JAK1 and JAK2 Modulate Myeloma Cell Susceptibility to NK Cells Through the Interferon Gamma (IFN-γ) Pathway,

Abstract 3960 Multiple myeloma (MM) is a B cell neoplasm characterized by clonal expansion of malignant plasma cells in the bone marrow. Despite the use of new drugs such as lenalidomide and bortezomib, MM remains an incurable disease. Successful treatment of MM with allogeneic stem cell transplantation suggests that MM is susceptible to immunologic approaches. NK cells are the primary effectors of the innate immune response against infectious pathogens and malignant transformation. Unlike T and B cells, NK cells do not recognize antigens in the context of classical major histocompatibility complex (MHC) but lyse target cells without specific antigen recognition. Nevertheless, MM cells have developed mechanisms to evade innate immune surveillance and the molecular basis for target resistance to NK cell-mediated lysis is not well understood. To identify novel pathways that modulate MM cell resistance to the immune system, we previously developed a genetic screen to detect cell-cell interactions using a large lentiviral shRNA library containing a total of 6,144 shRNAs targeting more than 1,000 human genes. Using this approach we found that silencing JAK1 and JAK2 results in significantly increased MM cell susceptibility to NK cell lysis. This effect was not noted when JAK3 and TYK2 were targeted. JAK1, JAK2 JAK3 and TYK2 are members of a family of tyrosine kinases that are constitutively associated with many membrane cytokine receptors. After activation, JAK proteins regulate phosphorylation/activation of STAT proteins, which subsequently initiate gene transcription. To understand JAK1 and JAK2 involvement in MM resistance to NK cells, we undertook a series of experiments to analyze the JAK signaling pathway in MM cells. We first analyzed the activation status of STAT proteins in a series of MM cell lines (IM-9, KM12BM, RPMI 8226, U266) in which JAK1 and JAK2 expression was reduced by specific shRNAs. Constitutive activation of STAT proteins was not affected by JAK1 or JAK2 gene silencing suggesting that these kinases were not activated in the absence of cytokine receptor-mediated signaling. Since JAK1 and JAK2 are associated with the IFN-γ receptor and we previously showed that JAK1 and JAK2 silencing induces increased secretion of IFN-γ from NK cells, we pre incubated MM cell lines with NK activated supernatant or recombinant IFN-γ and tested them for STAT activation. 15 min incubation was sufficient to initiate phosphorylation of STAT1 but no other STATs were activated. Silencing of JAK1 or JAK2 with specific shRNAs prevented STAT1 activation. To validate this finding, we tested primary MM cells treated with different concentrations of Jak inhibitor 1 (0 nM, 10 nM, 30 nM and 40 nM). These cells had a similar STAT profile at their basal level when compared with the previously tested MM cell lines. Pre-incubation with NK activated supernatant or IFN-γ also induced rapid activation of STAT1, which was completely inhibited when cells were pre-treated with Jak inhibitor 1. Treatment of MM cells with 10, 30 and 40 nM of Jak inhibitor enhanced killing by NK cells by 46.6%, 51% and 53%, compared to untreated cells (p=0.0036, p=0.0011 and p=0.0010 respectively). These findings demonstrate that IFN-γ signals rapidly enhance resistance of MM cells to NK cells but inhibition of this pathway at the level of JAK1 and JAK2 reverses this effect and induces susceptibility to NK cell mediated lysis. Disclosures: No relevant conflicts of interest to declare.