Diffuse Large B Cell Lymphoma (DLBCL)-Released NM23-H1 Promotes Monocyte Survival and Inflammatory Cytokine Release: A Mechanistic Link between the Dual Impacts of NM23-H1 Expression and Reduced Lymphocyte:Monocyte Ratio in DLBCL Prognosis

Elevated circulating levels of NM23-H1 have been associated with poor prognosis in haematological malignancies including AML and non-Hodgkin's lymphomas. In diffuse large B-cell lymphoma (DLBCL), several studies have demonstrated that both elevated circulating plasma levels and intra-tumoural levels of NM23-H1 correlate with poorer prognosis. These observations bring into question whether the mechanistic link of NM23-H1 expression with DLBCL prognosis depends an intrinsic intracellular mechanisms or is mediated by release of NM23-H1.

We have shown that DLBCL lymphoma cell lines (Farage, HT, OCI-LY1, OCI-LY3, OCI-LY7, SUDHL4, SUDHL5 SUDHL6, U2932) express both NM23-H1 protein and the highly related protein NM23-H2. We also demonstrated that DLBCL cell lines release significant levels of NM23-H1 into their extracellular environment but not NM23-H2. Release of NM23-H1 was highly variable between cell lines with some releasing very high levels and some releasing much lower levels. Thus, DLBCL cell lines represent appropriate models to investigate the role of high and low levels of NM23-H1 on prognosis. The DLBCL cell lines HT and OCI-LY1 where selected for further study as representative of high and low level releasers of NM23-H1, respectively. CRISPR-Cas9 knockout of NM23-H1 (KO-NM23-H1) in HT and OCI-LY1 DLBCL cells had no impact on cell growth, viability or immuno-phenotype either in normoxic or hypoxic cultures. Thus NM23-H1 appears to not be required intrinsically by DLBCL cell lines. We therefore considered that the link between higher expression and release of NM23-H1 with prognosis is mediated via tumour-host environment interactions. We used an NSG mouse model transplanted subcutaneously with 1x10⁶ CRISPR control (CTRL)-HT, CTRL-OCIL-Y1, KO-NM23-H1-HT or KO-NM23-H1-OCI-LY1 cell lines. We did not observe significant differences in tumour growth between CTRL and KO-NM23-H1 cells in the low NM23-H1 expressing OCI-LY1 DLBCL cell line. However, the high-expressing KO-NM23-H1-HT cells had significantly slower tumour progression and increased host survival when compared to CTRL-HT cells. We interpret to indicate that NM23-H1 release above a certain threshold provides a tumour growth advantage.

Reduced lymphocyte:monocyte ratios (LMR) are also associated with poor prognosis in DLBCL. To investigate a potential functional link between NM23-H1 release and monocyte behaviour we first exposed peripheral blood leucocytes to Alexa 647 labelled fluorescent recombinant-NM23-H1 and found that monocytes but not neutrophils, T-cells or B-cells bound NM23-H1. We also observed that monocyte viability and survival were elevated in serum free cultures when supplemented with rNM23-H1, an observation that might indicate that elevated circulating NM23-H1 and reduced LMR in poor prognosis DLBCL may be mechanistically linked. We next co- cultured CTRL-HT or koNM23-H1-HT in a 1:1 ratios with purified monocytes from healthy human donors. Principle component analyses of 27 cytokines simultaneously measured by luminex assays identified that IL-1β, IL-6, IL-8, MIP-1α, MIP-1β and TNF-α were elevated when monocytes were co-cultured with CTRL-HT cells compared to co-culture with koNm23-H1-HT cells. In contrast, IP-10 was found elevated in the co-culture with koNm23-H1-HT cells. These observations indicate potentially important cross talk between the malignant DLBCL cells and innate immune cells of the host. Consistent with this, IL-6 and IL-8 serum levels have been shown to be elevated in pretreatment DLBCL patients compared to control subjects and MIP-1α, IL-6, and IL-8 serum levels have a greater association with DLBCL than follicular lymphoma.

In conclusion ours is the first study to investigate potential mechanistic links between the associations of elevated NM23-H1 and poor prognosis in DLBCL and indicate a role for cross-talk between tumour cells and innate immune cells. Our data also indicate that NM23-H1 release from DLBCL cells may be a driving component in driving reduced LMRs that are also associated with poor prognosis. Whether reduced LMR and NM23-H1 release are casually related or not, the possibility that patients with both elevated monocytes and elevated circulating NM23-H1 levels are likely to represent a group with particularly poor prognosis requires urgent investigation.