Functional exploration of ZAP70 and LPL in chronic lymphocytic leukemia

A typical hallmark of chronic lymphocytic leukemia (CLL) is its highly variable clinical course, with life expectancies ranging from months to decades. Although the majority of patients presents with low-grade CLL at diagnosis, a significant subset will eventually develop a more aggressive and life-threatening disease. Identification of these patients with an elevated risk of progression is of utmost importance in the context of individual risk-adapted clinical management. Over the last decades many markers have been identified that allow predicting a patient’s prognosis in an early disease stage, among them zeta-chain- associated protein of 70kDa (ZAP70) and lipoprotein lipase (LPL). Although their prognostic value has repeatedly been reported before, whether and how ZAP70 and LPL contribute to an aggressive phenotype is not elucidated yet. Unraveling the functional role of these and other prognostic markers in CLL disease biology might provide more insights into oncogenic pathways and could unravel new therapeutic targets. By performing whole genome expression profiling in CLL cells overexpressing the protein of interest, being either ZAP70 or LPL, we aimed at further unraveling the role of these prognostic markers in the pathogenesis of CLL. In summary, we were able to demonstrate that ZAP70 induces enhanced nuclear factor-kappa B (NF-κB) signaling upon B cell receptor (BCR) stimulation. Given that NF-κB is involved in protecting cells from spontaneous and drug induced apoptosis, this could represent a mechanism by which ZAP70 contributes to an aggressive disease course. Our data concerning the functional implications of LPL overexpression in CLL require further validation, but point to involvement of LPL in epigenetic gene regulation. As such, LPL could modulate disease-promoting pathways and contribute to an aggressive disease course. Besides this, we studied the prognostic implications of three single nucleotide polymorphisms (SNPs) in the LPL gene and found that the presence of LPL SNPs rs301 and rs13702 was correlated with prolonged overall survival. Interestingly, several prognostic markers in CLL, including ZAP70 and probably also LPL, relate to the BCR, which has been demonstrated to exert a critical role in the crosstalk between the leukemic clone and the surrounding tissue in the lymphoid organs. Accumulating evidence suggests that activation of the BCR within this tissue microenvironment, along with other co-stimulatory signals, promotes the survival and expansion of the leukemic clone. Exactly how this is achieved and which downstream pathways are involved is not entirely clear yet. To gain more insight in the role of BCR signaling in CLL, we analyzed whole genome mRNA and microRNA (miRNA) expression profiles obtained in CLL cells upon BCR stimulation in vitro. Both the BCR driven mRNA and miRNA signatures are involved in cell cycle entry and progression and point to a MYC driven proliferative response. Next, we re-evaluated our in vitro BCR stimulation method and compared it to other protocols in an attempt to provide a standardized way to perform CLL BCR stimulation in vitro. We found a dramatic difference in respect to immobilized versus soluble triggers of the BCR and show that both IGHV mutated and unmutated CLL cells respond equally efficient to BCR triggering