ANALYSIS OF SIGNALING PROFILES IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course that reflects biological differences driven by intrinsic defects as well as external cues from the microenvironment. These events converge on the activation of regulatory signaling pathways involved in processes promoting resistance to apoptosis and overturning the control of proliferation. Indeed, CLL physiopathology is entwined with its microenvironment and the statuses of proteins involved in signal transduction represent a unique series of markers dynamically determined from microenvironment, indicating the cell capability to transduce information. To gain insights into the pathogenic role and clinical significance of microenvironment signals in CLL, we analyzed the phosphorylation status of proteins on the road of B-cell receptor (BCR) pathway, which represents a prominent pathogenic mechanism in CLL, using a multiparametric flow cytometry-based assay. This method allows to measure, simultaneously and quantitatively, at the single cell level, both extracellular surface markers and changes in signaling proteins in response to extracellular stimuli, thus providing a dynamic view of signaling responses to microenvironment. We showed that higher responsiveness of BCR signaling-protein was associated with poor clinical outcomes and signal profiles distinguished patients with indolent disease from those with a poor clinical course. Time-to-event modeling utilizing phosphorylation-status data identified Erk1/2 responsiveness as a significant, independent predictor of CLL disease progression. This result was independently confirmed in other test cohorts of CLL patients. Of interest, the presence of CXCR4 co-stimulation, another key pathway of CLL microenvironment cross- talk, induced a higher Erk1/2 phosphorylation response than the BCR alone. Also, Erk1/2 responsiveness induced by the BCR and CXCR4 co-stimulation was more powerful in discriminating different disease courses. Our data showed the utility of this flow cytometry-based assay for identification and independent verification of signaling profiles associated with prognosis in CLL. Moreover, these data support the pathogenic role and clinical relevance of microenvironment signals, highlighting the prominent role of BCR signaling, and suggest a critical role of Erk activation in CLL physiopathology.