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.

scholarly journals Stromal CYR61 Confers Resistance to Mitoxantrone Via Spleen Tyrosine Kinase Activation in Human Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2228-2228
Author(s):  
Xin Long ◽  
Laszlo Perlaky ◽  
Tsz-Kwong Chris Man ◽  
Michele S. Redell
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Chemotherapy Resistance ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a life-threatening bone marrow malignancy with a relapse rate near 50% in children, despite aggressive chemotherapy. 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. The factors that contribute to stroma-induced chemotherapy resistance are largely undetermined in AML. Our goal is to delineate the mechanisms underlying stroma-mediated chemotherapy resistance in human AML cells. We used two human bone marrow stromal cell lines, HS-5 and HS-27A, to study stroma-induced chemotherapy resistance. Both stromal cell lines are equally effective in protecting AML cell lines and primary samples from apoptosis induced by chemotherapy agents, including mitoxantrone, etoposide, and cytarabine. By gene expression profiling using the Affymetrix U133Plus 2 platform, we previously found that CYR61 was among the genes that were commonly upregulated in AML cells by both stromal cell lines. CYR61 is a secreted matricellular protein that is expressed at relatively low levels by AML cells, and at higher levels by stromal cells. CYR61 binds and activates integrins and enhances growth factor signaling in AML cells, and it has been associated with chemoresistance in other malignancies. Our current data provide functional evidence for a role for this protein in stroma-mediated chemoresistance in AML. First, we added anti-CYR61 neutralizing immunoglobulin (Ig), or control IgG, to AML-stromal co-cultures, treated with chemotherapy for 24 hours, and measured apoptosis with Annexin V staining and flow cytometry. In THP-1+HS-27A co-cultures treated with 50 nM mitoxantrone, the apoptosis rate was 33.0 ± 3.7% with anti-CYR61 Ig v. 16.3 ± 4.2% with control IgG; p=0.0015). Next, we knocked down CYR61 in the HS-5 and HS-27A stromal cell lines by lentiviral transduction of two individual shRNA constructs, and confirmed knockdown (KD) at the gene and protein levels for both cell lines. These CYR61-KD stromal cells provided significantly less protection for co-cultured AML cells treated with mitoxantrone, compared to stromal cells transduced with the non-silencing control. For example, the apoptosis rate for THP-1 cells co-cultured with CYR61-KD HS-27A cells was 10.8 ± 0.8%, compared to 6.8 ± 1.1% for THP-1 cells co-cultured with control HS-27A cells (p=0.02). Similar results were obtained with NB-4 AML cells. These results demonstrate that CYR61 contributes to stroma-mediated chemoresistance. CYR61 binds to integrin αvβ3 (Kireeva, et al, J. Biol. Chem., 1998, 273:3090), and this integrin activates spleen tyrosine kinase (Syk) (Miller, et al, Cancer Cell, 2013, 24:45). Using intracellular flow cytometry, we found that activated Syk (pSyk) increased in THP-1 and NB-4 cell lines, and in primary AML patient samples, upon exposure to control HS-27A cells. In primary samples, the mean fluorescence intensity (MFI) for pSyk averaged 11.7 ± 1.3 in co-culture v. 6.6 ± 0.6 for cells cultured alone (p=0.004, n=10). In contrast, pSyk did not significantly increase in AML cells co-cultured with CYR61-KD HS-27A cells (MFI for primary patient samples: 8.6 ± 0.8). This result implicates Syk as a downstream signaling mediator of CYR61. To determine the role of CYR61-induced Syk signaling in chemotherapy resistance, we treated AML-stromal cell co-cultures with 3 uM R406, a potent Syk inhibitor, or DMSO, then added 300 nM mitoxantrone, and measured apoptosis after 24 hours. In AML cells co-cultured with control HS-27A cells, mitoxantrone-induced apoptosis was significantly increased by Syk inhibition (THP-1 cells: 13.7 ± 0.7% with R406 v. 10.0 ± 0.3% with DMSO, p<0.05), consistent with reduced chemoresistance. Notably, R406 did not further increase mitoxantrone-induced apoptosis in AML cells co-cultured with CYR61-KD HS-27A stromal cells (THP-1 cells: 15.7 ± 0.2% with R406 v. 16.9 ± 0.4% with DMSO). Similar results were seen with NB-4 cells, as well. These results support the notion that CYR61 signals through the integrin-Syk pathway to protect AML cells from chemotherapy. Therefore, the CYR61 - integrin - Syk pathway may be a potential therapeutic target for overcoming stroma-induced chemotherapy resistance in AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Acute myeloid leukemia induces protumoral p16INK4a-driven senescence in the bone marrow microenvironment

Blood ◽  
2019 ◽  
Vol 133 (5) ◽  
pp. 446-456 ◽  
Author(s):  
Amina M. Abdul-Aziz ◽  
Yu Sun ◽  
Charlotte Hellmich ◽  
Christopher R. Marlein ◽  
Jayna Mistry ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Stromal Cell ◽  
Cell Senescence ◽  
Senescent Phenotype ◽  
Age Related ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. With increasing age, tissues accumulate senescent cells, characterized by an irreversible arrest of cell proliferation and the secretion of a set of proinflammatory cytokines, chemokines, and growth factors, collectively known as the senescence-associated secretory phenotype (SASP). Here, we report that AML blasts induce a senescent phenotype in the stromal cells within the BM microenvironment and that the BM stromal cell senescence is driven by p16INK4a expression. The p16INK4a-expressing senescent stromal cells then feed back to promote AML blast survival and proliferation via the SASP. Importantly, selective elimination of p16INK4a+ senescent BM stromal cells in vivo improved the survival of mice with leukemia. Next, we find that the leukemia-driven senescent tumor microenvironment is caused by AML-induced NOX2-derived superoxide. Finally, using the p16-3MR mouse model, we show that by targeting NOX2 we reduced BM stromal cell senescence and consequently reduced AML proliferation. Together, these data identify leukemia-generated NOX2-derived superoxide as a driver of protumoral p16INK4a-dependent senescence in BM stromal cells. Our findings reveal the importance of a senescent microenvironment for the pathophysiology of leukemia. These data now open the door to investigate drugs that specifically target the “benign” senescent cells that surround and support AML.

scholarly journals Identification of CHD4 As a Potential Therapeutic Target of Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1648-1648 ◽  
Author(s):  
Yaser Heshmati ◽  
Gözde Turköz ◽  
Aditya Harisankar ◽  
Sten Linnarsson ◽  
Marios Dimitriou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Loss Of Function ◽  
Potential Therapeutic Target ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

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

Oncogene ◽  
2019 ◽  
Vol 39 (6) ◽  
pp. 1198-1212
Author(s):  
Farah Kouzi ◽  
Kazem Zibara ◽  
Jerome Bourgeais ◽  
Frederic Picou ◽  
Nathalie Gallay ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Gap Junctions ◽  
Gap Junction ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Acute Myeloid

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

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

2019 ◽  
Vol 60 (8) ◽  
pp. 2042-2049
Author(s):  
Irina N. Shipounova ◽  
Nataliya A. Petinati ◽  
Alexey E. Bigildeev ◽  
Tamara V. Sorokina ◽  
Larisa A. Kuzmina ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Myeloid Leukemia ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Acute Myeloid

c-Myc plays part in drug resistance mediated by bone marrow stromal cells in acute myeloid leukemia

2015 ◽  
Vol 39 (1) ◽  
pp. 92-99 ◽  
Author(s):  
Bing Xia ◽  
Chen Tian ◽  
Shanqi Guo ◽  
Le Zhang ◽  
Dandan Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Myeloid Leukemia ◽  
Marrow Stromal Cells ◽  
Acute Myeloid

scholarly journals Human acute myeloid leukemia cells bind to bone marrow stroma via a combination of beta-1 and beta-2 integrin mechanisms

Blood ◽  
1993 ◽  
Vol 82 (10) ◽  
pp. 3125-3132 ◽  
Author(s):  
LJ Bendall ◽  
K Kortlepel ◽  
DJ Gottlieb
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Acute Myeloid Leukemia ◽  
Cell Adhesion ◽  
Myeloid Leukemia ◽  
Extracellular Matrix Proteins ◽  
Matrix Proteins ◽  
Leukemic Cells ◽  
Beta 2 ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) cells respond to exogenous stimulation from myeloid growth factors that may be secreted by cells of the bone marrow (BM) stroma and retained by glycosaminoglycans in the extracellular matrix. We have analyzed the capacity of malignant cells from patients with AML to maintain close proximity to sites of growth factor production and retention by binding to BM stromal elements, including fibroblasts and extracellular matrix proteins. Leukemic cells from all cases of AML adhered to BM fibroblast (BMF) monolayers (mean +/- standard error [SE] percentage binding, 30.9% +/- 2.5%; n = 23) and to fibronectin and laminin (mean +/- SE percentage binding, 28.0% +/- 4.1% [n = 11] and 21.5% +/- 2.3% [n = 8], respectively). Binding to bovine and human collagen type 1, vitronectin, hyaluronic acid, and albumin was minimal. Analysis of binding mechanisms indicated that very late antigen-4 (VLA-4) and VLA-5 were responsible for AML cell binding to fibronectin. Binding to laminin could be inhibited by antibody to the alpha chain of VLA-6. In contrast, AML cell adhesion to BMF monolayers was not impaired by blocking antibodies to either beta 1 or beta 2 integrins used alone, although the combination of anti-CD11/CD18 and anti-VLA-4 inhibited binding in more than 50% of cases. When anti- VLA-5 was added in these cases, mean +/- SE inhibition of binding of 45.5% +/- 9.1% (P < .001) was observed. Binding of AML cells to extracellular matrix proteins fibronectin and laminin is predominantly beta 1-integrin-dependent, but AML cell adhesion to BMF relies on the simultaneous involvement of beta 1 and beta 2 integrins as well as other currently unrecognized ligands.

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