ARC Suppresses Cell Death By Antagonizing p53 Function and Suppressing TRAIL In AML Cells
Authors: Mak Po Yee , Mak Duncan H , Kojima Kensuke , Dilip Archana , Ruvolo Vivian R , Jacamo Rodrigo , Andreeff Michael , Carter Bing Z 
Published: October 2019

Abstract
The apoptosis repressor with caspase recruitment domain (ARC) protein is a unique antiapoptotic protein as it can suppress the activation of both the intrinsic and extrinsic apoptosis pathways. ARC levels are prognostic in AML (Carter BZ et al., Blood 2011) and inhibition of ARC sensitizes leukemic cells to chemotherapy-, to the Bcl-2 inhibitor ABT-737-, and to TRAIL-induced apoptosis in AML (Mak PY et al., ASH 2012 abstract #893). Here we investigated the connection between p53 and TRAIL in the antiapoptotic function of ARC in AML.
Confocal microscopic analysis of wild type p53 OCI-AML3 and Molm13 AML cells showed co-localization of p53 and ARC, suggesting possible functional interactions between the two proteins. Contrary to a previous report (Li YZ, Mol Cell Biol, 2008), the activation of p53 by nutlin3a did not decrease ARC levels in AML cells. This was confirmed by Western blot and RT-PCR analysis.
ARC knockdown (KD) in OCI-AML3 and Molm13 cells resulted in increased expression of basal and nultin3a-induced p53 and p53 target proteins, suggesting that ARC inhibits p53 expression and antagonizes its function. Consistent with this finding, ARC KD in AML cells sensitized these cells to nutlin3a-induced cell death (EC50 for ARC KD and control OCI-AML3 cells was 2.06 ± 0.50 µM and 4.86 ± 0.05 µM, respectively, and for ARC KD and control Molm13 cells 0.59 ± 0.03 µM and 1.09 ± 0.05 µM, respectively, at 48 hours) (p<0.05).
CRM1 is known to transport p53 from the nucleus into the cytoplasm and decrease p53 transcriptional activity (Kojima K et al., Blood 2013). DR5, the TRAIL receptor is also a known transcriptional target of p53. ARC also regulated the expression of CRM1 and TRAIL. CRM1 protein levels are lower in ARC KD cells than in their controls. Conversely, TRAIL mRNA levels were lower in ARC overexpressing (OE) and higher in ARC KD cells. The combination of nutlin3a and TRAIL synergistically induced cell death in OCI-AML3 and Molm13 cells cultured alone or co-cultured with mesenchymal stromal cells (MSCs) (CI < 0.1), and this combination induced statistically significantly (P < 0.05) more cell death in ARC KD cells than in controls even when cells were co-cultured with MSCs. Similarly, combinations of nutlin3a and ABT-737 or TRAIL and ABT-737 synergistically induced cell death in AML cells cultured alone or co-cultured with MSCs (CI < 0.1). Cell death was more pronounced in ARC KD cells than in their respective controls (P < 0.05). Furthermore, the combination of TRAIL and ABT-737 synergistically induced cell death in KG-1 cells, and the cell death was significantly reduced in ARC OE KG-1 than the control cells.

Conclusions
This study identifies novel mechanisms by which ARC suppresses cell death in AML cells. Specifically, ARC antagonizes p53 function by suppressing its expression and that of its target proteins and by increasing CRM1 levels, and ARC inhibits TRAIL expression. These findings suggest that combined p53 activation and ARC inhibition can augment apoptosis induction and sensitize AML cells to chemotherapeutic agents.

Disclosures:
No relevant conflicts of interest to declare.