Wilm's Tumor 1 Protein Confers TRAIL Resistance in Myeloid Leukemia Cells Through Bcl-Xl

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive therapeutic agent because of its selective potential to induce apoptosis in variety of tumors but not in normal cells. However, many cancers including myeloid leukemias exhibit resistance to TRAIL and the underlying mechanisms are not fully understood. Wilms' tumor 1 (WT1) protein is an aberrantly overexpressed protein that is associated with leukemia blast survival, chemoresistance and shortened disease-free survival in acute myeloid leukemia (AML). Here, we report a previously unknown protective role of WT1 against TRAIL-induced apoptosis in leukemia cells.

Various myeloid leukemia cell lines with variable WT1 expression levels were tested for TRAIL sensitivity. The effect of pharmacological or short hairpin RNA (shRNA)-mediated inhibition of WT1 on TRAIL-induced apoptosis was evaluated by AnnexinV/PI staining method. The mRNA levels of WT1 and Bcl-xL were measured by reverse transcription-PCR (RT-PCR) and the expression of apoptosis regulators were analyzed by western blotting.

We observed a strong correlation between higher levels of WT1 and increased TRAIL resistance in myeloid leukemia cell lines. WT1 downregulation by shRNA significantly sensitized leukemia cells to TRAIL-induced apoptosis and importantly, ectopic expression of shRNA resistant WT1 (WT1^SR) in WT1-knockdown cells restored the TRAIL resistance. WT1 influences apoptosis through transcriptional regulation of Bcl-2 family members such as Bcl-2 and Bak. We found for the first time, a positive correlation between WT1 and Bcl-xL expression in leukemia cell lines and primary AML samples. Furthermore, using chromatin immunoprecipitation (ChIP) analysis, we show that Bcl-xL is a bonafide WT1-target gene as WT1 transctivates Bcl-xL by binding to its promoter. We have (Bansal et al., Blood 2010) recently shown that the expression and oncogenic functions of WT1 could be abrogated by heat shock protein 90 (Hsp90) inhibitors in myeloid leukemia cells. In order to establish whether Hsp90- mediated regulation of WT1 has any functional significance in TRAIL-resistance we treated leukemia cell with the Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG). The results confirm that Hsp90 inhibition was able to overcome TRAIL resistance, by reducing levels of WT1 and Bcl-xL and by increasing the levels of the proapoptotic protein Bak. Taken together, our results reveal that increased expression of Bcl-xL, along with low Bak, confer WT1-mediated TRAIL resistance.

Our data indicate that TRAIL resistance in myeloid leukemia cells is mediated by overexpression of the panleukemic marker WT1 and this can be overcome by either silencing WT1 or by pharmacologic inhibitors such as 17-AAG. of Hsp90. Our study highlights the potential therapeutic benefit of the combining TRAIL and Hsp90 inhibitors for the treatment of myeloid leukemias.

No relevant conflicts of interest to declare.