ZAP-70 Integrates Tonic B Cell Receptor and Chemokine Signaling in IGHV unmutated Chronic Lymphocytic Leukemia

Abstract

The importance of B cell receptor (BCR) signaling for the progression of Chronic Lymphocytic Leukemia (CLL) has impressively been demonstrated through the clinical success of small molecule inhibitors interfering with this pathway, providing effective treatment options for a vast majority of patients. The scientific evidence which supported the development of drugs like BTK and PI3K inhibitors largely originate from studies of antigen-induced BCR-signaling. Notably, the presence of a constitutive, so-called tonic BCR-signal was long postulated to co-exist besides antigen-driven activation and is based on the identification of constitutively activated signaling molecules down-stream of the BCR. We here provide direct evidence for the presence of a tonic BCR-signal, which critically relies on the expression of the tyrosine kinase ZAP-70. We have previously established a method to inhibit ZAP-70 expression in primary CLL cells usingRNA-interference in combination with an extended co-culture system. Our previous work demonstrated that ZAP-70 is required for the constitutive gene expression of T cell chemokines CCL3 and CCL4 and contributes to the dysregulation of global protein synthesis and MYC expression in CLL ( Blood, 2021). Applying this method, we here provide evidence for the presence of a constitutive tonic BCR signal, which is dependent on ZAP-70 expression and exclusively found in IGHV unmutated CLL, but not in IGHV mutated cases discordantly expressing ZAP-70. We demonstrate that ZAP-70 is directly involved in the constitutive activation of AKT and GSK-3beta. Furthermore, ZAP-70 expression increases steady state activation of CD19 and the downstream kinase SRC, providing direct evidence for the existence of a ZAP-70 dependent tonic BCR signal in I GHV unmutated CLL only. To identify the underlying signaling events driving this signal, we performed mass spectrometry in combination with proximity ligation assays on CLL cells in the absence of an induced BCR-signal. Unexpectedly, we identified that the majority of ZAP-70 binding proteins do not related to BCR-signaling components, but to cytoskeleton organizing proteins involved in cell migration. Indeed, migration assays demonstrated that the loss of ZAP-70 directly and significantly impaired cell migration induced by CCL19 and CCL21, in keeping with previous data indication an association between ZAP-70 and cell migration. These results were confirmed using a switchable model of ZAP-70, which allowed us to toggle ZAP-70 expression on and off at any given time. To further define the mechanisms of ZAP-70 mediated cell migration, we studied CCL19 and CCL21 signaling in ZAP-70 proficient and deficient cells. Unexpectedly, we identified that ZAP-70 is required for chemokine-mediated activation of AKT. In summary, we here demonstrate that a tonic BCR-signal is present only in IGHV-unmutated CLL and dependent on the expression of ZAP-70. Furthermore, ZAP-70 converges two seemingly independent signaling events, namely chemokine- and tonic BCR signaling. Our data provide further explanations for the more aggressive clinical course of ZAP-70 positive CLL.