ARC Supports Leukemia-Stromal Interactions By Regulating Multiple Receptor-Chemokine Axes In AML
Authors: Carter Bing Z , Mak Po Yee , Chen Ye , Jacamo Rodrigo , Ruvolo Vivian R , Andreeff Michael 
Published: October 2019

Abstract
We have reported that “apoptosis repressor with caspase recruitment domain” (ARC), an antiapoptotic protein promotes leukemia-stromal interactions in part by increasing the expression of CXCR4 in AML cells and of CXCL12 (SDF-1) in mesenchymal stromal cells (MSCs) (Carter et al., ASH 2012). To further understand the mechanisms of action of ARC in mediating leukemia-stromal interactions, we examined the expression of multiple chemokines by quantitative cytokine PCR array in ARC knockdown (KD) and control MSCs. We found that among 6 C-X-C motif and 22 C-C motif chemokines, CXCL12, CCL2 and CCL4 are highly expressed and upregulated by ARC in MSCs.
We then determined the protein levels of CCR2 and CCR5, the respective receptors for CCL2 and CCL4, in bone marrow samples from 8 AML patients and found that CCR2 was highly expressed in all samples and CCR5 was detectable in 75 % suggesting that the CCR2/CCL2 and CCR5/CCL4 axes may also contribute to leukemia-stromal interactions in AML. Trans-well migration assays showed that CCL2 and CCL4 induced the migration of AML cells which was antagonized by blocking antibodies or small molecule inhibitors. AML cells migrated less to ARC KD than to control MSCs. Primary leukemia cells from AML patient samples also migrated towards CCL2 and CCL4, and this migration correlated with the expression of the respective receptors in the leukemia cells.
Co-culture of MSCs with leukemia cells greatly increased the levels of CXCL12, CCL2, and CCL4 in MSCs and the increase was diminished when co-cultured with ARC KD and increased when co-cultured with ARC overexpressing (OE) cells was compared to their respective controls, suggesting that chemokine expression in MSCs is in part regulated by ARC-mediated signaling from AML cells. Cytokine PCR array revealed that IL1β expression is increased in ARC OE and decreased in ARC KD cells. Conversely, the level of the IL1 receptor antagonist IL1RN is lower in ARC OE and higher in ARC KD. ARC OE cells also secreted more IL1β while ARC KD cells secreted less. Furthermore, co-cultures with MSCs increased the expression of IL1β in AML cells. IL1β greatly increased the levels of CCL2, CCL4, and CXCL12 in MSCs and the increase was largely diminished by co-treating the MSCs with IL1β antagonist IL1βRA. Similarly, treating MSCs with conditioned media from AML cells increased the expression of the chemokines in MSCs, and the increase was reduced with conditioned medium from ARC KD cells; this increase was diminished by IL1βRA. Furthermore, IL1βRA blocked the migration of these cells toward CCL2 and CCL4.
Using a 3-D model in which cancellous bone was covered with MSCs, we found fewer OCI-AML3 cells attached to MSCs when ARC was knocked down in these cells further supporting the role of ARC in leukemia-stromal interactions.
ARC is a member of caspase recruitment domain (CARD) containing proteins that have diverse functions such as antiapoptosis and regulation of NFκB activity. ARC participation in cell death suppression and leukemia-stromal interactions suggests that ARC may interact not only with apoptosis regulators but also with signaling proteins to regulate multiple cellular functions in AML.

Conclusions
Our findings suggest that multiple ARC-regulated receptor/ligand pairs play important roles in leukemia-stromal interactions, and that there is reciprocal crosstalk between malignant cells and microenvironment cells that is in part mediated through ARC-regulated inflammatory cytokine IL1β. ARC is therefore a novel target not only for direct apoptosis induction in leukemia cells but also for disruption of protective leukemia-stromal interactions to further sensitize leukemia cells to their elimination by chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.