Abstract
Chronic lymphocytic leukemia (CLL) has a high incidence and a steeply growing prevalence in the Western world. The heterogeneity of the disease necessitates individual mapping of biology and predicted drug response in each patient as basis for administration of tailored treatments. Cell signaling aberrations may serve as biological indicators for suitable therapy. By applying phospho-specific flow cytometry, we mapped basal and induced phosphorylation levels of 20 phospho-epitopes on proteins relevant to B-cell signaling in B cells from 22 CLL patients and 25 normal controls. The signaling response of the cytostatic drugs fludarabine, doxorubicin and vincristine was also investigated. CLL cells exerted similar or lower basal phosphorylation levels compared to normal B cells, with the exception of STAT3 (pY705) which was increased. Interestingly, STAT3 inhibitors normalized the STAT3 (pY705) level and reduced cell viability. Vincristine treatment significantly modulated phosphorylation levels in CLL cells, while no effect was observed in controls or after fludarabine or doxorubicin treatment. After BCR stimulation, CLL cells showed a tendency towards impaired phosphorylation levels, significant for several of the analyzed proteins. However, the level of Akt (pS473) was more potently induced in IgHV unmutated CLL (UM-CLL) patient samples and was significantly higher than in M-CLL samples. Importantly, the PI3Kδ inhibitor idelalisib potently reversed the effect of anti-IgM on Akt (pS473). Thus, signaling aberrations could be identified by phosphoflow cytometry and aberrant signaling could be normalized by small molecule drugs. This approach can identify relevant drug targets as well as drug effects in the individual patient.
Highlights
Chronic lymphocytic leukemia (CLL) is one of the most prevalent B-cell neoplasias in the Western world [1]
Characterization of basal phosphorylation levels in CLL and normal B cells In order to identify signaling aberrations in CLL cells relative to normal B cells, we investigated both basal and induced phosphorylation levels of 20 different phospho-epitopes on signaling proteins relevant for the B cell receptor (BCR) signaling pathway
Basal and anti-IgM induced phospho-protein levels were characterized in B cells from healthy donors and CLL patients
Summary
Chronic lymphocytic leukemia (CLL) is one of the most prevalent B-cell neoplasias in the Western world [1]. Immunoglobulin heavy-chain variable gene (IgHV) mutational status, www.impactjournals.com/oncotarget chromosomal abnormalities and expression of CD38 and Zeta-chain-associated protein kinase of 70 kDa (ZAP70) are well established markers [3]. There is some redundancy in signaling, ZAP70, which is highly expressed in CLL cells with an aggressive course, may contribute [5]. BLNK serves as a docking site for Bruton’s tyrosine kinase (Btk), Phospholipase Cγ2 (PLCγ2) and the adaptor protein Growth factor receptorbound protein 2 (GRB2). This BCR signalosome generates a wide variety of downstream effects, including activation of the PI3K-Akt-mTOR pathway and the Ras-Raf-MEKERK pathway [6]
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