R01AI164692

Syk and ZAP70 Kinases in Lymphocyte Selection - Abstract

While B-cells are under intense selective pressure to eliminate autoreactive and pre-malignant clones, we identified Syk as the central kinase that sets 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-lymphocytes, respectively. Even though the two kinases are almost identical and serve analogous functions, their expression in B- and T-cells is strictly segregated throughout evolution.

Whereas the reason for separation of the two kinases is not known, aberrant coexpression of Syk and ZAP70 was previously reported in B-cell chronic lymphocytic leukemia (CLL) and in peripheral T-cell lymphoma (PTCL). Our group recently demonstrated that aberrant ZAP70 expression is a common feature in pre-germinal center (GC) B-cell malignancies (B-ALL, CLL, MCL; Sadras et al., Mol Cell 2021). In genetic mouse models for B-ALL and B-CLL, inducible coexpression of ZAP70 accelerated disease onset, while genetic deletion impaired malignant transformation. Likewise, inducible expression of ZAP70 during early B-cell development subverted negative selection of autoreactive B-cells to promote pervasive autoantibody production.

Mechanistically, ZAP70 competes with Syk and exerts a dominant-negative effect on Syk-dependent Ca2+-signaling. By occupying but not phosphorylating BLNK, BTK, and PLC2 substrates upstream of calcium signaling, 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-B and B-cell survival and proliferation.

We test here 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. At high frequencies, Ca2+-oscillations activate NFATC1 to initiate B-cell anergy and clonal deletion. Conversely, ZAP70 slows down Ca2+-oscillations to activate NF-B instead of NFATC1 and enables autoreactive and premalignant B-cell clones to persist and eventually give rise to autoimmune disease or pre-GC B-cell malignancies.

We are proposing three aims to:
1. Elucidate the mechanisms by which ZAP70 slows down Syk-dependent high-frequency Ca2+-oscillations.
2. Determine how coexpression of ZAP70 subverts negative selection of autoreactive B-cells in autoimmune disease.
3. Uncover how ZAP70 enables oncogenic signaling and overt malignant transformation of pre-malignant B-cells.

Yale Univ

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93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

New Haven, Connecticut 065116624 United States

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NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 397% from $617,340 to $3,068,179.