Hypoxia Regulates Exosomal Microrna Content, Trafficking and Function Of Key Elements In The AML Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 742-742 ◽  
Author(s):  
Noah Hornick ◽  
Jianya Huan ◽  
Natalya A Goloviznina ◽  
Amiee Potter ◽  
Peter Kurre
Keyword(s):  
Bone Marrow ◽  
Tissue Culture ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Oxygen Levels ◽  
Oxygen Conditions ◽  
Bystander Cells

Abstract Small, non-coding micro RNA (miRNA) are recognized for their potent regulatory capacity. Several recent studies indicate the prognostic value of miRNA profiling in acute myelogenous leukemia (AML), although a more mechanistic understanding of the role miRNA play in AML biology is still lacking. We recently demonstrated that patient-derived AML blasts release exosomes (nanometer-size, extracellular vesicles) that traffic a non-random subset of miRNA to stromal bystander cells, eliciting changes in transcriptional activity and growth factor secretion (Huan et al., Cancer Res. 2013). Here we hypothesized that exosome miRNA provide a candidate mechanism for the adaptation of the bone marrow to a specialized leukemic niche. As oxygen levels in the bone marrow are substantially lower than those commonly used in tissue culture, we undertook a systematic study of miRNA incorporation and exosome trafficking in AML under physiological oxygen conditions. In carefully calibrated tissue culture conditions we initially observed an up to 7-fold net increase in exosome number released by Molm14 (Flt3-ITD+ AML cell line) leukemia cells at 1% O2versus 21% O2. Nanoparticle tracking analysis and RNA bioanalyzer tracings suggested that the decreased O2 did not alter vesicle composition, average RNA amount per exosome, or global RNA profiles. Further emphasizing the critical nature of appropriate compartmental oxygenation in exosome trafficking, both murine and human stromal cells demonstrated increased uptake of Molm14 exosomes under hypoxia. Low-oxygen conditions alter transcriptional profiles, phenotypic behavior and drug resistance in AML. Therefore, we next evaluated the miRNA expression of leukemic cells and their incorporation in exosomes at 1% versus 21% O2, utilizing the Affymetrix microarray platform containing >5,000 human (hsa) miRNA probesets, followed by select qRT-PCR validation. Array experiments showed broad differences between cellular and exosomal miRNA and revealed that certain miRNA were selectively regulated in an oxygen-responsive manner. For example, hematopoiesis relevant hsa-miR-124, -146a, and -155 increased an average of 4.6-, 5.5-, and 4.9-fold, respectively, in exosomes from hypoxia-conditioned cells. Intriguingly, several known, non-AML specific, hypoxia-responsive miRNA substantially increased in cells cultured at 1% O2 (e.g. miR-210 by 33-fold), but changed less than 2-fold in exosomes. Several recent reports show that leukemia cells actively convert the bone marrow microenvironment and contribute to the erosion of hematopoiesis by modulating hematopoietic-stromal interactions, in part via decrease in SDF1a, SCF, and Angpt1. We investigated the ability of AML-derived exosomes to regulate these transcripts, and found a 50% decrease in SCF and over 90% decrease in Angpt1 in murine stromal cells after in vitro exposure to leukemia exosomes, again with relatively greater differences for exosomes from hypoxia-conditioned AML cells. These experiments were complemented by observations of altered clonogenicity (CFU-C) of murine lin-negative hematopoietic cells after AML exosome exposure, whereby hypoxia conditioning prompted a decline in colony count to 46% from vesicle-free media baseline, compared with 31% decrease at 21 % O2. Exosomes equilibrate across biological fluids and can be recovered from serum. To translate our observations to an in vivo setting, we developed a xenograft model using Molm14 cells in immune-deficient NSG mice. Early after grafting animals, exosomes could be reproducibly isolated from as little as 20 microL serum and candidate miRNA (hsa-miR-146, -150, 155, 210) were amplified, allowing us to quantitatively track leukemia progression via a unique miRNA signature even before circulating leukemia cells were detectable in the peripheral blood. A comparison of leukemic animals to NSG controls bearing cord blood MNC grafts revealed that changes in circulating miRNA were disease specific and resembled those in the hypoxia setting in vitro. In sum, our work demonstrates that physiologic oxygen levels not only increase AML exosome trafficking between cells, but selectively alter the miRNA profile contained therein. These changes produce phenotypic alterations in stromal and hematopoietic bystander cells that correlate with the functional conversion of the bone marrow to a leukemic niche. Disclosures: No relevant conflicts of interest to declare.

Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1372-1372
Author(s):  
Hendrik W. Van Deventer ◽  
Robert Mango ◽  
Jonathan Serody
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Mesenchymal Cells ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Wild Type

Abstract Abstract 1372 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results: We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells. Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis. To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease: Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

Stroma-Mediated Chemotherapy Resistance in Acute Myeloid Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 242-242
Author(s):  
Xin Long ◽  
Tsz-Kwong Man ◽  
Michele S. Redell
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Chemotherapy Resistance ◽  
Physical Contact ◽  
Leukemia Cells ◽  
Soluble Factors

Abstract Abstract 242 Overall objective: AML is a devastating malignancy with a relapse rate near 50% in children, despite very toxic chemotherapy. Once a child relapses, the chance of survival is very low. Therefore new, rational therapies for AML are desperately needed. Accumulating evidence shows that the bone marrow stromal environment protects a subset of leukemia cells and allows them to survive chemotherapy, eventually leading to recurrence. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in AML cells, which could potentially lead to new therapies for AML. Methods, Results & Conclusions: We used two human bone marrow stromal cell lines, HS-5 and HS-27 for our studies. Both provide physical contact with AML cells, while HS-5 cells secrete many more cytokines and growth factors than HS-27 stromal cells. To verify the difference between HS-5 and HS-27 in their secreted soluble factors, both stroma-conditioned media were harvested and soluble factors were quantified by multiplex cytokine assay for 42 individual soluble factors. We detected 23 factors in HS-5 conditioned medium, including G-CSF, IL-6, and MCP-3 at very high levels. HS-27-conditioned medium contained only a few cytokines at similar levels as HS-5, e.g., VEGF and Fractalkine. Next, we performed co-culture experiments to determine the ability of each stromal cell line to confer resistance to chemotherapy. Human AML cell lines (NB-4, THP-1 and Kasumi-1) were cultured alone or co-cultured with HS-5 or HS-27 cells, and treated with etoposide, mitoxantrone or cytarabine for 48 hours. Cells were then harvested and labeled with annexin V-FITC. Stromal cells were identifiable by stable mOrange expression, and the percentage of apoptotic AML cells (FITC positive and mOrange negative) was determined by FACS. Both HS-5 (p<0.001) and HS-27 (p<0.05) cells protected NB-4 and THP-1 cells from etoposide-induced apoptosis (apoptosis rate at the 3 uM dose: 86.6±1.4% NB-4 alone vs. 33.9±2.9% with HS-5 vs 60.7±2.5% with HS-27). The results with THP-1 were similar to NB-4. Using the same method, we demonstrated that both stromal cells protected NB-4 and THP-1 from the toxic effects of all three chemotherapy agents; Kasumi-1 were resistant to all three agents, even when cultured alone. To delineate if the protection induced by stromal cells against chemotherapy was dependent on adhesion pathways and/or soluble factors, we performed Transwell co-culture assays. Different from regular co-culture, there is no physical contact between AML and stromal cells, while soluble factors secreted by stromal cells can reach AML cells. In the absence of physical contact, both stromal cells provided little protection for NB-4 and THP-1 against etoposide and cytarabine; while both NB-4 and THP-1 were still protected against mitoxantrone. Those results suggest that the protection provided by both stromal cells against etoposide and cytarabine mostly relies on cell-cell contact; as for mitoxantrone, soluble factors secreted by both stromal cells seem more important. Surprisingly, HS-5 and HS-27 provided similar degrees of protection against all three chemotherapies. To discover genes in AML cells that are induced by interaction with stromal cells and may contribute to chemotherapy resistance, oligonucleotide microarray analysis was done using total RNA extracted from NB-4 and THP-1 cells cultured alone or co-cultured with stromal cells. We found that 43 genes were upregulated by HS-5, and over 1000 genes were either up- or down-regulated by HS-27. Among them, eighteen genes were upregulated by both stromal cell lines. Since HS-5 and HS-27 provided similar degrees of protection against chemotherapy, those eighteen commonly upregulated genes are likely to be important for stroma-induced chemotherapy resistance. Excitingly, seven out of those eighteen genes, e.g., including CYR61, CAV1, TM4SF1, have been reported to contribute to chemotherapy resistance in various cancer types. Further studies are underway to determine if those genes are responsible for stroma-induced chemotherapy resistance. This study suggests that distinct pathways in the microenvironment mediate resistance to different chemotherapy drugs. Elucidating the precise drug-specific mechanisms involved is likely to result in promising combination therapies to reduce chemotherapy resistance and relapse, and thereby improve survival for children with AML. Disclosures: No relevant conflicts of interest to declare.

EphB4/ephrinB2 Interaction Mediate Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Spreading and Osteogenic Differentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4772-4772
Author(s):  
Lin Li ◽  
Na Xu ◽  
Xuan Zhou ◽  
Jixian Huang ◽  
Yuling Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Direct Contact ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Qrt Pcr ◽  
Sh Group ◽  
Normal Human

Abstract Background and Objective: Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment.Recent studies have indicated interaction between acute leukemia cells and MSCs has a major role in cancer progression and resistance to treatment.Our previous study found that EphB4 receptor was over expressed in CML-Blast Crisis (BC) patients and resistant cell lines. Furthermore, we performed the experiment to prove that aberrant over expressed of EphB4 play an important role to change characterize of Imatinib-resistant in chronic myeloid leukemia cells. However,the contribution of over expressed of EphB4 molecules in leukemia cells to change MSCs function remains to be determined. Therefore,we hypothesis that the change of EphB4/ephrinB2 molecules on leukemia cells might play an important role to transform characterize of MSCs through direct contact, which finally support to leukemia progression and disruption of normal hematopoiesis in microenvironment of the bone marrow. Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or patients' BM and cultures in CyagenBone marrow culture medium at 37 °C, 5% CO2 incubator. EphrinB2(2.628±0.2303 n=3; P<0.05), ALP\RUNX2 (early osteogenesis differentiation genes)(6.430±0.1343, n=3P<0.001; 4.948±0.1418,n=3P<0.001)were over expressed in MSCs (CML patients)in contrast to normal human MSC by QRT-PCR. After osteogenic induced for 2 weeks,MSCs from CML-initial patient showed significantly higher osteogenic differentiation (Osteogenesis Score 4.5P<0.01) and protein (later period osteogenesis differentiation)(2.1669%±0.1443, n=3P<0.001)was overexpressed in MSCs (CML patients) in contrast to normal human MSC(0.2993%±0.1612n=3) by western blot. In functional spreading assay, cultured MSC (CML patient) exposed to EphB4-Fc (5 μg/mL) were significantly rounder (p<0.001) and smaller (p<0.001) as demonstrated with F-actin staining, compared to control and human-Fc.No difference inmorphology was observed when MSC were cultured in the presence of ephrin-B2-Fc.Incubation of MSC with signaling pathway-specific inhibitors before the spreading assay, the PI3Kinase pathway (LY294002) (p<0.001),not the Src kinase pathway (PP2), inhibited MSCs (CML) attachment and spreading. The results revealed that the PI3Kinase pathway was activated upon ephrinB2 reverse signaling in response to EphB4-Fc to promote the contraction and rounding up of MSC. Activation of ephrin-B2 molecules expressed by MSCs (CML)by EphB4-Fc(5 μg/mL), human-Fc(5 μg/mL) or blank control, which cultured in osteogenic media for 2 weeks, more mineral in the presence of EphB4-Fc was visualized by Alizarin Red staining compared to the control human-Fc.Furthermore, after co-cultured respectively with K562-R or K562-R-EPHB4-SH for 72h,ALP\RUNX2 in MSCs (CML) were increased significantly in K562-R group(15.544±2.647; 6.378±2.0775 n=3)compared to K562-R-EPHB4-SH group (6.014±1.19273; 4.045±2.0273 n=3) and control group by QRT-PCR. However, ALP\RUNX2 in normal MSC was unaffected when respectively co-cultured with K562-R(0.741±2.2121; 0.129±0.9194 n=3) or K562-R-EPHB4-SH(0.171±2.062; 0.232±2.0474 n=3) for 72h. The co-cultured assays were also performed using transwells. ALP\RUNX2 in MSCs (CML) were no difference in K562-R group (6.28±1.7875; 4.232±2.4533 n=3) compared to K562-R-EPHB4-SH group (6.107±2.158; 4.139±1.9727 n=3). Conclusion: Our study illustrated that the change of EphB4/ephrinB2 molecules on leukemia cells may transform MSCs functional spreading and osteogenic differentiation through direct contact involved in PI3Kinase pathway. Disclosures No relevant conflicts of interest to declare.

AT9283, A Novel Aurora Kinase/Jak2 Inhibitor Demonstrates Activity against Refractory Infant Leukemia Cells: Studies On Growth Inhibition, Biological Correlates, Drug Synergy and Effects On Leukemia Stem-Like Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3078-3078
Author(s):  
Shamim Lotfi ◽  
Aarthi Jayanthan ◽  
Victor A. Lewis ◽  
Greg Guilcher ◽  
Matthew S Squires ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Aurora Kinase ◽  
Marrow Stromal Cells ◽  
Leukemia Cells ◽  
Combination Studies ◽  
Infant Leukemia

Abstract Abstract 3078 Poster Board III-15 Leukemia in children less than 1 year of age confers a poor prognosis, despite intensification of therapy. These leukemias possess unique biologic characteristics including the presence of mixed-lineage leukemia (MLL) gene rearrangement and high expression of Fms-like tyrosine kinase 3 (FLT3). AT9283, a potent inhibitor of Aurora A and B kinases, JAK2, JAK3, and mutant Abl Kinase, has demonstrated inhibition of multiple solid tumor cell lines in vitro and in mouse xenograft models. Aurora kinase inhibition has been shown to inhibit cancer cell growth by interfering with the mitotic apparatus. We investigated the activity of AT9283 against cell lines derived from refractory infant leukemia cells to identify its efficacy in a future treatment protocol. Method Five cell lines derived from infant leukemia cells were used (ALL: BEL1, KOPN8, KCCF2, B1 and AML: TIB202). We also included the cell line SEM that was derived from a 5 year old child with t (4;11) MLL-AF4 preB-ALL. Normal bone marrow stromal cells were used to evaluate cytotoxicity against non-malignant cells. AT9283 was provided by Astex Therapeutics Ltd. (Cambridge, UK). Approximately 1×104 cells per well were seeded in 96-well plates and incubated with increasing concentrations of AT9283, alone or in combination with a panel of conventional and novel therapeutic agents. After four days, cell survival was measured by Alamar blue assay and IC50 values and combination indices were calculated. Stem-like cells were quantified by the distribution of ALDH bright cells by Aldefluor assay (Stem cell technologies) and characterized by conventional clonogenic assays. Alterations in cell-signaling pathways and survival proteins were measured by Western blot analysis using total and phospho-specific antibodies. Results AT9283 inhibited the growth of all five cell lines with a 10 fold variation in IC50 within cell lines (IC50 range, 0.1 to 0.01 μM). There was a corresponding increase in the number of cells displaying a polyploid phenotype, an effect of aurora kinase inhibition. No significant cytotoxicity against bone marrow stromal cells was seen under the experimental conditions used in this study (IC50 > 10 μM). Changes in the activation and expression of a variety of intracellular proteins were noted, including the down regulation of activated ERK1/2, MYC and AKT within 10 minutes of exposure to the agent. An increase in the activated form of RAF and ATF2 was observed immediately after drug exposure. Importantly, a significant decrease in the level of constitutive pFLT-3 was demonstrated. A concurrent increase in cleaved PARP was also noted, indicating the initiation of apoptosis. In combination studies, the HDAC inhibitor Apicidin showed synergy across all cell lines (CI range: 0.07 to 0.62). A decrease in ALDH bright stem-like cells was observed in a dose dependent manner, up to 50% over 24 hours at IC50 concentrations. Conclusions Our in vitro studies show that AT9283 significantly decreases the growth and survival of infant leukemia cell lines. Importantly, AT9283 potently induces FLT3 de-phosphorylation, inhibiting a critical growth stimulatory pathway of infant ALL cells. We have identified changes in a number of signaling and apoptotic molecules that can provide a panel of markers for biological correlative analysis for drug activity in vivo. Also, the drug combination studies demonstrate the potential of HDAC inhibition to synergize with the activity of this agent. Finally, the effect on stem-like cells provides a rationale and critical preclinical data for the formulation of an effective clinical trial for the treatment of infants with refractory ALL. Disclosures Squires: AstexTherapeutics Ltd: Employment.

Proteomic, Gene Expression, and Micro-RNA Analysis Of Bone Marrow Mesenchymal Stromal Cells In Acute Myeloid Leukemia Identifies Pro-Inflammatory, Pro-Survival Signatures In Vitro and In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3685-3685
Author(s):  
Michael Andreeff ◽  
Rui-yu Wang ◽  
Richard E. Davis ◽  
Rodrigo Jacamo ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Micro Rna ◽  
Leukemia Cells ◽  
Rna Analysis ◽  
Nsg Mice ◽  
Acute Myeloid

Abstract The bone marrow microenvironment (BME) in acute myeloid leukemia (AML) generates resistance signals that protect AML cells/stem cells from chemotherapy. The mechanisms how the BME might support leukemia cell survival are unclear but elucidation of this process could prove useful for therapy design. Here we report new insights specific to stroma functionality in AML. A series of novel experimental approaches were developed including : 1) nanostring micro-RNA and proteomic analysis using reverse-phase protein arrays (RPPAs) of MSC derived from AML patients and normal donors; 2) genome-wide RNA analysis of FACS-sorted MSCs using Illumina arrays of genetically-defined human and murine AML cell lines/primary AML samples after co-culture with normal MSC in vitro; 3) in vivo interaction between genetically-defined murine AML and stromal cells in syngenic C57BL/6J mice, followed by harvesting and FACS-isolation of specific MSC after leukemia engraftment; 4) use of genetically-modified human MSC in vivo in our ectopic humanized bone marrow model in NSG mice (Blood 2012 : 119,4971), followed by bioluminescence growth and homing analysis of human leukemia cells. This model allows the study of in vivo effects of altered MSC on human AML development. 1) Proteomic and transcriptomic analysis of primary MSC from AML patients (n = 106) and normal MSC (n = 71) by RPPA using validated mAbs to 150 proteins and phospho-proteins demonstrated major differences by hierarchical clustering analysis: GSKA, STAT1, PDK1, PP2A, CDKN1A, CDK4, and STAT5AB were significantly over-expressed in AML- vs. normal MSC, while STMN1, SIRT1, SMAD1, SMAD4, HSP90 and EIF2S1 were under-expressed. Differences were observed between MSC from newly diagnosed vs. relapsed AML-MSC. Nanostring analysis of 38 AML-MSC and 24 normal MSC identified differential expression of numerous miRs, a select group of which has been validated so far by qRT-PCR. AML MSC express reduced levels of let-7g, let-7c, miR 21 and miR93, and elevated levels of miR410 compared to normal MSC. Pathways were identified in MSC that might contribute to leukemia survival. 2) Analysis OCI-AML3 cells co-cultured with normal -MSC revealed upregulation of a variety of genes in MSC encoding cytokines and chemokines and gene set enrichment analysis (GSEA) identified activation of NFkB in MSC as a potential cause of these changes. When the ectopic humanized bone marrow model system in NSG mice was used, suppression of NFkB in MSC resulted in a ∼ 50% reduction of AML burden. When murine MSC cultured with wt p53 MLL/ENL-Luc-FLT3-ITD cells were compared to isogenic cells with deleted p53, striking differences were seen in the MSC transcriptome: 429 differentially expressed genes were identified that distinguished co-cultures with p53wt and p53-/- cells, suggesting that AML cells may communicate signals to their microenvironment in a p53-dependent manner. GSEA identified NFkB and HIF-1a as targets, data were confirmed independently, and HIF-1a knockout MSC were found to be inhospitable for AML in the ectopic in vivo model. 3) These syngenic cells were introduced into B57BL/6J mice and MSC were isolated after leukemia engraftment: 147 genes were consistently upregulated and 236 genes downregulated in MSC by their interactions with AML in vivo. Upregulated genes included CTGF, CXCL12, genes related to complement (C4A, C4B, Serpin G1), and IGFBP5, an inhibitor of osteoblast differentiation. Identification of CXCL12 was intriguing as Link's group recently reported the critical role of CXCL12 produced by early MSC in normal hematopoiesis (Nature 2013 : 495,227). Both AML-ETO and MLL-ENL leukemias caused upregulation of CTGF, metalloproteinases, adhesion molecules, and NFkB-related genes in vivo. IPA analysis showed responses in BM-MSC associated with inflammation, cellular movement, cell-cell signaling, cellular growth and proliferation and immune cell trafficking. Conclusion AML cells induce changes in MSC, in short term co-cultures in vitro, or in syngenic systems in vivo, that are consistent with pro-survival, anti-apoptotic, and growth-stimulatory signals that mimic inflammatory responses. Large-scale analysis of primary AML-derived MSC confirms and extends this data. Results facilitate the development of therapeutic strategies to render the BM microenvironment inhospitable to leukemia cells but supportive of normal hematopoiesis. Disclosures: Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

scholarly journals AML Genotype-Specific and Non-Specific Regulation of Mesenchymal Stromal Cell Transcriptome in the Bone Marrow Microenvironment

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1586-1586 ◽  
Author(s):  
Rodrigo Jacamo ◽  
Xiaoyang Ling ◽  
Zhiqiang Wang ◽  
Wencai Ma ◽  
Min Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Matrix Gla Protein ◽  
Leukemia Cells ◽  
Receptor Interaction ◽  
Normal Donor ◽  
Specific Regulation ◽  
Upregulated Genes

Abstract Acute myeloid leukemia (AML) cells can modify the bone marrow (BM) microenvironment and “educate” it to promote leukemia progression; e.g., leukemia cells induce transcriptional changes in BM mesenchymal stromal cells (BM-MSC) that support growth advantage and chemoresistance of leukemia cells (Jacamo, R et al., Blood 2014). Given the genetic heterogeneity of AML, and the variable responses of individual patients to therapy, a better understanding of how different AML genotypes can influence their BM microenvironment is needed. We inoculated C57BL/6 mice with syngeneic murine leukemia cells harboring one of the following human AML genotypes: AML1-ETO, MLL-ENL, or MLL-ENL/FLT3-ITD (further divided into p53 wt or p53-/-) (Zuber, J et al., Genes Dev 2009). Leukemia engraftment was confirmed by live imaging (IVIS) and allowed to progress for 2 to 4 weeks, according to leukemia burden. Multiple mice from each condition were sacrificed to allow isolation of sufficient BM-MSC by flow cytometry based on cell surface markers: CD105+, Sca1+, CD106+, PDGF-Rα+ (CD140), and CD45-. Gene expression profiling with Illumina mouse WGv2 BeadArrays was performed on duplicate pools of mice to identify differences between BM-MSC from various AML-bearing mice and BM-MSC from control mice. Heat mapping of highly-variant genes showed that samples clustered according to AML genotype, confirming data quality and suggesting that changes induced in BM-MSC differ with AML genotype: the two BM-MSC samples derived from the MLL-ENL/FLT3-ITD leukemia-bearing mice were nearest neighbors, and also clustered next to the sample from mice injected with MLL-ENL alone. 942 genes were upregulated in the AML1-ETO BM-MSC samples at least 2-fold as compared to control BM-MSC and those from other AML genotypes. The most highly upregulated genes among these included Tenm4 (an inhibitor of chondrogenesis also known as ODZ4) and Plxnd1, a regulator of angiogenesis. For both MLL-ENL/FLT3-ITD samples, 632 genes were upregulated at least 2-fold as compared to control MSC and other AML genotypes, notably including Cited4 (a repressor of HIF-1α transactivation), Bmp1, and Spon2(a TGF-beta-regulated extracellular matrix (ECM) protein upregulated in multiple types of cancer). Ingenuity Pathway Analysis (IPA) implicated upregulation of genes involved in aryl hydrocarbon receptor signaling, a potential inhibitor of HIF-1α by competition for HIF-1β. For genes differing between BM-MSC exposed to p53 WT and -/- forms of MLL-ENL/FLT3-ITD leukemia, IPA suggested that p53 status differentially affects the BM-stroma transcriptome through effects possibly mediated by CFS3, IL-3 and TGF-β1. We also observed sets of genes that were consistently up- or downregulated in BM-MSC by all four AML genotypes. At a minimal 2-fold change, 57 BM-MSC genes were upregulated in all 4 samples, and 60 genes downregulated, by exposure to AML in vivo. Notable among the upregulated genes were Ctgf (12 and 33 fold increase [FI] by 2 array probes), genes related to complement (C4a, C4b, Serping1), Igfbp5 (FI = ~12 by 3 probes), Mgp (matrix Gla protein, an ECM regulator of multiple types of mesenchymal differentiation, FI = ~8) and Cxcl12 (FI = ~6). The latter, the ligand for CXCR4, is expressed by a subset of BM stromal cells and is essential for the maintenance of normal hematopoietic precursors. Gene Set Enrichment Analysis (GSEA) implicated upregulation of genes involved in ECM receptor interaction and cancer stem cells. Common downregulated genes included Ltf, a tumor suppressor and inhibitor of AKT signaling, and PDCD4, a suppressor of tumor-permissive stromal changes downregulated by miR-21, consistent with our finding of increased mir-21 in AML-derived BM-MSC compared to normal donor-derived BM-MSC. In summary, these results support the hypothesis that AML regulates the transcriptomes of BM-MSC, in both shared and genotype-specific ways. Further analysis of the data is ongoing. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
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.

Myeloblasts Convert into Fibroblastoid Stromal Cells To Create a Microenvironment for Proliferation of Leukemia Blasts in Acute and Chronic Myelogenous Leukemia Cases.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4247-4247
Author(s):  
Haruko Tashiro ◽  
Mitsuho Noguchi ◽  
Ryosuke Shirasaki ◽  
Moritaka Gotoh ◽  
Kazuo Kawasugi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myelogenous Leukemia ◽  
Stromal Cells ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Myelogenous Leukemia ◽  
Differentiated Cells ◽  
Bone Marrow Fibroblasts ◽  
Molecular Expression

Abstract Object: Cancer stem cell has been analyzed in leukemia, which behaves similarly to normal stem cells on their self-renewal and self-conversion into abnormally differentiated cells to make an exact recapitulation of the original heterogeneous leukemia cells. We observed a primary in vitro culture of non-adherent leukemia blasts prepared from various kinds of acute leukemia and chronic myelogenous leukemia cases, and biological and biochemical characteristics were analyzed. Method: Leukemia blast-rich fractions were prepared from patients’ blood or bone marrow after gradient sedimentation method, which were cultured for a long term. When the appearance of the cultured cells converted into fibroblastoid cells, cells were divided into clones, analyzed molecularly to identify whether they were originated from leukemia clone, and their histochemical, biochemical and functional characterizations were determined. Results: Morphological changes into fibroblastoid stromal cells were observed in AML with t (11; 19) (p23; p13.1), M4E, Ph-positive biphenotype and CML (chronic and myeloid blast phase) cases but neither in ALL nor in CML-lymphoid blast cases. The generated fibroblastoid cells had enough functions equal to those of the normal bone marrow fibroblasts on their molecular expression (CD106, fibronectin), production of cytokines (VEGF, IL-7) and giving the activity of proliferation to normal hematopoietic cells. These cells maintained their characteristics observed in the original leukemia blasts (expression of CD13, CD33 and myeloperoxidase), which cells also expressed CD 34 and 133. Leukemia blasts proliferated extensively when cultured on the expanded fibroblastoid cells derived from leukemia blasts. Discussion: These results indicate that leukemia blasts can create their own microenvironment for proliferation.

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