B-Cell Receptor Signaling Drives Glycolysis in Chronic Lymphocytic Leukemia Cells
Authors: Clear Andrew James , D'Avola Annalisa , Agrawal Samir G. , Rassenti Laura Z. , Kipps Thomas J. , Gribben John G. , Riches John 
Published: October 2019

Abstract
A key feature of tumor cell energetics is preferential metabolism of glucose to lactate even in the presence of oxygen (aerobic glycolysis). While this is an inefficient way of producing energy, it enables cancer cells to use glucose to generate biomass to support cellular proliferation. It is unclear whether this process is playing a role in the pathogenesis of chronic lymphocytic leukemia (CLL). Previous studies have noted that CLL cells have increased numbers of mitochondria when compared with healthy B cells (Carew Leukaemia 2004, Jitschin Blood 2014). As CLL cells have increased expression of lipoprotein lipase it has been hypothesized that CLL cells adopt a predominantly mitochondrial metabolism.
In this study, we investigated the role of metabolism in the pathogenesis of CLL. While initial analysis of mitochondrial mass by flow cytometry showed that CLL cells do have increased mitochondrial content compared to healthy B cells, these and previous observations were comparing a monoclonal population of CLL cells with a CD27+ activated/memory phenotype with polyclonal healthy B cells predominantly composed of naïve CD27- cells. When we investigated this further we found that healthy CD27+ memory B cells have a higher mitochondrial mass when compared with healthy CD27- naïve B cells. CLL cells actually had reduced mitochondrial mass when compared healthy CD27+ memory B cells, which fell further with disease progression.
B-cell receptor (BCR) signaling plays a vital role in the pathogenesis of CLL, as shown by the clinical efficacy of inhibitors of this pathway. Despite this little is known regarding the impact of BCR-signaling on CLL-cell metabolism. Primary human CLL cells were stimulated with either anti-IgM, anti-IgD or anti-IgG (isotype control) for up to 72 hours before measuring the residual glucose concentration of the media to assess glucose uptake. Anti-IgM treated CLL cells showed an increase in glucose uptake compared to control. The impact of anti-IgM on the expression of enzymes involved in glycolysis including glucose transporters (GLUTs), hexokinase, phosphofructokinase, enolase, and pyruvate kinase was assessed by immunoblotting. As myc is induced by BCR-signaling in CLL cells we focused on known myc targets hexokinase 2 (HK2), enolase 1 (ENOL-1), lactate dehydrogenase A (LDHA) and the heterogenous nuclear ribonuclearproteins (hnRNPs) A1, A2/B1 and PTBP1. GLUT3, HK2 and the hnRNPs were all expressed at low levels in resting CLL cells but increased significantly (at 24 hours) after anti-IgM stimulation. The other myc targets ENOL-1 and LDHA were constitutively expressed and did not increase further after stimulation. There was significant heterogeneity in response to anti-IgM stimulation with IGHV unmutated cases showing a trend toward greater glucose uptake and enzyme induction, while anti-IgD stimulation had a similar but weaker effect. Immunohistochemistry on lymph node biopsies from CLL patients showed a significant increase in expression of GLUT3 and HK2 within CLL proliferation centres. hnRNP induction has been shown to promote a switch to use of the M2 isoform of pyruvate kinase (PKM2): a key feature of many cancers. Interestingly, both circulating and lymph node CLL cells were already switched to using PKM2 and so anti-IgM stimulation had little further effect. However, when the relative levels of PKM1 and PKM2 were compared between early- and advanced-stage patients there was a significant shift to use of PKM2 with disease progression. Treatment of CLL cells in vitro by ibrutinib, idelalisib and the MEK inhibitor U0126 all blocked the anti-IgM induced increase in glucose uptake and GLUT3, HK2 and hnRNP expression. Investigation of the expression of GLUT3 and HK2 in CLL cells obtained from ibrutinib-treated patients also showed a reduction in the expression of these proteins demonstrating that ibrutinib is metabolically reprogramming CLL cells in vivo.
We conclude that previous observations regarding an increase in mitochondrial mass in CLL cells compared to healthy B cells reflects their differentiation states. In contrast, we show that BCR-signaling increases glucose uptake and glycolytic enzyme expression in a myc-dependent manner as part of a switch to aerobic glycolysis. Treatment with BCR inhibitors block this effect. Therefore, we anticipate that many of the novel anti-glycolytic therapies currently in development will prove useful in the treatment of CLL.

Disclosures
Kipps: F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Gribben:Acerta Pharma: Honoraria, Research Funding; Novartis: Honoraria; Wellcome Trust: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; TG Therapeutics: Honoraria; Cancer Research UK: Research Funding; Unum: Equity Ownership; Medical Research Council: Research Funding; Janssen: Honoraria, Research Funding; NIH: Research Funding; Roche: Honoraria; Pharmacyclics: Honoraria.