Abstract 994: PU.1 inhibition confers resistance to TRAIL- and anthracycline-mediated apoptosis through NF-κB activation and TRAIL receptor downregulation in acute myeloid leukemia cells
Authors: Haimovici Aladin , Kaufmann Thomas , Tschan Mario P. 
Published: August 2015

Earlier studies found that acute myeloid leukemia (AML) cells show resistance to TRAIL, but did not reveal the exact mechanism. The myeloid master regulator PU.1 is a key molecule for myeloid and B-cell development. Its inactivation results in a differentiation block and contributes to the pathogenesis AML. On the other hand, several reports directly linked PU.1 to cell death. We now aimed at analyzing the role of PU.1 in TRAIL-mediated apoptosis of AML cells. We found that inhibiting PU.1 significantly increased resistance to TRAIL-induced apoptosis in several AML cell lines (NB4, HL60, MOLM13) as seen by significantly decreased AnnexinV/PI staining and caspase-3/7 activation. Interestingly, this resistance was specific to TRAIL-induced apoptosis since TNFα induced similar levels of cell death in control and PU.1 knockdown AML cells. AML PU.1 knockdown cells were further characterized by increased expression of anti-apoptotic genes such as FLIPShort, XIAP, Bcl-2 and Mcl-1 upon TRAIL treatment. Given that some of these genes are regulated by NF-κB we investigated if knocking down PU.1 activates this transcription factor. Indeed, PU.1 knockdown AML cells showed increased nuclear translocation of the NF-κB subunit p65 and increased activity of NF-κB specific reporters possibly accounting for FLIP, XIAP and Bcl-2 induction. In line with these findings, blocking the NF-κB pathway with the pharmacological NF-κB inhibitors JSH-23 and BAY 11-7085 or co-depleting FLIP restored TRAIL sensitivity in AML PU.1 knockdown cells. As a possible mechanism we found that PU.1 interacts with and inactivates the p65 protein directly. As an alternative mechanism of TRAIL-resistance in AML PU.1 knockdown cells, we observed a significant down-regulation of TRAIL receptor 1 (-R1) and -R2 levels as well as increased expression of the NF-κB-activating TRAIL decoy-receptor 2 (DcR2). To extend our study to a more clinical setting, we tested if AML PU.1 knockdown cells are resistant to anthracyclines currently used in AML therapy. As with TRAIL treatment AML PU.1 knockdown cells displayed significantly less cell death than control cells upon anthracycline treatment. This resistance was paralleled by increased expression of the above-listed anti-apoptotic factors. In summary, our results reveal a novel function of PU.1 that is keeping NF-κB in check and modulating TRAIL receptor expression. Our data suggest that PU.1 functions in resistance mechanisms not only against TRAIL- but also against anthracycline-based therapies in AML.

Citation Format: Aladin Haimovici, Thomas Kaufmann, Mario P. Tschan. PU.1 inhibition confers resistance to TRAIL- and anthracycline-mediated apoptosis through NF-κB activation and TRAIL receptor downregulation in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2015-994