Mesenchymal stromal cells from myelodysplastic and acute myeloid leukemia patients display in vitro reduced proliferative potential and similar capacity to support leukemia cell survival

Bone marrow (BM) microenvironment contributes to the regulation of normal hematopoiesis through a finely tuned balance of self-renewal and differentiation processes, cell-cell interaction and secretion of cytokines that during leukemogenesis are altered and favor tumor cell growth. In pediatric acute myeloid leukemia (AML), chemotherapy is the standard of care, but still >30% of patients relapse. The need to accelerate the evaluation of innovative medicines prompted us to investigate the mesenchymal stromal cells (MSCs) role in the leukemic niche to define its contribution to the mechanisms of leukemia escape. We generated humanized three-dimensional (3D) niche with AML cells and MSCs derived from either patients (AML-MSCs) or healthy donors. We observed that AML cells establish physical connections with MSCs, mediating a reprogrammed transcriptome inducing aberrant cell proliferation and differentiation, and severely compromising their immunomodulatory capability. We confirmed that AML cells modulate h-MSCs transcriptional profile promoting functions similar to the AML-MSCs when co-cultured in vitro, thus facilitating leukemia progression. Conversely, MSCs derived from BM of patients at time of disease remission showed recovered healthy features, at transcriptional and functional levels, including the secretome. We proved that AML blasts alter MSCs activities in the BM niche, favoring disease development and progression. We discovered that a novel AML-MSCs selective CaV1.2 channel blocker drug, Lercanidipine, is able to impair leukemia progression in 3D niche both in vitro and when implanted in vivo, if used in combination with chemotherapy, supporting the hypothesis that synergistic effects can be obtained by dual targeting approaches.

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Protein Kinase C-ß Dependent IL-8 Release Promotes Acute Myeloid Leukemia Blast Cell Survival in Co-Cultures with Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v126.23.3064.3064 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3064-3064

Author(s):

Amina M Abdul-Aziz ◽

Manar S Shafat ◽

Matthew J Lawes ◽

Kristian M Bowles ◽

Stuart A Rushworth

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Protein Kinase ◽

Cell Survival ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Leukemia Cells ◽

Acute Myeloid

Abstract Introduction: Acute myeloid leukemia (AML) cells exhibit a high level of spontaneous apoptosis when cultured in vitro but have a prolonged survival time in vivo indicating that the tissue microenvironment plays a critical role in promoting AML cell survival. Knowledge of the complexity of the bone marrow microenvironment is increasing especially with respect to the bone marrow mesenchymal stromal cells [BM-MSC] which are considered a major protective cell type. Other studies have demonstrated the ability of BM-MSC to protect leukemia cells from spontaneous and chemotherapy-induced apoptosis. Increasing evidence suggests the existence of crosstalk between leukemia cells and BM stromal cells to create a leukemia-promoting environment. Recently our group and others have shown that this crosstalk is achieved by a complex communication system that involves multiple bidirectional signals which enhance AML survival and proliferation. Here we report a novel interaction between AML blasts and BM-MSC which benefits AML proliferation and survival. Methods: To investigate the interaction between primary AML blasts and BM-MSC we isolated AML and BM-MSC from the same patient and used an autologous in vitro culture assay to analyze the cytokine profile. Conditioned medium was collected from cultures of primary human AML alone or cultured with autologous BM-MSC and analyzed using Proteome Profiler Human XL Cytokine Array and target specific ELISAs. Real-time PCR was also used to verify the array data. MIF-Receptor inhibitors (SB 225002- CXCR2, AMD3100 - CXCR4 and CD74 blocking antibody - CD74) and signaling kinase inhibitors (LY294002- PI3K/AKT, PD098059 - MAPK, Ro-31-8220 - PKC) were used for initial determination of MIF signaling pathways in BM-MSC. Specific PKC isoform inhibitors (Go6976-PKCα/ß and enzastaurin -PKCß) were then used to determine isoform specific activation. Western blot and siRNA were used to confirm the role of AML derived MIF in regulating downstream BM-MSC signaling pathways including MAPK, PI3K/AKT, and PKC. Results: We initially examined the cytokine profile in cultured human AML compared to AML cultured with autologous BM-MSC or BM-MSC alone and found that MIF was highly expressed by primary AML and that IL-8 was increased in AML/BM-MSC co-cultures. The observed changes in IL-8 were confirmed by ELISA assays. RT-PCR was used to measure MIF and IL-8 gene expression from RNA extracted from primary AML or BM-MSC cultured alone or in combination. Results confirmed that MIF is highly expressed at the RNA and protein level by AML blasts and IL-8 transcription and cytokine release was upregulated in BM-MSC in response to co-culture with AML. Next we found that recombinant MIF increases IL-8 mRNA and protein expression in BM-MSC. Moreover, the MIF inhibition by, ISO-1, inhibits AML induced IL-8 expression and secretion by BM-MSC. Next we sought to determine which kinase signaling cascade is activated by MIF. We used a panel of protein kinase inhibitors and found that the pan-PKC inhibitor Ro-31-8220 completely inhibits AML and MIF induced IL-8 mRNA at sub micromolar concentrations. To further identify the specific PKC isoform responsible for linking AML induced MIF to IL-8 we used PKC isoform specific inhibitors (Go6976 and enzastaurin) which significantly inhibited MIF induced IL-8 expression and protein in BM-MSC. The introduction of PKCß siRNA dramatically inhibited MIF induced IL-8 mRNA expression in BM-MSC confirming that PKCß regulates AML induced BM-MSC derived IL-8 expression. Finally, inhibition of AML/BM-MSC co-cultures with the PKCß inhibitor enzastaurin inhibits BM-MSC induced AML survival in vitro. Conclusions: These results reported here show a novel bidirectional survival mechanism between AML blasts and BM-MSC. Furthermore this work identifies the PKC-ß-IL8 pathway in the BM-MSC of patients with AML as a novel target for future treatment strategies. Disclosures No relevant conflicts of interest to declare.

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Upregulation of Indoleamine 2,3-Dioxygenase Enzymes in Leukemic Mesenchymal Stromal Cells (MSCs) Can Influence MSC/Acute Myeloid Leukemia Cell Cross Talk

Blood ◽

10.1182/blood.v126.23.1191.1191 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1191-1191

Author(s):

Marilena Ciciarello ◽

Giulia Corradi ◽

Carmen Baldazzi ◽

Nicoletta Testoni ◽

Roberto Massimo Lemoli ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Mesenchymal Stromal Cells ◽

Cross Talk ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Leukemia Cell ◽

Essential Element ◽

Fish Analysis ◽

Hematopoietic Stem ◽

Acute Myeloid

Abstract Mesenchymal stromal cells (MSCs), an essential element of both normal and leukemic hematopoietic microenvironment, are multipotent cells with a unique immune-modulating ability. Thus, MSCs play a crucial role for both the proliferation and differentiation of hematopoietic stem cells (HSCs) and induce an immune-tolerant milieu. Indoleamine 2, 3-dioxygenase (IDO1 and IDO2) enzymes catabolize tryptophan to kynurenines and play a key role in the induction of immune tolerance in different settings, including acute myeloid leukemia (AML). Furthermore, IDO1/IDO2 pathway is a well described mechanism by which MSCs exert their mmunomodulatory properties. We hypothesized that: 1) MSC-dependent mechanisms are involved in leukemia initiation, maintenance and progression; 2) the expression of IDO1 and IDO2 by MSCs is part of a MSC-dependent mechanism able to create a tumor-supportive milieu. To this aim, we isolated MSCs from the bone marrow of AML patients (AML-MSCs) at diagnosis. We first analyzed their phenotypic and functional properties compared to that of healthy donor-derived MSCs (HD-MSCs). We found that AML-MSCs showed a reduced proliferative capacity but normal immunophenotype, differentiative and immunomodulatory capacity as compared to HD-MSCs. Furthermore, AML-MSCs did not show the chromosomal abnormalities identified in the primary blast counterpart (FISH analysis). We next investigated IDO1/2 expression and functions in MSCs. We demonstrated that IDO enzymes are expressed in AML-MSCs as well as in HD-MSCs. IDO1 is efficiently upregulated by different inflammatory stimuli, and IDO1 protein expression parallels mRNA in both HD-MSCs and AML-MSCs. Interestingly, IDO2 mRNA is expressed at low basal level in all analyzed conditions in HD-MSCs, while it is upregulated, in particular after IFN-gamma stimulation, in AML-MSCs, although the level of induction varies between different patients. When T-cell proliferation was tested in MSC co-cultures, w/or w/out IDO1/2 inhibitor, 1-methyltryptophan, we found that MSC immunomodulatory potential is IDO-dependent both in HD-MSCs and AML-MSCs. Finally, we found that in co-culture assay with primary AML blasts, MSCs stimulated blast proliferation and this effect is, at least in part, IDO-mediated. These data suggested that IDO enzymes, in particular IDO2, may be differentially expressed in AML-MSCs as compared to HD-MSCs and IDO inhibition has an impact on MSC/AML cell cross talk. These findings may help to discover novel niche-target prognostic/therapeutic factors and to provide novel applications for drugs already under active clinical investigation (i.e. IDO-inhibitors). Disclosures Cavo: Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

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Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells

Oncogene ◽

10.1038/s41388-019-1069-y ◽

2019 ◽

Vol 39 (6) ◽

pp. 1198-1212

Author(s):

Farah Kouzi ◽

Kazem Zibara ◽

Jerome Bourgeais ◽

Frederic Picou ◽

Nathalie Gallay ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Gap Junctions ◽

Gap Junction ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Leukemic Cells ◽

Acute Myeloid

Abstract The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell–cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.

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Alterations in multipotent mesenchymal stromal cells from the bone marrow of acute myeloid leukemia patients at diagnosis and during treatment

Leukemia & Lymphoma ◽

10.1080/10428194.2018.1554861 ◽

2019 ◽

Vol 60 (8) ◽

pp. 2042-2049

Author(s):

Irina N. Shipounova ◽

Nataliya A. Petinati ◽

Alexey E. Bigildeev ◽

Tamara V. Sorokina ◽

Larisa A. Kuzmina ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Multipotent Mesenchymal Stromal Cells ◽

Acute Myeloid

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Anti-leukemia properties of cadmium nanoparticles in the in vitro and in vivo condition: A chemobiological study

Archives of Medical Science ◽

10.5114/aoms/142465 ◽

2021 ◽

Author(s):

Yudi Miao ◽

Behnam Mahdavi ◽

Mohammad Zangeneh

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Lines ◽

Aqueous Extract ◽

Myeloid Leukemia ◽

Antioxidant Properties ◽

Leukemia Cell ◽

Sem Images ◽

Acute Myeloid

IntroductionThe present study investigated the anti-acute myeloid leukemia effects of Ziziphora clinopodides Lam leaf aqueous extract conjugated cadmium nanoparticles.Material and methodsTo synthesize CdNPs, Z. clinopodides aqueous extract was mixed with Cd(NO3)2 .4H2O. The characterization of the biosynthesized cadmium nanoparticles was carried out using many various techniques such as UV-Vis. and FT-IR spectroscopy, XRD, FE-SEM, and EDS.ResultsThe uniform spherical morphology of NPs was proved by FE-SEM images with NPs the average size of 26.78cnm. For investigating the antioxidant properties of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, the DPPH test was used. The cadmium nanoparticles inhibited half of the DPPH molecules in a concentration of 196 µg/mL. To survey the cytotoxicity and anti-acute myeloid leukemia effects of Cd(NO3)2, Z. clinopodides, CdNPs, and Daunorubicin, MTT assay was used on the human acute myeloid leukemia cell lines i.e., Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr. The IC50 of the cadmium nanoparticles was 168, 205, and 210 µg/mL against Murine C1498, 32D-FLT3-ITD, and Human HL-60/vcr cell lines, respectively. In the part of in vivo study, DMBA was used for inducing acute myeloid leukemia in mice. CdNPs similar to daunorubicin ameliorated significantly (p≤0.01) the biochemical, inflammatory, RBC, WBC, platelet, stereological, histopathological, and cellular-molecular parameters compared to the other groups.ConclusionsAs mentioned, the cadmium nanoparticles had significant anti-acute myeloid leukemia effects. After approving the above results in the clinical trial studies, these cadmium nanoparticles can be used as a chemotherapeutic drug to treat acute myeloid leukemia in humans.

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The Vitamin E Derivative Gamma Tocotrienol Promotes Anti-Tumor Effects in Acute Myeloid Leukemia Cell Lines

Nutrients ◽

10.3390/nu11112808 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2808 ◽

Cited By ~ 6

Author(s):

Ghanem ◽

Zouein ◽

Mohamad ◽

Hodroj ◽

Haykal ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Vitamin E ◽

Cell Lines ◽

Myeloid Leukemia ◽

Apoptotic Cell ◽

Leukemia Cell ◽

Therapeutic Effects ◽

Apoptotic Pathway ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro. For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway.

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