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. 2021 Nov 1;106(11):2995-2999.
doi: 10.3324/haematol.2021.278644.

IL4-STAT6 signaling induces CD20 in chronic lymphocytic leukemia and this axis is repressed by PI3Kδ inhibitor idelalisib

Veronika Sandova  1 , Gabriela Mladonicka Pavlasova  2 , Vaclav Seda  1 , Katerina Amruz Cerna  2 , Sonali Sharma  2 , Veronika Palusova  2 , Yvona Brychtova  3 , Sarka Pospisilova  3 , Stacey M Fernandes  4 , Anna Panovska  3 , Michael Doubek  3 , Matthew S Davids  4 , Jennifer R Brown  4 , Jiri Mayer  3 , Marek Mraz  5
Affiliations

IL4-STAT6 signaling induces CD20 in chronic lymphocytic leukemia and this axis is repressed by PI3Kδ inhibitor idelalisib

Veronika Sandova et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
IL4 upregulates CD20 expression via STAT6. (A) Cell surface level of CD20 after interleukin 4 (IL4) treatment (20 ng/mL, PeproTech) for 24 hours (hrs) (n=25), 48 hrs (n=23), or 72 hrs (n=22) in comparison to untreated control cells (ctrl). (B) Representative immunoblot of CD20 and pSTAT6 (Tyr 641) protein levels in chronic lymphocytic leukemia (CLL) cells after IL4 stimulation (24-72 hrs). (C) Densitometric quantification of CD20 protein levels for independent replicates of the experiment described in (B) (n=17; 24-72 hrs). Untreated control (ctrl) without IL4 was set as 1 and compared to the other samples. (D) Normalized cell surface CD20 levels in primary CLL cells treated by SDF1a (100 ng/mL, PeproTech), IL4 (20 ng/mL), or their combination (SDF1a + IL4) for 24-48 hrs (n=7). The untreated ctrl was set as 1. (E) Peripheral blood CLL cells were electroporated with small interfering RNA (siRNA) against STAT6 (siSTAT6, 500 nM; Dharmacon) or negative control (Neg.Ctrl). IL4 (20 ng/ml) was added 48 hrs after transfection and cells were cultured for another 24 hrs. For the immunoblot, β-actin was used as a loading control and pSTAT6 (Tyr 641)/tSTAT6 levels were assessed. (F) Densitometric quantification of CD20 protein levels for independent replicates (n=4) of the experiment described in (E). Neg.Ctrl without IL4 was set as 1 and compared to the other samples. (G) Peripheral blood CLL cells were pretreated with pSTAT6 inhibitor (AS1517499, 1 mM, Selleckchem) for 12 hrs. Subsequently, IL4 (20 ng/mL) was added to the media, and cells were cultured for another 24 hrs. CD20 expression was determined by real-time quantitative polymerase chain reaction (TaqMan, ABI), and the expression of CD20 was normalized to endogenous control HPRT (n=6). (H) Chromatin immunoprecipitation analysis of samples that were immunoprecipitated with anti-STAT6 antibody in comparison with immunoglobulin G (IgG) antibody before (-) and after (+) IL4 stimulation (n=5; 40 ng/mL, 30 minutes). IgG antibody was used as an isotype control. For all in vitro experiments in Figure 1 and 2 CLL cells were purified by RosetteSep Human B Cell Enrichment Cocktail (Stemcell Technologies) to obtain purity ≥95% of CD5+CD19+ cells. For immunoblots, b-actin was used as a loading controls. In all experiments, the statistical difference was tested using a paired t-test, and the error bars indicate standard error of the mean.
Figure 2.
Figure 2.
CD20 is downmodulated by idelalisib and embedded in the IL4 pathway. (A) Cell-surface CD20 levels and (B) relative expression of CD20 mRNA in paired samples before (Pre) and after 5 weeks (idelalisib-week 5) and 9 weeks (idelalisib-week 9) of idelalisib therapy in vivo (cell surface CD20: n=1 week 4, n=6 week 5, n=6 week 9; CD20 mRNA: n=6 week 5 and week 9). Chronic lymphocytic leukemia (CLL) cells were isolated by density centrifugation (Ficoll-Paque) followed by magnetic anti-CD3 MicroBeads separation (Miltenyi Biotec) or in some cases negative selection with RosetteSep Human B Cell Enrichment Cocktail (Stemcell Technologies) was used to obtain purity of ≥95% of CD5+19+ cells. (C) Representative examples (n=3) of CD20 protein levels in CLL cells obtained before (Pre) and during idelalisib therapy in vivo (week 4/5 and 9). (D) CLL cells (purity ≥95%) were pretreated with idelalisib (2 mM, Selleckchem) for 4 hours (hrs) or plerixafor (5 mg/mL, Selleckchem) for 4 hrs and then SDF1 (100 ng/mL, CXCR4 ligand) or interleukin 4 (IL4) (20 ng/mL) were added into the media for 24 hrs (for the result of the 48 hrs treatment with SDF1/IL4 see the Online Supplementary Figure S3A). Cell surface CD20 levels were measured and the results are visualized as a fold-change to untreated control (ctrl) (n=12). Viable CLL cells were gated for assessment of cell surface CD20 levels. The pretreatment of CLL cells by idelalisib or plerixafor (CXCR4 inhibitor) for 4 hrs was performed to ensure a full inhibition of the pathways before exposure to the receptor ligands. (Ei) Representative immunoblot of CLL cells treated in vitro with idelalisib (2 mM; 48 hrs) and subsequently stimulated by IL4 (40 ng/mL; 3 minutes [min]). (Eii) Densitometric quantification of pSTAT6 (Tyr 641) protein levels for independent replicates (n=7) of the experiment described in (Ei). (Eiii) Representative immunoblot of CLL cells treated in vitro with idelalisib (2 mM; 48 hrs) then washed twice with clean culture media and stimulated by IL4 in full media (20 ng/mL; 24 hrs). (Eiv) Densitometric quantification of CD20 protein levels for independent replicates of the experiment described in (Eiii) (n=7). (Fi) Representative immunoblot of pSTAT6 (Tyr 641) downmodulation after pretreatment of CLL cells with idelalisib (2 mM; 4 hrs) followed by IL4 stimulation (40 ng/mL; 3 and 5 min). (Fii) Densitometric quantification of pSTAT6 (Tyr 641) protein level for independent replicates (n=5) of the experiment described in (Fi). (G) Representative immunoblot of CLL cells transfected with small interfering RNA (siRNA) against the PI3Kδ isoform (siPI3Kδ, 500 nM, Dharmacon) or a negative control (Neg.Ctrl). Seventy-two hrs after transfection, cells were stimulated by IL4 (40 ng/mL; 3 min) and STAT6 phosphorylation (Tyr 641) was assessed. Phosphorylation of AKT (Ser 473) was used as a surrogated marker for PI3Kδ downmodulation after siPI3Kδ since we were not able to detect PI3Kδ protein due to issues with anti- PI3Kδ primary antibody. (Hi) Representative immunoblot of MEC1 cells transfected with siRNA against CD20 (siCD20, 500 nM, Thermo Fisher Scientific) or negative control (Neg.Ctrl). Forty-eight hrs after the transfection, cells were stimulated by IL4 (40 ng/mL; 3 min). (Hii) Densitometric quantification of pSTAT6 (Tyr 641) protein level for independent replicates (n=4) of the experiment described in (Hi). Neg.Ctrl without IL4 was set as 1 and compared to the other samples. (I) Representative immunoblot of primary CLL cells transfected with siRNA against CD20 (siCD20) or negative control (Neg.Ctrl), cultured for 48 hrs, and then stimulated by IL4 (40 ng/mL; 3 min). For immunoblots, β-actin or GAPDH were used as a loading controls. In all experiments, the statistical difference was tested using a paired t-test, and the error bars indicate standard error of the mean.
Figure 3.
Figure 3.
Schematic overview for CD20 downmodulation by idelalisib via impaired IL4-STAT6 axis. In chronic lymphocytic leukemia (CLL), IL4-STAT6 axis upregulates CD20 gene expression through the STAT6 phosphorylation and its direct binding to CD20 (MS4A1) promoter. The IL4-STAT6-CD20 axis is inhibited by PI3Kδ inhibitor idelalisib. NFκB and FoxO1 represent other two known regulators of CD20 transcription in CLL (NFκB is a positive regulator, and FoxO1 is an indirect negative regulator).1,4,5 This figure was created with tools at BioRender.com.
See this image and copyright information in PMC

References

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