Anti-apoptotic ARC protein confers chemoresistance by controlling leukemia-microenvironment interactions through a NFκB/IL1β signaling network

Abstract

To better understand how the apoptosis repressor with caspase recruitment domain (ARC) protein confers drug resistance in acute myeloid leukemia (AML), we investigated the role of ARC in regulating leukemia-mesenchymal stromal cell (MSC) interactions. In addition to the previously reported effect on AML apoptosis, we have demonstrated that ARC enhances migration and adhesion of leukemia cells to MSCs both in vitro and in a novel human extramedullary bone/bone marrow mouse model. Mechanistic studies revealed that ARC induces IL1β expression in AML cells and increases CCL2, CCL4, and CXCL12 expression in MSCs, both through ARC-mediated activation of NFκB. Expression of these chemokines in MSCs increased by AML cells in an ARC/IL1β-dependent manner; likewise, IL1β expression was elevated when leukemia cells were co-cultured with MSCs. Further, cells from AML patients expressed the receptors for and migrated toward CCL2, CCL4, and CXCL12. Inhibition of IL1β suppressed AML cell migration and sensitized the cells co-cultured with MSCs to chemotherapy. Our results suggest the existence of a complex ARC-regulated circuit that maintains intimate connection of AML with the tumor microenvironment through NFκB/IL1β-regulated chemokine receptor/ligand axes and reciprocal crosstalk resulting in cytoprotection. The data implicate ARC as a promising drug target to potentially sensitize AML cells to chemotherapy.

Keywords: AML; ARC; NFκB; chemoresistance.

Figure 1. ARC regulates leukemia-stromal interactions

(A) Adhesion and migration of ARC KD OCI-AML3 and ARC OE KG-1 or their control cells to MSCs. (B) Adhesion and migration of OCI-AML3 and BM cells from AML patients to ARC KD or control MSCs. Adhesion was determined at 24 h and migration at 6 h for OCI-AML3 and 24 h for patient samples. (C) ARC KD or control OCI-AML3 cells (expressing GFP) were co-cultured with MSCs (expressing RFP) growing on the surface of bone chips. Attachment of leukemia cells to MSCs was measured at 24 h by confocal microscopy. A representative image and the quantifications from four images are shown on the left and viability and growth curves of ARC KD and control OCI-AML3 cells are shown on the right. (D) Molm13 cells (1 × 10⁶) stably expressing a dual firefly luciferase–GFP reporter were injected into the tail vein of NSG mice harboring extramedullary bone/BM developed from either ARC KD or control human MSCs on each flank. Leukemia burden was monitored by IVIS imaging. Vec, vector control.

Figure 2. ARC regulates CXCL12, CCL2, and CCL4 production in MSCs and promotes chemokine-mediated leukemia-stromal interactions

(A) Expression of various C-X-C and C-C motif chemokines in ARC KD and control MSCs determined by quantitative PCR array. (B) Chemotaxis of OCI-AML3 cells to CCL2 and CCL4 in the presence or absence of antibodies or inhibitors against CCL2, CCR2, CCL4, and CCR5 for 4 h. (C) Correlation of CCR2, CCR5, and CXCR4 expression in BM cells from patients with AML (n = 8) and migrations to CCL2, CCL4, and CXCL12. (D) Migration of OCI-AML3 to ARC KD or control MSCs in the presence or absence of antibodies or inhibitors against CCL2, CCR2, CCL4, and CCR5 for 6 h. Chemotaxis to CXCL12 (100 ng/ml) was used as positive and random migration as negative controls. hCCL2 and hCCL4, neutralizing antibodies for CCL2 and CCL4, respectively.

Figure 3. ARC in AML modulates CCL2, CCL4, and CXCL12 expression in MSCs

(A) MSCs were cultured alone or with ARC KD OCI-AML3, ARC OE KG-1, or the respective control cells for 48 h and MSCs were FACS sorted conservatively as marked in the boxes for CD45⁻CD90⁺ cells. (B) CCL2, CCL4, and CXCL12 levels in MSCs were determined by quantitative RT-PCR.

Figure 4. ARC regulates IL1β in AML cells and triggers chemokine productions by MSCs

(A) Cytokine expressions in ARC KD, ARC OE, and the control AML cells determined by real time RT-PCR. (B) The expression of IL1β in the supernatant of ARC KD and control OCI-AML3 cells determined by ELISA. (C) The expression of human IL1β in the serum of NSG mice three weeks after the mice were injected with ARC KD or control Molm13 cells determined by ELISA. (D) The expression of CCL2, CCL4, and CXCL12 in MSCs treated with IL1β (100 ng/ml) or conditioned medium from control or ARC KD OCI-AML3 cells with or without IL1βRA for 24 h. *P < 0.05.

Figure 5. MSCs induce the expression of IL1β in AML cells

OCI-AML3 cells were cultured alone or co-cultured with ARC KD or control MSCs for 48 h. CD45⁺CD90⁻ AML cells were FACS-sorted from the floaters (collected from cells in the suspension and after PBS wash) and attached (collected by trypsinization) cells. IL1β RNA levels were determined in sorted OCI-AML3 cells and the cells cultured alone by real-time RT-PCR.

Figure 6. Inhibition of IL1β suppresses adhesion and chemotaxis of leukemia cells to MSCs and abolishes chemoprotection of MSCs to leukemia cells

(A) OCI-AML3 cells were added to MSCs which were set the night before and treated with ILβRA (100 ng/ml). Cell adhesion was determined after 24 h. OCI-AML3 cells were pre-treated with ILβRA (100 ng/ml) for 1 h and their migration towards MSCs was determined at 6 h. (B) OCI-AML3 cells, cultured alone or with MSCs were treated with Ara-C in the presence or absence of ILβRA (100 ng/ml). Apoptosis was assessed at 48 and 72 h by flow cytometry after staining cells with annexin V (AnnV) and 7-aminoactinomycin D (7AAD).

Figure 7. ARC regulates NFκBp65 signaling

(A) N-terminal region of ARC adopts an atypical CARD domain fold where the sixth helix is unfolded. The model shown is color-ramped from the N-terminus (blue, residue 1) to the C-terminus (red, residue 95). The program SwissModel was used to model residues 87-90 which are not included in the PDB model (entry 4UZ0). (B) The expression levels of NFκBp65 protein in cytoplasmic/nuclear fractions determined by western blot and the expression levels of p-NFκBp65 and ARC proteins by CyTOF in ARC KD and OE cells. (C) The C-terminal of ARC is most likely disordered. YDPP (positions 105–108) is a SH2-domains binding motif and TPEE (positions 149–152) is a TRAF2-binding consensus motif (both underlined with blue). Asterisks (*) identify the start and end of the stably folded CARD domain of ARC in the crystallographic structure 4UZ0. (D) A putative mechanism of ARC-regulated leukemia-stromal interactions through NFκB/IL1β signaling.