The Role of Tumour Microenvironmental Signals in TP53-dependent Therapeutic Strategies for Chronic Lymphocytic Leukaemia (CLL)

Abstract

Chronic lymphocytic leukaemia (CLL) is a haematologic malignancy of B cells that shows a highly heterogeneous clinical course, with approximately 90% of patient CLL samples being wild type for the TP53 tumour suppressor gene at diagnosis. CLL accounts for approximately 1000 deaths annually in the United Kingdom alone, highlighting the need to unravel the molecular mechanisms that could be used to produce more effective treatment options. Since TP53 disruption by either mutation and/or deletion occurs in only 10-15% of CLL cases, targeting MDM2 to activate TP53 in a non-genotoxic manner is a potential treatment strategy for wild type TP53 CLL. In this study, the combination of WIP1 inhibitor (GSK2830371) with RG7388 was investigated to potentiate the stabilization and pro-apoptotic effects of wild type TP53 at lower concentrations. The potential effect of the microenvironment on the response to MDM2 and WIP1 inhibitors was modelled ex-vivo, including the stimulation of CLL cells with CD40 ligand (CD40L), IL-4 and B cell receptor (BCR) signalling with anti-IgM treatment. The results identified that WIP1 inhibitor potentiated the effect of the MDM2-p53 binding antagonist (RG7388) in wild type TP53 haematological cell lines and non-proliferative CLL cells. The stabilization activity of TP53 was confirmed by the induction of downstream target genes. Furthermore, ex-vivo combined CD40L/IL-4 stimulation showed the proliferative CLL cells became more sensitive to RG7388 as a single agent treatment. The WIP1 inhibitor potentiated the effect of RG7388 and increased the expression of TP53 dependent pro-apoptotic genes. Both immobilized anti-IgM antibody and IL-4 signalling induced proliferation and cell survival signals, which was associated with the ex-vivo primary CLL cells becoming less sensitive to RG7388, and under these conditions the combination with WIP1 inhibitor also did not significantly potentiate the TP53 stabilization by RG7388. However, in the presence of IL-4 the expression of TP53 downstream target genes showed a transcriptional induction of the TP53-dependent pro-apoptotic gene (PUMA) and negative regulator of TP53 (MDM2). In summary, the WIP1 inhibitor (GSK2830371) significantly potentiated the effect of non-genotoxic small molecule MDM2 inhibitor (RG7388) in functional TP53 CLL cells. Modelling the ex-vivo microenvironment with CD40L, IL-4 and BCR activation with anti-IgM antibody was found to reduce the sensitivity of the CLL cells to both single agent RG7388 and when in combination with GSK2830371. This highlighting the importance of the in-vivo microenvironment and the need to develop combination strategies that overcome its limiting effect on the response to p53-dependent therapies.