Regulation of B-Cell Receptor Signalling By the Tumour Microenvironment in Chronic Lymphocytic Leukemia (CLL) and Its Impact on Adhesion and miRNA Expression
Authors: Blunt Matthew D , Dobson Rachel , Smith Lindsay D , Strefford Jonathan C. , Conley Pamela B. , Pandey Anjali , Davies Andrew J , Johnson Peter M , Packham Graham , Stevenson Freda K , Forconi Francesco , Coffey Greg P , Steele Andrew J 
Published: October 2019

Abstract
B-cell receptor (BCR) mediated signalling is pivotal to the pathogenesis of chronic lymphocytic leukaemia (CLL), which is broadly split into two major subsets based on the immunoglobulin heavy chain variable (IGHV) gene mutational status. Progressive disease is characterised by unmutated IGHV genes (U-CLL) and more indolent disease by mutated IGHV genes (M-CLL). Antigen/autoantigen engagement is crucial for BCR-driven CLL cell survival, disease progression and resistance to therapy. Drugs that target this pathway are revolutionising the treatment of this disease. However, these inhibitors are not curative and some patients develop resistance to ibrutinib following mutation of the BTK or PLCγ2 genes or for as yet unknown reasons. We have shown that IL-4 pre-treatment induced sIgM expression especially in cases with unmutated IGHV genes, augmented anti-IgM induced signalling and compromised the ability of ibrutinib (1 µM) and idelalisib (1 µM) to inhibit BCR-mediated signalling. Previous reports highlighted a role of miR-155 (Cui Blood 2014) and miR150 (Mraz Blood 2014) in regulating BCR signalling in CLL cells by targeting SHIP1 (negative regulator of BCR signalling) and GAB1 and FOXP1 (positive regulators of BCR signalling) respectively. In this study we investigated the effect of IL-4 on expression of these miRNA and demonstrated that IL-4 augments and inhibits expression of miR155 and miR150 respectively (Figure 1), but had no effect on the house keeping control gene RNU6, demonstrating a mechanism whereby IL-4 augments BCR signalling. Furthermore, since BCR engagement can promote adhesion in CLL cells, we investigated the effect of IL-4 alone and in combination with anti-IgM on integrin expression such as CD49d, CD49e and CD29 and on adhesion. IL-4 pre-treatment augmented CD49d (α4) and CD44 expression and promoted anti-IgM as well as PMA mediated adhesion of CD5\CD19+ CLL cells to fibronectin. Interestingly in contrast to greater sIgM increases by IL-4 in U-CLL, IL-4 promoted greater anti-IgM-induced adhesion largely in the M-CLL subset. This indicates that IL-4 may have distinct effects in the two subsets.
Given our results, we hypothesised that dual inhibition of IL-4 and BCR signalling may promote greater responses in patients. At ASH 2015 we demonstrated that cerdulatinib, a dual JAK/SYK inhibitor, now in phase II clinical trials in CLL, can inhibit IL-4 and anti-IgM/anti-IgD induced signalling at concentrations achievable in patients (Cmin 1μM). We have now extended these studies and shown that cerdulatinib treatment prevented IL-4-induced effects such as increased sIgM expression and subsequent downstream signalling, and prevented downmodulation of CXCR4 and to a greater extent than the specific JAK3 inhibitor tofacitinib. Furthermore, inhibition of JAK1/3 by cerdulatinib also reduced IL-6, IL-10, IL-15, IL-21 and IFNγ-induced signalling, which have all been shown to play a role in CLL biology. More importantly, cerdulatinib killed CLL cells under basal conditions, particularly in cases with progressive disease. However, CLL cells within the patient lymph nodes likely receive signals from the BCR, IL-4 and possibly CD40L, which results in the elevation of pro-survival proteins Mcl-1 and Bcl-XL, providing protection from basal and ibrutinib or venetoclax induced killing. Following simultaneous treatment with bead-immobilised anti-IgM, 10ng/ml IL-4 and 300ng/ml CD40L, cerdulatinib was still able to induce a modest amount of apoptosis, however in combination with venetoclax there was a synergistic increase in killing. Together these data provide evidence of how IL-4 increases BCR signalling by directly modulating miRNA expression, but also additional effects on adhesion which may subsequently retain the tumour cells within the lymph nodes. JAK inhibition may prevent these effects. Therefore, these findings indicate that JAK inhibition in combination with BCR kinase inhibition maybe of value for the treatment of CLL.
Figure. Q-PCR analysis of miR-155 and mIR-150 expression following treatment with or without IL-4 for 24h. Figure. Q-PCR analysis of miR-155 and mIR-150 expression following treatment with or without IL-4 for 24h.

Disclosures
Conley: Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Pandey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Davies:Bayer: Research Funding; Karyopharma: Honoraria, Research Funding; GSK: Research Funding; Janssen: Honoraria; Pfizer: Research Funding; Mundipharma: Honoraria; Gilead: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel to scientific conferences, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodation, expenses, Research Funding. Johnson:Celldex Therapeutics: Research Funding. Packham:Karus Therapeutics: Other: Share Holder & Founder; Aquinox Pharmaceuticals: Research Funding. Coffey:Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Steele:Portola Pharmaceuticals: Honoraria.