B-Cell Receptor Pathway Inhibitors Affect CD20 Levels and Impair Antitumor Activity of Anti-CD20 Monoclonal Antibodies

Letters to the Editor 1163 13 Kuruvilla J, Gutierrez M, Shah BD, Gabrail NY, de Nully Brown P, 14 Yu L, Mohamed AJ, Simonson OE, Vargas L, Blomberg KE, Bjorkstrand B et al. Stone RM et al. Preliminary evidence of anti tumor activity of selinexor Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter (KPT-330) in a phase I trial of a first-in-class oral selective inhibitor via NF-kappaB. Blood 2008; 111: 4617–4626. of nuclear export (SINE) in patients (pts) with relapsed/refractory non 15BurgerJA,BurgerM,KippsTJ.Chronic lymphocytic leukemia B cells Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Blood 2013; express functional CXCR4 chemokine receptors that mediate spontaneous 122: 90. migration beneath bone marrow stromal cells. Blood 1999; 94: 3658–3667. Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) B-cell receptor pathway inhibitors affect CD20 levels and impair antitumor activity of anti-CD20 monoclonal antibodies Leukemia (2014) 28, 1163–1167; doi:10.1038/leu.2014.12 also tested a primary MCL sample and upon treatment with BCR inhibitors observed a significant downregulation of surface CD20 levels and a trend towards impaired R-CDC and O-CDC (Supplementary Figure 1b). Moreover, we determined the Signaling via the aberrantly activated B-cell receptor (BCR) has a influence of BCR inhibitors on CD20 surface levels in a critical role in the pathogenesis of B-cell tumors by promoting series of 15 tumor cell lines, including Burkitt’s lymphoma (Ramos, survival and clonal expansion of malignant B cells.1,2 Multiple Daudi and BJAB), ALL (NALM-6), diffuse large B-cell lymphoma preclinical studies indicate that blocking various components of (BCR-dependent Ly-1, Ly-7, Ly-10, DHL-6, HBL-1, U2932 and the BCR signaling pathway holds a great therapeutic potential in BCR-independent Ly-4, Ly-19, Pfeiffer) and CLL (EHEB and MEC-1). the treatment of B-cell leukemias and lymphomas.3,4 This is further We observed a significantly decreased surface CD20 levels in most supported by clinical data from recent and ongoing clinical trials, CD20-positive tumor cells irrespectively of the tumor type/subtype which demonstrate considerable activity of SYK inhibitors (Figure 1e). Surprisingly, as opposed to the primary CLL samples we (fostamatinib and GS-9973),5 BTK inhibitors (ibrutinib, AVL-292, did not observe a significant CD20 downregulation in either of CC-292 and ONO-4059)6,7 and PI3Kd inhibitor (CAL-101)8 as single the two tested CLL cell lines (Figure 1e). Since anti-CD20 mAbs are agents or in combination with other therapies in patients with known to also mediate antibody-dependent cell-mediated cytotoxi- 10 B-cell tumors, for whom rituximab-based regimens have become a city (ADCC), we carried out additional analyses to measure the standard of care. However, tumor relapses occur in a significant effects of R406, ibrutinib or CAL-101 on ADCC in various settings. percentage of patients treated even with the most effective Pre-incubation of Raji cells with the increasing concentrations of modalities. Therefore, combinations of anti-CD20 monoclonal inhibitors, followed by a coculture with rituximab and NK effector antibodies (mAbs) with targeted therapeutics inhibiting BCR cells in the absence of tested drugs, failed to influence R-ADCC, as signaling pathways have recently become an active area of determined by CD107a mobilization (Figure 1f). However, in a investigation. For example, ibrutinib and CAL-101 are currently clinically more relevant co-incubation model, where both target Raji being investigated in combination with chemotherapy and/or cells and effector NK cells were cocultured for 4 h in presence of BCR anti-CD20 mAbs (rituximab and ofatumumab) in several phase I, II inhibitors, rituximab-induced degranulation of NK cells was severely and III clinical trials (summarized in Kharfan-Dabaja et al.9). impaired at 1 mM of R406 or ibrutinib (Figure 1f). Similarly, in a Conspicuously, the antitumor effects of these combinations have co-incubation model R406 and ibrutinib inhibited NK cytotoxicity not been studied in the preclinical setting. Therefore, we decided against rituximab- (Figure 1g) and ofatumumab-opsonized to investigate the molecular interactions between anti-CD20 (Supplementary Figure 1c) carboxyfluorescein succinimidyl ester mAbs and R406 (active metabolite of fostamatinib), ibrutinib (CFSE)-labeled Raji cells as assessed using flow cytometry. Moreover, and CAL-101. the release of tumor necrosis factor (TNF) and interferon (IFN)-g from Startlingly, we observed a significant impairment of ritux- NK cells incubated with either R406 or ibrutinib was severely imab-(R-CDC, Figure 1a) and ofatumumab-induced comple- impaired (Figure 1h). Noteworthy, even the highest concentrations of ment-dependent cytotoxicity (O-CDC) (Supplementary Figure 1a) in all tested BCR inhibitors did not affect the viability of NK cells in a 48- Raji cells pre-incubated for 48 h with increasing concentrations of h culture (Supplementary Figure 1d). Conspicuously, CAL-101 exerted R406, ibrutinib or CAL-101, when compared with controls. The no antagonistic effect on NK cell cytotoxicity, CD107a mobilization highest tested concentration (1 mM) of R406 and ibrutinib almost and cytokine secretion in a co-incubation model. This is in 11 completely abrogated both R-CDC and O-CDC, whereas CAL-101 accordance with a study by Herman et al., where CAL-101 was affected anti-CD20 mAb-mediated CDC to a lesser extent. Flow reported not to interfere with rituximab-mediated or alemtuzumab- cytometry studies further revealed that pre-incubation of Raji cells mediated ADCC, although it diminished IFN-g production by NK cells. with BCR pathway inhibitors severely impaired the binding of The levels of complement regulatory proteins (CD46 and CD55) various anti-CD20 mAbs (fluorescein isothiocyanate-conjugated L27 as well as other B-cell surface antigens (CD38 and CD19) remained clone (Figure 1b), rituximab and ofatumumab (Figure 1c)). All three roughly unchanged upon treatment with BCR inhibitors compounds induced a downregulation of surface CD20 levels in a (Supplementary Figure 2). However, we observed a significant panel of 26 primary chronic lymphocytic leukemia (CLL) samples downregulation of CD21 and CD22, of which CD22 is currently pre-incubated for 48 h with 1 mM of each BCR signaling inhibitor used as a target for toxin-conjugated anti-CD22 mAbs (inotuzu- (Figure 1d). Since ibrutinib has been recently approved as a second- mab ozogamicin). Thus, our results may suggest that CD20 line monotherapy for patients with mantle cell lymphoma (MCL), we downregulation is not an isolated phenomenon and BCR Accepted article preview online 10 January 2014; advance online publication, 4 February 2014 & 2014 Macmillan Publishers Limited Leukemia (2014) 1129 – 1174 Letters to the Editor 1164 Leukemia (2014) 1129 – 1174 & 2014 Macmillan Publishers Limited Letters to the Editor 1165 inhibitors could potentially reduce the activity of other mAbs- incubation with R406, ibrutinib or CAL-101, we observed a based modalities. significant decrease in luciferase activity (Figure 2e, black bars). The selectivity of small-molecule signaling inhibitors is rather Since stimulation of BCR signaling is followed by recruitment and limited, and most inhibitors target various kinases. Therefore, to activation of SYK, we co-transfected Raji cells with the CD20 investigate whether modulation of CD20 levels results from promoter reporter and the TEL–SYK fusion gene ensuring inhibition of selected components of BCR signaling, we efficiently constitutive autophosphorylation and activation of SYK. knocked down SYK and BTK with short hairpin RNA (shRNA) Expression of TEL–SYK protected Raji cells from R406- or (Figure 2a) and using flow cytometry observed significantly ibrutinib-mediated CD20 promoter downregulation but had no decreased surface CD20 levels when compared with controls influence on CAL-101 activity. Furthermore, given the role of AKT modified with vector-encoding non-silencing shRNA (Figure 2a). in propagation of signals from BCR,13 we co-transfected Raji cells These results indicate that the effects of R406 and ibrutinib are with the CD20 promoter reporter plasmid and a constitutively likely caused by inhibition of SYK and BTK, respectively. active AKT1 expression vector (pMIG-myrAKT). We observed that Immunoblotting revealed a significant, dose-dependent down- myrAKT1 reversed downregulatory effects of BCR inhibitors regulation of total CD20 at the protein level in Raji cells pre- on CD20 promoter activity (Figure 2e, gray bars). The rescue incubated with increasing concentrations of all tested BCR effect was most pronounced for CAL-101 but was also inhibitors (Figure 2b). Accordingly, quantitative reverse transcrip- significant for both ibrutinib and R406. Similarly, Raji cells tion polymerase chain reaction (qRT-PCR) analyses revealed that retrovirally modified to express myrAKT1 (Raji pMIG-myrAKT) CD20 is downregulated at the mRNA level in a dose-dependent were protected from BCR inhibitor-induced surface CD20 decrease manner for all tested drugs (Figure 2c). Noteworthy, we to a higher extent than cells modified with a control plasmid reanalyzed the microarray data originally published by Chen (Raji pMIG-con) (Figure 2g). et al.12 and concluded that the MS4A1 (CD20)genewas Collectively, these results indicate that BCR signaling inhibitors significantly downregulated upon treatment with R406 in a downregulate CD20 levels

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