scholarly journals Ceritinib Enhances the Efficacy of Substrate Chemotherapeutic Agent in Human ABCB1-Overexpressing Leukemia Cells In Vitro, In Vivo and Ex-Vivo

2018 ◽  
Vol 46 (6) ◽  
pp. 2487-2499 ◽  
Author(s):  
Li Yang ◽  
Manjun Li ◽  
Fang Wang ◽  
Chen Zhen ◽  
Min Luo ◽  
...  
Keyword(s):  
Chemotherapeutic Agent ◽  
Ex Vivo ◽  
Leukemia Cell ◽  
Surface Protein ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Intracellular Accumulation ◽  
In Vivo Models

Background/Aims: Multidrug resistance (MDR) triggered by ATP binding cassette (ABC) transporters, such as ABCB1, ABCC1, and ABCG2, is a key obstacle for successful cancer chemotherapy. There is currently no FDA-approved MDR modulator that can be used in clinic. Ceritinib, a selective ALK inhibitor, has been approved as the second-line treatment for ALK-positive non-small cell lung cancer. Here, we examined the role of ceritinib in leukemia associated MDR in therapy. Methods: The cell proliferation was detected by MTT assay. The flow cytometry was used to detect the expression of cell surface protein and to detect the accumulation and efflux of rhodamine 123 (Rh123) or doxorubicin (Dox) in cells. The RT-PCR and Western blot were performed to detect the gene expression and protein expression levels, respectively. Results: We found that ceritinib enhanced the efficacy of substrate chemotherapeutic agent in ABCB1-overexpressing K562/adr leukemia cells both in vitro and in vivo models, but neither in sensitive parental K562 leukemia cells nor in ABCC1-overexpressing HL-60/adr leukemia cells. Mechanistically, ceritinib significantly increased the intracellular accumulation of Rh123 or Dox but did neither alter ABCB1 expressions at both protein and mRNA levels nor block the phosphorylations of AKT and ERK1/2 at the concentration of MDR reversal. Importantly, ceritinib also increased the intracellular accumulation of Dox and enhanced the efficacy of Dox in primary leukemia cells in ex-vivo. Conclusion: Our results suggested that ceritinib enhanced the efficacy of substrate chemotherapeutic agent on inhibition of leukemia cell growth in vitro, in vivo and ex-vivo, which linked to block ABCB1 function, pumping out its substrate conventional chemotherapeutic agent, thereby increasing the intracellular accumulation. These suggest the combination of ceritinib and substrate chemotherapeutic drugs maybe an effective treatment of resistant leukemia patients with ABCB1-mediated MDR.

scholarly journals Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s)

2018 ◽  
Vol 315 (5) ◽  
pp. C653-C663 ◽  
Author(s):  
Kasin Yadunandam Anandam ◽  
Omar A. Alwan ◽  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Significant Inhibition ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Uptake Inhibition ◽  
Tnf Α ◽  
Vitro Model ◽  
Factor Α

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

scholarly journals In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 758-758
Author(s):  
◽  
Fatima Al-Shahrour ◽  
Kimberly A. Hartwell ◽  
Lisa P Chu ◽  
Jaras Marcus ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Growth ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Growth And Survival ◽  
Integrin Beta 3 ◽  
Shrna Screen ◽  
Primary Leukemia

Abstract Abstract 758 Primary leukemia stem cells (LSCs) reside in an in vivo microenvironment that supports the growth and survival of malignant cells. Despite the increasing understanding of the importance of niche interactions and primary cell biology in leukemia, many studies continue to focus on cell autonomous processes in artificial model systems. The majority of strategies to-date that attempt to define therapeutic targets in leukemia have relied on screening cell lines in culture; new strategies should incorporate the use of primary disease within a physiologic niche. Using a primary murine MLL-AF9 acute myeloid leukemia (AML) model highly enriched for LSCs, we performed an in vivo short hairpin RNA (shRNA) screen to identify novel genes that are essential for leukemia growth and survival. LSCs infected with pools of shRNA lentivirus were transplanted and grown in recipient mice for 2 weeks, after which bone marrow and spleen cells were isolated. Massively parallel sequencing of infected LSCs isolated before and after transplant was used to quantify the changes in shRNA representation over time. Our in vivo screens were highly sensitive, robust, and reproducible and identified a number of positive controls including genes required for MLL-AF9 transformation (Ctnnb1, Mef2c, Ccna1), genes universally required for cell survival (Ube2j2, Utp18), and genes required in other AML models (Myb, Pbx1, Hmgb3). In our primary and validation screens, multiple shRNAs targeting Integrin Beta 3 (Itgb3) were consistently depleted by more than 20-fold over two weeks in vivo. Follow up studies using RNA interference (RNAi) and Itgb3−/− mice identified Itgb3 as essential for murine leukemia cells growth and transformation in vivo, and loss of Itgb3 conferred a statistically significant survival advantage to recipient mice. Importantly, neither Itgb3 knockdown or genetic loss impaired normal hematopoietic stem and progenitor cell (HSPC) function in 16 week multilineage reconstitution assays. We further identified Itgav as the heterodimeric partner of Itgb3 in our model, and found that knockdown of Itgav inhibited leukemia cell growth in vivo. Consistent the therapeutic aims or our study, flow cytometry on primary human AML samples revealed ITGAV/ITGB3 heterodimer expression. To functionally assess the importance of gene expression in a human system, we performed another RNAi screen on M9 leukemia cells, primary human cord blood CD34+ cells transduced with MLL-ENL that are capable of growing in vitro or in a xenotransplant model in vivo. We found that ITGB3 loss inhibited M9 cell growth in vivo, but not in vitro, consistent with the importance of ITGB3 in a physiologic microenvironment. We explored the signaling pathways downstream of Itgb3 using an additional in vivo, unbiased shRNA screen and identified Syk as a critical mediator of Itgb3 activity in leukemia. Syk knockdown by RNAi inhibited leukemia cell growth in vivo; downregulation of Itgb3 expression resulted in decreased levels of Syk phosphorylation; and expression of an activated form of Syk, TEL-SYK, rescued the effects of Itgb3 knockdown on leukemia cell growth in vivo. To understand cellular processes controlled by Itgb3, we performed gene expression studies and found that, in leukemia cells, Itgb3 knockdown induced differentiation and inhibited multiple previously published LSC transcriptional programs. We confirmed these results using primary leukemia cell histology and a model system of leukemia differentiation. Finally, addition of a small molecule Syk inhibitor, R406, to primary cells co-cultured with bone marrow stroma caused a dose-dependent decrease in leukemia cell growth. Our results establish the significance of the Itgb3 signaling pathway, including Syk, as a potential therapeutic target in AML, and demonstrate the utility of in vivo RNA interference screens. Disclosures: Armstrong: Epizyme: Consultancy.

220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS

2015 ◽  
Vol 27 (1) ◽  
pp. 200
Author(s):  
J.-S. Lee ◽  
E.-B. Jeung
Keyword(s):  
Mrna Expression ◽  
Therapeutic Effects ◽  
Mrna Levels ◽  
Oestrogen Receptors ◽  
In Vivo Models ◽  
Ru 486 ◽  
Final Injection ◽  
M 10

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.

An Innovative Ex Vivo Vascular Bioreactor as Comprehensive Tool to Study the Behavior of Native Blood Vessels Under Physiologically Relevant Conditions

2019 ◽  
Vol 2 (4) ◽  
Author(s):  
Noemi Vanerio ◽  
Marco Stijnen ◽  
Bas A. J. M. de Mol ◽  
Linda M. Kock
Keyword(s):  
Shear Stress ◽  
Blood Vessels ◽  
Ultrasound Imaging ◽  
Ex Vivo ◽  
Biological Response ◽  
Vessel Diameter ◽  
Tissue Growth ◽  
In Vivo Models

Abstract Ex vivo systems represent important models to study vascular biology and to test medical devices, combining the advantages of in vitro and in vivo models such as controllability of parameters and the presence of biological response, respectively. The aim of this study was to develop a comprehensive ex vivo vascular bioreactor to long-term culture and study the behavior of native blood vessels under physiologically relevant conditions. The system was designed to allow for physiological mechanical loading in terms of pulsatile hemodynamics, shear stress, and longitudinal prestretch and ultrasound imaging for vessel diameter and morphology evaluation. In this first experience, porcine carotid arteries (n = 4) from slaughterhouse animals were cultured in the platform for 10 days at physiological temperature, CO2 and humidity using medium with blood-mimicking viscosity, components, and stability of composition. As expected, a significant increase in vessel diameter was observed during culture. Flow rate was adjusted according to diameter values to reproduce and maintain physiological shear stress, while pressure was kept physiological. Ultrasound imaging showed that the morphology and structure of cultured arteries were comparable to in vivo. Histological analyses showed preserved endothelium and extracellular matrix and neointimal tissue growth over 10 days of culture. In conclusion, we have developed a comprehensive pulsatile system in which a native blood vessel can be cultured under physiological conditions. The present model represents a significant step toward ex vivo testing of vascular therapies, devices, drug interaction, and as basis for further model developments.

scholarly journals How Can Biomolecules Improve Mucoadhesion of Oral Insulin? A Comprehensive Insight using Ex-Vivo, In Silico, and In Vivo Models

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 675 ◽  
Author(s):  
Mariana Amaral ◽  
Ana Sofia Martins ◽  
José Catarino ◽  
Pedro Faísca ◽  
Pradeep Kumar ◽  
...  
Keyword(s):  
In Silico ◽  
Ex Vivo ◽  
Polymeric Nanoparticles ◽  
Spherical Shape ◽  
Diabetic Rats ◽  
Laser Scattering ◽  
In Vivo Models ◽  
Mean Size

Currently, insulin can only be administered through the subcutaneous route. Due to the flaws associated with this route, it is of interest to orally deliver this drug. However, insulin delivered orally has several barriers to overcome as it is degraded by the stomach’s low pH, enzymatic content, and poor absorption in the gastrointestinal tract. Polymers with marine source like chitosan are commonly used in nanotechnology and drug delivery due to their biocompatibility and special features. This work focuses on the preparation and characterization of mucoadhesive insulin-loaded polymeric nanoparticles. Results showed a suitable mean size for oral administration (<600 nm by dynamic laser scattering), spherical shape, encapsulation efficiency (59.8%), and high recovery yield (80.6%). Circular dichroism spectroscopy demonstrated that protein retained its secondary structure after encapsulation. Moreover, the mucoadhesive potential of the nanoparticles was assessed in silico and the results, corroborated with ex-vivo experiments, showed that using chitosan strongly increases mucoadhesion. Besides, in vitro and in vivo safety assessment of the final formulation were performed, showing no toxicity. Lastly, the insulin-loaded nanoparticles were effective in reducing diabetic rats’ glycemia. Overall, the coating of insulin-loaded nanoparticles with chitosan represents a potentially safe and promising approach to protect insulin and enhance peroral delivery.

scholarly journals Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
L. I. Nagy ◽  
L. Z. Fehér ◽  
G. J. Szebeni ◽  
M. Gyuris ◽  
P. Sipos ◽  
...  
Keyword(s):  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Great Promise ◽  
Human Leukemia ◽  
Human Leukemia Cell Line ◽  
Apoptotic Effects

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

Rig-I Activates Expression of Interferon-Stimulated Genes (ISGs) and Inhibits the Proliferation of Acute Myeloid Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2846-2846 ◽  
Author(s):  
Nan-Nan Zhang ◽  
Lei Chen ◽  
Wu Zhang ◽  
Xian-Yang Li ◽  
Lin-Jia Jiang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Mrna Levels ◽  
Terminal Part ◽  
Granulocytic Differentiation ◽  
Acute Myeloid Leukemia Cells ◽  
Acute Myeloid

Abstract Acute promyelocytic leukemia (APL) is initiated by the formation of PML/RARα oncogenic fusion protein, a potent transcriptional repressor. Retinoid acid (RA) at pharmacological dosage can physically bind to the PML/RARα protein, ushering in the unfolding of downstream programs normally regulated by the wild type RARα. However, through what particular regulatory pathways RA inhibits APL malignant hematopoiesis has remained largely obscured. Rig-I is one of the genes whose mRNA levels were highly up-regulated, along with all-trans-RA (ATRA)-induced terminal granulocytic differentiation of APL cell line NB4 cells in vitro. Based on the analysis of a Rig-I−/− mouse model, recently we have reported a critical regulatory role of Rig-I in normal granulopoiesis. To understand the functional contribution of Rig-I induction in RA-mediated leukemia cell differentiation, we converted a pair of previously reported Rig-I RNAi-duplex sequences into a miR30a-based small hairpin-encoding sequence, which was expressed under the CMV enhancer/promoter within a lentiviral vector. As expected, Rig-I shRNAmir30 infection induced a significant knockdown of Rig-I protein level, and accordingly its delivery into HL-60 cells partially inhibited ATRA-induced granulocytic differentiation, growth inhibition/cell cycle arrest and apoptosis induction, suggesting that Rig-I upregulation participates in RA-induced granulocytic differentiation of acute myeloid leukemia cells. In order to investigate the effect of Rig-I induction on the proliferation of APL cells in vivo, we transduced PML/RARα-harboring leukemic cells with vector or Rig-I-expressing retrovirus, and then transplanted these cells into the syngeneic mice. The vector-transduced APL cells readily expanded in vivo, but the proliferation of Rig-I-transduced cells was apparently prohibited. Moreover, we found that the forced expression of Rig-I induced the expression of numerous ISGs in APL cells, which was recapitulated by the transduction of the C terminal part of Rig-I, but not by the N terminal part. In line with this, during the in vitro short-term culture post-IFNγ or IFNα stimulation, Stat1 phosphorylation at p701 in Rig-I−/− granulocytes was significantly inhibited. In parallel, the induction of multiple ISGs by IFNs was also significantly impaired. In conclusion, our findings indicate that the Rig-I induction inhibited APL reconstitution potentially through up-regulating a number of ISGs via regulating Stat1Tyr701 phosphorylation.

Comparison of the Effects on Reversal of Multi-Drug Resistance of 5-Bromotetrandrine and Tetrandrine in K562/A02 Cell Line in Vivo and in Vitro

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5059-5059
Author(s):  
Bao-An Chen ◽  
Jue-qiong Wang ◽  
Jian Cheng ◽  
Feng Gao ◽  
Wen-lin Xu ◽  
...  
Keyword(s):  
Cell Line ◽  
Nude Mice ◽  
Leukemia Cell Line ◽  
Multi Drug Resistance ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Intracellular Accumulation

Abstract Objective This study was to compare the reversal effect of 5-bromotetrandrine (BrTet) with Tetrandrine (Tet) when combined with ADM on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this new derivative. Methods The protein levels of P-glycoprotein (P-gp) were detected by fluorospectrophotometry and Western blot. The mRNA levels of P-gp were determined by RT-PCR. The in vivo effect of Tet was investigated using nude mice grafted with sensitive human leukemia cell line K562 and MDR cell line K562/A02. Results Flow cytometry assay showed that 1.0 μMol/L BrTet significantly increased the apoptosis percentage. BrTet also enhanced the intracellular accumulation of ADM in K562/A02 cells and its potency was greater than that of Tet at the same concentrations. BrTet inhibited the overexpression of P-gp and down regulated MDR1 mRNA expression in K562/A02 cells in a dose-dependent manner. In nude mice bearing K562 xenografts on the left flank and K562/A02 xenografts on the right flank, i.p. injection of 10 mg/kg BrTet significantly enhanced the antitumor activity of ADM against K562/A02 xenografts with inhibitory rates of 26.1%, while ADM alone inhibited the growth of KBv200 xenografts by only 5.8%. Conclusion BrTet showed significant MDR reversal activity in vitro and in vivo. Its activity may be related to the inhibition of P-gp overexpression and the increase in intracellular accumulation of anticancer drugs, which lead to more K562/A02 cells apoptosis.

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.

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