Biological Significance of B Cell Receptor Mediated Regulation of Autophagy in Chronic Lymphocytic Leukemia
Authors: Smith Lindsay , Hogg Elizabeth , Haynes Angus , Strefford Jonathan C , Forconi Francesco , Stevenson Freda K , Packham Graham , Cragg Mark S , Tumbarello David , Blunt Matthew D , Steele Andrew J 
Published: October 2019

Abstract
Chronic lymphocytic leukaemia (CLL) is characterised by an accumulation of B cells which is broadly split into two groups representing a progressive IGHV unmutated (U-CLL) and a more indolent IGHV mutated (M-CLL) disease. Activation of the B cell receptor (BCR) by antigen/autoantigen engagement is crucial for CLL cell survival, disease progression and resistance to therapy, however further research is required to better understand how BCR signalling impacts on CLL biology. Autophagy is known to play a role in tumorigenesis and resistance to therapy in solid tumors, however whether autophagy has a role in CLL biology and how it is regulated has not been fully investigated. Autophagy is important for normal B cell development and is known to be regulated by various drug treatments in vitro in CLL samples. A previous study showed that activation of the BCR on murine splenic B cells with soluble or bead immobilised (BI) anti-IgM induced autophagy and subsequent apoptosis, however, the role of BCR-induced autophagy has not been explored in B cell malignancies and particularly CLL. Firstly, we assessed basal protein expression of key autophagy markers LC3BII, and ATG3 in CLL samples and age-matched normal donor B cells (NDB). CLL cells expressed significantly more LC3BII (p=.014, n=57) and ATG3 (p=.04, n=58) compared with NDB (n=8), with a greater LC3BII protein expression in U-CLL compared to M-CLL (p=.039, n=57), indicating more autophagy occurs in U-CLL. Furthermore basal increases in autophagy markers GABARAPL2 (LC3B family member) (p=.0004, n=34) and ATG4A (p=.04, n=20) at the RNA level were significantly associated with the ability of CLL cells to flux calcium (>10%) in response to anti-IgM. This indicated a possible role of the BCR in the regulation of autophagy in CLL samples and a possible association with progressive disease. Activation of the CLL BCR with BI anti-IgM significantly induced expression of autophagy markers ATG3 (p=.002, n=22), LC3BII (p<.0001, n=23) and p62 (p=.0011, n=9) at the protein level and ATG3 (p=.04, n=8) and GABARAPL2 (p=.03, n=6) at the RNA level in a time dependent manner. Next, CLL samples were treated with BI anti-IgM in the presence or absence of the autophagy inhibitor hydroxychloroquine (HCQ) to confirm the increase in autophagic flux indicated at the RNA level. Addition of HCQ in combination with BI anti-IgM significantly increased accumulation of LC3BII protein expression compared with HCQ or anti-IgM alone (p=.01 and p=.02 respectively, n=5) indicating an increase in autophagic flux. Treatment with ibrutinib or R406 prevented BI anti-IgM dependent increases in LC3BII (p=.02 and p=.009 respectively, n=9) and p62 expression (p=.02 and p=.006 respectively, n=9). Confirming the role of the BCR in the regulation of autophagy in CLL samples and indicating that inhibition of autophagy may contribute to the clinical responses seen with these BCR kinase inhibitors. Finally we showed that BI anti-IgM increased CLL cell viability compared to BI isotype control (p=.002, n=7), whilst the addition of HCQ significantly reversed this effect (p=.001, n=7). More importantly treatment with BI anti-IgM protected CLL cells against fludarabine induced cell death (p=.0004, n=3) which again was reversed by the addition of HCQ (p=.05, n=3). These data emphasise the importance of BCR signalling in the regulation of autophagy and its impact on therapy resistance and suggest a possible role for autophagy inhibitors in the treatment of CLL.

Disclosures
Strefford: Roche: Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.