Enhancement of Tumor Cell Susceptibility to NK Cell Activity Through Inhibition of the PI3K Signaling Pathway.

Abstract 2152

NK cells are the primary effectors of the innate immune response against cells that have undergone malignant transformation. Unlike T cells, NK cells recognize their targets through a complex array of activating and inhibitory receptors, which regulate the intensity of the NK response against individual cells. Tumor cells have developed ways to escape killing by NK cells through a variety of mechanisms that are poorly understood. Using shRNA libraries, we previously demonstrated that several common signaling pathways modulate susceptibility of various tumor cells to NK cell activity. In this study we focused on one of the PI3K sub-units (PI3KCB, p110β isoform) that was identified in this genetic screen. The PI3K pathway has been linked to diverse cellular functions including cell growth, proliferation, survival and intracellular trafficking but PI3K signaling has not previously been associated with susceptibility to NK cells.

To examine the mechanisms of PI3KCB involvement in tumor susceptibility to NK cell lysis, we tested 4 tumor cell lines representing different hematologic malignancies: IM9-multiple myeloma, U937-acute myeloid leukemia, K562-chronic myeloid leukemia and Jurkat-acute T cell leukemia. Stable cell lines were established expressing 4 independent shRNAs (P1, P2, P3, P4-shRNA) and 2 irrelevant shRNAs as controls. Western blot and RT-PCR showed different silencing activity of individual PI3KCB-shRNAs with efficient silencing of PI3KCB by 3 of 4 shRNAs in IM9 (P1, P2, P4-shRNA), 2 of 4 in K562 and U937 (P1, P2-shRNA) and 2 of 4 in Jurkat (P2, P4-shRNA). No PI3KCB gene silencing or protein down regulation was observed with P3-shRNA that was used as an additional negative control. PI3KCB silencing did not affect the proliferative capacity of the 4 tumor cell lines tested over a 5 day period. When compared with irrelevant shRNAs or P3-shRNA, effective silencing of PI3KCB in IM-9, K562 and U937 induced increased IFNγ secretion when target cells were incubated with primary purified NK effector cells and 2 different human NK cell lines (NKL and NK-92). Silencing of PI3KCB in Jurkat target cells did not affect IFNγ secretion by NK cells. To determine whether increased IFNγ secretion by NK cells correlated with increased lytic activity, we incubated target cells with NK effectors overnight and assessed cytotoxicity using AnnexinV/7AAD. Lysis of IM9-PI3KCB-P1, P2 and P4-shRNA was increased by 10%-15% compared to irrelevant controls (P=0.04, P=0.04 and P=0.03 respectively); lysis of U937-PI3KCB-P1 and P2-shRNA was increased by 20% and 22% (P=0.02 and P=0.04 respectively); and lysis of K562-PI3KCB-P1 and P2-shRNA was increased by 10% and 20% (P=0.04 and P=0.04, respectively). Correlating with IFNγ assays, increased lysis was not detected for the Jurkat-PI3KCB-silenced cell lines.

To define mechanisms responsible for increased susceptibility to NK cells, we examined the expression of several activating/inhibitory ligands on the membrane of PI3KCB-silenced tumor cells. Flow cytometry demonstrated increased expression of MICB (NKG2D ligand) and CD48 (2B4 ligand) in IM9-PI3KCB-KO lines; increased expression of MICA, MICB, CD48 and CD112/CD115 (DNAM-1 ligands) in K562-PI3KCB-KO lines; and increased expression of CD48, CD112 in U937-PI3KCB-KO lines. Reduced expression of MHC class I was also found in IM9 and U937 lines after PI3KCB silencing. Importantly, none of the activating/inhibitory ligands tested were modulated in Jurkat cells after PI3KCB silencing. Co-culture experiments with NK cells and blocking antibodies targeting NKG2D significantly reduced NK IFNγ secretion against IM9-PI3KCB-KO and K562-PI3KCB-KO targets when compared to controls. In contrast, DNAM-1 blocking in U937-PI3KCB-KO produced a uniform decrease of IFNγ secretion in U937-PI3KCB-KO and controls suggesting that in this target, modulation of other NK ligands could also be involved. These data demonstrate that the beta sub-unit of PI3K (PI3KCB) can modulate tumor susceptibility in different hematologic malignancies and that this effect is, at least in part, due to modulation of several activating/inhibitory ligands. These findings identify a new and important role of PI3KCB in modulating tumor cell susceptibility to NK cells and open the way to future combined target immunotherapies.