Separation of B- and T-Cell-Specific Signaling Molecules Prevents Oncogenic Transformation in Lymphoid Malignancies

Abstract

Background and significance: Despite the highly homologous structure and function of proteins controlling signal transduction from the BCR and TCR, B- and T-cells have evolved separate sets of linker and kinase molecules and mechanisms to ensure that their expression is strictly separated between B- and T-lymphoid lineages. While this is the case among normal B- and T-cells, we and others showed that the separation of B- and T-cell-specific signaling molecules is often blurred in lymphoid malignancies. For instance, B-ALL, CLL, and MCL often aberrantly express the T-cell kinase ZAP70, whereas PTCLs and γδTCR CTCLs often express SYK or the ITK-SYK fusion. Here we investigated the mechanisms driving the aberrant cross-lineage expression of BCR- and TCR-downstream signaling molecules and how the promiscuous expression of these molecules contributes to the malignant transformation of lymphoid malignancies. Results: We have previously shown that the B-cell-specific kinase SYK and the T-cell-specific kinase ZAP70 are segregated to prevent autoimmunity and malignant transformation of pre-germinal center B-cells. In pre-malignant B-cells, SYK drives negative selection through Ca 2+-signaling via BTK-BLNK phosphorylation and downstream activation of NFAT. The aberrant expression of ZAP70 diverts SYK signaling towards CD19-PI3K, weakening negative selection, and consequently permitting the expansion of pre-malignant cells. This represents a novel key mechanism of aberrantly expressed signaling molecules in the etiology and persistence of B-lymphoid malignancy. We previously showed that co-expression of the T-cell kinase ZAP70 with the B-cell kinase SYK dramatically accelerated the development of fatal disease in a mouse model of CLL, consistent with poor clinical outcomes of ZAP70 + CLL in patients. To elucidate the role of ZAP70 co-expression in the initiation of B-ALL, pre-B-cells were isolated from Zap70 -/- mice and transformed ex vivo with BCR-ABL1 and NRAS G12D to model B-ALL. These cells showed significantly impaired colony-forming capacity compared to transformed wild-type cells. To corroborate these results, we crossed BCR-ABL1-transgenic with Zap70 -/- mice. Next, patient-derived B-ALL and MCL xenografts (PDX) were engineered to carry a homozygous knock-in of dTAG at the C-terminus of ZAP70. Upon degradation of ZAP70-dTAG, consequences on cellular fitness and proliferation were assessed, as well as the downstream activation of Ca 2+-signaling and NFAT activation ( Figure 1). Additionally, a mouse strain carrying a bulky ATP-analog-sensitive variant of Zap70 (M413A) was crossed with the p190 strain to assess the effect of ZAP70 inhibition after the manifestation of B-cell leukemia. Since the expression of ZAP70 increases upon malignant transformation of pre-germinal center B-cells, we used Genomic Locus Proteomics (GLoPro) to identify transcription factors driving this transcriptional upregulation ( Figure 1). By comparing malignant B- and T-cell lines, which either express only SYK or ZAP70 or co-express them, we were able to isolate specific mediators of aberrant transcription of ZAP70, including β-catenin/WNT signaling. ChIP-qPCR, CRISPR/Cas9-mediated knockout, as well as dTAG, were used to validate the binding of transcription factors identified by GLoPro to the ZAP70 enhancers and their role in ZAP70 expression. Apart from ZAP70, we identified multiple aberrantly co-expressed T-cell signaling proteins by comparative proteomic analysis of malignant B- and T-cell lines. These included LCK, PLCγ1, and SLP-76, while the BTK-homolog ITK is completely absent in B-cell lines. In future work, we aim to elucidate the transcriptional regulation and consequences of the loss of these aberrantly co-expressed proteins. Further, we will perform a CRISPR/Cas9-based switch of BTK and ITK to explore the effects of forced co-expression of these segregated proteins. Conclusions: We conclude that malignant pre-germinal center B-cells increase the expression of ZAP70 through the formation of aberrant transcription machinery. Furthermore, our work elucidates the role of ZAP70 not only for the initiation but also for the maintenance of B-cell leukemia. Other T-cell signaling molecules are also aberrantly expressed in B-cell malignancies and further investigation will highlight their transcriptional regulation and role during and after malignant transformation.