Deciphering The Molecular Impact Of Myc Overexpression On B Cell Receptor Signaling and Its Implications For Burkitt Lymphoma Treatment

B cell antigen receptors (BCR) transduce signals and thereby control important cell fate decisions of the B-lineage including differentiation, proliferation and B cell survival. The intracellular signaling networks are complex and mainly regulated through reversible protein phosphorylation on tyrosine, serine or threonine residues. This process is highly controlled to ensure regular B cell function. However, dysregulation of BCR signaling can induce immunodeficiency, autoimmune disorders and malignant transformation. Recently, it was demonstrated that besides chronic lymphocytic leukemia and diffuse large B cell lymphomas of the activated B cell-type also subgroups of Burkitt lymphomas (BL) critically depend on BCR signaling that is enhanced due to TCF3 or ID3mutations. Thus, targeting of BCR signaling pathways by small molecule inhibitors is a promising therapeutic strategy for BCR-dependent BL.

In order to identify putative novel drug targets, comprehensive knowledge about BCR effectors in BL cells is essential. We combined quantitative mass spectrometry (MS) and next generation sequencing (NGS) to globally characterize the dynamics of BCR-induced signaling and its impact on the (phospho)proteome and transcriptome of BL cells. We identified and quantified 8360 phosphorylation sites, of which 885 were regulated by BCRs in BL cells. Of these, about 85% have not been described before in the context of B- or lymphoma cells. Thus, the approach uncovered many unknown BCR effectors. The identified effector proteins belong to various functional groups, of which kinases, transcription factors and cytoskeleton regulators are most represented. Phosphoproteomic analyses revealed strong activatory phosphorylation patterns in several kinases. To complement and verify these data, we performed a kinobead-based kinase activity profiling in resting and activated BL cells. The combined data reveal the activation of known tyrosine kinases, but also of kinases with yet unknown function in BCR signaling. Particularly, certain MAP-kinases and other cell cycle regulators, such as kinases of the NEK family were highly activated in BL cells. Utilizing transcriptome sequencing, we furthermore identified about 150 genes that are differentially transcribed up to two hours after BCR stimulation. Correlating these data with expression proteomics, we investigated, how this genetic regulation affects protein expression in BL cells. Taken together, our large-scale study elucidates the molecular effects of BCR activation in BL in a comprehensive manner and reveals for the first time the dynamic interplay between receptor-triggered kinase activation, phosphorylation events and mRNA-/protein expression changes in these cells. Furthermore, the approach to combine proteomic, genetic and cell biological techniques also allowed us to decipher the influence of Myc overexpression on BCR signaling and provides data on possible molecular drug targets for the treatment of Burkitt’s lymphoma that have to be validated in further independent experiments. In case Myc was overexpressed Ca2+ signaling pathways were suppressed, while activity of other BCR-dependent pathways was enhanced. Manipulation of these altered activation patterns by certain small molecule inhibitors turned out to efficiently suppress BL cell growth.