The role of SYK and ZAP70 kinases in the etiology of B-cell malignancies

• Applicant: Dr. Franz Ketzer
• Subject Area: Hematology, Oncology
• Term: from 2023 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 513549849

B-cell neoplasia are the most common malignancies in children and young adults. B-cells are under intense selective pressure to eliminate autoreactive and pre-malignant clones. Specifically, SYK was identified as a central kinase to set the thresholds for negative selection. SYK and its highly homologous relative ZAP70 initiate B-cell receptor (BCR) and T-cell receptor (TCR) signaling in B- and T-cells, respectively, and are strictly evolutionarily segregated. The Müschen lab recently demonstrated that aberrant ZAP70 expression is a common feature in pre-germinal center (GC) B-cell malignancies (B-ALL, CLL, MCL). In genetic mouse models for B-ALL and B-CLL, inducible co-expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Mechanistically, ZAP70 competes with SYK and exerts a dominant-negative effect on SYK-dependent Ca2+-signaling. By occupying but not phosphorylating BLNK, BTK and PLCγ2 substrates upstream of Ca2+ release, ZAP70 dramatically reduces the frequency of autonomous Ca2+-oscillations. Fast oscillations in the sole presence of SYK (4.5 mHz) are decoded by NFATC1 and result in anergy and cell death. In contrast, slow Ca2+-oscillations in the presence of ZAP70 (0.25 mHz) promote selective activation of NF-κB and B-cell survival. In this project, I set to test the central hypothesis that B-cells sense pathological signaling downstream of an autoreactive BCR or a transforming oncogene through SYK-dependent high-frequency Ca2+-oscillations. First, I will perform genetic studies of structural features of SYK and ZAP70 and phosphoproteomic analyses of analog-sensitive mutants of both kinases, and will test the hypothesis that ZAP70 redirects SYK from BLNK, BTK and PLCγ2 substrates of calcium signaling to PI3K activity. Furthermore, I will analyze the functional consequences of Ca2+-oscillations on NFATC1/NF-κB activation in enabling the persistence of pre-malignant B-cell clones via a proprietary optogenetic platform for Ca2+-release and RNA-sequencing. Second, based on a genetic knock-in allele that drives Zap70-expression from the Syk promoter, I will test whether inducible co-expression of Zap70 accelerates malignant progression. In genetic loss-of-function studies of Zap70 in mouse models for pre-GC B-cell tumors (B-ALL, CLL, MCL) and in patient-derived xenografts carrying analog-sensitive Zap70, I will test whether ZAP70-mediated diversion of Ca2+-signaling from NFATC1 to NF-κB is required for malignant transformation. Finally, the Müschen lab identified small molecule inhibitors of the SHIP1 phosphatase, which opposes SYK. These compounds induced NFATC1-activation and cell death in ZAP70+ pre-GC but not ZAP70- post-GC B-cell tumors. Pre-clinical testing will establish feasibility and efficacy of this approach and determine whether ZAP70-expression represents a useful biomarker to identify patients who benefit from targeted hyperactivation of SYK, e.g. by SHIP1 inhibition.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Dr. Markus Müschen