Apoptosis Repressor with Caspase Recruitment Domain (ARC) Increases AML Cell Migration and Adhesion in Vitro and in Vivo by Regulating Leukemia-Stroma Interactions.
Authors: Carter Bing Z , Mak Po Yee , Chen Ye , Jacamo Rodrigo , Ruvolo Vivian , Mak Duncan H , McQueen Teresa , Konopleva Marina , Kornblau Steven M. , Andreeff Michael 
Published: October 2019

Abstract
Abstract 2626
Hematopoietic cells express a wide range of adhesion molecules and bone marrow (BM) stroma cells produce their corresponding ligands. Through these ligand-receptor pairs, hematopoietic cells interact with their BM microenvironment. The same system is hijacked by AML and often adhesion molecules in leukemia cells and/or their ligands in stroma cells are upregulated, promote leukemia-stroma interactions, and protect leukemia cells from therapeutic agents. Understanding the underlying mechanisms is critical for therapeutic strategies aimed at disrupting leukemia-stroma interactions. For example, pharmacological blockage of the CXCR4-SDF1a axis has been shown to result in chemosensitization of AML cells in vitro, in vivo and in clinical trials (Zeng Z et al., Blood 2009; Uy GL et al., Blood 2012; Andreeff M et al., ASCO 2012).
ARC (Apoptosis repressor with caspase recruitment domain) is an antiapoptotic protein. We recently determined ARC expression in samples from 511 newly diagnosed AML patients by reverse-phase protein array and reported that ARC is one of the strongest adverse prognostic markers in AML (Carter BZ et al., Blood 2011). In the same sample set, we also probed the expression of more than 200 additional proteins enabling us to correlate ARC expression with the expressions of other proteins. Surprisingly, ARC expression was correlated with multiple proteins involved in cell adhesion and migration. We generated stable ARC overexpressing (O/E) KG-1 cells, ARC knock down (K/D) OCI-AML3 and Molm13 cells, and ARC K/D BM derived mesenchymal stroma cells (MSCs) to investigate ARC's roles in leukemia-stroma interactions. Expression levels of FAK, integrinb3, fibronectin, and VLA4 were increased in ARC O/E and decreased in ARC K/D cells, compared to controls. CXCR4 mRNA and cell surface CXCR4 protein were higher in ARC O/E KG-1 (3.80- and 1.53-fold, P<0.01; respectively) and lower in ARC K/D cells (OCI-AML3: 0.43- and 0.70-fold, P=0.01, and Molm13: 0.46- and 0.74-fold, P=0.02, respectively), while levels of SDF1a mRNA as well as secreted SDF-1a protein determined by ELISA (P=0.013) were lower in ARC K/D as compared to control MSCs. Interestingly, MSCs co-cultured with AML cells significantly increased SDF1a secretion and this increase was greatly diminished when ARC was knocked down in MSCs.
Migration of leukemic cells towards MSCs was significantly higher for ARC O/E and lower for ARC K/D AML cells (P<0.001) and migration of leukemic cells to ARC K/D MSCs was significantly diminished compared to control MSCs for both, OCI-AML3 (P<0.001) and primary AML cells (18.1±0.8% vs. 32.8±0.5%, n=4, P<0.001). In addition, adhesion to MSCs and to VCAM1 coated plates was increased for ARC O/E and decreased for ARC K/D AML cells (P<0.01). Furthermore, adhesion to ARC K/D MSCs was decreased compared to control MSCs for AML cells for both cell lines (KG-1, 43.6±1.2% vs. 58.2±1.7%; OCI-AML3, 34.8±1.0% vs. 48.7±0.7%; Molm13, 9.2±2.2% vs. 16.8±2.7%) and primary patient samples (52.1±1.6% vs. 66.3±6.4%, P=0.02 and 42.1±5.0% vs. 59.6±7.4%, P=0.03).
The role of ARC in leukemia-stroma interactions was further assessed using a novel human extramedullary BM model in mice recently developed by our group (Chen Y et al., Blood 2012). Luciferase/GFP Molm13 cells were injected into Nod/ScidIL2Rg−/− mice after extramedullary human BM containing either ARC K/D or control MSCs was established in the flanks of mice. Significantly fewer AML cells engrafted in the human extramedullary BM developed from ARC K/D than from control MSCs as determined by immunohistochemistry staining for human CD45+ cells and quantitative image analysis (P<0.01).
In conclusion, we here demonstrate that antiapoptotic ARC regulates migration and adhesion of leukemia cells to BM stroma in vitro and in vivo via activation of multiple mechanisms, not only in AML cells but also in MSCs, thus enhancing its anti-apoptotic activity. Given the fact that both intrinsic apoptosis resistance and extrinsic environmental factors contribute to drug resistance and relapse in AML, ARC may play a central role in AML and be an excellent therapeutic target: ARC inactivation may disrupt leukemia-stroma interactions and increase leukemia cell sensitivity to chemotherapy by increasing susceptibility to apoptosis. We speculate that ARC, a CARD containing protein, acts at least in part via activating NF-kB signaling, which is known to be regulated by CARD proteins.

Disclosures:
No relevant conflicts of interest to declare.