Introduction:NK cells play an important role in the immune response to cancer due to their ability to induce T-cell independent apoptosis of neoplastic cells. NK cells recognize tumor cells through the interaction of several distinct cell surface receptors, with the net result of inducing NK cell activation and cytotoxic attack on tumor cells. It is hypothesized that a failure of NK surveillance is a contributing factor in the development of acute myeloid leukemia (AML). The discovery of pathways that regulate AML cell apoptosis in response to NK cells activation may represent a potential therapeutic target. It is known that the Wnt/beta-catenin pathway regulates cellular proliferation, differentiation and resistance to apoptosis in AML cells. However, its contribution to the resistance of AML cells in response to NK cell-induced apoptosis resistance is currently unknown.
Objective:The objective of the current study is to determine if inhibition of the Wnt/beta catenin pathway impacts NK cell-induced apoptosis of AML cells.
Methods:HL-60 leukemia cells were treated with different concentrations of the Wnt/beta-catenin pathway inhibitor XAV-939 (tankyrase inhibitor) to determine the lowest dose capable of inhibiting the Wnt/beta-catenin pathway without inducing cell apoptosis. Inhibition of the pathway was confirmed by evaluating beta-catenin protein by intracellular flow cytometry and real-time PCR for downstream factors MYC and TCF4. Next, differentiation of HL-60 cells into mature granulocytes was evaluated by analyzing expression of CD11b by flow cytometry after stimulation with dimethyl sulfoxide (DMSO), as another marker of Wnt/Beta-catenin pathway inhibition. After confirming pathway inhibition, HL-60 cells were incubated with healthy donor derived NK cells in a 2:1 ratio for 24 hours in the presence or absence of XAV-939. Apoptosis was evaluated by annexin staining. Expression of NK cell inhibitory proteins were determined by flow cytometry. A panel of 84 genes for hematopoietic transcription factors was evaluated by PCR array (RT2 Profiler PCR Array Gene Expression - Qiagen) in HL-60 cells after exposure to XAV-939 for 2 and 6 hours. Expression of apoptosis related genes in HL-60 cells after exposure to XAV-939 was determined by real-time PCR.
Results:The dose of 1mM XAV-939 was selected to evaluate is effect in the HL-60 cell line. Suppression of pathway signaling was confirmed by a decrease in expression of Beta-catenin,MYC,TCF4and blockade of HL-60 differentiation into neutrophils. After coincubation with NK cells, there was a decrease in HL-60 cell death with concomitant exposure to XAV-939 (70% [untreated] vs. 30% [XAV-939]; p<0.05) (figure 1). There was no change in NK cell surface receptors or their putative ligands in HL-60 cells as evaluated by flow cytometry. Analysis of expression of hematopoietic transcription factor in HL-60 cells showed a downregulation of several transcription factors after exposure to XAV-939, includingJUNDandTCF7L2. Since there was a decrease in apoptotic cell death, we next evaluated expression of genes related to apoptotic pathways and could demonstrate a decrease in expression ofFASandTNFR1after exposure to XAV-939 (p<0.05) (figure 2A and 2B).
Conclusions:These results suggest that inhibition of the Wnt/beta-catenin pathway decreases apoptosis of myeloid leukemia cells mediated by NK cells, and this effect may be secondary to a decreased expression ofFASandTNFR1in the leukemic cells.
Santos:Novartis:Other: Speaker fee;Bristol-Myers Squibb:Other: Speaker fee.
