3181 - Diffuse Large B-Cell Lymphoma Cells Influence Cytokine Secretion by Monocytes via Release of Nm23-H1

Abstract

High circulating levels of the nucleoside diphosphate kinase (NDPK) Nm23-H1 are associated with worse prognosis in both Acute Myeloid Leukaemia and high grade lymphomas including high grade DLBCL. In DLBCL, the relationship with poorer prognosis is further associated with elevated intracellular Nm23-H1 as revealed by immunohistology; raising the possibility that Nm23-H1 may affect outcome by ether cell intrinsic and/or extrinsic mechanisms. We analysed a panel of DLBCL cell lines (Farage, HT, OCI-LY1, OCI-LY3, OCI-LY7, SUDHL4, SUDHL5 SUDHL6, U2932) for expression and secretion of Nm23-H1 and the closely related NDPK Nm23-H2. Western blotting of cell lysates revealed that all cell lines expressed both Nm23-H1 and Nm23-H2 at equivalence and at similar levels as seen in the AML CD34+ cell line KG1a. Analysis of DLBCL conditioned medium revealed high levels of extracellular Nm23-H1, but little if any Nm23-H2. In contrast, KG1a secreted neither Nm23-H1 nor Nm23-H2. KG1a cells bind and take up exogenous Nm23-H1 however, exposure of DLBCL cell lines to exogenous fluorescent recombinant Nm23-H1 (rNm23-H1) did not result in increased fluorescence by flow cytometry or microscopy suggesting that DLBCL cells do not significantly bind Nm23-H1. Furthermore CRISPR-Cas9 knockout of Nm23-H1 in HT DLBCL cells had no impact on cell growth, viability or immuno-phenotype. Thus it appears that expression and release of Nm23-H1 is not directly necessary for DLBCL cells, suggesting that the impact of Nm23-H1 on prognosis may be mediated by cell extrinsic mechanisms affecting the tumour microenvironment. To test this hypothesis we purified monocytes from normal donors and co-cultured them with wild type (WT) or Nm23-H1 knock-out (KO) HT cells for 48 hours. Of 27 cytokines analysed from the conditioned media IL-1, IL-6, IL-8, MIP-1, and MIP-1 were elevated when monocytes were co-cultured with WT-HT cells compared to co-culture with Nm23-H1 KO-HT cells. Thus, we hypothesise that the association of elevated Nm23-H1 with poor prognosis in DLBCL may be mediated by crosstalk between tumour cells and monocyte/macrophages either in the immediate tumour environment or beyond.