Single cell profiling of phospho-protein levels in chronic lymphocytic leukemia

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.

The Important Role of STAT3 in Chronic Lymphocytic Leukaemia Biology

“…In addition, in CLL cells, STAT3 can be activated easily through Y705 phosphorylation because CLL cells reside in the bone marrow, blood and lymph nodes and are thus exposed to various cytokines, chemokines and growth factors and therefore capable of potentiate through this phosphorylation a magnitude of STAT3 activation. Although it is not constitutive in CLL cells, increased pY705 STAT3 levels were described by Myhrvold et al [20]. In CLL cells, the basal phosphorylation level of proteins involved in B-cell signalling was similar or lower compared to normal B cells, with the exception of pY705 STAT3, which was increased.…”

“…The blocking of STAT3 led to downregulation of an anti-apoptotic protein XIAP. STAT3 inhibitors WP1066 and niclosamide significantly reduced both pY705 STAT3 levels and cell viability [20]. STAT3 inhibition reduced cellular metabolism before cell death was induced.…”

New Protein Markers of Chronic Lymphocytic and Acute Lymphocytic Leukemia

“…After BCR stimulation, CLL cells have shown a tendency toward impaired phosphorylation levels. Higher basal phosphorylation levels of PLCγ2 (pY759), p44/42 MAPK (pT202/Y204), p38 MAPK (pT180/Y182), NF-κB p65 (pS529), STAT5 (pY694), and STAT6 (pY641) were detected in CLL cells compared to normal B cells, predicting their impaired function [12]. As such, these markers may represent some of the novel protein targets involved in the development of efficient therapeutics.…”

“…New specific protein markers connected with CLL and ALL which have been discovered in the last 10-15 years represent novel potential targets for highly personalized treatments of leukemia. These proteins, associated with different cellular signaling events, mostly include surface receptors/transmembrane proteins-CD5, CD19, CD20, CD22, CD23, CD52, and many others [9,11,31,33,38]-where protein phosphorylation may play an important role in protein activity regulation connected to the progression of disease and regulation of pathological events [12][13][14]. Focusing on such specific modifications presents key opportunities to further facilitate efficient and precise drug strategy design [55][56][57][58].…”

The Nordic EMBL Partnership – a paragon of collaboration

“…This has now evolved into a Nordic, multi‐centre, double‐blinded, randomised, placebo‐controlled http://www.asac.no/, where the first patients of 800 are currently being recruited at sites in Norway, Sweden and Denmark. NCMM's work with cancer drug sensitivity screening may also fit into future precision medicine approaches that can provide benefits to patients . DANDRITE A particular landmark achievement for DANDRITE was receiving a five‐year prolongation grant from the Lundbeck Foundation, to continue its ambitious research in the second funding period, up until 2023.Another highlight was the launch of a new Danish National Research Foundation Centre of Excellence called http://promemo.au.dk/, which is led by DANDRITE Group Leader Professor Anders Nykjær. The centre will work towards identifying and understanding the function of memory‐associated proteins that determines the persistence of a memory, with a view to improving the treatment of psychiatric disorders in the future.An important achievement is also the development of a ‘DANDRITE spirit’.…”