Identification of Posttranvslationally Modified Neoantigens As Targets of B Cell Receptors of Burkitt Lymphoma

Introduction: Burkitt lymphoma represents the most aggressive neoplasm of mature sIg+ B cells. Besides the characterisitc translocation of the MYC gene with an immunoglobulin heavy or light chain gene locus, activating mutations in the TCF3 gene and inactivating mutations in the ID3 gene represent the key events in the pathogenesis of Burkitt lymphoma. These TCF3/ID3 mutations result in tonic and antigen-independent B cell receptor (BCR) pathway activation. Additionally, chronic BCR activation by antigens might play a role in Burkitt lymphoma pathogenesis and we set out to identify such potential target antigens of BCRs of Burkitt lymphoma. Methods: BCRs were expressed as recombinant Fabs in TG1 E.coli based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen sporadic Burkitt lymphoma specimens. Additionally, natural Fabs and recombinant Fabs were produced of 8 established Burkitt lymphoma cell lines by Papain digestion and BCR expression cloning. The purified pooled Fabs were screened for reactivities against non-modified and differently posttranslationally modified human protein macroarrays. Reactivities were verified by ELISA with coated N-terminally FLAG-tagged candidate antigens, each separately for the posttranslationally modified and non-modified isoforms. Recombinant Fabs derived of mantle cell lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma served as controls. Moreover the functional effects on proliferation and BCR pathway activation after addition of the identified target antigens to Burkitt lymphoma cell lines with and without reactive BCRs were analyzed by proliferation assays and western blots. Finally, mutation status, methylation status and expression level of identified target antigens were analyzed in ICGC MMML-Seq lymphoma databases for differences in Burkitt lymphoma versus distinct lymphoma entities. Results: The Burkitt lymphoma derived Fabs were tested on posttranslationally modified protein arrays. The BCR of CA46 line showed specific reactivity against sumoylated Bystin, the Fab of the BL41 line reacted specifically against acetylated HSP40. Recombinant Fabs of diffuse large B cell lymphoma, primary central nervous system lymphoma and mantle cell lymphoma did neither bind sumoylated Bystin nor acetylated HSP40. Addition of the posttranslationally modified cognate antigens to respective Burkitt lymphoma