The p90RSK Inhibitor BI-D1870 Induces Apoptosis in CLL Cells

Abstract 2492

Chronic Lymphocytic leukaemia (CLL) is the most common leukemia in the western world but is currently incurable using conventional therapies. Therefore novel agents which can kill CLL cells either alone or in combination with conventional agents are being continually sought. Activation of the MAPK pathway has been implicated as one of the protective signaling pathway in CLL and is characterised by expression of phosphorylated ERK. In CLL cells ERK can be activated by phosphorylation after BCR activation with αIgM. However in a proportion of CLL isolates ERK is constitutively active. MEK inhibitors (MEKi) are currently being used in clinical trials to treat relapsed or refractory leukemia including CLL (www.clinicaltrials.gov, NCT00920140). Therefore we hypothesised that inhibiting the ribosomal S6 kinase p90^RSK which is immediately downstream of the MAPK pathway, may be of therapeutic value for the treatment of CLL. Since p90^RSK regulates the majority of the anti-apoptotic signaling of the ERK pathway, we investigated the effect of inhibiting p90^RSK with its specific inhibitor BI-D1870 (BI-D) on apoptosis in CLL.

Immunoblotting was carried out to investigate phosphorylation of components of the ERK/p90RSK pathway using phosphorylation state-specific and pan-reactive antibodies. BI-D toxicity was evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form.

BI-D treatment of CLL cells decreased S6 phosphorylation, and this down regulation correlated with apoptosis. BI-D killed CLL cells rapidly, with a mean IC₅₀ of 12μM following 18h incubation. Treatment of CLL cells with BI-D induced cytochrome C release indicating inhibition of p90^RSK induced apoptosis by the intrinsic death pathway. However its toxicity was not due to inhibition of protein translation since inhibition of mTOR, a parallel pathway which also regulates S6 phosphorylation, was minimally toxic to CLL cells at concentrations as high as 500nM. However apoptosis did coincide with a reduction of the antiapoptotic protein Mcl-1. We further demonstrated that BI-D augmented apoptosis induction by cytotoxic drugs, as shown by PARP cleavage and by annexin V/PI binding assays. Analysis of cytotoxicity data using CalcuSyn software provided evidence for synergistic killing of the majority of CLL isolates by combinations of BI-D with either chlorambucil, fludarabine or nutlin 3.

Constitutive or antigen induced activation of p90^RSK via the MAPK pathway in CLL cells may contribute to apoptosis resistance and its inhibition may be of value in devising novel therapeutic strategies, either alone or in conjunction with conventional cytotoxic drugs. p90^RSK inhibition led to apoptosis independently of protein translation inhibition and may be attributable in part to downregulation of Mcl-1. Given the recent interest in using MEK inhibitors (MEKi) for the treatment of CLL, these data suggests MEKi may induce apoptosis in CLL in part by inhibiting pathways downstream of p90^RSK. In addition BI-D is a highly specific inhibitor of p90^RSK and therefore may negate the off target effects shown with some MEKi. This is the first report on the consequences of inhibition of p90^RSK in CLL cells and suggests that inhibiting p90^RSK may be of considerable interest for the treatment of CLL.