ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia.

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Viral transduction of primary human lymphoma B cells reveals mechanisms of NOTCH-mediated immune escape.

Hotspot mutations in the PEST-domain of NOTCH1 and NOTCH2 are recurrently identified in B cell malignancies. To address how NOTCH-mutations contribute to a dismal prognosis, we have generated isogenic primary human tumor cells from patients with Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), differing only in their expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. Our data demonstrate that both NOTCH-paralogs facilitate immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-γ signaling. In addition, NOTCH-activation causes silencing of the entire HLA-class II locus via epigenetic regulation of the transcriptional co-activator CIITA. Notably, while NOTCH1 and NOTCH2 govern similar transcriptional programs, disease-specific differences in their expression levels can favor paralog-specific selection. Importantly, NOTCH-ICD also strongly down-regulates the expression of CD19, possibly limiting the effectiveness of immune-therapies. These NOTCH-mediated immune escape mechanisms are associated with the expansion of exhausted CD8+ T cells in vivo.