leukemia. In accordance, 7 of the 8 ALL cell lines (the exception was 697) we tested showed high expression levels of Sox4, but AML cell lines, normal mature B cells, T cells, and bone marrow CD34+ cells had low levels of expression. Since the majority of clinical B-ALL cases correspond to the pre-B cell stage, we investigated the role of Sox4 in a pre-B cell line (Nalm6) by lentivirus-mediated RNAi. Remarkably, knockdown of Sox4 in Nalm6 cells caused 70% reduction in the formation of leukemic cell clusters under the monolayer of co-cultured M2-10B4 bone marrow stromal cells, a phenomenon known as pseudo-emperipolesis. Similar results were obtained with ex vivo cultured bone marrow cells from conditional Sox4 knockout mice that displayed B cell developmental arrest at the transition from pro-B to pre-B cell stage and an absence of pre-B cells. These findings suggested that Sox4 is required for the interaction of the developing B cells or leukemic cells with bone marrow stromal cells, a component of the bone marrow niche. Since CXCR4/SDF1-mediated “homing” is known to be required for pseudo-emperipolesis, we tested the effect of Sox4 on Nalm6 cell migration toward SDF1 gradient and found that Sox4 did not affect the migration, suggesting that Sox4 is not acting through “homing”. Instead, our data indicated that the role of Sox4 in the interaction of leukemic cells with stromal cells is most likely mediated by its ability in enhancing the adhesion of the leukemic cells because we found that lentivirus-medicated overexpression of Sox4 in the 697 B cell line caused the suspension cells to display a spindle and adhesive morphology. In addition, 21% of the putative Sox4 downstream genes that we identified by multiple sets of gene expression microarray experiments are known to be involved in cell adhesion. Moreover, we found that the changes in gene expression profile of leukemic cells upon Sox4 knockdown or overexpression significantly overlap with the changes in response to the presence of bone marrow stromal cells in co-culture, indicating that Sox4 pathways are involved in leukemic cell response to stromal cell signaling. Based on these findings we hypothesize that deletion of Sox4 abolishes the interaction between the developing lymphocytes and their niches during lymphopoiesis. Conversely, overexpression of Sox4 may enforce these cells to over-interact with the niches so that they are overexposed to local growth factor stimuli. If superimposed with other genetic and/or epigenetic changes in the developing lymphocytes, such over-interaction may result in the development of leukemia. In case of established leukemia, such over-interaction may lead to the enhanced protection of leukemic cells by their niches. Therefore, the role of Sox4 in the interaction of developing lymphocytes or leukemic cells with their niches is like “rooting into the soil” of a growing tree, abbreviated as “rooting”.

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Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

Blood ◽

10.1182/blood-2008-05-158311 ◽

2009 ◽

Vol 113 (24) ◽

pp. 6215-6224 ◽

Cited By ~ 339

Author(s):

Zhihong Zeng ◽

Yue Xi Shi ◽

Ismael J. Samudio ◽

Rui-Yu Wang ◽

Xiaoyang Ling ◽

...

Keyword(s):

Bone Marrow ◽

Stromal Cells ◽

Fetal Liver ◽

Bone Marrow Microenvironment ◽

Leukemic Cells ◽

In Vivo Studies ◽

Protective Effects ◽

Cxcr4 Signaling ◽

Induced Apoptosis

Abstract SDF-1α/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow–derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1α (SDF-1α)–induced and stroma-induced chemotaxis and inhibited SDF-1α–induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor– and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.

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Bone Marrow Stromal Cells Protect Both FLT3-ITD and FLT3-WT Primary AML Cells from the Anti-Leukemic Activity of the FLT3 Inhibitor AC220

Blood ◽

10.1182/blood.v124.21.4377.4377 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4377-4377

Author(s):

Cedric Dos Santos ◽

Georges Habineza Ndikuyeze ◽

Michael Nisssan ◽

Chenghui Zhou ◽

Xiaochuan Shan ◽

...

Keyword(s):

Bone Marrow ◽

Growth Inhibition ◽

Stromal Cells ◽

Research Funding ◽

Critical Role ◽

Annexin V ◽

Dependent Manner ◽

Induced Apoptosis ◽

The Impact ◽

Dose Dependent

Abstract FTL3 mutations are found in about 30% of AML patients, conferring a leukemic blast growth advantage, drug therapy resistance in the bone marrow (BM) and poor outcome. Mesenchymal stem/stromal cells (MSCs) are essential components of the bone marrow microenvironment, and growing evidence suggest that MSCs play a critical role in AML chemo-resistance, although the molecular mechanisms involved are poorly understood. The purpose of the study was to (1) establish an novel in vitro co-culture system between primary AML blasts and healthy donor BM-MSCs (HD-MSCs) or AML patient-derived MSCs (AML-MSCs), (2) evaluate the impact of culture with BM-MSCs on the sensitivity of AML cells to AC220 using patients samples with FLT3-ITD (n=4) or FLT3-WT (n=3). We first cultured HD-MSCs (n=5) and AML-MSC (n=3) and observed no phenotypical differences (CD14- CD34- CD45- CD73+ CD90+ CD105+), although HD-MSCs grew faster. We evaluated the effect of co-culturing AML samples (n=6) with HD-MSCs or AML-MSCs for 5 and 12 days on leukemic cell growth and found that both types of MSCs significantly and equally enhanced AML cell proliferation while maintaining blast phenotype. Using clonogenic assays on 4 AML specimens cultured alone or with either HD- or AML-MSCs for 5 and 12 days, we found that co-culture with either source of BM-MSCs drastically increased colony-forming cells number at day 5 and day 12 while CFC number decreased in the absence on BM-MSCs (no colonies at day 12 for the 4 samples), indicating that AML co-culture with HD/AML-MSCs supports the survival and/or proliferation of AML stem/progenitor cells. We next assessed the effect of increasing doses of AC220 (1, 10, 50, 100 and 500nM) on the apoptosis of FLT3-ITD (n=3) and FLT3-WT (n=4) AML cells cultured alone or with HD-MSCs. Exposure to AC220 for 72 hours significantly, and in a dose-dependent manner, increased the apoptosis of AML FLT3-ITD cells in monoculture (n=3, 21±1% of Annexin V positive cells for control, AC220 1nM 29±3.7%, 10nM 31±2.5%, 50nM 32±1.5%, 100nM 34±1.7% and 500nM 38±3.6%). In contrast, AML FLT3-ITD cells co-cultured with HD-MSCs were resistant to the drug (n=3, 21±2.6% of Annexin V positive cells for control, AC220 1nM 23±3%, 10nM 22±3%, 50nM 25±5.7%, 100nM 30±8.3% and 500nM 33±9.5%). Interestingly, we found that AML FLT3-WT are much less sensitive to increasing doses of AC220 compared to ITD samples (n=4, 27±3.9% of Annexin V positive cells for control, AC220 1nM 30±6.5%, 10nM 35±14%, 50nM 37±11%, 100nM 39±13% and 500nM 43±11%), and co-culture with BM-MSCs further decreased the sensitivity of AML FLT3-WT cells to AC220-induced apoptosis (n=4, 19±3.2% of Annexin V positive cells for control, AC220 1nM 17±3.9%, 10nM 20±3.4%, 50nM 19±3.7%, 100nM 21±4.5% and 500nM 26±1%). AC220 treatment for 3 days significantly, and in a dose-dependent manner, inhibited CFCs in AML FLT3-ITD (n=4, with 26±8%, 46±6%, 60±9%, 69±10% and 86±3% inhibition with 1, 10, 50, 100 and 500nM of AC220 respectively) while AML FLT3-ITD co-culture with HD-MSCs were less sensitive (n=4, with 9±10%, 30±6%, 42±9%, 57±11% and 72±7% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Similarly to the AC220-induced apoptosis, we observed that AML FLT3-WT CFCs are less sensitive to AC220-induced growth inhibition compared to ITD samples, although a 3 days exposure to AC220 significantly, and in a dose-dependent manner, inhibited AML FLT3-WT CFCs (n=3, with 38±16%, 44±14%, 58±12%, 70±21% and 81±19% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Interestingly, we observed that co-culture of AML FLT3-WT with stromal cells were significantly more resistant to increasing doses of AC220 (n=3, with 22±7%, 36±5%, 43±8%, 46±8% and 57±6% inhibition with 1, 10, 50, 100 and 500nM of AC220, respectively). Altogether, these results suggest that AML FLT3-ITD cells in monoculture are more sensitive to AC220 treatment compared to AML FLT3-WT primary cells, but more importantly, upon interaction with primary HD-MSCs, both WT and FLT3-ITD primary samples are protected from apoptosis and growth inhibition induced by AC220, indicating a critical role for the BM microenvironment in AC220 resistance. We are currently testing the impact of BM-MSCs co-culture on leukemic stem cell sensitivity to AC220 using transplantation in NSG mice. We will also evaluate if this co-culture model can be predictive of the response to in vivo treatment with AC220 in a patient-derived xenograft model. Disclosures Dos Santos: Janssen R&D: Research Funding. Danet-Desnoyers:Janssen R&D: Research Funding.

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