Researchers suggest anticancer kinase inhibitor candidates may have additional immunological effects in vivo.

Researchers have demonstrated that silencing the JAK1 and JAK 2 genes in cancer cells increases the susceptibility of a range of tumor types to the cytolytic activity of natural killer cells. A team at the Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and Harvard Medical School designed a lentiviral shRNA screen to identify which of more than 1,000 genes in a multiple myeloma tumor cell line were involved in protecting the cells from NK cell lysis.

The results identified a set of 83 genes that, when silenced, increased the susceptibility of the tumor cells to the activity of different NK cell lines with which they were subsequently incubated. Of the genes identified in the screen, 79% were kinases, 14.4% were proteins with nonkinase functions, and just 4.8% were phosphatases.

Many of the 83 genes identified were found to be involved in common intracellular pathways including MAPK, PIK3, IGF1R, JAK1, and JAK2, Dana-Farber’s Jerome Ritz, M.D., and colleagues, report. In fact 18% of the total gene set and 22% of the 66 kinases identified were involved in the MAPK pathway. Subsequent studies showed that silencing a number of the 83 genes or inhibiting proteins directly using small molecule compounds was often enough to increase sensitivity to NK cell activity.

Interestingly, the authors state in their paper in The Journal of Clinical Investigation, of all the genes identified, “none of them have previously been known to modulate susceptibility of human tumor cells to immunologic destruction.” Their published paper is titled “Tyrosine kinase pathways modulate tumor susceptibility to natural killer cells.”

NK cells don’t express clonal recognition receptors and also don’t recognize unique target antigens, but the cells do play a key role in immune surveillance and coordinating the responses of other immune cells, the authors explain. Although most tumor cells express surface molecules that can be recognized by activating receptors on NK cells, they often develop as yet unidentified mechanisms to evade innate immune surveillance.

The researchers thus established their screen to identify which genes may be involved in these NK cell evasion mechanisms. “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,” they explain.

The lentiviral library used for the shRNA screen was a kinase/phosphatase subset of The RNAi Consortium (TRC) library, which targeted 1,028 genes, including more than 88% of the known human protein kinases and phosphatases. After gene knockdown, the tumor cells were incubated with either a functional human NK cell line NKL or an additional NK effector cell line NK-92.

 Of the 83 genes identified through the screen, two that had the most significant effect on NK cell activity were JAK1 and JAK2. Analyses indicated that reduced expression of either JAK1 of JAK2 in the tumor cells resulted in significantly higher levels of IFNγ production by NK effector cells and higher levels of NK cell-related tumor cell lysis and apoptosis. Notably, knocking down other members of the same gene family, including JAK3 and TYK2, didn’t impact on tumor cell susceptibility to NK cell-mediated death.

The team went on to confirm that shRNA-mediated JAK1 or JAK2 knockdown similarly increased the susceptibility of multiple myeloma tumor cells to lysis when incubated with peripheral blood mononuclear cells PBMCs or purified NK cells from healthy human donors. Notably, using shRNAs to knock down the two genes in other tumor cell lines also led to increased INF-γ secretion by NK cells and tumor cell lysis, to varying degress. Cell lines tested included additional myeloma cell lines, acute myeloid leukemia, chronic myeloid leukemia, and acute T-cell leukemia cell lines. And importantly, treating different cancer cell lines with small molecule JAK inhibitors also increased their susceptibility to the cytolytic effects of NK cells including purified primary human NK cells.

To test the relevance of their findings to a clinical setting, the researchers examined the effects of JAK inhibitors on primary tumor cells from patients with different hematologic malignancies including multiple myeloma, acute myeloid leukemia, and acute lymphoblastic leukemia. Tumor cells were incubated with different concentrations of a JAK1 inhibitor and then incubated with an NK cell line. In each case the JAK inhibitor-treated cancer cells were rendered more susceptible to NK activity, resulting in 20–53% increases in tumor cell apoptosis.

Gene expression analysis indicated that 34 genes were highly differentially expressed (either up- or downregulated) in the tumor cells after JAK1 silencing, although these included none of the common activating or inhibitory NK cell ligands that are known to play a role in modulating NK cell activity. Two genes of particular interest found to be highly upregulated in the JAK1-knockdown cells were TNFRSF10A (Trail-R1) and CXCL10, which have both been shown to play key roles in NK cell recognition and activation. Increased expression of both genes was also confirmed in JAK2-knockout tumor cells. And in fact, when JAK1- and JAK2- knockdown cells were incubated with NK cells in the presence of antibodies targeting Trail-R1 and CXCL10, NK cell reactivity was markedly reduced.

“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 authors conclude. “Importantly, many of these pathways are also being targeted by specific inhibitors for potential use as therapeutic agents … These findings may also have important clinical implications and suggest that small molecule inhibitors of these kinases that are being developed as direct therapeutic antitumor agents may also have important immunologic effects in vivo.”

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