scholarly journals Bone Marrow Stromal Cell Mediated Resistance to Mertk Inhibition in Acute Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2819-2819 ◽  
Author(s):  
Katherine A Minson ◽  
Madeline G Huey ◽  
Amanda A Hill ◽  
Irene Perez ◽  
Xiaodong Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Leukemia ◽  
Cell Cultures ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Wild Type ◽  
Equity Ownership ◽  
Induced Apoptosis

Abstract MERTK is a receptor tyrosine kinase of the TAM family (TYRO-3, AXL, MERTK) that is ectopically expressed in 30-50% of newly diagnosed pediatric acute lymphoblastic leukemia (ALL) patient samples and aberrantly expressed in 80-100% of pediatric and adult primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by shRNA or a small molecule inhibitor, MRX-2843, decreased colony-forming potential and induced apoptosis in leukemia cell cultures. Moreover, MERTK inhibition prolonged survival in mouse xenograft models of acute leukemia, but was not curative. In these models, treatment with MRX-2843 effectively reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. Additionally, Gas6, a MERTK ligand, is a poor prognostic factor in AML, mediates increased resistance to cytotoxic chemotherapy in leukemia cells, and is expressed in the bone marrow. To determine the role of Gas6 produced by bone marrow stromal cells in mediating resistance to MERTK inhibition by MRX-2843, acute leukemia cell lines were cultured in the presence of a Gas6-producing fibroblast-like cell line (HS27) or bone marrow derived stromal cells (BMDSCs) from wild type or Gas6 knockout mice and induction of apoptosis and cell death was determined by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with the HS27 cell line significantly reduced cell death in Kasumi-1 AML cell cultures in response to treatment with 300nM MRX-2843 compared to leukemia cells alone (29.4% versus 60.5%, p=0.002). Similar results were observed in Nomo1 AML and 697 pre-B ALL cell cultures. To evaluate whether soluble factors mediated this protective effect, Kasumi-1 cells were cultured in HS27-conditioned medium in the presence or absence of MRX-2843. Interestingly, conditioned medium was not sufficient to provide protection from MRX-2843 induced apoptosis (86.0% vs. 85.5% in unconditioned medium). To more directly assess the role of Gas6, BMDSCs isolated from wild-type and Gas6 knockout C57Bl/6 mice were co-cultured with 697 leukemia cells and sensitivity to MRX-2843 was determined. BMDSCs from wild-type mice protected 697 leukemia cells from MRX-2843 induced cell death much more effectively than BMDSCs from Gas6 knockout mice (4.3% apoptotic and dead cells versus 72.4%, respectively). To investigate biochemical mechanisms of Gas6-mediated protection, Kasumi-1 AML cells were cultured with 200nM MRX-2843 or vehicle in the presence or absence of HS27 cells and expression and activation of MERTK, AXL, TYRO-3, and downstream signaling effectors STAT5, AKT, and ERK1/2 were determined by immunoblot. AXL was not expressed in Kasumi-1 cells with or without co-culture. Treatment with MRX-2843 mediated robust inhibition of MERTK activation indicated by reduced levels of phosphorylated protein in both the presence and absence of stromal cell co-culture. In contrast, activation of TYRO-3 was increased after treatment with MRX-2843 in leukemia cells co-cultured with HS27 stromal cells. Similarly, in the absence of co-culture MRX-2843 inhibited activation of STAT5, AKT, and ERK1/2. However, in the presence of HS27 cells there was robust activation of STAT5 that was sustained even after treatment with MRX-2843. In contrast, MRX-2843 inhibited activation of AKT and ERK1/2 in HS27 co-cultures, although higher doses were required. Together these data support a model whereby Gas6 produced by stromal cells mediates leukemia cell resistance to MERTK inhibition in the bone marrow by inducing activation of TYRO-3, thereby promoting downstream signaling and cell survival despite MERTK inhibition. Thus, combined treatment with MRX-2843 and a TAM ligand sink (eg MERTK-Fc), a TYRO-3 inhibitor, or a bone marrow mobilizing agent may be particularly effective therapeutic strategies. Disclosures Wang: Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Frye:Meryx, Inc: Equity Ownership, Patents & Royalties: MRX-2843. Earp:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. DeRyckere:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK. Graham:Meryx, Inc: Equity Ownership, Patents & Royalties: targeting MERTK.

TGF-β Neutralizing Antibody 1D11 Inhibits LIF-JAK-Stat3 Signaling and Enhances Cytarabine Induced Apoptosis in AML Cells in Bone Marrow Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 926-926
Author(s):  
Yoko Tabe ◽  
Yuexi Shi ◽  
Zhihong Zeng ◽  
Linhua Jin ◽  
Yixin Zhou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Molecular Mechanisms ◽  
Leukemia Cell ◽  
Neutralizing Antibody ◽  
Leukemia Cells ◽  
U937 Cells ◽  
Induced Apoptosis ◽  
Cells Cultured

Abstract Abstract 926 We have previously reported pro-survival effects of TGF-β1 in myelo-monocytic leukemia cells (Xu et al.,Br J Haematol.2008). Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of leukemic BM microenvironment that promote leukemia cell survival and chemoresistance. Our preliminary data indicate that TGF-β neutralizing antibody 1D11 (Genzyme) prevents accumulation of AML cells in a quiescent G0 state under co-culture condition with BM-derived mesenchymal stromal cells (MSC) (Jin et al., ASH abstract 2010). In turn, the chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. In this study, we investigated the anti-leukemic effects and molecular mechanisms of action of TGF-β neutralizing antibody 1D11 under hypoxic conditions. We further investigated the anti-leukemic efficacy of 1D11 combined with CXCR4 antagonist plerixafor in the in vivo leukemia models. AML cells (MV4;11 and U937) were propagated under 1% O2 for at least 14 days to assure their sustained proliferation and survival. Isotype control antibody 13C4 combined with ara-C induced no significant change in apoptosis or cell cycle progression. In MV4;11 cells cultured with 2ng/mL rhTGF-β1, 1D11 (10 μg/mL) induced only minimal apoptosis by itself, yet enhanced low-dose cytarabine (AraC, 0.5 μM) induced apoptosis. This effect was more prominent under hypoxia compared to normoxia (% of subG1 fraction, 21% O2: ara-C, 2.6 ± 0.2%, ara-C + 1D11, 10.8 ± 2.5%, p=0.03; 1% O2: ara-C, 11.3 ± 2.7%, AraC + 1D11, 21.4 ± 0.5%, p=0.001). 1D11 with ara-C abrogated rhTGFβ1-induced accumulation of cells in G0/G1 phase (21% O2; cont, 73.8 ± 4.1, rhTGFβ, 82.2 ± 3.2, rhTGFβ + AraC, 65.4 ± 2.5, rhTGFβ + AraC + 1D11, 50.3 ± 1.9, p=0.001: in 1% O2; cont, 71.8 ± 1.3, rhTGFβ, 85.4 ± 1.4, rhTGFβ + AraC, 79.3 ± 5.1, rhTGFβ + AraC + 1D11, 67.1 ± 4.0, p = 0.03). The anti-leukemic efficacy of 1D11 was next examined in an in vivo leukemia model. 1D11 administered at 5 mg/kg IP every other day in combination with ara-C (50 mg/kg IP weekly) decreased leukemia burden of nude mice injected with Baf3/ITD-luciferase leukemia cells (p=0.002). Administration of small molecule CXCR4 inhibitor plerixafor, which successfully diminished cell migration to CXCL12 in vitro, in combination with 1D11 decreased leukemia burden in vivo (p=0.05), and co-administration of ara-C, plerixafor and 1D11 was most effective (bioluminescence intensity, ×107 photons/sec) control, 1.2 ± 0.2; ara-C, 0.94 ± 0.3; plerixafor + 1D11, 0.56 ± 0.1; plerixafor + 1D11 + ara-C, 0.23 ± 0.09, p=0.003). We next examined the molecular mechanisms responsible for chemosensitization through blockade of TGFβ with 1D11. Treatment with rhTGF-β1 induced upregulation of p21 expression as well as pro-survival phosphorylation of Stat3 in MV4;11 and U937 cells, and these effects were abrogated by 1D11. Knock-down of Stat3 by siRNA increased apoptosis induction in U937 cells cultured in the presence of rhTGFβ1. Notably, 4-fold upregulation of the established TGFβ target, leukemia inhibitory factor (LIF) gene mRNA, was observed after rhTGF-β1 treatment and this was reversed by 1D11. These results indicate that 1D11 inhibits rhTGF-β1-induced autocrine stimulation of pro-survival LIF-JAK-Stat3 signal transduction pathway in AML cells. In summary, blockade of TGF-β by 1D11, and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment. These findings warrant further investigations in human clinical trials. Disclosures: Konopleva: Genzyme: Research Funding.

Image-Based RNA Interference Screening Identifies Microenvironmental Signals Supporting Primary Acute Lymphoblastic Leukemia Cell Survival.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2348-2348
Author(s):  
Beat C Bornhauser ◽  
Jeannette Boutter ◽  
Peter Horvath ◽  
Martin Stanulla ◽  
Jean-Pierre Bourquin
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Rna Interference ◽  
Cell Survival ◽  
Candidate Genes ◽  
Stromal Cells ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Down Regulation ◽  
Stromal Support

Abstract Abstract 2348 Leukemia cells are critically dependent upon interactions with the microenvironment in the bone marrow and at extramedullary sanctuary sites, which is likely to provide a protective mechanism to escape chemotherapy. In vitro, co-culture of primary ALL cells on human bone marrow derived mesenchymal stromal cells (MSCs) provides survival cues allowing long-term cultures. In contrast, primary ALL cells rapidly undergo cell death when cultured without stromal support. We developed an automated microscopy-based approach to identify pro-survival signals by RNA interference of candidate genes in MSCs and subsequent evaluation of leukemia cell survival, enabling us to functionally profile primary leukemia cells. We took advantage of our leukemia xenograft system as a renewable source of well characterized samples derived from cases with very high risk (VHR) ALL, which were selected based on clinical resistance to chemotherapy. Based on gene expression and cell surface proteomic data that we had obtained from both cellular compartments, we generated a customized siRNA library for 110 candidate genes with a potential function in stromal support. Primary ALL cells were seeded on reversely transfected MSC cells and ALL cell viability was assessed with a fluorescent vital dye after 6 days. Image analysis and machine learning algorithms were developed for quantification of surviving ALL cells on top of MSC. Evaluating three VHR-ALL cases we observed a strong decrease of viability when interfering with the expression of 14 candidate genes in 2 out of 3 patients. Interestingly, in validation studies with 7 additional cases, the pattern of dependence on the genes tested were confirmed to be only partially overlapping between patients, indicating the existence of functional differences in distinct subsets. As a proof of concept, we could show that down-regulation of VCAM1 or the VEGF pathway in MSCs decreased ALL survival supporting earlier studies. Concomitantly, inhibitors of VEGF signalling recapitulated ALL cell viability decrease for patients that showed to be dependent on VEGFC expression in MSCs. One of the strongest effects on ALL survival was achieved by down-regulation of the membrane protein Basigin (CD147). Specifically, 13 out of 17 ALL cases were affected by the modulation of Basigin on MSC level. Basigin has been implicated in cell signalling, in interactions with extracellular matrix and serves as chaperone to different membrane carrier proteins. Among putative Basigin interactors we identified the heteromeric amino acid transporter SLC3A2 (CD98) to be required for ALL survival in the same set of ALL cases. The down-regulation of Basigin, SLC3A2 or both together in MSC cells induces an increase in ROS in ALL cells resulting in apoptotic cell death, which indicates that Basigin/SLC3A2 function is important for the integrity of ALL cell metabolism in this model of the leukemia niche. We are now investigating which metabolites are implicated in the mechanism of action. Taken together, we have established a robust platform for systematic functional investigation of primary ALL survival in a 2-D model of the microenvironment and obtained evidence for patient-specific dependence of leukemia cell survival on stromal support. Critical interactions between ALL cells and bone marrow stromal cells can be identified with this approach, which will be useful for unbiased higher throughput screening and combinatorial testing. This platform will also be of great interest for preclinical drug profiling on clinically relevant patients samples in the context of protective bone marrow signals. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256708
Author(s):  
Christopher von Beek ◽  
Linnéa Alriksson ◽  
Josefine Palle ◽  
Ann-Marie Gustafson ◽  
Mirjana Grujic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Tumor Formation ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Bone Marrow Biopsies

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

Vitamin K2 Induces Autophagy and Apoptosis Simultaneously in Leukemia Cells and Bcl-2 Expression Level Determines the Phenotype of Their Cell Death.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3478-3478
Author(s):  
Keisuke Miyazawa ◽  
Tomohisa Yokoyama ◽  
Munekazu Naito ◽  
Juri Toyotake ◽  
Testuzo Tauchi ◽  
...  
Keyword(s):  
Cell Death ◽  
Leukemia Cell ◽  
Vitamin K2 ◽  
Expression Level ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Potent Inducer ◽  
Acridine Orange Staining ◽  
Induced Apoptosis

Abstract Vitamin K2 (menaquinone-2: VK2) is now known to be a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of human bcl-2 gene into HL-60 leukemia cell line, show 5-fold greater expression of Bcl-2 protein compared with that in HL-60neo cells, a control clone transfected with vector alone. Although HL-60neo cells are induced apoptosis in response to VK2, HL-60bcl-2 cells are resistant against apoptosis induction but still show cell growth inhibition along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed autophagosomes and autolysosomes formation in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVO) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported enhanced autophagy induction in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, autophagosome formation was rather prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells were protected from rapid apoptotic death by higher expression level of Bcl-2.

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

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

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

HDAC and LSD1 Inhibitors Synergize to Induce Cell Death in Acute Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1427-1427 ◽  
Author(s):  
Lorimar Ramirez ◽  
Melissa Singh ◽  
Joya Chandra
Keyword(s):  
Cell Death ◽  
Monoamine Oxidase ◽  
Acute Leukemia ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Hdac Inhibitors ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines

Abstract Abstract 1427 Histone deacetylase inhibitors (HDACi) are a class of emerging epigenetic therapies which are being used to treat cancer. Two HDACi (vorinostat and romidepsin) are FDA approved for cutaneous T-cell lymphoma. HDACi have been employed in clinical trials for acute leukemia, but single agent activity has been limited. Improved efficacy is observed when combined with other anticancer agents. In the current study we addressed acute leukemia models using vorinostat, a pan-HDACi that inhibits HDAC class I, II, and IV and entinostat, a newer HDACi that inhibits HDAC class I more specifically. These HDACi were combined with inhibition of another histone modifying enzyme: lysine specific demethylase 1 (LSD1). The LSD1 gene encodes a favin-dependent monoamine oxidase, which demethylates mono- and di-methylated lysines, specifically lysines 4 and 9 on histone 3 (H3K4 and H3K9), thus it is also involved in gene regulation through post-translational histone modification. LSD1 overexpression has been linked to human carcinogenesis in bladder carcinomas, lung cancer, and poorly differentiated neuroblastoma. However, it has not been studied in hematologic malignancies. Because LSD1 is structurally similar to monoamine oxidase (MAO), it has been shown that nonselective MAO inhibitors also inhibit LSD1. Here we employed tranylcypromine, a monoamine oxidase inhibitor (MAOi), as an irreversible LSD1 inhibitor. Recently published work from our laboratory has shown synergistic effects of combined HDAC and LSD1 inhibition in brain tumors (glioblastoma multiforme). Similar results have been published in breast cancer cells, but no work has been done in hematological malignancies. The objective of this study was to investigate the possible synergy of HDAC and LSD1 inhibitors in acute leukemia cells. LSD1 protein expression in several leukemia cells lines was analyzed by Western blot analysis. LSD1 was expressed in all leukemia cell lines tested, which included T-cell ALL (Jurkat, Sub-T1, MOLT4), B-cell ALL (JM-1,697), and Philadelphia chromosome positive ALL (Z33, Z119, Z181). To determine whether synergy exists between HDACi and LSD1 inhibitors, Jurkat cells were exposed to different concentrations of tranylcypromine and vorinostat or entinostat. After 24 hr, DNA fragmentation was assessed by propidium iodide (PI) staining followed by flow cytometric analysis. A combination index (CI) less than 1.0 is representative of synergism as measured by Calcusyn software. Results showed a synergistic effect on DNA fragmentation when combining the 2.5 μM dose of vorinostat with a range of tranylcypromine doses (1 mM CI= 0.78, 1.5 mM CI= 0.49, and 2 mM CI= 0.39). The same effect was observed with the combination of 2.5 μM entinostat with 2 mM tranylcypromine (CI=0.52). Viability studies performed with the same drug concentrations in conbination also showed statistically significant cell death. Additional acute leukemia cell lines, 697 and MOLT-4, also demonstrated significantly increased cell death with the combination relative to treatment with either agent alone. Since these agents inhibit histone deacetylation and lysine demethylation, we tested whether these histone modifications were promoted by combination treatment. Jurkat cell lysates were generated by acid extraction of histones and Western blot analysis was conducted. We demonstrated that in fact histone acetylation was increased with combination treatment, indicating that these modifications coordinately regulate each other in acute leukemia cells. A molecular target for LSD1 is p53, a tumor suppressor protein whose activity is regulated by lysine methylation and demethylation. Western blot analysis showed that p53 is downregulated in leukemia cells after exposure to the combination of HDAC and LSD1 inhibitors. Future studies will address if p53 downregulation is a trigger for the synergistic cell death. Taken together, our data shows the efficacy of combining LSD1 inhibitors with HDAC inhibitors in multiple acute leukemia models. Since tranylcypromine is also a FDA-approved agent, these results urge the design of a feasible and effective clinical trial combining LSD1 and HDAC inhibitors for acute leukemia. Disclosures: No relevant conflicts of interest to declare.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 893-893
Author(s):  
Po Yee Mak ◽  
Duncan H Mak ◽  
Yuexi Shi ◽  
Vivian Ruvolo ◽  
Rodrigo Jacamo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Apoptosis Resistance ◽  
Control Mscs ◽  
Extrinsic Apoptosis ◽  
Induced Apoptosis ◽  
And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

Uncovering Cardioprotective Strategies For Anthracycline Therapy By Analysis Of Redox and Apoptotic Effects

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1293-1293
Author(s):  
Daniela E. Egas Bejar ◽  
Joy M. Fulbright ◽  
Fernando F. Corrales-Medina ◽  
Mary E. Irwin ◽  
Blake Johnson ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Vitamin E ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
Soluble Form ◽  
Leukemia Cell Lines

Abstract Anthracyclines are among the most powerful drugs used for the treatment of leukemia, however their use has been associated with cardiotoxicity. Reactive oxygen species (ROS) are generated in both cancer and normal cells after anthracycline exposure and have been implicated in both early and late onset cardiotoxicity. Counteracting this ROS generation are intracellular antioxidants such as the ubiquitous antioxidant glutathione (GSH), levels of which are depleted upon anthracycline exposure. Basal expression of GSH pathway components and other antioxidants vary greatly between different cell types. Due to this differential expression of cellular antioxidants in cardiomyocytes versus leukemia cells, we posit that anthracyclines exert distinct effects on oxidative stress and consequent apoptosis induction in leukemia cells and nontransformed hematopoietic cells (PBMC) relative to cardiomyocytes. As a result, we expect potentially varied mechanisms of cell death induction in these cell lines after anthracycline treatment. To test this hypothesis, the acute leukemia cell lines Jurkat and ML-1 and the cardiomyocyte line H9C2 were used. Dose responses with the anthracyclines, doxorubicin and daunorubicin, were carried out and trypan blue exclusion and propidium iodide staining followed by flow cytometry were used to assess viability and DNA fragmentation respectively. Cardiomyocytes had a 25-150 fold higher IC50 value than the acute leukemia cell lines, indicating selectivity. To assess whether apoptosis was induced by anthracyclines, caspase 3 activity was measured and found to be increased at 24 hours in Jurkat cells which preceded decreases in viability, supporting an apoptotic mechanism of cell death. GSH levels also decreased markedly after 24 hours of treatment with anthracyclines in this cell line, however, a pan-caspase inhibitor did not block GSH depletion, indicating that these events occur independent of each other. To evaluate whether antioxidants conferred protection against loss of viability in all cell types, cells were pretreated for at least 30 minutes with antioxidants and then treated with doxorubicin and daunorubicin for 24 hours. Antioxidants used were N-acetylcysteine (NAC, a GSH precursor and amino acid source), GSH ethyl ester (cell permeable form of GSH), tiron (free radical scavenger) and trolox (a water soluble form of vitamin E). GSH ethylester did not prevent cytotoxicity of anthracyclines in acute leukemia lines or cardiomyocytes. Therefore boosting GSH levels in leukemia cells does not reverse cytotoxicity. Trolox, however, did block anthracycline induced cell death in ML-1 cells, suggesting that vitamin E supplementation would counteract leukemia cell specific effects of anthracyclines on AML cells. Tiron protected PBMC from doxorubicin cytotoxicity but did not protect leukemia cells or cardiomyocytes, hinting at a protective strategy for normal non-leukemia blood cells. Interestingly, NAC did not interfere with the cytotoxic effects of anthracyclines on acute leukemia cells or PBMC, but protected H9C2 cells from daunorubicin cytotoxicity. Taken together, these data reveal differential protective effects of antioxidants in cardiomyocytes and PBMCs relative to ALL and AML cells. Our work indicates that NAC can protect cardiomyocytes without interfering with anthracycline cytotoxicity in acute leukemia cells. In humans, one randomized control trial tested the addition of NAC to doxorubicin therapy, detecting no evidence of cardioprotective activity by chronic administration of NAC. However, the schedule used for administration of NAC in that study may not have been optimal, and biomarkers for oxidative stress reduction by NAC were not incorporated into the trial. Previously, other antioxidants have been used with very limited clinical success and possible contributing factors include inadequate sample size, choice of agent, dose used, duration of intervention and the lack of biomarker endpoints. Designing a cardioprotective and antioxidant strategy with attention to these factors may prove to be efficacious in protecting cardiac cells without interfering with the antitumoral effect of anthracyclines. To this end, our data suggests that trolox and vitamin E analogues should not be used in acute leukemia as they may interfere with the cytotoxic action of anthracyclines but NAC or cysteine may be used as cardioprotectants. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone Marrow Stromal Cells Modulate Mouse ENT1 Activity and Protect Leukemia Cells from Cytarabine Induced Apoptosis

PLoS ONE ◽  
2012 ◽  
Vol 7 (5) ◽  
pp. e37203 ◽  
Author(s):  
Patricia Macanas-Pirard ◽  
Andrea Leisewitz ◽  
Richard Broekhuizen ◽  
Kelly Cautivo ◽  
Francisco M. Barriga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemia Cells ◽  
Induced Apoptosis

scholarly journals MBNL1 As a New Therapeutic Target in MLL-Fusion Gene Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 462-462 ◽  
Author(s):  
Svetlana S Itskovich ◽  
Jason Clark ◽  
James C. Mulloy ◽  
Matthew D Disney ◽  
Ashish R Kumar
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Fusion Gene ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Transformed Cells ◽  
Wild Type

Abstract Translocations of the Mixed Lineage Leukemia (MLL) gene located on chromosome 11 are commonly found in infants with AML or ALL and in secondary leukemia at all ages. A majority of patients with these translocations have a poor prognosis. Gene expression profiling studies demonstrate that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is muscleblind-like 1 (MBNL1). Further, MBNL1 was also identified as a direct transcriptional target of MLL-fusion proteins. An RNA-binding protein, MBNL1 is known to be a key factor in the pathophysiology of Myotonic Dystrophy Type I (DM), where sequestration of MBNL1 leads to splicing defects in muscle and neuronal cells. However, the role of MBNL1 in hematopoiesis and leukemogenesis is unknown. To determine the role of MBNL1 in normal hematopoiesis we studied MBNL1-/- mice. Compared to littermate controls, MBNL1-/- mice showed no differences in peripheral blood counts or bone marrow cellularity. When challenged with 5-FU, both MBNL1-/- and wild type mice displayed similar kinetics of peripheral blood cytopenia and recovery. Next we examined the role of MBNL1 in hematopoietic stem cell function using a competitive transplantation assay. Lethally irradiated mice were transplanted with a 1:1 mix of CD45.1 and CD45.2 bone marrow, with the latter being wild-type or MBNL1-/-. Flow cytometry analysis of peripheral blood at 4 weeks post-transplant showed donor chimerism being 53±4.14% in recipients of wild type marrow and 25±5.41 % in the MBNL1-/- recipients. Successive analyses every 4 weeks showed the chimerism to be stable over the next 16 weeks. To determine the role of MBNL1 in leukemia, we transformed MBNL1-/- or wild type bone marrow cells with various oncogenes delivered via retroviral transduction and compared them in methylcellulose colony replating assays. Absence of MBNL1 significantly reduced colony formation in MLL-AF9 and E2A-HLF transformed cells by 59.5% (± 27.1) and 50.7% (± 23) respectively, compared to controls. To assess the role of MBNL1 in leukemia in vivo, we transplanted MLL-AF9-transformed wild type or MBNL1-/- cells into irradiated mice. All recipients injected with wild-type MLL-AF9-transformed cells succumbed to leukemia with a median time of 106 days. In contrast, the majority of recipients of MBNL1-/- cells survived leukemia-free for at least 140 days post-transplantation (p=0.0017, log rank test). We next assessed the role of MBNL1 in human leukemia cells. Lentiviral-shRNA knockdown of MBNL1 in leukemia cell lines (MV4;11, THP-1) significantly inhibited cell growth, both in liquid culture and methylcellulose colony forming assays. To determine the requirement of MBNL1 for leukemia propagation in vivo, we used cord blood-derived leukemia cells bearing the MLL-AF9 fusion gene and mutant NRAS (MA9NRAS). MA9NRAS cells transduced with MBNL1-specific or control (non-targeting, NT) shRNA were transplanted into immunodeficient mice. Six weeks after transplant, bone marrow aspirates showed persistence of lentiviral-transduced cells in 85% of the NT-group. On the other hand, MBNL1-shRNA transduced cells were not detected in any of the recipient mice. These results suggest that MBNL1 is essential for leukemia cell propagation in vivo. Finally, we tested therapeutic targeting of MBNL1 in MLL-fusion gene leukemia. A lead inhibitor that prevents binding of MBNL1 to its targets was recently identified. Treatment of MA9NRAS cells with the inhibitor for 48 hours led to significant apoptosis whereas normal cord blood CD34+ cells were relatively less sensitive. Blockade of MBNL1 in leukemia cells either by shRNA-knockdown or by the inhibitor showed identical changes in splicing patterns of known MBNL1 target genes. Collectively, our data suggest that MBNL1 is required for the initiation and propagation of MLL-fusion gene leukemia while it appears relatively dispensable for normal hematopoiesis. Further, we have identified a promising lead inhibitor that could be developed for novel treatments for therapy-resistant leukemias. Disclosures No relevant conflicts of interest to declare.

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