Oncogenic Role for the Lck/ZAP70/PLCG2 Signaling Pathway in Pre-B-ALL Pathogenesis

Although the treatment and prognosis of patients with pediatric acute lymphoblastic leukemia (ALL) have improved during the last decades, there is still a clinical need for more effective/selective and less toxic therapies.

To address this, we have interrogated various signaling pathways in human ALL cells and mouse strains that express E2A-PBX1, which is present in 5-7% of pediatric ALL. Phospho-flow analysis revealed basal hyper-phosphorylation levels of PLCγ2 in mouse E2A-PBX1 leukemias, consistent with hyper-activation of upstream signaling pathways. Efficient shRNA-mediated depletion of PLCγ2 reduced colony formation of mouse E2A-PBX1+ leukemias in vitro and increased disease-free survival after secondary bone marrow transplantation in vivo. Furthermore, PLCγ2-depleted human ALL cell lines including E2A-PBX1+ cells, showed reduced proliferation. These data suggest a pathogenic role of hyperactivated PLCγ2 in pre-B-ALL.

Bioinformatics analysis of E2A-PBX1 target genes in human ALLs revealed an enrichment of B- and T-cell activation pathways, which include the SRC-family kinase LCK and the cytoplasmic kinase ZAP70, upstream of PLCγ2. Comparative analyses of global transcriptional profiles in human primary and mouse leukemias and preleukemias induced by the E2A-PBX1 oncogene identified the signaling kinase ZAP70 as one of the earliest and most consistently up-regulated genes in E2A-PBX1 leukemias. Using a candidate gene approach, we identified LCK with increased expression levels in E2A-PBX1 leukemia cells compared to normal B-cell progenitors.

Mouse and human E2A-PBX1 leukemia cells were dependent on the E2A-PBX1 target genes ZAP70 and LCK for proliferation and survival as confirmed by shRNA knock-down experiments. Hence, efficient depletion of these genes resulted in a decrease of phosphorylated PLCγ2, suggesting therapeutic targets in E2A-PBX1 leukemias. Combined suppression of ZAP70 and LCK using double-shRNA experiments showed an additive effect on inhibition of cell proliferation and decrease of phosphorylated PLCγ2. These results provide a rationale for combination therapy to block this hyper-activated signaling pathway at different levels.

Several small molecule inhibitors were evaluated for their effects on PLCγ2 upstream pathways in E2A-PBX1 leukemia cells. SRC-family kinase inhibitors including dasatinib were most effective in reducing phosphorylation of PLCγ2 and inhibiting cell proliferation. Furthermore, dasatinib showed promising preclinical efficacy in vitro in colony forming assays and in vivo after secondary bone marrow transplantation of leukemias.

In summary, our studies demonstrate that the proliferation and survival of E2A-PBX1 leukemias are dependent on PLCγ2 and upstream signaling pathways, which are suitable for pharmacological inhibition.