A Molecular Signature of Anergy Detected in a Subset of CLL Patients.
Authors: Ghia Paolo , Muzio Marta , Apollonio Benedetta , Scielzo Cristina , Frenquelli Michela , Vandoni Irene , Boussiotis Vassiliki , Caligaris-Cappio Federico 
Published: November 2019

Abstract
It has been long assumed that BCR signaling plays a relevant role in the natural history of Chronic Lymphocytic Leukemia (CLL), by regulating various and possibly opposing mechanisms. A proportion of CLL cases carry a surface Immunoglobulin (sIg) which is responsive to crosslinking, resulting in effective activation of leukemic cells in vitro. In clear contrast, the remaining cases show a typical functional unresponsiveness through the Immunoglobulin, as indicated by a lack of tyrosine phosphorylation following BCR cross-linking. This feature together with the characteristic decreased expression of the BCR, have led to the time-honored hypothesis that these cells might have been anergized in vivo upon their encounter with a (self-) antigen. Of note, this functional feature tends to correlate with an indolent clinical course of the disease, which may derive from the quiescent state of the functionally silenced leukemic lymphocytes. Despite extensive studies, the concept of B cell anergy in the human immune system and in particular of the functional unresponsiveness in CLL lacks a molecular and biochemical definition. These observations prompted us to examine the expression and activation of key molecules involved in the signaling pathways originating from the BCR in 37 CLL patients and to investigate potential association of the distinct signaling patterns with clinical prognostic features. Our studies showed that in those CLL patients characterized by cellular unresponsiveness to sIg ligation, the MAP kinases ERK1/2 were constitutively phosphorylated and remained unchanged after stimulation with anti-IgM. These cases also displayed constitutive phosphorylation of MEK1/2 and Raf-1 and increased NF-AT transactivation; interestingly, these biochemical features were associated with a lack of AKT activation. In contrast, PMA that bypasses BCR proximal events was able to induce phosphorylation of ERK of these CLL cases indicating that the MAPK signaling machinery is intact but is selectively silenced and unresponsive to BCR-mediated signals. Notably, CLL cases with a constitutive ERK1/2 activation showed a more favorable clinical presentation as compared to the ERK1/2 negative cases. Constitutive activation of MAPK, along with NF-AT transactivation and lack of AKT activation has been previously described as a hallmark of anergic B lymphocytes in the murine system. Taking together all the above, we propose that the “molecularly anergic” subset of CLL described in our work may represent a human cellular model of anergic human B cells aberrantly expanded as part of the malignant process. These molecular features of constitutive BCR-mediated activation may provide evidence for BCR engagement by a putative anergizing antigen occurring in vivo. In addition, these data raise the intriguing hypothesis that this molecular signature of in vivo anergization may also be responsible for the quiescent state of the leukemic cells resulting in the indolent clinical course. In conclusion, our results may provide a molecular basis for the concept of human B-cell anergy and for the distinct functional and clinical behavior observed in a subset of CLL patients.