Kinome Profiling Identifies Phospholipase C Gamma 1 (PLCγ1) As a Potential Target for t(8;21) AML

The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML). Still about 30% of patients with t(8;21) AML struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to identify new potential druggable targets. The aim of this study was to perform kinome profiling on 29 AML patients (cytogenetically normal, CN-AML, n=17 and t(8;21), n=12) and 4 normal bone marrow (NBM) samples to identify new potential druggable targets relevant to t(8;21) AML. We defined 130 peptides differentially phosphorylated between CN-AML, t(8;21) and NBM (p<0.05). With this technique, phospholipase C gamma 1 (PLCγ1_Y783) was identified as one of the highest phosphorylated peptide in t(8;21) AML. The knowledge about the role of PLCγ1 in leukemia progression is unknown. Therefore, we selected PLCγ1 to study its functional role in t(8;21) AML cell line (Kasumi-1) and also to test its efficacy as a potential target and provide proof of principle for this approach. PLCγ1 peptide phosphorylation was significantly higher in all t(8;21) AML samples when compared to NBM or to CN-AML (p<0.001 and p<0.05 respectively). mRNA expression of PLCγ1 in a publicly available paediatric AML database (http://r2.amc.nl) also showed significantly higher expression in t(8;21) AML compared to other AML karyotypes (p<0.001). PLCγ1 silencing with the specific short hairpin RNAs (two constructs, shRNAs PLCγ1-A and shRNAs PLCγ1-B), showed 50% and 80% decrease in PLCγ1 mRNA level compared with the control (scramble shRNA). These results were confirmed by PLCγ1 protein level analysis by western blotting. The shRNA-mediated silencing of PLCγ1 leads to suppression of the Kasumi-1 cell proliferation and viability at day 8 after transduction (p<0.05). The percentage of apoptosis in PLCγ1 supressing kasumi-1 cells at day 4 was two-fold higher than in scramble (p<0.01). To understand mechanistic insights of inhibited cell proliferation of PLCγ1 knock down cells, human phosphokinase microarray was performed and higher phosphorylation of Chk2_T68 and AMPK-α1_T183 were found in these cells. Higher Chk2 phosphorylation may responsible for cell cycle arrest and induced cell death of PLCγ1 supressing kasumi-1 cells. Similarly, higher phosphorylation of AMPK-α1 may inhibited cell proliferation of PLCγ1 knockdown cells. In conclusion kinome profiling is an elegant approach to identify interesting potential druggable targets. The results from proof of principle experiments strongly support the notion that suppression of PLCγ1 inhibited cell growth and induced apoptosis in Kasumi-1 cells.