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.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2587-2587
Author(s):  
Yang Yang ◽  
Baohua Sun ◽  
Saradhi Mallampati ◽  
Zhen Cai ◽  
Xiaoping Sun
Keyword(s):  
Bone Marrow ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Apoptosis ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells ◽  
New Therapies ◽  
Induced Apoptosis

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

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.

Stroma cells and Monocytes make Multiple Myeloma Cells Active In Bone Marrow Microenviroment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5333-5333
Author(s):  
Hiroshi Ikeda ◽  
Tadao Ishida ◽  
Toshiaki Hayashi ◽  
Yuka Aoki ◽  
Yasuhisa Shinomura
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Cell Contact ◽  
Drug Induced ◽  
Myeloma Cells

Abstract The Bone marrow (BM) microenvironment plays crucial role in pathogenesis of multiple myeloma　(MM). Paracrine secretion of cytokines in BM stromal cells promotes multiple myeloma cell proliferation and protects against drug-induced cytotoxicity. In current study, monocytes, component of BM cells, can directly promote mesenchymal stem cells osteogenic differentiation through cell contact interactions. Down-regulation of inhibitors such as DKK1 drives the differentiation of mesechymal stem cells into osteoblasts. In this study, we examined the role of monocytes as a potential niche component that supports myeloma cells. We investigated the proliferation of MM cell lines cultured alone or co-cultured with BM stromal cells, monocytes, or a combination of BM stromal cells and monocytes. Consistently, we observed increased proliferation of MM cell lines in the presence of either BM stromal cells or monocytes compared to cell line-only control. Furthermore, the co-culture of BM stromal cells plus monocytes induced the greatest degree of proliferation of myeloma cells. In addition to increased proliferation, BMSCs and monocytes decreased the rate of apoptosis of myeloma cells. Our results therefore suggest that highlights the role of monocyte as an important component of the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Stromal Cells Enhance DNA Damage Signaling Independent Of Stat3 Activation In Pediatric AML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2548-2548
Author(s):  
Xin Long ◽  
Marcos J. Ruiz ◽  
Michele S. Redell
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Cell Lines ◽  
Stromal Cells ◽  
Strong Correlation ◽  
Stromal Cell ◽  
Chemotherapy Resistance ◽  
Soluble Factors ◽  
Dna Damage Signaling ◽  
Cells Cultured

Abstract Despite aggressive treatments, death from chemoresistant disease still occurs for almost half of children with AML. It is clear that the bone marrow microenvironment protects a subset of cells from chemotherapy, but the mechanisms of resistance remain unknown. We recently reported that patients whose AML blasts activated Stat3 in response to two bone marrow-derived ligands, G-CSF and IL-6, had a significantly superior survival rate, compared to patients whose blasts failed to activate Stat3 (Redell, et al, 2013, Blood121:1083). In this followup study of a subset of these patients, we further investigated the basis for the failure of the Stat3 response, as well as mechanisms of environment-mediated chemotherapy resistance. Our hypotheses were A) Stat3 responsiveness to G-CSF/IL-6 reflects the cell’s general ability to respond to ligand stimulation, rather than a specific defect in a Stat3 pathway; and B) Stat3 responsiveness correlates with other functional responses to stromal exposure, including survival, proliferation, and chemotherapy resistance. Second samples from 27 patients were studied by FACS analysis of intracellular markers. To study stroma-induced changes, AML cells were co-cultured on stromal cells overnight, or briefly stimulated with stroma-conditioned medium (CM). We used HS5 cells, which secrete high levels of many soluble factors, and HS27A cells, which secrete very few factors. The stromal cell lines express mOrange for exclusion by FACS. First, primary AML cells were stimulated with G-CSF (10 ng/ml) or IL-6 + soluble IL-6R (5 + 10 ng/ml) for 15 min, then pY-Stat3 (PE) and pERK1/2 (AlexaFluor488) were measured by FACS. Data were acquired on the LSRII (BD) and analyzed with FCSExpress4 (DeNovo). Ligand responses were expressed as the fold change in mean fluorescence intensity over unstimulated cells (ΔMFI). We found a strong correlation between G-CSF-induced pY-Stat3 ΔMFI and pERK1/2 ΔMFI (R=0.858, p<0.001), suggesting that failure to activate Stat3 in unresponsive samples was not due to a Stat3-specific block. As expected, HS5 CM strongly activated Stat3 in samples that also responded to G-CSF and IL-6, while HS27A CM did not activate Stat3 in any samples. We compared responses to G-CSF v. HS5 CM, and found a strong correlation for pY-Stat3 ΔMFI (R=0.886, p<0.001) and for pERK1/2 ΔMFI (R=0.907, p<0.001). Samples that failed to respond to G-CSF also failed to respond to a combination of multiple soluble factors, supporting the idea that signaling function or dysfunction is a generalized property of AML cells. To further investigate functional changes induced in AML cells by stromal cells, we co-cultured blasts from 17 samples overnight with HS5 or HS27A cells. We labeled cleaved PARP (AlexaFluor647) to measure viable cells (%cPARP-) in each condition. Within the viable AML cell population, we measured the proliferative fraction (Ki67-FITC+ or Ki67-AlexaFluor700+) and the fraction with active DNA damage signaling (γH2AX-AlexaFluor488+). We found a small increase in the %viable for co-cultured cells v. cultured alone (p<0.05 for HS27A v. alone; Wilcoxon Signed Rank Test) and in the %Ki67+ (p<0.005 for HS27A v. alone). Moreover, increases were seen in γH2AX, with 5.6 ±6%+ in cells cultured alone, v. 7.9 ±6% in HS5 co-cultures (p<0.05) and v. 11.5 ±9% in HS27A co-cultures (p<0.001). For 10 samples, cell number was sufficient for additional overnight cultures with etoposide (up to 10 mM). Both stromal cell lines enhanced DNA damage signaling, reflected by an increase in γH2AX from 7 ±10% in etoposide-treated cells cultured alone, to 18 ±19% in HS5 co-cultures (p<0.05) and 19 ±16% in HS27A co-cultures (p<0.005). To test whether HS5 CM-induced pY-Stat3 or pERK1/2 were related to these parameters, we examined bivariate correlations. We found no correlation in most cases, but an inverse relationship between both ΔMFIs and %viable after etoposide (pY-Stat3: R=-0.733, p<0.05 and pERK1/2: R=-0.683, p<0.05). In other words, samples characterized by signaling failure tended to have better survival after etoposide treatment. This result is consistent with our prior study showing that patients whose blasts failed to activate Stat3 had poorer outcome than those whose blasts activated Stat3 normally. In summary, we found that environment-induced Stat3 signaling did not promote chemoresistance, and that stromal cells may instead confer chemoresistance by enhancing DNA damage signaling. Disclosures: No relevant conflicts of interest to declare.

Mutual Education Between Hematopoietic Cells and Bone Marrow Stromal Cells Through Direct Cell-to-Cell Contact: Factors That Determine the Growth of Bone Marrow Stroma-Dependent Leukemic (HB-1) Cells

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 834-841 ◽  
Author(s):  
Huijie Jiang ◽  
Kenkichi Sugimoto ◽  
Hitoshi Sawada ◽  
Emi Takashita ◽  
Maki Tohma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Direct Contact ◽  
Leukemic Cells ◽  
Cell Contact ◽  
Type I ◽  
Dependent Cell ◽  
Direct Cell ◽  
Interleukin 1Α ◽  
American Society

Abstract A stroma-dependent cell line (HB-1) was established from myelogenous leukemic cells of CBA/N mouse. Characterization of the cells showed that HB-1 proliferated on hematopoietic supportive stromal cells (MS-10), but did not survive or proliferate on hematopoietic nonsupportive cells (MS-K). Direct contact between HB-1 and MS-10 appears to be necessary for HB-1 to proliferate on MS-10. We found that interleukin-1α (IL-1α) produced by MS-10 plays a major role in the survival and proliferation of HB-1. IL-11 did not support the proliferation of HB-1 cells by itself, but enhanced the proliferation of HB-1 cells in the presence of IL-1α. The expression of IL-1α and IL-11 was induced in MS-10 by the direct contact with HB-1 cells, and the expression of IL-1 receptor type I (IL-1RI) and interleukin-11 receptor (IL-11R) was induced in HB-1 cells by the attachment of the cells to MS-10. These findings show the existence of two-way interactions between HB-1 and MS-10. © 1998 by The American Society of Hematology.

scholarly journals B-Cell Precursor Acute Lymphoblastic Leukemia Instructs Mesenchymal Stromal Cells to Alter Interferon-Related Gene Expression and Cyto/Chemokine Secretion

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-4
Author(s):  
Mandy W.E. Smeets ◽  
Femke Stalpers ◽  
Myrthe M.P. Vermeeren ◽  
Alex Q. Hoogkamer ◽  
Stefan Nierkens ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Stromal Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Sequencing Data ◽  
Cell Precursor ◽  
And Migration ◽  
Pro Inflammatory Cytokines

Introduction: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells that are present in the bone marrow microenvironment are able to hijack the normal hematopoietic stem cell niches to create a leukemic niche. The importance of this microenvironment for leukemic cells is demonstrated by the protection that the niche provides against chemotherapeutic agents. Patient-derived mesenchymal stromal cells (MSCs) mimic this protective effect in vitro. Unraveling the mechanism of protection is important to provide potential novel options for therapeutic intervention. Therefore, our research is focused on the interaction between BCP-ALL cells and MSCs. We aimed to determine gene expression changes in BCP-ALL cells and MSCs after co-culture compared to mono-culture, and to investigate which cyto- and chemokines are differentially secreted upon contact between BCP-ALL cells and MSCs. Methods: We performed co-cultures of primary MSCs and BCP-ALL cells, and mono-cultures of MSCs or BCP-ALL cells for 40 hours to determine gene expression changes. Viable cells were sorted by FACS and RNA was isolated. Total-RNA sequencing data (Illumina) were analyzed using R. Supernatant was saved to determine cyto/chemokine profiles by Luminex technology, and to investigate the effect of these cyto/chemokines on the survival and migration of the leukemic cells. Moreover, migration experiments using transwells (3.0µm pore size) were performed to determine the level of BCP-ALL migration towards cyto/chemokines of interest. Results: RNA sequencing data from 15 independent co-culture experiments revealed that interferon (IFN)-related genes, such as IFI6, MX1, IFI27, and OAS1, were 2.5 to 3.1-fold upregulated in the MSCs after co-culture with BCP-ALL compared to MSC mono-culture. The type of upregulated pro-inflammatory genes and amount of upregulation varied between BCP-ALL patients. However, the observed changes were always similar when an ALL case was co-cultured with different MSC samples. This suggests that the observed changes are induced by the leukemic cells and that leukemic cells manipulate MSCs. Survival benefit (0.3 - 37.9%) was observed in BCP-ALL cells after co-culture with MSCs compared to BCP-ALL mono-culture. Moreover, pro-inflammatory cytokines, and several migration-related chemokines such as CCL2, CXCL8, and CXCL10/IP-10 were upregulated in 5 out of 15 co-cultures compared to the sum of the separate mono-cultures of BCP-ALL and MSCs. A gradient of CXCL10/IP-10 in transwell experiments showed that this chemokine did not enhance the migration of primary BCP-ALL cells, suggesting that the ALL-induced secretion of this chemokine serves a different role in BCP-ALL. The role of CXCL10/IP-10 and other cyto/chemokines in immune regulation at the time of overt leukemia is part of ongoing studies. Conclusion: Our data show that IFN-related genes, pro-inflammatory cytokines and migration-related chemokines become upregulated in bone marrow stromal cells upon exposure to BCP-ALL. These induced changes may be important for BCP-ALL cell survival, affecting the mobility of other immune cells, and/or ensure that leukemic cells remain in close contact with MSCs. We postulate that interference with these affected genes and cyto/chemokines may disrupt the direct contact between leukemic cells and their niche, and may provide an alternative way to eliminate leukemic cells more efficaciously. Functional studies addressing this concept are currently being executed and the results will be presented during the meeting. Disclosures No relevant conflicts of interest to declare.

Survivin in Multiple Myeloma: Prognostic and Therapeutic Implications

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 137-137
Author(s):  
Verena Wagner ◽  
Dirk Hose ◽  
Anja Seckinger ◽  
Ludmila Weitz ◽  
Tobias Meißner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stress Response ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Survivin Expression ◽  
Marrow Stromal Cells ◽  
Er Stress Response

Abstract Abstract 137 The IAP (inhibitor-of-apoptosis) family member, survivin, is one of the most significantly over-expressed genes in malignant cells. Survivin has been reported to inhibit apoptosis and regulate mitosis as well as cytokinesis. We therefore first determined the expression of survivin in CD138-purified multiple myeloma (MM) cells from previously untreated patients at our centers' trial group (TG) (n=246) and the UAMS Arkansas (n=345) validation group (VG). Using the PANP algorithm, survivin is aberrantly expressed in 27% (TG) of MM cell samples. It is not expressed in normal bone marrow plasma cell samples (n=7) while expression increases significantly from MM stage I-III (P<.001). Survivin expression correlates with proliferation as assessed by gene expression- (r=.8, P<.001) or propidium iodide (r=.7, P<.001). Presence of survivin expression correlates with inferior event-free and overall survival in patients undergoing high-dose chemotherapy in the TG (22.6 vs. 35.4 months, P<.001, 52.9 vs. n.r., P=.002) as well as in the VG (12.3 vs. 54.1 months, P<.001, and 17.4 vs. n.r., respectively). These results support the further evaluation of survivin as a therapeutic target in MM. We next assessed the effects of siRNA-mediated knock-down of survivin in vitro. Suppression of survivin by siRNA induced cell cycle arrest and apoptosis in MM cell lines. The small molecule suppressant of survivin, YM155, is currently in clinical development for the treatment of solid tumors. Here, we investigated YM155 for its anti-MM activity. YM155 abrogated proliferation and induced apoptosis in a panel of 10 human MM cell lines and MM cells isolated from multidrug-resistant patients at an IC50 of 4–50nM while the IC50 was not reached in primary bone marrow stromal cells up to 500nM. YM155 was also able to overcome the protective effect of IL-6, IGF-1 and the presence of bone marrow stromal cells, respectively. The induction of apoptosis by YM155 closely correlated with down-regulation of intracellular survivin protein expression within 24 to 36h of treatment with 50–100nM of YM155. However, inhibition of cell proliferation is already detectable at 12h at 5–10nM, suggesting two different dose- and time-dependent mechanisms of action. We therefore performed gene expression and protein profiling on YM155-treated MM cells. Strikingly, these data revealed early up-regulation of the ER stress response (PERK, phospho-eIF2a, ATF4, ATF3) followed by increased CHOP expression and a profound abrogation of proliferation. This appeared to be independent of cellular survivin levels, indicating that the early proliferation arrest at very low nanomolar concentrations is mediated primarily by the ER stress response. Moreover, gene signatures regulated by the IL-6/STAT pathway (CCND1, BCL2L1, MCL1, BIRC5A) were markedly altered upon YM155 treatment. Importantly, IL-6 profoundly sensitized IL-6 responsive MM cell lines to treatment with YM155. We therefore hypothesized that YM155 might abrogate upstream regulatory signaling pathways of survivin expression. Indeed, YM155 abrogated constitutive as well as IL-6 induced phosphorylation of STAT3, an important transcription factor for survivin expression in MM cells. In contrast, phosphorylation of ERK1/2 and AKT remained unchanged. Dephosphorylation of STAT3 closely correlated with the loss of intracellular survivin. In conclusion, we have demonstrated the prognostic significance of survivin expression and a potential therapeutic role for the small molecule suppressant of survivin YM155 in MM. Disclosures: No relevant conflicts of interest to declare.

Mutual Education Between Hematopoietic Cells and Bone Marrow Stromal Cells Through Direct Cell-to-Cell Contact: Factors That Determine the Growth of Bone Marrow Stroma-Dependent Leukemic (HB-1) Cells

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 834-841 ◽  
Author(s):  
Huijie Jiang ◽  
Kenkichi Sugimoto ◽  
Hitoshi Sawada ◽  
Emi Takashita ◽  
Maki Tohma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Direct Contact ◽  
Leukemic Cells ◽  
Cell Contact ◽  
Type I ◽  
Dependent Cell ◽  
Direct Cell ◽  
Interleukin 1Α ◽  
American Society

A stroma-dependent cell line (HB-1) was established from myelogenous leukemic cells of CBA/N mouse. Characterization of the cells showed that HB-1 proliferated on hematopoietic supportive stromal cells (MS-10), but did not survive or proliferate on hematopoietic nonsupportive cells (MS-K). Direct contact between HB-1 and MS-10 appears to be necessary for HB-1 to proliferate on MS-10. We found that interleukin-1α (IL-1α) produced by MS-10 plays a major role in the survival and proliferation of HB-1. IL-11 did not support the proliferation of HB-1 cells by itself, but enhanced the proliferation of HB-1 cells in the presence of IL-1α. The expression of IL-1α and IL-11 was induced in MS-10 by the direct contact with HB-1 cells, and the expression of IL-1 receptor type I (IL-1RI) and interleukin-11 receptor (IL-11R) was induced in HB-1 cells by the attachment of the cells to MS-10. These findings show the existence of two-way interactions between HB-1 and MS-10. © 1998 by The American Society of Hematology.

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.

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