The role of NM23-H1/H2 proteins in diffuse large B-cell lymphoma

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Al-Amere, Maiss Kasem (2019). The role of NM23-H1/H2 proteins in diffuse large B-cell lymphoma. University of Birmingham. Ph.D.

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Abstract

There is increasing evidence that NM23-H1 plays important roles in normal cell development, differentiation and in cancer progression. It was found in previous studies that high expression of NM23-H1 protein is associated with poor prognosis and worse outcomes in diffuse large B-cell lymphoma (DLBCL). One possible approach is to study the role of NM23-H1 in DLBCL is to use cell line models.
The expression of NM23-H1 and related NM23-H2 was investigated in a panel of DLBCL cell lines (Farage, HT, U2932, SUDHL4, SUDHL5, SUDHL6, OCILY1, OCILY3 and OCILY7) by Western blotting and quantitative RT-PCR. These approaches confirmed high expression of NM23-H1 and -H2 indicating that DLBCL cell lines are appropriate models to investigate the role of these proteins in DLBCL. Western blotting identified for the first time that DLBCL cells release NM23-H1 into their extracellular environment whilst NM23-H2 is not released. In contrast KG1a acute myeloid leukaemia cells did not secrete NM23-H1. To investigate possible autocrine signalling by NM23-H1, DLBCL cell lines were tested for endogenous binding of NM23-H1 and binding of exogenously added fluorescently labelled NM23-H1. Neither approach suggested strong binding by DLBCL cells. Together these results supported the hypothesis that the elevated NM23-H1 serum levels detected in DLBCL patients is derived, at least partially, from tumour cell clone and that NM23-H1 protein is secreted from DLBCL cells not to act on tumour clone directly but by acting upon other cells in the host and tumour microenvironment.
The CRISPR/Cas9 approach was used to generate NM23-H1 KO clones of the HT DLBCL cell line. HT NM23-H1 KO cells grew normally and maintained high viability when cultured in either normoxic or hypoxic conditions. Furthermore, their growth and viability was not altered when provided with recombinant NM23-H1. These findings indicated that NM23-H1 is not crucial for DLBCL cell survival and that the impact of NM23-H1 on DLBCL prognosis is most likely mediated via an extrinsic cellular mechanism affecting the tumour microenvironment.
Our lab has previously identified that monocytes stained positively when exposed to exogenous recombinant NM23-H1. Given the relationships between monocytes/macrophage infiltration in lymph nodes and monocytes/lymphocytes ratios on patients’ outcomes in DLBCL this study focused on a possible interaction of DLBCL cells with monocytes via the release of NM23-H1. HT NM23-H1 knockout cells were acclimatised to serum free culture and co-cultured with peripheral blood monocytes from normal donors purified by negative selection. Monocytes survived poorly as monocultures in serum free cultures but their survival was very much enhanced in the presence of HT WT and HT NM23-H1 KO cells and also WT and NM23-H1 KO KG1a cells. The levels of 27 cytokines were measured in the supernatants of co-cultures via Luminex multiplex technology. Whilst many cytokines showed no variation in levels, IL-1β, IL-6, IL-8, MIP-1α and MIP-1β were elevated in co-cultures of WT HT cells and monocytes compared to HT KO cells, KG1a WT or KO cells. Elevated secretion of these cytokines did not require cell-cell contact between HT cells and monocytes. The commonality between HT KO, KG1a WT and KG1a KO cells is that unlike HT WT cells they do not secrete NM23-H1. Thus it is possible that secreted NM23-H1 drove the elevated secretion of IL-1β, IL-6, IL-8, MIP-1α and MIP-1β. To test this recombinant Nm23-H1 was added HT KO cell/monocyte co-cultures. Addition of NM23-H1 enhanced the secretion of IL-6 and IL-1β and to a lesser degree IL-8. However, the addition of recombinant NM23-H1 had a negligible effect on MIP-1β and MIP-1α. In summary the experiments provided here demonstrate the mechanistic relationship between elevated serum NM23-H1 in DLBCL patients and provide evidence that the prognostic relationship relates to modification of the cytokine milieu via released NM23-H1 interaction with other cells including monocyte/macrophages.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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