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. 2020 Feb;34(2):640-644.
doi: 10.1038/s41375-019-0557-y. Epub 2019 Aug 28.

BCR signaling contributes to autophagy regulation in chronic lymphocytic leukemia

Lindsay D Smith  1 , Annabel R Minton  2 , Matthew D Blunt  2 , Laura I Karydis  2 , David A Dutton  2 , Karly-Rai Rogers-Broadway  2 , Rachel Dobson  2 , Rena Liu  2 , Faith Norster  3 , Elizabeth Hogg  2 , Margaret Ashton-Key  4 , Jonathan C Strefford  2 , Li Jia  3 , Dimitar G Efremov  5 , G Vignir Helgason  6 , Peter W M Johnson  2 , Freda K Stevenson  2 , Francesco Forconi  2 , Mark S Cragg  2 , David A Tumbarello  7 , Graham Packham  2 , Andrew J Steele  8   9
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BCR signaling contributes to autophagy regulation in chronic lymphocytic leukemia

Lindsay D Smith et al. Leukemia. 2020 Feb.
No abstract available

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Characterization of autophagy-marker protein levels at baseline and following BCR engagement. a Protein was extracted from snap-frozen PBMCs isolated from CLL patients or HDB purified by negative selection. The level of LC3B-II (n = 43), GABARAPL2 (n = 19), ATG3 (n = 42), and ATG7 (n = 40) was quantified by immunoblotting and normalized protein levels relative to the Hsc70 loading control are shown. Mean values are indicated. A Mann–Whitney test was used for statistical analysis. b Basal LC3B-II protein levels in CLL samples (n = 43) divided by IGHV mutational status into mutated (M-CLL) and unmutated (U-CLL) cases, and (c) BCR signaling capacity defined by U-CLL, M-CLL signallers (M-CLL-S) (>5% iCa2+ flux), and M-CLL low signallers (M-CLL-LS) (≤5% iCa2+ flux). Mean values are indicated. A Mann–Whitney test was used for statistical analysis. d CLL samples were treated with bead-bound isotype control antibody (IC), anti-IgM, or anti-IgD for 2, 4, or 24 h, and the level of LC3B-II (n = 21) assessed by immunoblotting. Blots were quantified and the mean fold change (±SEM) in the level of LC3B-II with anti-IgM normalized to the IC at 2 h is shown. A Wilcoxon’s matched-pairs signed-rank test was  used for statistical analysis. e A larger cohort of patient samples (n = 45) were treated with bead-bound anti-IgM for 24 h and LC3B-II levels divided according to IGHV status into M-CLL and U-CLL as previously described. Mean values are indicated. A Mann–Whitney test was used for statistical analysis
Fig. 2
Fig. 2
BCR-mediated autophagy is dependent on BCR signaling and provides a survival advantage to CLL cells. a CLL samples (n = 11) were treated with bead-bound isotype control antibody (IC) or anti-IgM with or without HCQ at indicated concentrations for 24 h and LC3B-II levels evaluated by immunoblotting. A representative immunoblot is shown. Blots were quantified and the mean fold change (±SEM) in LC3B-II level with each treatment vs. IC without HCQ is shown in the accompanying graph. A t-test was used for statistical analysis. b CLL samples (n = 6) were treated with or without IL-4 (10 ng/ml), in the presence or absence of HCQ, for 24 h before treatment with bead-bound IC or anti-IgM for 24 h. LC3B-II levels were evaluated by immunoblotting and the mean fold change (±SEM) in LC3B-II levels with each treatment vs. IC without HCQ is shown. A Wilcoxon’s matched-pairs signed-rank test was used for statistical analysis. c CLL samples (n = 10) were treated with HCQ and a SYK (tamatinib; Tam) or BTK (ibrutinib; Ibr) inhibitor (both 5 μM) for 1 h before stimulation with bead-bound IC or anti-IgM for 24 h and the LC3B-II level evaluated by immunoblotting. Hsc70 was used as a loading control. A representative immunoblot is shown. Blots were quantified and the mean fold change (±SEM) in LC3B-II level with each treatment vs. IC DMSO is shown in the accompanying graph. A Wilcoxon’s matched-pairs signed-rank test was used for statistical analysis. d CLL samples were treated for 6 h with bead-bound IC or anti-IgM before treatment with autophagy inhibitor, VPS34-IN1 (3 μM), either alone or in combination with venetoclax (5 nM n = 6, or 10 nM n = 8 where shown) for 24 h. All conditions were carried out with or without Q-VD-Ph (10 μM) to identify caspase-dependent drug-mediated cell killing. Cell viability was assessed by the CellTiter-Glo Cell Viability Assay. The mean (+SEM) percentage of viable cells relative to IC is shown. A Wilcoxon’s matched-pairs signed-rank test was used for statistical analysis. e Synergy between VPS34-IN1 (3 μM) and venetoclax (5 nm, left and 10 nM, right) was evaluated as detailed in the Supplementary Materials and Methods. XY line, observed survival = expected survival. Points below the line, synergistic interactions; points above the line, additive interactions
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References

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