Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Apoptosis. 2014 Apr;19(4):698–707. doi: 10.1007/s10495-013-0954-z

Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML

P Y Mak 1, D H Mak 2, H Mu 3, Y Shi 4, P Ruvolo 5, V Ruvolo 6, R Jacamo 7, J K Burks 8, W Wei 9, X Huang 10, S M Kornblau 11, M Andreeff 12, B Z Carter 13,*
PMCID: PMC3943601  NIHMSID: NIHMS549057  PMID: 24337870

Abstract

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 versus 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2R null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Keywords: ARC, apoptosis, MSCs, AML, kinase signaling

Introduction

The apoptosis repressor with caspase recruitment domain (ARC) protein was originally described in normal heart [1, 2], brain [3], and muscle [4] cells. It protects these cells from apoptosis induced by various stressors. ARC is unique since it suppresses the activation of both the intrinsic and extrinsic apoptosis pathways [4–8]. ARC is overexpressed in various malignant cells, and this expression is associated with disease progression and poor outcomes [5, 9–13]. Furthermore, ARC was found to be induced by RAS to promote tumorigenesis and metastasis in breast and other cancer cells [12–15].

As part of our ongoing search for prognostic markers and clinically relevant therapeutic targets in acute myeloid leukemia (AML), we recently analyzed ARC expression by reverse-phase protein array (RPPA) in samples obtained from patients (n = 511) with newly diagnosed AML. This analysis identified ARC as a novel adverse prognostic factor in AML with the highest negative impact. Specifically, high ARC protein expression predicted a shorter overall survival and poor treatment outcome in patients with AML [16].

In addition to intrinsic apoptosis resistance in leukemia cells, external factors in the bone marrow (BM) microenvironment where leukemic cells reside contribute to drug resistance and disease relapse. BM-derived mesenchymal stromal cells (MSCs) are known to secrete various growth factors, interact with leukemia cells, and provide a sanctuary to protect leukemia cells from apoptosis induction by various therapeutic agents via activation of survival signaling pathways [17] and induction of anti-apoptotic proteins [18–20]. Nevertheless, the mechanisms of ARC regulation in AML cells, and the role of ARC in AML cell survival and drug resistance are largely unknown.

In this study, we investigated the regulation of ARC in AML by signaling pathways and by MSCs from the BM microenvironment. Using AML cells in which ARC expression was genetically modified, we studied the role of ARC in drug resistance of AML cells. We establish that ARC is upregulated by MSCs through MAPK and PI3K signaling in AML cells. Furthermore, we demonstrate that ARC protects AML cells from apoptosis induced by either chemotherapy or agents that selectively induce intrinsic or extrinsic apoptosis, thus conferring survival advantage to AML both in vitro and in vivo in mouse models.

Materials and methods

Cells and cell cultures

OCI-AML3 cells were kindly provided by Dr. M. Minden (Ontario Cancer Institute, Ontario, Canada), KG-1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and Molm13 cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FSC), 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin. MSCs were isolated from BM of healthy subjects as previously described [21].

ARC knockdown in AML cells

ARC was knocked down by lentiviral transduction using gene-specific shRNAmir-green fluorescent protein (GFP)-expressing transfer vectors: clone V3LHS_337663, targeting residues 732–750 on RefSeq NM_003946.4, for OCI-AML3 cells and clone V3LHS_337662, targeting residues 217–235 on RefSeq NM_003946.4, for Molm13 cells (Open Biosystems, Huntsville, AL, USA). Lentivirus was prepared by co-transfection of HEK293T cells (American Type Culture Collection) with an equal molar mix of transfer vector and packaging plasmids (psPAX2 and pMD2.G; Addgene, Cambridge, MA, USA) using JetPrime transfection reagent as directed by the manufacturer (Polyplus, Illkirch, France). Fresh lentiviral supernatants were passed through 0.45-micron-pore surfactant-free cellulose acetate membranes and then used at once to infect leukemic cells by incubation overnight at 37°C in 5% CO2. Infected cells were subjected to selection with puromycin (Invivogen, San Diego, CA, USA) starting at 1 g/ml. In parallel, OCI-AML3 and Molm13 cells were transduced with lentivirus delivering a non-specific control vector (Open Biosystems). Knockdown (K/D) was verified by western blot analysis and by real-time RT-PCR.

ARC overexpression in KG-1 cells

The ARC coding sequence was excised from EGFP-Myp (kindly provided by Dr. S. Stamm, University of Kentucky, Lexington, KY, USA) with MluI-BglII and its ends filled in with Klenow before it was cloned into pCDH-CMV-MCS-EF1-copGFP (SystemBio, Mountain View, CA, USA) between the blunted NheI-NotI sites. The resulting lentiviral vector was designated pCDH-CMV-ARC-EF1-copGFP. KG-1 cells were infected with concentrated lentivirus transduced with either pCDH-CMV-ARC-EF1-copGFP or pCDH empty vector generated by a process similar to that just described; 8 μg/ml Polybrene (Sigma Chemical Co., St. Louis, MO, USA) was included to enhance lentiviral infections. A week after infection, stably transduced KG-1 cells were sorted by fluorescence activation (FACS) to obtain a homogeneous population of ARC overexpressing (O/E) CopGFP-positive cells.

Treatment of cells

OCI-AML3 cells were treated with MEK/ERK inhibitor PD0325901 (Cayman Chemical, Ann Arbor, MI, USA) or PI3K inhibitor LY294002 (Sigma) or BEZ235 (LC Laboratories, Woburn, MA, USA), and cells were collected for RNA purification, lysate preparation, or immunofluorescence staining. To transiently knockdown MAPK signaling, OCI-AML3 cells were transfected with MAP3K1siRNA (on Target Plus Smart pool; Thermo Scientific Dharmacon, Pittsburgh, PA, USA) by electroporation using an Amaxa apparatus (Amaxa Biosystems, Cologne, Germany) following the manufacturer’s instructions. For OCI-AML3-MSCs co-culture experiments, MSCs were pre-plated at 5×103/cm2 for 24 hours. OCI-AML3 cells were added (four times the number of MSCs). Cells were co-cultured for 24 hours and leukemia cells were then obtained by combining all cells in the suspension and cells collected after washing the wells twice with phosphate buffered saline (PBS), stained with antibodies against CD45 (CD45-PE) and CD90 (CD90-APC) (both from BD Biosciences, San Jose, CA, USA), and sorted by FACS. The co-cultured cells were also treated with PD0325901 or LY294002. Leukemia cells were collected at 24 hours by combining all cells in the suspension and cells collected after PBS washing.

ARC K/D and O/E cells were treated with cytarabine (Ara-C), ABT-737 (synthesized at the MD Anderson Cancer Center based on the published chemical structure [22], or TNF-related apoptosis inducing ligand (TRAIL, Alexis Biochemicals, San Diego, CA, USA).

Taq-Man RT-PCR

Cell pellets were lysed in Trizol (Invitrogen). Total RNA was extracted by chloroform extraction and isopropanol precipitation and re-suspended in nuclease-free water. cDNA was prepared from total RNA in a mixture containing dNTP, random hexamers, and AMV reverse transcriptase and RNase inhibitor (both, Roche Applied Science, Indianapolis, IN, USA) at 42°C for 1 hour. ARC was detected by using a TaqMan Gene Expression Assay (NOL3, Hs00358724_g1; Applied Biosystems, Inc. (ABI), Carlsbad, CA, USA) in an ABI 7900HT Fast RT-PCR system. Another TaqMan Gene Expression Assay (ABL1, Hs01104728_m1; ABI) was used as the loading control. The PCR reaction mixture contained cDNA, Taqman Gene Expression Assay reagent, and TaqMan Fast Universal PCR Master Mix (ABI). The reaction was initiated by a hold for 10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. The abundance of ARC transcript relative to that of ABL1 was calculated using the 2−ΔCt method, where ΔCt is the mean Ct of ARC transcript minus the mean Ct of the ABL1 transcript.

Immunofluorescence microscopy

Cells were spun onto slides using a Statspin Cytofuge 12 or cultured on microscope slides and fixed with 4% paraformaldehyde and permeabilized with 100% methanol. Cells were incubated in blocking solution (3% FBS/1% bovine serum albumin/PBS). ARC rabbit polyclonal antibody (Cayman Chemical) was added to cells at 1:200 dilution in blocking solution. Cells were washed and secondary antibody (Alexa 488-tagged donkey anti-rabbit; Invitrogen, Eugene, OR, USA) was added in blocking solution. Cells were washed, stained with 4′,6-diamidino-2-phenylindole (DAPI), washed again. Images were captured using an Olympus FV1000 laser scanning confocal microscope using a 1.45 NA 100× oil objective. Images were then imported and analyzed in Intelligent Imaging Innovations Slidebook 5.0 software. Briefly, masks were created from the 488 channel to identify the regions of cells. These masks were then watershed to separate neighboring cells. Once individual cells were identified in the segmentation mask the Sum was quantified for the 488 channel. This was then normalized per cell for three conditions (control, n = 165; PD0325901 treatment, n = 254; or LY294002 treatment, n = 148) for comparison purposes.

Phospho-flow analysis

Cells were stained with Live/Dead fixable yellow dead cell stain kit (Invitrogen, Eugene, OR, USA), then fixed with 1.6% formaldehyde, permeabilized with 0.1% Triton X-100, and stored in methanol at 20 C awaiting further processing. Cells were stained with p-ERK 1/2-Alexa 488 and p-AKT (Ser473)-Alexa 647 (Cell Signaling Technology, Danvers, MA, USA). AML cells (CD45+) were distinguished from MSCs (CD90+) by staining cells with CD45-APC-H7 and CD90-PerCP-Cy5.5 (both from BD Biosciences). Signals were determined on the gated live CD45+CD90− cells using a Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA) and expressed as median fluorescent intensity ([MFI of stained cells MFI of unstained cells] / MFI of unstained cells).

Cell viability assay

Apoptosis was estimated by flow cytometric measurements of phosphatidyl serine with the annexin-V-Cy5 (AnnV-Cy5) staining (BD Biosciences). Membrane integrity was simultaneously assessed by 7-amino-actinomycin D (7AAD) exclusion in the annexin V-stained cells. Viable AML cells were counted via flow cytometry using CountBright absolute counting beads (Invitrogen). Signals were determined by using a FACSArray Bioanalyzer (BD Biosciences).

Western blot analysis

Protein levels in treated cells were determined by western blot analysis, as described previously [19]. ARC antibody was purchased from Imgenex (San Diego, CA, USA). Antibodies against pERK, ERK, p-AKT (Ser473), and AKT were obtained from Cell Signaling Technology (Danvers, MA, USA). Signals were detected by using the Odyssey Infrared Imaging System and quantitated by Odyssey software version 3.0 (LI-COR Biosciences, Lincoln, NE, USA). -Actin was used as a loading control.

In vivo experiments

NOD/SCID mice, six weeks old, were irradiated (2.5 Gy) and divided into two groups of nine mice/group under the institution approved protocol. The mice in one group were injected with 4.2 × 106 ARC O/E KG-1 cells and the mice in the other group with the same number of vector control cells via tail vein. NOD/SCID IL2R null (NSG) mice, five weeks old, were divided into two groups of seven mice/group. The mice in one group were injected with 0.35 × 106 ARC K/D Molm13 cells and the mice in the other group with the same number of vector control cells via tail vein. Molm13 levels were assessed by flow cytometric measurement of human CD45+ cells in mouse blood samples. Mouse survival times were recorded and survival data were analyzed by the log-rank test.

Statistical analyses

All experiments were carried out in triplicate, and the results are expressed as the mean SEM, unless otherwise stated. The concentrations of agents that induced annexin-V positivity in 50% of cells (EC50) were calculated by using Calcusyn software (Biosoft, Ferguson, MO, USA). Expression of ARC in AML patient samples was summarized using mean, medium, standard deviation, and range. Comparison of ARC between patient groups (e.g., RAS mutation status) was performed using Wilcoxon rank sum test. Statistical analysis was carried out using SAS version 9 (SAS Institute, Cary, NC). Statistical plotting was done using Spotfire S+ version 8.2 (TIBCO, Somerville, MA). The Student t-test was used to compare differences between the groups. All tests were two-sided and P values ≤ 0.05 were considered statistically significant.

Results

ARC is upregulated by stromal cells via signaling pathways in AML cells

We first investigated whether ARC in AML cells is regulated by PI3K and/or MAPK signaling, which are two pathways that are constitutively active in AML. We treated OCI-AML3 cells with a MEK/ERK inhibitor PD0325901 (5 nM) or a PI3K inhibitor LY294002 (20 M) and found that blockade of either MAPK or PI3K signaling led to time-dependent decreases in both ARC RNA and protein levels (Fig. 1a). Similarly, inhibition of MAP3K1 expression by siRNA or inhibition of PI3K signaling by a dual PI3K and mTOR inhibitor BEZ235 currently under clinical development also decreased ARC expression (Fig. 1b). In contrast, no change in ARC levels was observed when SRC kinase was inhibited by dasatinib (not shown). To confirm this finding and to assess the cellular localization of ARC, we also determined ARC levels in PD0325901 and LY294002 treated OCI-AML3 by immunofluorescence microscopy. As shown in Fig. 1c, ARC was localized both in nucleus and cytoplasm and its expression was decreased by MAPK or PI3K inhibition (P < 0.0001).

Fig. 1.

Fig. 1

Open in a new tab

ARC expression is regulated by MAPK and PI3K signaling pathways in AML cells. a OCI-AML3 cells were treated with MEK/ERK inhibitor PD0325901 (5 nM) or PI3K inhibitor LY294002 (20 M). ARC mRNA and protein levels were determined at various time points by TaqMan RT-PCR and western blot, respectively. b OCI-AML3 cells were treated with MAP3K1siRNA (4 M) by electroporation or PI3K/mTOR inhibitor BEZ235 (50 nM) for 24 hours. ERK and ARC protein levels were determined by western blot. c OCI-AML3 cells were treated with MEK/ERK inhibitor PD0325901 (10 nM) or PI3K inhibitor LY294002 (40 M). ARC protein expression was determined by immunofluorescence microscopy. d OCI-AML3 cells were cultured alone or co-cultured with MSCs for 24 hours. ARC levels in FACS-sorted CD45+CD90− OCI-AML3 cells were determined by western blot. e OCI-AML3 cells were cultured alone or co-cultured with MSCs and treated with PD0325901 (10 nM) or LY294002 (40 M) for 24 hours. ERK, AKT, and ARC levels were determined by western blot in leukemia cells. p-ERK and p-AKT levels were also determined by phopho-flow in CD45+CD90− OCI-AML3 cells. Alone, leukemia cells are cultured alone in suspension; cocx, leukemia cells were co-cultured with MSCs; PD, PD0325901; LY, LY294002.

We next co-cultured OCI-AML3 cells with MSCs and FACS-sorted CD45+CD90− AML cells. As shown in Fig. 1d, co-cultures with MSCs strongly increased ARC expression in CD45+CD90− leukemic cells. To determine whether the induction of ARC expression by MSCs is mediated through MAPK or PI3K signaling, we cultured OCI-AML3 cells with or without MSCs and treated them with PD0325901 (10 nM) or LY294002 (40 M). We then examined MAPK and PI3K signaling and ARC expression by western blot analysis. As shown in Fig. 1e, co-culture greatly increased p-ERK and p-AKT levels in OCI-AML3 cells. PD0325901 or LY294002 decreased p-ERK or p-AKT levels in OCI-AML3 cells cultured alone and suppressed MSC co-culture-mediated induction of p-ERK or p-AKT in leukemia cells. Consistent with changes in MAPK and PI3K signaling, PD0325901 or LY294002 decreased ARC expression in OCI-AML3 cells and suppressed the MSC co-culture-mediated induction of ARC, suggesting that ARC is regulated by MAPK and PI3K signaling and upregulated by stroma via MAPK and PI3K signaling pathways. Unlike the data presented in Fig. 1d, we did not sort cells before performing Western blot analysis since the collected leukemia cells contained negligible amounts of CD90+ MSCs and the sorting procedure would diminish kinase activity. To confirm the levels of kinases we measured are from leukemia cells, and not from potentially contaminating MSCs, we also determined p-ERK and p-AKT by phospho-flow in CD45+CD90− cells (Fig. 1e) and confirmed the immune-blot data. Cell viability assessments for the cells treated with PD0325901 or LY294002 are shown below the graphs in Fig. 1e.

We then examined whether ARC is regulated by signaling pathways in samples from patients with AML. In the large sample set comprising 511 patients with newly diagnosed AML that we previously used to profile ARC expression [16], 435 had known RAS mutation status. We analyzed ARC levels in RAS mutation positive (N = 64) and negative (N = 371) samples and found that samples with RAS mutations expressed significantly higher levels of ARC than samples without RAS mutations (P = 0.016, Fig. 2). Taken together, these findings suggest that ARC expression is regulated, at least in part, by MAPK and PI3K signaling mechanisms.

Fig. 2.

Fig. 2

Open in a new tab

ARC levels were compared in AML patient samples with or without RAS mutation. ARC levels were determined by RPPA. Samples with a RAS mutation (N = 64) expressed significantly higher levels of ARC (P = 0.016) than samples without (N = 371).

ARC protects AML cells from apoptosis induced by chemotherapy or inducers of intrinsic or extrinsic apoptosis

Endogenous expression of ARC is extremely low in KG-1 and higher in OCI-AML3 and Molm13 AML cells. To investigate the role of ARC in apoptosis resistance in AML cells, we generated stable ARC O/E KG-1 and stable ARC K/D OCI-AML3 and Molm13 cells (Fig. 3a) and treated them with Ara-C or agents that selectively induce intrinsic (i.e., ABT-737) or extrinsic (i.e., TRAIL) apoptosis. We found that the ARC O/E cells were more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL than the respective controls (Fig. 3b), suggesting that ARC expression in AML cells protects these cells from both intrinsic and extrinsic apoptotic stimuli. The EC50 values from these assessments, relative to the respective vector controls, are shown in Fig. 3b.

Fig. 3.

Fig. 3

Open in a new tab

ARC in AML cells protects them from apoptosis. a ARC levels were measured in ARC O/E KG-1 and K/D OCI-AML3 and Molm13 AML cells by western blot. b ARC O/E KG-1 and ARC K/D OCI-AML3 and Molm13 cells were treated with Ara-C, ABT-737, or TRAIL. Cell death was quantified by annexin V/7AAD staining and flow cytometric analysis 48 hors after treatment. *P ≤ 0.05 and **P ≤ 0.01. EC50s of various treatments in ARC O/E or K/D over control cells are shown at the bottom of the graphs.

ARC confers in vitro and in vivo growth advantages to AML

To determine the role of ARC in cell growth, we compared the growth rates of KG-1 with ARC O/E KG-1 cells. As shown in Fig. 4a, ARC O/E KG-1 cells grew faster than their control cells. We injected equal numbers of ARC O/E or vector control KG-1 cells into NOD/SCID mice and recorded their survival times. ARC O/E in the AML cells was associated with shortened survival of mice. Indeed, the median survival for mice injected with vector control KG-1 was 111 days, versus 84 days for the animals injected with the ARC O/E KG-1 cells (P = 0.0002) (Fig. 4b). We also injected equal numbers of ARC K/D or vector control Molm13 cells into NSG mice and found that significantly fewer leukemia developed when NSG mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively) (Fig. 4c). There was a trend for prolonged survival of mice injected with ARC K/D cells, but it did not reach statistical significance (not shown). Limited knockdown of ARC may therefore not sufficiently change physiological apoptosis thresholds, which are regulated by many proteins, while overexpression resulted in significantly shorter survival of leukemia bearing mice.

Fig. 4.

Fig. 4

Open in a new tab

ARC enhances leukemia cell growth in vitro and in in vivo xenograft mouse models. a Growth curves of ARC O/E and control KG-1 cells. Results are expressed as mean ± standard derivation. b Survival curves of NOD-SCID mice injected with ARC O/E or control KG-1 cells. c Leukemia cells in NSG mice injected with ARC K/D or control Molm13 cells at 2 weeks and 3 weeks.

Interestingly, we previously reported that when newly diagnosed AML patients (n = 511) were grouped based on their ARC levels determined by RPPA, the patients with high ARC had significantly shorter overall survival and event-free survival than the patients with low ARC [16].

Discussion

In this study, we demonstrate that ARC is regulated by MAPK and PI3K signaling in AML cells, and that the BM microenvironment can increase ARC expression in AML cells through these signaling pathways. Our findings also indicate that ARC expression protects AML cells from apoptosis induction by chemotherapy and agents that selectively induce intrinsic or extrinsic apoptosis. Moreover, ARC conveys a survival advantage to AML cells in vitro and in in vivo mouse AML models.

Constitutive activation of MAPK and PI3K is common in AML, and this aberrant cell signaling is typically associated with poor clinical outcomes in AML patients [23–28]. These signaling pathways play critical roles in cancer cell growth and survival via various mechanisms. For example, they can induce the expression of multiple antiapoptotic proteins such as Mcl-1, XIAP, and survivin [18, 19, 29]. The BM microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from apoptosis induced by various therapeutic agents. It exerts its protective role in part by activating MAPK/PI3K signaling and by inducing the expression of antiapoptotic proteins. Our findings that ARC expression is regulated by MAPK and PI3K signaling, and that MSC co-culture stimulates MAPK/PI3K signaling and ARC expression, support the pro-survival role of ARC in AML cells.

RAS regulates multiple signaling pathways and it is frequently mutated in AML. RAS is reportedly mutated in 10 to 25% patients with AML [30–34]. Proteomic analysis revealed that RAS-Raf-MAPK and PI3K signaling pathways were upregulated in AML patients with RAS mutations [35]. We observed that ARC expression was increased in samples from patients with RAS-mutated AML compared to patients with RAS-wild type AML, supporting the in vitro finding that ARC is regulated by MAPK and PI3K signaling. Our results are also consistent with a report that ARC is induced by RAS in a MEK/ERK-dependent manner in epithelial cancers [14].

ARC was reported to be localized almost exclusively in the nuclei of various human and rat cancer cells. Conversely, in non-cancer cells it localizes to the cytoplasm [9]. We did not observe a restricted nuclear localization of ARC in OCI-AML3 cells, and it is not clear currently what this could mean for the relation to AML to other types of cancer. ARC’s role as a protector of AML cells from both intrinsic and extrinsic apoptotic cell death was validated using ARC O/E and ARC K/D cells. Indeed, we found that high ARC levels promoted resistance to chemotherapy and apoptotic stimuli in the AML cells. This implies that targeting of ARC with small molecule inhibitors could perhaps directly induce apoptosis and/or sensitizes chemotherapy in AML. Nevertheless, high nuclear ARC in OCI-AML3 cells may suggest that ARC is stored in the nucleus and released into the cytoplasm as needed, or ARC has roles in the nucleus such as protecting cells from DNA damage or affecting the expression of other genes through protein-protein interactions that need to be further defined.

Together, our results demonstrate that ARC plays critical roles in AML growth and survival. ARC levels are regulated in AML cells by BM MSCs through various survival signaling pathways, protects leukemia cells from apoptosis induced by chemotherapy and agents that selectively induce intrinsic or extrinsic apoptosis, and promotes leukemia cell growth in vitro and in vivo, all of which may contribute to ARC being a poor prognostic factor in AML. Results suggest that ARC could be a novel target for treatment of AML

Acknowledgments

This research was supported by the University Cancer Foundation via the Institutional Research Grant program at the University of Texas MD Anderson Cancer Center (BZC) and by grants from the National Institutes of Health grants (P01 CA55164 and P30 CA016672) and the Paul and Mary Haas Chair in Genetics (MA). We thank Deanna A. Alexander for assisting with manuscript preparation, Kathryn L. Hale and Numsen Hail, Jr. for editing the manuscript, and Anitha G. Somanchi for helping with data analysis.

Footnotes

Conflict of interest

The authors declare that they have no conflict of interest.

Contributor Information

P. Y. Mak, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

D. H. Mak, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

H. Mu, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Y. Shi, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

P. Ruvolo, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

V. Ruvolo, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

R. Jacamo, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

J. K. Burks, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

W. Wei, Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

X. Huang, Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

S. M. Kornblau, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

M. Andreeff, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

B. Z. Carter, Section of Molecular Hematology and Therapy, Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

References

  • 1.Li Y, Ge X, Liu X. The cardioprotective effect of postconditioning is mediated by ARC through inhibiting mitochondrial apoptotic pathway. Apoptosis. 2009;14:164–172. doi: 10.1007/s10495-008-0296-4. [DOI] [PubMed] [Google Scholar]
  • 2.Li YZ, Liu XH, Zhu XM, Cai LR. ARC contributes to the inhibitory effect of preconditioning on cardiomyocyte apoptosis. Apoptosis. 2007;12:1589–1595. doi: 10.1007/s10495-007-0094-4. [DOI] [PubMed] [Google Scholar]
  • 3.Hong YM, Jo DG, Lee JY, Chang JW, Nam JH, Noh JY, et al. Down-regulation of ARC contributes to vulnerability of hippocampal neurons to ischemia/hypoxia. FEBS letters. 2003;543:170–173. doi: 10.1016/s0014-5793(03)00444-7. [DOI] [PubMed] [Google Scholar]
  • 4.Koseki T, Inohara N, Chen S, Nunez G. ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases. Proc Natl Acad Sci USA. 1998;95:5156–5160. doi: 10.1073/pnas.95.9.5156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Heikaus S, Kempf T, Mahotka C, Gabbert HE, Ramp U. Caspase-8 and its inhibitors in RCCs in vivo: the prominent role of ARC. Apoptosis. 2008;13:938–949. doi: 10.1007/s10495-008-0225-6. [DOI] [PubMed] [Google Scholar]
  • 6.Zhang YQ, Herman B. ARC protects rat cardiomyocytes against oxidative stress through inhibition of caspase-2 mediated mitochondrial pathway. J Cell Biochem. 2006;99:575–588. doi: 10.1002/jcb.20946. [DOI] [PubMed] [Google Scholar]
  • 7.Nam YJ, Mani K, Ashton AW, Peng CF, Krishnamurthy B, Hayakawa Y, et al. Inhibition of both the extrinsic and intrinsic death pathways through nonhomotypic death-fold interactions. Mol Cell. 2004;15:901–912. doi: 10.1016/j.molcel.2004.08.020. [DOI] [PubMed] [Google Scholar]
  • 8.Gustafsson AB, Tsai JG, Logue SE, Crow MT, Gottlieb RA. Apoptosis repressor with caspase recruitment domain protects against cell death by interfering with Bax activation. J Biol Chem. 2004;279:21233–21238. doi: 10.1074/jbc.M400695200. [DOI] [PubMed] [Google Scholar]
  • 9.Wang M, Qanungo S, Crow MT, Watanabe M, Nieminen AL. Apoptosis repressor with caspase recruitment domain (ARC) is expressed in cancer cells and localizes to nuclei. FEBS Letters. 2005;579:2411–2415. doi: 10.1016/j.febslet.2005.03.040. [DOI] [PubMed] [Google Scholar]
  • 10.Mercier I, Vuolo M, Jasmin JF, Medina CM, Williams M, Mariadason JM, et al. ARC (apoptosis repressor with caspase recruitment domain) is a novel marker of human colon cancer. Cell Cycle. 2008;7:1640–1647. doi: 10.4161/cc.7.11.5979. [DOI] [PubMed] [Google Scholar]
  • 11.Ziegler DS, Wright RD, Kesari S, et al. Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins. J Clin Invest. 2008;118:3109–3122. doi: 10.1172/JCI34120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Mercier I, Vuolo M, Madan R, Xue X, Levalley AJ, Ashton AW, et al. ARC, an apoptosis suppressor limited to terminally differentiated cells, is induced in human breast cancer and confers chemo- and radiation-resistance. Cell Death Differ. 2005;12:682–686. doi: 10.1038/sj.cdd.4401631. [DOI] [PubMed] [Google Scholar]
  • 13.Wang JX, Li Q, Li PF. Apoptosis repressor with caspase recruitment domain contributes to chemotherapy resistance by abolishing mitochondrial fission mediated by dynamin-related protein-1. Can Res. 2009;69:492–500. doi: 10.1158/0008-5472.CAN-08-2962. [DOI] [PubMed] [Google Scholar]
  • 14.Wu L, Nam YJ, Kung G, Crow MT, Kitsis RN. Induction of the apoptosis inhibitor ARC by Ras in human cancers. J Biol Chem. 2010;285:19235–19245. doi: 10.1074/jbc.M110.114892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Medina-Ramirez CM, Goswami S, Smirnova T, Bamira D, Benson B, Ferrick N, et al. Apoptosis inhibitor ARC promotes breast tumorigenesis, metastasis, and chemoresistance. Can Res. 2011;71:7705–7715. doi: 10.1158/0008-5472.CAN-11-2192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Carter BZ, Qiu YH, Zhang N, Coombes KR, Mak DH, Thomas DA, et al. Expression of ARC (apoptosis repressor with caspase recruitment domain), an antiapoptotic protein, is strongly prognostic in AML. Blood. 2011;117:780–787. doi: 10.1182/blood-2010-04-280503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Tabe Y, Jin L, Tsutsumi-Ishii Y, Xu Y, McQueen T, Priebe W, et al. Activation of integrin-linked kinase is a critical prosurvival pathway induced in leukemic cells by bone marrow-derived stromal cells. Can Res. 2007;67:684–694. doi: 10.1158/0008-5472.CAN-06-3166. [DOI] [PubMed] [Google Scholar]
  • 18.Carter BZ, Milella M, Altieri DC, Andreeff M. Cytokine-regulated expression of survivin in myeloid leukemia. Blood. 2001;97:2784–2790. doi: 10.1182/blood.v97.9.2784. [DOI] [PubMed] [Google Scholar]
  • 19.Carter BZ, Milella M, Tsao T, McQueen T, Schober WD, Hu W, et al. Regulation and targeting of antiapoptotic XIAP in acute myeloid leukemia. Leukemia. 2003;17:2081–2089. doi: 10.1038/sj.leu.2403113. [DOI] [PubMed] [Google Scholar]
  • 20.Konopleva M, Konoplev S, Hu W, Zaritskey AY, Afanasiev BV, Andreeff M. Stroma cells prevent apoptosis of AML cells by upregulation of anti-apoptotic proteins. Leukemia. 2002;16:1713–1724. doi: 10.1038/sj.leu.2402608. [DOI] [PubMed] [Google Scholar]
  • 21.Studeny M, Marini FC, Champlin RE, Zompetta C, Fidler IJ, Andreeff M. Bone marrow-derived mesenchymal stem cells as vehicles for interferon-beta delivery into tumors. Can Res. 2002;62:3603–3608. [PubMed] [Google Scholar]
  • 22.Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA, et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature. 2005;435:677–681. doi: 10.1038/nature03579. [DOI] [PubMed] [Google Scholar]
  • 23.Ricciardi MR, McQueen T, Chism D, Milella M, Estey E, Kaldjian E, et al. Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia. Leukemia. 2005;19:1543–1549. doi: 10.1038/sj.leu.2403859. [DOI] [PubMed] [Google Scholar]
  • 24.Kim SC, Hahn JS, Min YH, Yoo NC, Ko YW, Lee WJ. Constitutive activation of extracellular signal-regulated kinase in human acute leukemias: combined role of activation of MEK, hyperexpression of extracellular signal-regulated kinase, and downregulation of a phosphatase, PAC1. Blood. 1999;93:3893–3899. [PubMed] [Google Scholar]
  • 25.Towatari M, Iida H, Tanimoto M, Iwata H, Hamaguchi M, Saito H. Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells. Leukemia. 1997;11:479–484. doi: 10.1038/sj.leu.2400617. [DOI] [PubMed] [Google Scholar]
  • 26.Min YH, Eom JI, Cheong JW, Maeng HO, Kim JY, Jeung HK, et al. Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable. Leukemia. 2003;17:995–997. doi: 10.1038/sj.leu.2402874. [DOI] [PubMed] [Google Scholar]
  • 27.Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute myeloid leukemia cells requires PI3 kinase activation. Blood. 2003;102:972–980. doi: 10.1182/blood-2002-11-3429. [DOI] [PubMed] [Google Scholar]
  • 28.Kornblau SM, Womble M, Qiu YH, Jackson CE, Chen W, Konopleva M, et al. Simultaneous activation of multiple signal transduction pathways confers poor prognosis in acute myelogenous leukemia. Blood. 2006;108:2356–2365. doi: 10.1182/blood-2006-02-003475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Konopleva M, Milella M, Ruvolo P, Watts JC, Ricciardi MR, Korchin B, et al. MEK inhibition enhances ABT-737-induced leukemia cell apoptosis via prevention of ERK-activated MCL-1 induction and modulation of MCL-1/BIM complex. Leukemia. 2012;26:778–787. doi: 10.1038/leu.2011.287. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  • 30.Bacher U, Haferlach T, Schoch C, Kern W, Schnittger S. Implications of NRAS mutations in AML: a study of 2502 patients. Blood. 2006;107:3847–3853. doi: 10.1182/blood-2005-08-3522. [DOI] [PubMed] [Google Scholar]
  • 31.Bos JL, Verlaan-de Vries M, van der Eb AJ, Janssen JW, Delwel R, Lowenberg B, et al. Mutations in N-ras predominate in acute myeloid leukemia. Blood. 1987;69:1237–1241. [PubMed] [Google Scholar]
  • 32.Coghlan DW, Morley AA, Matthews JP, Bishop JF. The Incidence and Prognostic Significance of Mutation in Codon 13 of the N-ras Gene in Acute Myeloid Leukemia. Leukemia. 1994;8 (10):1682–1687. [PubMed] [Google Scholar]
  • 33.Neubauer A, Dodge R, George SL, Davey FR, Silver R, Schiffer CA, et al. Prognostic Importance of Mutations in the ras Proto-Oncogenes in De Novo Acute Myeloid Leukemia. Blood. 1994;83:1603–1611. [PubMed] [Google Scholar]
  • 34.Radich JP, Kopecky KJ, Willman CL, Weick J, Head D, Appelbaum F, et al. N-ras mutations in adult de novo acute myelogenous leukemia: prevalence and clinical significance. Blood. 1990;76:801–807. [PubMed] [Google Scholar]
  • 35.Kadia TM, Kantarjian H, Kornblau S, Borthakur G, Faderl S, Freireich EJ, et al. Clinical and proteomic characterization of acute myeloid leukemia with mutated RAS. Cancer. 2012;118:5550–5559. doi: 10.1002/cncr.27596. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES