Aberrant Simultaneous ERK and CDK2 Kinase Activity Discloses Leukemia

Abstract 2487

Genome wide analyses of regulatory elements have identified successive waves of coherent transcriptional programs guiding through and controlling cell cycle progression. Each of these modules induces its successor and, at the same time, inactivates the key kinases active in the antecedent phase of the cycle. In an initial signaling module, the RAF-MEK-ERK pathway is central for phosphorylation of the tumor suppressor Rb (retinoblastoma), release of transcription factors of the E2F family and subsequent cell cycle entry. CDK2 is the driving force and guardian of the consecutive phase where DNA replication occurs. After having initiated CDK2 activity, ERK is inactive and non-inducible (Pouyssegur 2003). The pRB/CDK2 node thus is a bistable switch that converts mitogenic information into an irreversible cellular commitment to duplicate, and uncouples the cell's growth and division program from environmental or internal control. Comparing primary leukemic blasts with healthy CD34⁺ hematopoietic progenitor cells, we here show that simultaneous rather than serial activation of the canonical ERK and CDK2 pathways is a recurrent aberrant signaling motif indicative for malignant transformation. This phenomenon was observed in leukemic blasts and also in cancer cells of epithelial and mesenchymal provenience, but not in healthy or regenerating tissues using flow cytometry and confocal microscopy techniques and phospho-epitope specific antibodies. To further visualize this “forbidden ERK/CDK2 signal combination”, we developed a biosensor comprising both, the optimal peptide substrates for ERK and CDK kinases attached to a fluorescein tag and a membrane-penetrating nona-arginine (9R) moiety. This compound enables detection of simultaneous ERK and CDK2 activity on single cell level by means of electrophoretic mobility shift. Site directed mutagenesis of the two phospho sites confirmed mutual exclusive substrate recognition thus ascertaining the specificity of the read out system. In healthy CD34⁺ cells, single but never dual phosphorylation of the biosensor was detected even after stimulation with PMA/Ionomycin, the latter establishing a non-physiological, proliferative compartment. These findings confirm the serial and mutually exclusive piloting of ERK and CDK2 signaling pathways in non-transformed cells. In stark contrast, dual phosphorylation of the biosensor peptide was observed upon analysis of leukemic blasts. Thus, simultaneous and therefore aberrant recruitment of ERK and Rb/E2F/CDK2 cell cycle elements in transformed but not healthy cells discloses leukemia. Our data do not add to the increasing complexity of genetic insults associated with malignant transformation. Rather, we demonstrate that violations of the orderly recruitment of common signaling pathways are indicative for a malignant phenotype which can be detected on the basis of aberrant spatiotemporal organization of essential cell signaling nodes. Our findings do not only anticipate a novel way to diagnose malignancy. Based on the structural information provided by our template peptide, we further envisage a new class of cancer therapeutics where a nontoxic prodrug is converted into a tumoricidal substance exclusively in malignant cells after simultaneous double-phosphorylation by aberrantly active kinases.