Introduction: Chronic lymphocytic leukaemia (CLL) is characterised by the accumulation of malignant B-cells within the peripheral blood and secondary lymphoid organs. CLL patients can be broadly divided into two groups depending on the mutational status of the immunoglobulin variable region genes (IGHV), with unmutated and mutated V- genes associated with progressive and indolent disease respectively. B cell receptor (BCR) expression and signalling capacity is pivotal to CLL pathogenesis and whilst drug which targeting this pathway are revolutionising the treatment of this disease. Resistance is already occurring and therefore a better understanding of this pathway is urgently required if we are to identify novel therapeutic strategies to treat this largely incurable disease. Autophagy primarily acts as a protective mechanism against cellular stress induced by starvation, cytokine removal, hypoxia, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress and is important for normal B-cell development. However autophagy can also promote tumorigenesis and resistance to therapy. The role of autophagy in CLL cases in response to therapy has previously been investigated, however its role in CLL biology and how it is regulated has not previously been evaluated.

Results: We investigated basal expression of a number of autophagy markers between CLL cases and normal donor B cells (NDB). We demonstrated that basal expression of LC3B-II p=0.0005, GABARAPL2 p=0.0113, ATG3 p=0.0005 and ATG7 p=0.034 was greater in CLL samples compared to NDB. Moreover even greater LC3B-II expression was observed in U-CLL cases compared to M-CLL (p=0.02). Using a pre-determined cut-off, LC3B-II expression identified a subset that had a shorter time to first treatment from first lymphocytosis (p=.017) and identified samples particularly within the M-CLL subset which were more likely to progress. Next using tissue extracted from CLL lymph nodes we showed greater LC3B expression in areas with higher levels of Ki67 by immunohistochemistry, indicating an association between proliferation centers and autophagy. This suggested that autophagy might be induced following BCR signalling. Indeed basal expression of LC3B-II was greater in samples with increased BCR signalling capacity. Therefore we investigated the role of the BCR in regulating autophagy. Activation of the CLL BCR with bead immobilised (BI) anti-IgM significantly induced expression of LC3B-II (p<.0001) at 2, 4 and 24 hours, with greater levels induced in U-CLL compared to M-CLL cases. Similar changes were observed with anti-IgD but at reduced levels compared to anti-IgM treatment. Interestingly using confocal microscopy and transmission electron microscopy we demonstrated that the beads coated with anti-IgM were being endocytosed by the CLL cells and the autophagosomes formed around the beads. To confirm that the BI anti-IgM induced autophagy was not solely a result of bead endocytosis we also treated CLL and NDB with soluble anti-IgM and anti-IgD. Soluble anti-IgM significantly induced LC3B-II (p=0.006) and GABARAPL2 (p=0.04) expression compared to untreated controls in CLL cases, but at slightly reduced levels compared to BI anti-IgM. In contrast soluble anti-IgD induced minimal changes or no change at all. Similar changes were also observed in NDB. Next we examined autophagic flux to determine whether BI anti-IgM increased RNA and protein expression or simply inhibited autophagosome breakdown. CLL samples treated with BI anti-IgM increased LC3B and GABARAP RNA expression within 2h (p=.0017 & p=.022 respectively) whilst treatment with BI anti-IgM plus HCQ significantly increased LC3B-II protein expression up to 24 hours compared to HCQ or anti-IgM alone (p<.0001). Confirming that activation of the BCR increased autophagic flux. To confirm the role of the BCR in the regulation of autophagy we treated CLL cells with ibrutinib or fostamatinib to inhibit BCR signalling capacity or treated cells with IL-4 to increase sIgM expression and signalling as we have previously demonstrated. As expected BCR kinase inhibitors prevented anti-IgM induced autophagy. In contrast IL-4 increased sIgM and signalling and consequently induced autophagy highlighting the importance of the BCR in regulating autophagy in CLL. Together these data suggest that the autophagy pathway might be therapeutically targeted for the treatment of CLL.


Strefford: Roche: Research Funding. Forconi: AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Steele: Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria.

Author notes


Asterisk with author names denotes non-ASH members.