Authors
Jodie Hay, Karen Dunn, Barbara Cadden, Colin Nixon, Jiatian Li, Andrew Clear, John G. Gribben, Alison M. McCaig & Alison M. Michie.
Ibrutinib, a selective and irreversible inhibitor of the non-receptor tyrosine kinase BTK, is responsible for improving survival rates of poor prognostic CLL patients. However, BTK inhibitors are not suitable for all patients, with some being unresponsive from the outset, or developing resistance mutations after initial treatment responses, ultimately leading to relapse. To develop new treatment strategies, we must first gain a deeper understanding of pathways involved in CLL pathogenesis. Our previous studies highlighted the importance of mRNA translation elongation as a driver of disease progression, identifying inactivation of eEF2 activity as a novel therapeutic target for blocking CLL progression. eEF2 activity can be mediated by a number of post-translational modifications, most notably through phosphorylation by eEF2K. eEF2K inhibition, through phosphorylation at residue Ser366 by p90RSK and p70S6 kinase or by SAPK4/p38delta at Ser359, can promote protein synthesis. This occurs in part through the reduction in eEF2T56 phosphorylation, thereby increasing eEF2 activity and promoting translation elongation.
Analysis of CLL patient-derived lymph node (LN) biopsies (n=27) revealed that higher p-eEF2KS366 was associated with significantly less time to treatment, indicative of higher protein synthesis being associated with poorer clinical outcome. Elevated p-eEF2KS366 further correlated with increased eEF2 expression and higher phosphorylation of ribosomal protein S6, a marker of protein translation. These data support our hypothesis that increased protein synthesis can drive CLL tumorigenesis and identify eEF2K as a potential therapeutic target Analysis also highlighted that activated ERK-MAPK (p-p42/p44 ERK-MAPKT202/Y204) significantly correlated with faster relapse time, indicative of a more aggressive disease. Active ERK-MAPK also significantly aligned with high expression of eEF2 and was associated with eEF2K inactivation (p-eEF2KS366), indicative of active protein synthesis. Together these data support rationale for therapeutically targeting ERK-MAPK-mediated protein synthesis.
To determine the effects of targeting ERK-MAPK signalling, we combined AZ6197, a novel ERK1/2 inhibitor, alone and in combination with ibrutinib (ERKi IBR). In the proliferating primary CLL-NTL-CD40L/IL21 co-culture model ERKi IBR had anti-tumour activity, by significantly reducing protein synthesis, cell proliferation and cell survival. The efficacy of ERK-MAPK inhibition was also tested in our poor prognostic CLL mouse model, which is generated by retrovirally transducing murine HSPCs with a catalytically inactive PKC⍺ construct (PKC⍺-KR) producing CLL-like cells with elevated BCR signalling. In in vitro studies using CLL-like cells ERKi IBR also induced tumour suppressive effects by reducing protein synthesis and cell proliferation compared to control or single treatment arms.
To specifically investigate the role of the eEF2K/eEF2 signalling axis in the generation of CLL in our mouse model we used the HSPCs derived from eEF2K with reduced catalytic activity (Eef2kD273A/D273A) or constitutively active eEF2 (Eef2T56A/T56A) mice to generate CLL-like cells. In this way we established that both models proliferated faster and had increased protein synthesis compared to wild-type cells. In wild-type cells, treatment with ERKi IBR induced a block in proliferation and reduced protein synthesis compared to control conditions. However, these tumorigenic effects were only partially attenuated in Eef2T56A/T56A and Eef2kD273A/D273A derived CLL-like cells with ERKi IBR, suggesting that the eEF2/eEF2K signalling is a key mechanistic target of ERKi IBR.
Collectively, these studies indicate that reduced eEF2K activity is associated with poor outcome in CLL, through the promotion of protein synthesis. These analyses also identify ERK-MAPK as a novel target for modulating protein synthesis in CLL. Through in vitro CLL modelling, and in vivo studies, we have demonstrated that targeting both ERK and BTK signalling simultaneously, to therapeutically block protein synthesis via eEF2 signalling, is an attractive clinical strategy.
Keywords : protein synthesis
therapeutic
ERK/MAPK
Please indicate how this research was funded. : Project Grant
Please indicate the name of the funding organization.: Medical Research Council (MRC)