Cyclic-AMP Responsive Element Binding Protein 1 (CREB) As a New Potential Druggable Target in Pediatric Acute Lymphoblastic Leukemia

New treatment options are necessary to improve survival rates for patients with Acute Lymphoblastic Leukemia (ALL), especially for patients with unfavorable prognostic predictors. As a new therapeutic approach specific protein kinase inhibitors are being developed that can down-regulate vital signaling pathways in leukemic blasts (McCubrey et al, 2008). The main goal of the present study is to obtain a better understanding of the kinase signaling pathways active in ALL cells and to identify potential targets for therapeutic intervention, To identify active signaling pathways in ALL we have used kinase activity arrays containing 1024 peptides representing all major signaling pathways and human proteome profiler arrays containing 46 phospho-antibodies on lysates of primary ALL blasts. In 20 patient samples a total of 10.6% 109(1024) peptides were found to be phosphorylated in 90% of the samples. About 46% 50(109). Activities for kinases including PKC, PKA, Akt, CAMK2, CDC2, CDK2, ERK, GSK3beta, JAK and MAPK were detected in these lysates. The human proteome profiler array demonstrated high levels of protein phosphorylation of CREB and RSK. We constructed a provisional signal transduction scheme of active kinases and phosphorylated proteins in ALL cells (Fig. 1A). Consistent with earlier reports, we identified a prominent role for the Raf/MEK/ERK and the PI3K/Akt/mTOR pathways in these ALL cells. Based on this provisional signal transduction scheme we composed a list of possible new druggable targets. Two proteins were selected for further investigation, CREB and RSK. Inhibition of RSK by the p90 RSK inhibitor BI-D1870 had no effect on cell viability as measured with WST-1 cell viability assay in ALL cell lines. Interestingly, inhibition of CREB by the CREB inhibitor KG-501 showed a dose- and time-dependent decrease in cell viability in all cell lines tested (LC50 values after 24h: Jurkat: 18.55 mM, Molt 4: 13.02 mM, RCH-ACV: 38.11 mM, and RS4;11 45.36 mM (Fig. 1B). LC50 values after 48h: Jurkat: 7.36 mM, Molt 4: 6.53 mM, RCH-ACV: 31.73 mM, RS4;11 36.66 mM (Fig. 1C)). In addition, apoptosis measured by AnnexinV/ PI staining showed an increased percentage of apoptotic cells in a dose- and time-dependent manner in all cell lines upon treatment with the CREB inhibitor (apoptosis after 24h: Jurkat 35.83% to 79.7%, Molt 4: 12.19% to 48.5%), RCH-ACV 11.30% to 45.9%, and RS4;11 9.84% to 19.16. Apoptosis after 48h: Jurkat 53.40% to 86.4%, Molt 4: 27.70% to 92.9%, RCH-ACV 14.07% to 63.32%, and RS4;11 7.11% to 20.75%) (Fig. 1D). To investigate the downstream effect of CREB inhibition we measured the mRNA expression of a know CREB target gene: BCL-2. Upon inhibition of CREB (50 mM KG-501) mRNA levels of BCL-2 were found to be significantly decreased compared to vehicle treated cells. In conclusion we have identified the transcription factor CREB in vitro as a potential druggable target for ALL. It is known that CREB plays an important role as a downstream target of hematopoietic growth factor signaling in hematopoiesis (Cheng et al, 2008). Based on these results, we propose CREB as a promising potential druggable target in ALL.