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

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 vs 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2Rγ 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.

Fig. 1

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.

Fig. 2

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).

Fig. 3

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.

Fig. 4

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.