scholarly journals Targeting Extracellular Vesicle Secretion As a Strategy to Overcome Microenvironment Mediated Drug Resistance in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3733-3733
Author(s):  
Palani Kumar Kumar ◽  
Saravanan Ganesan ◽  
Nithya Balasundaram ◽  
Sachin David ◽  
Arvind Venkatraman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Extracellular Vesicle ◽  
Akt Signaling ◽  
Leukemic Cells ◽  
Stromal Microenvironment

Increasing evidence suggests that bone marrow microenvironment act as a sanctuary site for acute myeloid leukemia (AML) cells and provides protection from conventional chemotherapy agents. Recently, extracellular vesicles (EVs) have attracted substantial attention as a carrier of complex intercellular information by transferring microRNA, mRNA and proteins. We undertook a study to delineate the molecular mediators and potential role of extracellular vesicles in stromal microenvironment mediated drug resistance in AML. We performed a series of in vitro experiments with AML cell lines (U937, THP-1, Kasumi-1) and primary cells to evaluate their response to daunorubicin (DNR) and cytarabine (AraC) with stromal cells (HS-5 cell line). Towards this we co-cultured the leukemic cells with stromal cells in a contact dependent and contact independent (transwell plates) system and with EVs derived from HS-5 culture media using well established methods (Suzanne et al, Blood 2015). The percentage of viability was calculated using Annexin V/7AAD staining by flow cytometry. Gene expression profiling was done using Agilent Human Whole Genome 8x60K Gene Expression Array. The quantification of extracellular vesicle was performed using NanoSight LM10. Direct stromal co-culture experiments with AML cells demonstrated a significant stromal cell mediated protective effect against AraC and DNR in cell lines (figure 1A) and primary cells [AraC p < 0.01; DNR p < 0.001 (n=50)]. A similar significant protective effect was also seen in contact independent system and EVs alone treated leukemic cells (supplemented in place of HS-5 co-culture). Gene expression profiling analysis of leukemic cells (U937) and stromal cell (HS-5) post co-culture revealed a bidirectional enrichment of genes involved in extracellular vesicle biogenesis and secretion (p < 0.001) along with a significant dysregulation of PI3K-AKT signaling in leukemic cells. We have previously reported that stromal EVs activates PI3K-AKT signaling and mediates drug resistance in leukemic cells similar to direct stromal co-culture (Blood 2017 130:1160). In addition to PI3K-AKT signaling, our qPCR validation also confirmed the significant up regulation of genes which are involved in EVs secretion (RAB27A, RAB35 and VAMP7) in leukemic cells as well as stromal cells post co-culture (figure 1B). Hence, we quantified the amount of EVs production in leukemic and stromal cells post 48hrs of co-culture where the number of EVs showed a trend towards increase in co-cultured leukemic and stromal cells when compared to the cells cultured alone (figure 1C). We also noted that treatment with neutral sphingomyelinease inhibitor GW4869 a known inhibitor of EVs secretion was able overcome the stroma mediated drug resistance significantly in leukemic cell lines (figure 1D) and also in primary AML cells [AraC p < 0.01; DNR p < 0.001 (n=6)]. Our results illustrate that reciprocal interaction of leukemic and stromal cells influences the secretion of extracellular vesicles and plays a significant role in mediating drug resistance. We further demonstrated that inhibiting extracellular vesicles secretion was able to overcome the stromal microenvironment mediated drug resistance in AML illustrating a potentially novel therapeutic strategy. Additional studies are required to explore and characterize the cargo (microRNA and proteins) in detail of these EVs and the mechanism/s by which they mediate drug resistance. Disclosures No relevant conflicts of interest to declare.

scholarly journals Changes in the Multipotent Stromal Mesenchymal Cells from the Bone Marrow of the Patients with Hematological Diseases in Debut and after the Treatment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5014-5014
Author(s):  
Irina N. Shipounova ◽  
Nataliya A. Petinati ◽  
Nina J. Drize ◽  
Aminat A. Magomedova ◽  
Ekaterina A. Fastova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Marker Gene ◽  
Malignant Cells ◽  
Hematopoietic Stem ◽  
Stromal Microenvironment ◽  
Expression Of Genes

Introduction. Stromal microenvironment of the bone marrow (BM) is essential for normal hematopoiesis; the very same cells are involved in the interaction with the leukemic stem cells. The aim of the study was to reveal the alterations in stromal microenvironment of patients in debut and after the therapy using multipotent mesenchymal stromal cells (MSC) as a model. Methods. MSC of patients with acute myeloid leukemia (AML, N=32), acute lymphoblastic leukemia (ALL, N=20), chronic myeloid leukemia (CML, N=19), and diffuse large B-cell lymphoma without BM involvement (DLBCL, N=17) were isolated by standard method from the patients' BM. Each BM sample was acquired during diagnostic aspiration after the informed signed consent was obtained from the patient. Groups of BM donors comparable by age and gender were used as controls for each nosology. Gene expression was analyzed with real-time RT-PCR. The significance of differences was evaluated with Mann-Whitney U-test. Results. The results of gene expression analysis are summarized in Table. The expression of genes regulating hematopoietic stem and precursor cells (JAG1, LIF, IL6) was significantly upregulated in MSC of the patients in debut, except for DLBCL. The latter was characterized with upregulation of osteogenic marker gene SPP1 and downregulation of FGFR1 gene. The upregulation of the expression of genes regulating proliferation of stromal cells (PDGFRA, FGFR1) and adipogenic marker gene (PPARG) was common for AML and CML. Both acute leukemias were characterized by the upregulation of genes associated with inflammation and regulation of hematopoietic precursors (CSF1, IL1B, IL1BR1) and by the downregulation of chondrogenic differentiation marker gene (SOX9). CML and DLBCL demonstrated the upregulation of FGFR2. BM of the DLBCL patients did not contain any malignant cells; nevertheless, stromal precursors from the BM were significantly affected. This indicates the distant effects of DLBCL malignant cells on the patients' BM. Myeloid malignancies seem to affect MSC more profoundly then lymphoid ones. Effect of leukemic cells on stromal microenvironment in case of myeloid leukemia was more pronounced. The treatment significantly affected gene expression in MSC of patients. In all studied nosologies the IL6 gene expression was upregulated, which may reflect the inflammation processes ongoing in the organism. The expression of LIF was upregulated and ICAM1, downregulated in MSCs of AML, ALL, and CML patients. In the MSC of patients with AML, who had received the highest doses of cytostatic drugs to achieve remission, a significant decrease in the expression of most studied genes was found. In patients with ALL with long-term continuing treatment in combination with lower doses of drugs, IL1B expression was increased, while the decrease in expression was detected for a number of genes regulating hematopoietic stem cells (SDF1, TGFB1), differentiation and proliferation (SOX9, FGFR1, FGFR2). Treatment of CML patients is based on tyrosine kinase inhibitors in doses designed for long-term use, and is less damaging for MSC. The upregulation of TGFB1, SOX9, PDGFRA genes and downregulation of IL1B gene was revealed. MCS of DLBCL patients, unlike the other samples, were analyzed after the end of treatment. Nevertheless, significant upregulation of IL8 and FGFR2 genes was found. Thus, both the malignant cells and chemotherapy affect stromal precursor cells. The changes are not transient; they are preserved for a few months at least. MSCs comprise only a minor subpopulation in the BM in vivo. When expanded in vitro, they demonstrate significant changes between groups of patients and healthy donors. Conclusions. Leukemia cells adapt the stromal microenvironment. With different leukemia, the same changes are observed in the expression of genes in MSC. MSC of patients with acute forms have a lot of changes which coincide among these two diseases. MSC of AML patients are most affected both in debut and after the therapy. Treatment depends on the nosology and in varying degrees changes the MSC. This work was supported by the Russian Foundation for Basic Research, project no. 17-00-00170. Disclosures Chelysheva: Novartis: Consultancy, Honoraria; Fusion Pharma: Consultancy. Shukhov:Novartis: Consultancy; Pfizer: Consultancy. Turkina:Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy, Speakers Bureau; fusion pharma: Consultancy.

Acute Myeloid Leukemia Alters The Mesenchymal Stem Cell Potential Of The HSC Niche: Evidence For Modulation By β-Adrenergic Signals

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 342-342
Author(s):  
Maher Hanoun ◽  
Dachuan Zhang ◽  
Sandra Pinho ◽  
Toshihide Mizoguchi ◽  
Julie Lacombe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stromal Cells ◽  
Adrenergic Receptor ◽  
Myeloid Leukemia ◽  
Fold Increase ◽  
Leukemic Cells ◽  
Bm Niche ◽  
Β2 Adrenergic Receptor ◽  
Acute Myeloid

Abstract Hematopoietic stem cells (HSC) reside in specific bone marrow niches comprised of perisinusoidal Nestin-GFP+ (Nes-GFP+) and leptin receptor (LepR)+ stromal cells which highly overlap with each other, as well as osteolineage and endothelial cells. These different cellular constituents regulate HSC maintenance and retention in the bone marrow (BM). Recently, our laboratory further identified rare periarteriolar Nes-GFPbright cells which have been identified to be pericytes and functionally crucial for HSC quiescence and maintenance, from the more abundant reticular Nes-GFPdim population (Kunisaki et al, unpublished data). The function of Nes-GFP+ niche cells is tightly regulated by the sympathetic nervous system (SNS) via β2- and β3-adrenergic receptors. In acute myeloid leukemia (AML), BM infiltration by leukemic blasts is known to lead to hematopoietic failure. Although cytopenias are thought to result from the BM occupation by AML, the mechanisms remain unclear. In this study, we investigated the impact of AML on BM niche constituents, and evaluated the influence of SNS signals in AML progression using a syngeneic murine MLL-AF9 transplantation model. We observed a ∼4.4-fold expansion of Nes-GFP+ stromal cells (p<0.05) in leukemic BM, which positively correlated with the degree of leukemic marrow infiltration (r=0.49, p<0.01). However, while we observed a massive expansion of reticular Nes-GFPdim cells, we detected a ∼2.2-fold reduction in pericytic Nes-GFPbright cells (p<0.05). Phenotypically, Nes-GFPdim cells showed similar expression of LepR, PDGFRα and CD51 to healthy controls, markers enriching for BM mesenchymal stem cells. Interestingly, Nes-GFP+ cells in leukemic BM had a ∼1.5-fold higher colony-forming units-fibroblast (CFU-F) and ∼2.6-fold higher CFU-Osteoblast capacity. The gene expression profile of Nes-GFP+ cells using Affymetrix Gene 1.0 ST microarrays revealed a profound dysregulation when the BM was infiltrated with AML, including most notably activated TGFβ signaling pathways and upregulation in the expression of inflammatory response genes. Nes-GFP+ niche cells were previously shown to be highly enriched for HSC-maintenance gene expression. In leukemic BM, Nes-GFP+ cells showed a significant reduction in the expression of Cxcl12, Scf and Vcam1 (p<0.01), while no significant differences were observed for Opn. In line with these data, we observed a significant reduction of HSC-enriched Lin−Sca1+c-Kit+Flt3− and progenitor cells in the BM (p<0.05, except for granulocyte-macrophage progenitors) and their mobilization into peripheral blood and spleen (p<0.01). To further analyze if Nes-GFP+ niche cells in leukemic BM still depended on adrenergic signals, we performed in vivo chemical sympathetic denervation with 6-hydroxydopamine. Sympathectomized leukemic mice showed a ∼1.5-fold increase in Nes-GFP+ cells, a ∼1.8-fold increase of IL-7R-Lin-c-KithiCD34+FcγRII/IIIhi leukemic stem cells (LSC, p<0.05) which was associated with significantly shorter survival (p=0.017). Denervated mice did not show differences in homing, proliferation or apoptosis of leukemic cells. We confirmed our findings in a xenograft model, in which sympathectomized mice were transplanted with primary human AML samples and showed a higher leukemic BM infiltration (p<0.05). Further analyses using specific adrenergic β2- and β3-receptor antagonists (ICI118,551 hydrochloride and SR59230A) suggested that adrenergic signals were predominantly mediated by the β2-adrenergic receptor. Preliminary data indicate that activation of the β2-adrenergic receptor leads to decreased leukemia burden. In summary, our data identify that leukemic cells severely transform the BM niche by gradually expanding and directing mesenchymal stem cell differentiation into the osteoblastic lineage and simultaneously decreasing numbers of pericytic niche cells. Ultimately, this creates a microenvironment with impaired HSC maintenance capacity and favors myeloid expansion. This transformed niche, however, remains regulated by signals from the SNS which in turn modulates leukemic BM infiltration. These results thus uncover a novel approach for niche-targeted therapeutic strategies in AML. Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Proteomic Characterization of Circulating Extracellular Vesicles Identifies Novel Serum Myeloma Associated Markers

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1814-1814
Author(s):  
David Ciarlariello ◽  
Sean Harshman ◽  
Alessandro Canella ◽  
Erinn Hade ◽  
Alberto Rocci ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Protein Content ◽  
Cell Lines ◽  
Extracellular Vesicles ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Predictive Biomarker ◽  
Total Serum ◽  
Label Free ◽  
Β2 Microglobulin

Abstract Introduction: Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells (PCs) in the bone marrow (BM). MM cells are dependent on the BM microenvironment (e.g. BM stromal cells, macrophages etc) and create a network with surrounding cells. These cells play a pivotal role in the regulation of MM cell survival and drug resistance bydirect interactions through adhesion molecules causing cell adhesion mediated drug resistance (CAM-DR) or soluble factors including supportive cytokines (e.g. IL-6, IL-8, and VEGF) or exosomes (or extracellular vesicles). Exosomes (EV) are endosome-derived membrane-covered cell fragments, which contain specific protein and RNA cargo. The protein content of EVs in MM has not been readily explored. Recently our group established the use a global label-free quantification method to determine the relative amount of proteins identified from EVs obtained from MM cell lines. Here we are reporting a systematic proteomic analysis of EVs derived from MM cell lines, blood from MM patients, and BM from MM patients. We also report preliminary data showing the biological importance of specific EV enriched proteins in MM cell lines and MM patients. Methods: Liquid Chromatography Mass Spectrometry (MS) and Label-free relative quantitation were used to assess the protein content of MM EV and cellular lysates. Cryo-Transmission Electron Microscopy (cryo-TEM) and Nanoparticle Tracking Analysis were used to assess size distribution and specific EV surface markers in EV isolated from the supernatant of MM cell lines and from the serum of non-cancer, MGUS, smoldering MM and active MM donors. Western blot and enzyme-linked immunosorbent assays were used to validate MS data in a bigger cohort of primary MM patients and to assess the biological effect of MM derived EV in the BM stromal cells. Results: Our data show that the Major Histocompatibility Complex Class I (MHCI) and its associated binding protein β2-microglobulin (β2-MG) are the most abundant communally enriched proteins in the EV derived from MM cell lines and from the serum of MM patients. Although it is well known that the serum level of β2-MG is an important prognostic factor in MM, our data indicate that β2-MG represents only a small percentage of the total serum β2-MG, suggesting that two separate β2-microglobulin populations coexist in the serum of patients. Additionally, we show that the main receptor of hyaluronic acid, CD44, is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R, and is a protein that is differentially expressed in EVs isolated from newly diagnosed MM patients and which seems to play a key role in communicating with the BM microenvironment. By using a large cohort of serum obtained from MM patients prospectively treated on a randomized phase 3 trial (233), we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as easily accessible MM biomarker. Conclusions: Our results generate a foundation for the potential use of circulating EVs as novel serum markers of MM and provide the rationale to further explore previously unconsidered molecular players associated with MM disease. Disclosures Palumbo: Array BioPharma: Consultancy; Onyx Pharmaceuticals: Consultancy; Millennium Pharmaceuticals Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Genmab A/S: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy; Sanofi Aventis: Consultancy.

scholarly journals Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Saravanan Ganesan ◽  
Hamenth Kumar Palani ◽  
Vairavan Lakshmanan ◽  
Nithya Balasundaram ◽  
Ansu Abu Alex ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Tlr2 Expression ◽  
Molecular Events ◽  
Acute Myeloid

Abstract Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

scholarly journals Global Gene Expression Analysis Reveals Genotoxic Effect of Nrf2 Pharmacological Inhibitor Brusatol in Myeloid Leukemia Cells- a Promising Novel Anticancer Agent

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5150-5150
Author(s):  
Raveen Stephen Stallon Illangeswaran ◽  
Sreeja Karathedath ◽  
Bharathi M Rajamani ◽  
Poonkuzhali Balasubramanian
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Drug Resistance ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Global Gene Expression ◽  
Molar Concentration ◽  
Differentially Expressed ◽  
Micro Array ◽  
Common Target

Abstract Combating drug resistance is a paramount task in treating acute myeloid leukemia (AML). NFE2 related factor 2 (NRF2) is a major player in inducing chemo resistance in both AML and solid tumors. We have previously reported that inhibition of NRF2 both by shRNA knockdown and pharmacological inhibition using brusatol sensitizes inherent resistant AML cell lines and patient samples to chemotherapy (Karathedath et al, Plos one, 2017). Recent studies have shown that brusatol acts as a potent protein translation inhibitor in A549 lung cancer cell line and this deleterious effect is due to the molar scale concentrations of the drug (Stokoe, et al., 2016, Harder et al, 2016). As the available reports was in solid tumor lines and at higher concentrations, we undertook this study to elucidate the mechanism of action of brusatol in our experimental setup utilizing Nano molar concentration of brusatol in AML cell lines. We compared the global gene expression changes induced by brusatol in AML cell lines resistant to Ara-C, Dnr and ATO namely THP-1 and U937. Total RNA was extracted from both cell lines with and without brusatol treatment (100nM for 6 hours) using Qiagen RNeasy mini kit, cDNA was synthesized and subjected to a One -color 8X60K Agilent micro-array analysis. Data normalization, and analysis was performed using Gene Spring GX (V 12.0) software using the 75th percentile shift. Using student t-Test, 3070 genes were identified to be differentially expressed between THP-1 control and brusatol treated while 6856 genes were identified to be differentially expressed between U937 control and brusatol treated samples. About 1242 differentially expressed genes were common between brusatol treated THP-1 and U937. Differentially expressed genes(DEGs) were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category using biological analysis tool DAVID. The enriched genes in both THP-1 and U937 after brusatol treatment fell into the following; Molecular function: protein binding (1396 and 1701 genes), Biological function: regulation of transcription (350 and 502 genes), Cellular component: Mitochondrion (231 and 249 genes) and so on. KEGG analysis was performed to identify enriched pathways after brusatol treatment in DEGs. DEGs in THP1 and U937 were enriched in 33 and 57 pathways were respectively, combining the pathways revealed 20 common target pathways between THP1 and U937. Pathways involved in cellular stress response such as NFkB, TNF and MAPK pathways were selectively up-regulated in the common target pathways and genes involved in cell cycle pathway were down regulated. Select genes from these target pathways and few genes DOCK3, KLF10, NR4A3, GADD45B, KDM4B, GPR155, H2FAY, from the top 100 DEGs were validated by RQ-PCR (SyBr green) all the genes screened showed results consistent with the micro-array data. Interestingly DNA damage response genes GADD45B, KDM4B, H2AFY were in the top up-regulated genes indicating that brusatol might also cause DNA damage in leukemic cells. NR4A3 the orphan nuclear receptor is an established tumor suppressor in AML and is generally silenced in both AML cell lines and primary samples. One of the key finding from our data is that brusatol strikingly up-regulated NRA4A3 expression in both THP1 and U937. This study has shed light into other modes of action of brusatol apart from being a modulator of the protein translational machinery. Mechanism of chemo sensitization by brusatol could be tissue, time and concentration specific. Our data reveals brusatol at nano-molar concentration acts as a potent DNA damaging agent and reactivates NR4A3 expression in AML apart from being an inhibitor of NRF2. We have also reported at nano-molar concentration brusatol has minimal toxicity towards normal Peripheral blood mono-nuclear cells. Brusatol could be an ideal compound to combat drug resistance in AML. Further experimental validation is warranted to substantiate these finding of the mode of action of brusatol in chemo sensitizing AML cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Stroma Cells Promote a S100A8/A9high-Subset of AML Blasts with Distinct Metabolic Features in a Jak/STAT3-Dependent Manner

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2807-2807
Author(s):  
Martin Böttcher ◽  
Konstantinos Panagiotidis ◽  
Andreas Mackensen ◽  
Dimitrios Mougiakakos
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
S100 Protein ◽  
Leukemic Cells ◽  
Cell Contact ◽  
Soluble Factors ◽  
Underlying Mechanisms ◽  
Upregulated Genes

Abstract Introduction: It is well established that the bone marrow stromal niche can serve as a protective environment in hematological malignancies such as AML by multiple cell contact-dependent and independent mechanisms. Intensive research of the bidirectional interactions between leukemic cells and mesenchymal stromal cells already highlighted numerous modes-of-action how malignant cells are capable of hijacking or altering their surroundings to their own favor. However, the entirety of underlying mechanisms is still incompletely understood. We found two small intracellular calcium-sensing molecules, S100A8 and S100A9, among the top upregulated genes in primary AML cells upon stromal contact. S100A8/A9 are members of the S100 protein family that, by functioning both as intracellular Ca2+ sensors and as extracellular mediators, can modulate cellular responses such as proliferation, migration, inflammation, and differentiation. Dysregulation of S100 protein expression is described as a common feature in several human cancers. Specifically in AML expression of S100A8 in leukemic cells predicts poor survival in de novo AML patients. Thus, we aimed to elucidate the underlying mechanisms of stroma-mediated S100A8/A9 upregulation as well as the consequences, and characterized S100A8/A9high AML cells in comparison to their S100A8/A9low counterparts in terms of gene expression pattern, differentiation, metabolic profiles, and chemoresistance. Methods: We co-cultured both AML cell lines and primary AML blasts in a contact-dependent and -independent manner with human bone marrow stromal cells. After co-culture AML cells were re-purified and analyzed by RNA sequencing, flow cytometry and quantitative real-time PCR. In some experiments, AML cells were sorted based on their S100A8/A9 protein levels and S100A8/A9high cells were compared to S100A8/A9low cells for their transcriptome. Results: We found S100A8 and S100A9 among the top upregulated genes in an unbiased transcriptome analysis of primary AML cells cultured in the presence of HS-5 cells compared tothe controls. Upregulation of S100A8/A9 could be confirmed in AML cell lines and was shown to be reversible. We could demonstrate that S100A8/A9 upregulation is mediated by soluble factors as cell-to-cell contact was not necessary and exosome-free conditioned medium from HS-5 cells did not induce S100A8/A9 gene expression. We found the Jak/STAT3 signaling being one major responsible pathway. The S100A8/A9high population was characterized by increased surface levels of maturation markers (such as CD14 and CD11b) as well as altered metabolically important transporters (e.g. for glucose, fatty and amino acids). Finally, we could demonstrate an increased chemoresistance of the S100A8/A9high cells. Conclusion: We could demonstrate bone marrow stroma-induced S100A8/A9 upregulation in AML cells is mediated by soluble factors activating the Jak/STAT3 pathway. S100A8/A9 leads to metabolic alterations and increased differentiation of AML cells conferring enhanced chemoresistance and thus represents a potential therapeutic target against AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Influence of Bone Marrow Stromal and Leukemic Cells on Cytarabine Chemo-Toxicity in Acute Myeloid Leukemia (AML)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2492-2492
Author(s):  
Liana E Gynn ◽  
Elizabeth Anderson ◽  
Gareth M Robinson ◽  
Sarah Anne Wexler ◽  
Gillian Upstill-Goddard ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Drug Induced ◽  
Acute Myeloid

Mesenchymal stromal cells (MSC) are known to protect leukemic cells from drug-induced toxicity within the bone marrow (BM) niche, however, less is known about leukemic impact on supportive MSC. The nucleoside-analogue, cytarabine (Ara-C), is a front-line therapy for acute myeloid leukemia (AML), entering cells via the human equilibrative nucleoside transporter (hENT1). Over a third of AML patients do not show continued response to Ara-C-based regimens, with chemo-resistance linked with repressed hENT1 availability in some patients, while other mechanisms remain unknown. In addition to chemo-resistance, DNA damage caused by chemotherapeutics such as Ara-C can persist in BM-MSC, which remain of host origin following allogeneic stem cell transplantation. This genotoxicity hinders cellular functionality, and may be implicated in long-term hematopoietic failure and secondary malignancies. This study aimed to further elucidate chemo-resistance mechanisms, with particular focus on the contribution of leukemic cells to stromal cell toxicity; aiming to uncover potentially targetable features of resistant AML and reduce treatment burden on the BM. Primary MSC were isolated from BM aspirates from patients both at diagnosis and post-treatment; following ethical approval and informed consent. MSC cultures were confirmed by immunophenotype (flow cytometry) and differentiation capacity (cytological staining) and used in a similar manner to that of cell lines. AML (HL-60, K562) and stromal (HS-5) cell lines were mono- or co-cultured using trans-well inserts for 24h, prior to 1-24h treatment with 25µM Ara-C (equivalent to in vivo standard dose; 100-200mg/m2). Cytotoxicity was monitored by viability and proliferation (CFSE tracing) assays, and chemo-sensitivity assessed with a drug-efficacy screening tool (bacterial bioluminescent biosensor). Genotoxicity was determined by micronucleus and alkaline comet assays, assessing division abnormalities and DNA fragmentation respectively. Differential cytokine secretion utilised an array, with quantification by ELISA. In co-culture, stromal cells were sensitised to drug-induced cytotoxicity, while leukemic cells were themselves protected from treatment. Genotoxicity was also significantly increased in stromal cells (p=0.0397), while being significantly decreased in leukemic cells when co-cultured (p=0.0089), conferring with cytotoxicity findings. Similarly, BM-MSC from previously treated patients had significantly higher genotoxicity than patients at diagnosis (p=0.0138). While stromal cell proliferation remained unchanged regardless of intervention, data suggest increased proliferation in co-cultured leukemic cells compared to cells cultured alone. Chemo-sensitivity also increased in stromal cells in co-culture, while the opposite was seen for leukemic cells. Seven of 32 cytokines were differentially secreted by cell lines in co-culture compared to combined values from mono-cultured cells; CCL2, CXCL1, G-CSF, GM-CSF, IL-6, MIF and Serpin E1. Of these, the inflammatory cytokine MIF, macrophage migration inhibitory factor, was decreased in co-culture (p<0.0001), and has been implicated in the progression of other malignancies. Separation of cells following co-culture and treatment uncovered opposing MIF secretion profiles in cells with high (HL-60) and low (K562) sensitivity to Ara-C. Despite differential secretion, neither MIF, nor the MIF inhibitor ISO-1, had significant effects on chemo-sensitivity when cells were cultured in each condition for 24 hours. Chemo-resistance is evidently a network of complex, interlinking mechanisms which are not easily identified in vitro. MIF remains a likely candidate for studies into AML chemo-resistance, with research ongoing. This study shows for the first time that the co-culture of AML and MSC alters the genotoxic effect of chemotherapy. Future research utilising larger patient cohorts is required to fully understand how cells in the BM micro-environment can be targeted. This could potentially improve not only the overall outcome for AML patients, but reduce the cytotoxic and long-term genotoxic complications of current therapies. Disclosures No relevant conflicts of interest to declare.

Galectin-3 Is the Molecular Target for Overcoming Multidrug Resistance Due to the Cell Protection by Bone Marrow Leukemia Microenvironment in Chronic Myeloid Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3746-3746
Author(s):  
Mio Yamamoto ◽  
Junya Kuroda ◽  
Tsutomu Kobayashi ◽  
Nana Sasaki ◽  
Hisao Nagoshi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Death ◽  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Chemotherapeutic Agents ◽  
Leukemic Cells ◽  
Galectin 3 ◽  
The Impact

Abstract Abstract 3746 Recent advances in molecular targeted therapy using tyrosine kinase (TK) inhibitors (TKIs) for Bcr-Abl TK have greatly improved treatment outcomes for chronic myeloid leukemia (CML). However, complete elimination of CML clones has been rarely achieved by TKIs due to both intrinsic and extrinsic cell mechanisms. In this study, we investigated the molecular mechanisms for bone marrow microenvironment (BMME)-mediated leukemia progression and drug resistance in CML. CML cell lines MYL and K562 acquired drug resistance to imatinib mesylate (IM), dasatinib (Das), doxorubicin (DOX), cytarabine (CA), etoposide (VP16), or vincristine (VCR) through co-culture with HS-5, an immortalized human bone marrow stromal cell (BMSC)-derived cell line. We used microarray-based assays to investigate the changes in gene expression profiles in Philadelphia (Ph)-positive MYL as a result of co-culture with HS-5 and of adhesion to fibronectin (FN). In MYL with HS-5 or with FN, 902 and 910 genes, respectively, were upregulated more than 2.0-fold compared with control. Among the 284 genes commonly upregulated in MYL with HS-5 and with FN, we focused on galectin-3 (Gal-3) as one of the candidate mediators of BMME-mediated leukemia progression because of its pleiotropic cellular function. The levels of galectin-3 mRNA increased 3.84-fold as a result of co-culture with HS-5, and 2.83-fold as a result of adhesion to FN in MYL. The induction of Gal-3 by the co-culture with HS-5 or the adhesion to FN was also confirmed at the protein level, not only in MYL, but also in all Ph+ leukemic cell lines examined (K562, KBM5, KCL22, BV173), while Gal-3 protein expression was either absent or extremely low in normal liquid culture. To investigate the clinical significance of Gal-3 in CML, we examined its expression in BM-derived primary CML cells. Approval was obtained from the institutional review board at Kyoto Prefectural University of Medicine for a study using patient-derived samples, and the study was conducted in accordance with the ethical principles of the Declaration of Helsinki. Of the leukemic cells of 20 CML patients, those of all but one CML-blast crisis phase patient were positive for Gal-3. Ph+ cells from the CML-chronic phase were especially highly positive for Gal-3. In contrast, the frequency of Gal-3-positive cells from most acute leukemia patients was as low as that of BM hematopoietic cells from healthy volunteers. These results suggest that in the clinical setting Gal-3 expression in the BM milieu is more predominant in CML. To further characterize the role of Gal-3 in CML, Gal-3 overexpressing subcell lines of MYL and K562 were generated by transfection of pEF1Galec3.neo plasmid and designated as MYL/G3 and K562/G3, respectively. Gal-3 overexpression was found to confer moderately higher in vitro proliferation potency in medium containing 10% FCS as well as in low nutrient 1% FCS-containing medium compared with the cells transfected with the mock plasmid. We also examined the impact of Gal-3 overexpression on cell death induced by chemotherapeutic agents. Both MYL/G3 and K562/G3 were less sensitive than their parental cells to cell death induced by chemotherapeutic agents. This diminished sensitivity to cell death caused by chemotherapeutic agents was due to a reduction in apoptosis, as reflected by the reduced frequency of subG1 populations detected in DNA content analyses (FACS SubG1 analysis of results for 48-hour treatment: MYL 54.9%, MYL/G3 15.7% with IM 0.25uM; MYL 47.1%, MYL/G3 23.1% with Das 1.0nM; MYL 40.2%; MYL/G3 13.4% with DOX 200nM; MYL 45.7%; MYL/G3 19.4% with CA 150nM; MYL 50.9%; MYL/G3 19.1% with VP16 4uM; MYL 75.0%; MYL/G3 49.5% with VCR 8nM). We next examined the effect of an inhibitor for Gal-3, fractionated citrus pectin powder (FPP), on MYL and MYL/G3. As expected, MYL and MYL/G3 showed similar sensitivity to cell death induced by FPP, while the addition of FPP overcame resistance to IM-induced cell death in MYL/G3. Furthermore, the addition of FPP was found to have overcome HS-5-induced resistance against IM in MYL. Molecular sequelae of Gal-3 overexpression in leukemic cells were also investigated. Both co-culture with HS-5 and Gal-3 overexpression activated Akt and Erk, induced accumulation of Mcl-1 in MYL and K562. Collectively, our study disclosed that leukemia microenvironment-specific Gal-3 may be a candidate therapeutic target to help overcome BMME-mediated therapeutic resistance in CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Myeloid-Derived Suppressor Cells and Mesenchymal Stem/Stromal Cells in Myeloid Malignancies

2021 ◽  
Vol 10 (13) ◽  
pp. 2788
Author(s):  
Suncica Kapor ◽  
Juan F. Santibanez
Keyword(s):  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Chemotherapy Resistance ◽  
Leukemia Cell ◽  
Suppressor Cells ◽  
Leukemic Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Adaptive Immune Systems

Myeloid malignancies arise from an altered hematopoietic stem cell and mainly comprise acute myeloid leukemia, myelodysplastic syndromes, myeloproliferative malignancies, and chronic myelomonocytic leukemia. Myeloid neoplastic leukemic cells may influence the growth and differentiation of other hematopoietic cell lineages in peripheral blood and bone marrow. Myeloid-derived suppressor cells (MDSCs) and mesenchymal stromal cells (MSCs) display immunoregulatory properties by controlling the innate and adaptive immune systems that may induce a tolerant and supportive microenvironment for neoplasm development. This review analyzes the main features of MDSCs and MSCs in myeloid malignancies. The number of MDSCs is elevated in myeloid malignancies exhibiting high immunosuppressive capacities, whereas MSCs, in addition to their immunosuppression contribution, regulate myeloid leukemia cell proliferation, apoptosis, and chemotherapy resistance. Moreover, MSCs may promote MDSC expansion, which may mutually contribute to the creation of an immuno-tolerant neoplasm microenvironment. Understanding the implication of MDSCs and MSCs in myeloid malignancies may favor their potential use in immunotherapeutic strategies.

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