Unlike T and B cells of the adaptive immune system, NK cells do not express clonal recognition receptors and do not recognize unique target antigens. Nevertheless, these cells play an important role in immune surveillance and coordinating responses of other immune cells. Most tumor cells express surface molecules that can be recognized by activating receptors on NK cells (25
). The expression of these receptors make such cells susceptible to endogenous NK cells, but malignant cells have developed mechanisms to evade innate immune surveillance (26
). The goal of our studies was to begin to characterize these resistance mechanisms in a broad and unbiased approach. To accomplish this goal, we designed a high-throughput genetic screen to assess interactions between tumor cell targets and NK effector cells. In this assay, tumor cell targets were first transduced with individual lentiviral shRNAs. After integration of shRNAs, NKL effector cells were added to each well, and the interaction between genetically modified target cells and NK effector cells was assessed by measurement of IFN-γ release into the cell culture supernatant. Since our goal was to identify genes that, when silenced, would increase susceptibility to NK cell–mediated lysis, assay conditions were optimized to identify shRNAs that resulted in increased IFN-γ secretion. The lentiviral library we used was a subset of the TRC library that targeted 1,028 genes, including more than 88% of the known human protein kinases and phosphatases (29
). We initially focused on protein kinases and phosphatases, since these genes are involved in many cellular functions and their deregulated activity occurs frequently in cancer, where this class of proteins regulates many aspects of cell growth, differentiation, adhesion, and death. Interestingly, 79% of the 83 genes that modulated tumor susceptibility to NK activity were protein kinases, while only 4.8% were phosphatases, suggesting a predominant role of protein kinases rather than phosphatases in possible mechanisms of tumor resistance. While several studies have shown that kinases play important roles in immune cell activation (30
), no previous studies have suggested that these genes also play a central role in modulating tumor cell susceptibility to elimination by immune cells. Our library also contained shRNAs targeting 372 non-protein kinases, and 12 of the 83 selected genes (14.4%) belonged to this category, suggesting that future studies using a whole genome-wide screening approach could identify many other proteins involved in tumor susceptibility to innate immune surveillance.
Our screening approach was based on the ability of shRNAs to silence the expression of individual genes in tumor cell targets. To avoid off-target effects, the shRNAs included in the TRC library were designed to contain at least 3 mismatches to all known cDNAs in the human genome. We further limited the influence of off-target effects by applying strict selection criteria (11
). Among the genes that scored in the top 5th percentile, we only selected genes that induced increased IFN-γ secretion by NKL cells when silenced by at least 2 independent shRNAs, with the second shRNA scoring within the top 20th percentile. Although we employed relatively strict criteria, our approach identified a large set of 83 genes that appear to modulate target cell susceptibility to NK cells. Notably, many of these genes represent common membrane and intracellular signaling pathways that are often activated in malignant cells. For example, 15 of the 83 genes are connected to the MAPK pathway. This pathway has been shown to be involved in many cellular functions, including cell proliferation, cell cycle regulation, cell survival, angiogenesis, and cell migration and is often activated in response to cytokines and growth factors (12
). Our screen also identified several membrane receptors such as IGF1R and INSR that can signal through the MAPK and the PIK3 pathways (20
). Taken together, these results suggest that many genes can play an important role in tumor susceptibility to immune surveillance and tumor cells can engage multiple pathways and mechanisms to prevent recognition and destruction by endogenous NK cells in vivo.
Independent experiments conducted with stable cell lines incorporating individual shRNAs targeting 5 different genes identified in our screen (MAPK1, IGF1R, INSR, JAK1, and JAK2) confirmed that increased IFN-γ secretion by NKL cells was specifically associated with reduced expression of the gene in IM-9 target cells. Moreover, this association was also observed with different tumor cell targets and an additional NK effector cell, NK-92, as well as primary NK cells. Finally, increased susceptibility could be measured by increased lysis of target cells as well as by increased secretion of IFN-γ. Overall, 14 of 15 different shRNAs targeting 5 different genes were validated, and only 1 shRNA (INSR-4) was found to induce increased secretion of IFN-γ by NKL cells without measurable downregulation of protein expression. These results support the overall design of our genetic screen and the strict criteria we established to identify genes with functional activity in our assay.
Additional studies focused on JAK family genes, since 2 of the 4 members of this family (JAK1 and JAK2) were identified in our screen. This family of kinases has been functionally well characterized and is known to be associated with cell surface receptors for growth factors, cytokines, chemokines, and immune modulators (32
). These kinases play a critical role in cell growth, survival, and development, and activating mutations have been associated with malignant transformation (34
). These genes have not previously been associated with tumor cell susceptibility, but because of their importance in many pathways, several specific inhibitors of JAK activity have been developed. For example, a JAK3 inhibitor (CP-690 550) has been found to have immune-suppressive activity in organ transplantation models (36
), and clinical trials are underway to test its efficacy in rheumatoid arthritis, psoriasis, and renal transplant rejection. JAK2 inhibitors have potent antitumor activity in solid tumor models (37
) and can induce apoptosis of acute lymphoid leukemia and AML cells (38
) in combination with other agents. In our studies, we found that silencing of JAK1
genes increased tumor cell susceptibility to NK cells but silencing the other 2 members of this family (JAK3
) did not have any effect. These results were confirmed in independent experiments where 3 of 4 JAK3 shRNAs and 2 of 4 TYK2 shRNAs selectively downregulated specific protein expression but had no effect on target cell susceptibility to either NKL or NK-92 effector cells. In contrast, silencing of either JAK1 or JAK2 enhanced susceptibility of various tumor cell lines, demonstrating for the first time to our knowledge that these proteins play an important role in tumor cell susceptibility to NK cell lysis. Gene expression profiling experiments showed increased expression of TRAIL-R1 and CXCL10 in IM-9-JAK1-KO cells. However, many known inhibitory/activating ligands such as HLA class I, HLA-A, HLA-C, NKG2D or NCR ligands, CD48 (2B4 ligand), CD155 (DNAM-1 ligand), CD112 (PVR ligand), CD95 (FAS ligand), and adhesion molecules important for cell-cell interactions such as ICAM-1, VCAM-1, CD49d, CD49b and CD49e were not modulated by JAK1 silencing. TRAIL-R1 and CXCL10 have been associated with NK cell recognition and activation (22
), and their overexpression was confirmed in JAK2-KO as well as JAK1-KO cells. Blocking experiments showed that while CXCL10 antibodies significantly blocked only the reactivity against JAK1- and JAK2-KO lines, TRAIL-R1 equally blocked the reactivity against JAK1-KO, JAK2-KO, as well as irrelevant controls. These findings suggest that the increased susceptibility of JAK1-KO and JAK2-KO cells could be mostly related to factors secreted by target cells rather than upregulation of activating ligands. CXCL10 antibodies did not completely block the reactivity to the level of the control lines, suggesting that other factors may still contribute to the mechanism. Further experiments will be necessary to gain an understanding of how and whether other molecules are related to the mechanism whereby JAK1 and JAK2 regulate the susceptibility of tumor cells to killing by human NK cells.
To establish the activity of JAK1 and JAK2 as modulators of susceptibility to NK cell lysis, we also tested 2 small molecule inhibitors of JAK1 and JAK2 kinase activity. These studies confirmed that inhibition of these genes in various target cells enhances their susceptibility to apoptosis induced by NK cells. This included primary tumor cells from patients with MM, AML, and ALL, as well as tumor cell lines. This effect of JAK inhibitors was mediated entirely through their inhibition of JAK1 and JAK2 signaling, since they had no effect in tumor cell lines that had already been silenced for these genes. Previous studies have shown that various kinase inhibitors such as dasatinib, which targets SFK and Abl, can also suppress T and NK functions in vivo, suggesting that they could be used as immunomodulatory drugs in autoimmune diseases when administered at higher doses (39
). In contrast, kinase inhibitors approved for treatment of renal cell carcinoma such as sorafenib and sunitinib (41
) showed differential effects on immune cells activity, especially NK cells (43
). Although the JAK inhibitors we used in our experiments did not influence the function of NK cells in vitro, the choice and dose of inhibitors used for antitumor treatment should be carefully evaluated when they are combined with immunotherapeutic approaches in patients with cancer.
Taken together, our studies have identified a large set of genes representing several common signaling pathways that appear to modulate tumor cell susceptibility to human NK cells. The unexpected functional role of these genes was uncovered in an unbiased genetic screen, suggesting that many signaling pathways can be utilized by tumor cells to escape immune surveillance. Importantly, many of these pathways are also being targeted by specific inhibitors for potential use as therapeutic agents. Our studies suggest that targeting specific members of these pathways may also enhance the susceptibility of such agents to immune destruction in vivo and this additional activity may enhance the antitumor efficacy of these new therapies.