Regulation Of CD20 Expression Research Articles

Cd20 Expression and Effects on Outcome of Relapsed/ Refractory Diffuse Large B Cell Lymphoma after Treatment with Rituximab

Introduction:Down regulation of CD20 expression has been reported in diffuse large B cell lymphoma (DLBCL)). Therefore, it is important to determine whether chemotherapy with rituximab induces CD20 down regulation and effects survival.Objectives:To determine the incidence of down regulation of CD20 expression in relapsed DLBCL after treatment with rituximab and to compare outcomes and assess pattern of relapse between CD20 negative and CD20 positive cases.Methodology:We retrospectively reviewed patients with relapsed DLBCL who received rituximab in the first line setting at Aga Khan University Hospital between January 2007 and December 2014. Data were recorded on predesigned questionnaires, with variables including demographics, details regarding date of diagnosis and relapse, histology, staging, international prognostic index, treatment and outcomes at initial diagnosis and at relapse. The Chi square test was applied to determine statistical significance between categorical variables. Survival curves were generated by the Kaplan–Meier method.Results:A total of 54 patients with relapsed DLBCL were included in our study, 38 (70 %) males and 16(30%) females. Some 23 (43%) patients were at stage IV at the time of diagnosis and 34 (63%) had B symptoms. The most frequent R-IPI at diagnosis was II in 24 (44%) patients. Only 6 (11%) did not show CD20 expression on re-biopsy for relapsed/refractory disease, 2 with CD20 negative DLBCL responding to second line chemotherapy. A complete response after salvage chemotherapy was noted in 16 (29.6%) cases with relapsed/refractory DLBCL. Seven (13%) patients underwent an autologous bone marrow transplant as consolidation after second line treatment. Median overall survival was 18 months in CD20 positive vs. 13 months in CD20 negative patients.Conclusion:This study demonstrated that a small percentage of patients treated with rituximab lose their CD20 expression at the time of relapse. However, it is unclear whether this is associated with an inferior outcome.

Abstract A34: The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies

Abstract Introduction: It was shown that BCR inhibitors such as ibrutinib interrupt microenvironmental interactions and mobilize malignant B cells of chronic lymphocytic leukemia (CLL) from immune niches into the blood stream. It has been suggested that this mobilization will sensitize malignant B cells to other drugs, providing a possibility to introduce synthetically lethal combinations of drugs with an acceptable toxicity profile. This might be limited to a subgroup of CLL patients with specific underlying molecular mechanisms of disease biology or reaction to BCR inhibitors. The aim of this study was to test for the effect of ibrutinib on the expression of genes and activity of pathways that could be potentially targeted by clinically available drugs to achieve highly potent combinations. Results: We performed gene expression profiling in samples obtained from CLL patients treated with ibrutinib as a single agent (pre-ibrutinib vs. day 15 and/or week 5/12/15; N=12 pairs) to identify potential therapeutic targets. We have primarily focused on analyzes of cell-surface proteins and anti-apoptotic Bcl2 family members, since therapeutic antibodies and small molecular inhibitors for these molecules are available. We have analyzed changes in >30 cell-surface molecules, 8 members of the Bcl2 family, and members of other potentially relevant pathways (chemokines, adhesion, Akt, Erk, Ras, NFkB). Notably, we have observed that the mRNA and B cell-surface levels of CD20 are strongly down-modulated with ibrutinib, which indicates that the previously suggested combination of ibrutinib and anti-CD20 antibodies (such as rituximab or ofatumumab) is likely not ideal. Indeed, we showed that CLL cells with lower levels of CD20 are more resistant to rituximab in vitro, but also in vivo (P<0.01). We also showed that the effect of ibrutinib on CD20 levels is mediated by inhibition of CXCR4/SDF-1 axis, which impairs the SDF-1α (ligand for CXCR4) mediated regulation of CD20 expression (P<0.01). Notably, it has been shown that addition of rituximab to ibrutinib in vivo is able to partially eliminate CLL cells despite lower levels of CD20 suggesting that there must be other mechanism of ibrutinib action that allows for the effect of rituximab. The screening of anti-apoptotic molecules revealed that Mcl1 levels were significantly down-modulated after ibrutinib treatment in vivo (P=0.002), and ibrutinib also inhibited Mcl1 induction in CLL cells co-cultured with stromal cells (HS-5). As Mcl1 was described to directly protect CLL cells from rituximab, we suggest that ibrutinib-mediated Mcl1 reduction is a mechanism that facilitates rituximab efficacy. On the other hand we have shown that several members of Bcl2 family are up-regulated in CLL samples obtained after >3 weeks on ibrutinib therapy. We have also shown that interactions of stromal cells (HS-5) with CLL cells lead to up-regulation of these molecules while ibrutinib inhibits this in vitro (P<0.01). This suggest that the observed in vivo up-regulation of specific Bcl2 family members is due to an adaptation of B cells or selection of malignant B cells with high-level expression of anti-apoptotic molecules sufficient to survive the BCR signalling inhibition. We observed that the survival of CLL cells treated with ibrutinib is dependent on Bcl2, and subsequent Bcl2 inhibition can act synergistically with ibrutinib in B cell lines and CLL samples. These anti-apoptotic molecules therefore represent primary candidates with synthetically lethal potential in combination with ibrutinib. Additionally, we have identified dependence of a subtype of CLL cases on integrin signalling. The inhibition of integrin VLA-4 with anti-VLA4 antibody or integrin-associated kinase overcame the resistance of CLL cells to chemotherapy drugs and also to anti-CD20 antibodies, and synergized with the effects of ibrutinib. Conclusion: We have performed gene expression analysis of primary samples obtained from patients on ibrutinib, and identified gene expression changes that are associated with adaptation of B cells and/or selection of B cell resistant to BCR inhibition. We have shown that malignant B cells are dependent on integrin-signalling and the up-regulation of Bcl2 protein, which makes them synergistically lethal targets. Supported by: The results of this research have been acquired within CEITEC 2020 (LQ1601) project with financial contribution made by the Ministry of Education, Youths and Sports of the Czech Republic within special support paid from the National Programme for Sustainability II funds. Citation Format: Gabriela Pavlasova, Katerina Musilova, Vaclav Seda, Katerina Cerna, Eva Vojackova, Veronika Svobodova, Marek Mraz. The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A34.

Abstract 3291: The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions

Abstract The introduction of anti-CD20 antibodies has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin lymphomas. The aim of this study was to analyze molecular pathways that influence expression of CD20 since this is largely unknown. This is of a great clinical interest, as combinatorial therapy of novel BCR-signaling inhibitors ibrutinib and idelalisib currently focuses mainly on the use with anti-CD20 antibodies. Firstly, we analyzed samples obtained from CLL patients treated with ibrutinib and showed that administration of ibrutinib in vivo leads to CD20 down-modulation (P<0.0001). That implies that CD20 expression might be regulated by a yet unknown mechanism in the context of immune niches. Therefore, we focused on determining the effect of microenvironmental interactions on CD20 expression on malignant B cells. We co-cultured primary CLL cells with the bone marrow stromal cell line HS-5. This revealed that CLL cells co-cultured with HS-5 had higher surface CD20 expression compared to control cells cultured on plastic (P<0.01). Moreover, CLL cells that have been recently released from the lymph node microenvironment to the peripheral blood (CXCR4dimCD5bright) had ∼2-fold higher surface CD20 expression when compared to CLL cells circulating in blood of the same patient for a longer time (CXCR4brightCD5dim cells; P<0.0001). Further, sorted CXCR4dimCD5bright CLL cells had also ∼2 times higher CD20 mRNA expression (P = 0.002) suggesting that changes in CD20 expression were rather due to changes in its mRNA levels than surface modulation. As CD20 expression was decreasing with the transition from CXCR4dim to CXCR4bright CLL cells, we hypothesized that the CXCR4/SDF-1 axis is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 produced by stromal cells, significantly up-regulated surface CD20 (P<0.05). On the contrary, the treatment with plerixafor, CXCR4 antagonist, inhibited this SDF-1α mediated up-regulation of CD20 which proves that SDF-1α binding to CXCR4 is directly involved in CD20 regulation. Altogether, the CD20 levels are up-regulated within the context of microenvironmental interactions through the CXCR4/SDF-1 axis, and the impairment of microenvironmental interactions mediated by ibrutinib down-regulates CD20 expression. This study reveals a novel regulation of CD20 expression in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684; NGS-PTL(306242); EHA Fellowship award; Ministry of Health of CR (16-29622A); Academy of Sciences of CR (16-13334Y); Ministry of Education, Youth and Sports, grant LD15144 (COST CZ); MUNI/A/1028/2015; CZ.1.05/1.1.00/02.0068; G.P. is supported by Ostrava city. Citation Format: Gabriela Pavlasova, Marek Borsky, Vaclav Seda, Katerina Cerna, Jitka Osickova, Michael Doubek, Yvona Brychtova, Jiri Mayer, Sarka Pospisilova, Matthew S. Davids, Jennifer R. Brown, Marek Mraz. The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3291.

Microenvironmental Interactions up-Regulate CD20 Expression in CLL B Cells through the CXCR4/SDF-1 Axis: Implications for CD20-Targeting Antibodies and the Use of BCR-Inhibitors in Combination

Abstract The aim of this study was to investigate the role of microenvironmental interactions in the regulation of CD20 expression, since down-modulation of CD20 in the context of immune niches would be a straightforward explanation for the stroma-mediated rituximab resistance described by us and others (Mraz et al., BJH, 2011; Buchner et al., BJH, 2010; Marquez et al., BJH, 2015). Firstly, we co-cultured primary CLL cells with the bone marrow stromal cell line HS-5 for 24/48/72 hrs and analyzed CD20 surface expression by flow cytometry. Suprisingly, CD20 levels were significantly up-regulated (P=0.03) on CLL cells co-cultured with HS-5 in comparison to CLL cells cultured on plastic. The changes in CD20 expression levels are likely to play a direct role in microenvironmental interactions and especially BCR signaling in immune niches (Uchida et al., Int Immunol, 2004). To test this hypothesis we assessed the CD20 expression on CLL cell populations defined according to CXCR4 and CD5 levels. The low-level surface expression of chemokine receptor CXCR4 and high-level expression of activation molecule CD5 can characterize B cells that have recently exited the lymph nodes (Calissano et al., Mol Med, 2011). CXCR4dim CD5bright cells had ~2-fold higher surface CD20 expression when compared to CXCR4bright CD5dim CLL cells circulating in the blood of the same patient for a longer time (P<0.0001, N=21) and CD20 expression clearly decreased with the transition of CLL cells from CXCR4dim to CXCR4bright together with the decrease of CD5 expression. Further, sorted CXCR4dim CD5bright CLL cells had also ~2 times higher CD20 mRNA expression (P=0.002, N=8) suggesting that changes in CD20 expression were due to changes in its mRNA levels rather than a surface modulation. This led us to hypothesize that CXCR4/SDF-1 is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 which is produced by HS-5, up-regulated surface CD20 (P=0.03). We also observed that CLL cells with higher expression of CD20 had higher levels of surface Ig (P=0.0015, N=12) and this was coupled with higher responsiveness to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Therefore, we investigated the effect of ibrutinib on CD20 levels as ibrutinib is a BCR-signaling pathway inhibitor and prevents CLL cells from responding to microenvironmental stimuli, including chemokine signaling. We observed that treatment of CLL cells with ibrutinib in vitro resulted in CD20 down-modulation (P<0.001). The levels of CD20 in CLL cells were also strongly down-modulated in samples obtained before vs. during ibrutinib treatment of CLL patients in vivo (pre- ibrutinib vs. day 15 and week 5/12, N=8; P=0.02, P=0.01, P=0.07, respectively). These data suggest that a reduction of the target antigen (CD20) is not the cause for rituximab resistance in the context of immune niches, and the sustained activity of ibrutinib and rituximab combinations in clinical trials (Burger et al., Lancet Oncol, 2014) suggests that ibrutinib has other mechanisms that allow for rituximab efficacy. We focused on the regulation of anti-apoptotic molecules, namely Mcl1, since that was shown to protect from rituximab-induced apoptosis and to be rapidly down-modulated by rituximab infusion in vivo (Byrd et al., Blood, 2002; Hussain et al., Clin Cancer Res, 2007). Indeed, we observed higher levels of Mcl1 mRNA in the CXCR4dim CD5bright CLL subpopulation (N=8, P=0.04), and in CLL cells co-cultured with stromal cells. Importantly, Mcl1 was down-regulated after ibrutinib treatment both in vitro and in ibrutinib treated patients. Conclusion: Microenvironmental interactions up-regulate CD20 expression in CLL B cells through the CXCR4/SDF-1 axis, and the ibrutinib-mediated impairment of microenvironmental interactions down-regulates CD20 expression. This study reveals a novel regulation of CD20 levels in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684 (cofinanced by the European Union and the South-Moravian Region); NGS-PTL(306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; MUNI/A/1180/2014; CZ.1.05/1.1.00/02.0068; G.P. is a city of Ostrava scholarship holder. #G.P. and V.S. contributed equally to this study. contact: marek.mraz@email.cz Disclosures Mayer: Janssen: Research Funding. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy.

Abstract LB-242: PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies

Abstract The therapeutic strategies currently used in B cell malignancies include the treatment with monoclonal antibodies (rituximab and ofatumumab) directed against CD20 antigen. These antibodies specifically eliminate B cells by triggering indirect effector mechanisms of the immune system, like complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. In many patients, the reduced level of CD20 antigen on the surface of tumor B cells leads to the resistance to anti-CD20 therapy. The aim of this study was to explore the molecular mechanisms governing the regulation of CD20 expression in lymphoma cells as a potential explanation of the resistance to rituximab/ofatumumab therapy. We previously observed that CD20 mRNA expression is significantly affected by the SRC family inhibitors. Here, we report that also PTEN tumor suppressor is a negative regulator of CD20 expression. To uncover the transcriptional mechanisms governing the CD20 expression we employed the construct encoding the promoter region of CD20 cloned upstream of the firefly luciferase gene. Overexpression of wild-type PTEN (but not the phosphatase-deficient mutant) strongly affected the promoter activity and the expression of CD20, leading to decreased binding of rituximab and ofatumumab and increased resistance of tumor cells to complement-dependent cytotoxicity. Using the truncated versions of the CD20 promoter we identified a particular region (-313/-198) as the major region sensitive to PTEN overexpression. We found that the negative regulation of CD20 promoter activity by PTEN was mediated by inhibition of AKT signaling. We observed that the overexpression of constitutively active AKT1 (CA-AKT1) overcame the negative effect of PTEN and sensitized cells to rituximab/ofatumumab treatment. The results of our studies indicate that PTEN status in tumor cells should therefore be considered when analyzing the mechanisms of resistance of B cell malignancies to anti-CD20 therapies. Citation Format: Beata Pyrzynska, Kamil Bojarczuk, Marta Siernicka, Michal Dwojak, Malgorzata Bobrowicz, Nina Miazek, Piotr Zapala, Agnieszka Zagozdzon, Jakub Golab, Magdalena Winiarska. PTEN regulates the CD20 antigen expression and affects rituximab-based therapy of lymphoma malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-242. doi:10.1158/1538-7445.AM2015-LB-242

Src Family Tyrosine Kinases Are Involved in the Transcriptional Regulation of CD20 Levels

Abstract 1661 Introduction:Anti-CD20 monoclonal antibodies (mAbs) have considerably improved the outcomes of patients with B-cell malignancies and reveal promising therapeutic activity in some autoimmune diseases. They eliminate B cells by triggering indirect effector mechanisms of the immune system, namely complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Unfortunately, the resistance to anti-CD20 mAb-based first line therapies has been a considerable clinical problem. The mechanisms of this resistance are still poorly understood.In an elegant in vitro study by van Meerten et al. a direct positive correlation between rituximab antitumor activity and CD20 levels has been demonstrated. Although for many years CD20 has been described as a stable antigen, accumulating evidence indicates that CD20 can be modulated at several levels, both transcriptional and posttranscriptional. The processes that lead to CD20 downregulation could potentially impair antitumor activity of rituximab-based therapies and lead to rituximab resistance.Src family tyrosine kinases (SFTKs) including Lyn, Fyn and Lck have been already reported by Deans et al. to associate with CD20 localized to lipid rafts. They were shown to be activated during anti-CD20 mAb-mediated apoptosis upon clustering of rafts. However, to the best of our knowledge, the role of SFTKs in the regulation of CD20 expression has not been studied so far. Objectives:The aim of this study was to explore the molecular basis for Src family tyrosine kinases- dependent regulation of CD20 levels in lymphoma cells. Results:In the initial experiments performed using flow cytometry we observed a significantly reduced binding of anti-CD20 mAb to Raji cells incubated for 48h with various inhibitors of Src kinases (dasatinib, PP2, nilotynib, bosutinib, saracatinib) (Fig.1A-E). Dasatinib also impaired the binding of rituximab to Raji cells (Fig.1F). Decreased binding of anti-CD20 mAb upon dasatinib treatment was observed in three additional lymphoma cell lines and primary cells isolated from patients with chronic lymphocytic leukemia. All tested SFTKs inhibitors impaired rituximab-mediated CDC (R-CDC) over a dose range of rituximab concentrations (1–100 ug/ml) in all lymphoma cell lines. Interestingly, in Raji cells incubated for 48h with dasatinib we also observed a dose-dependent reduction of total CD20 protein levels, when assayed by Western blotting (Fig.2A). Moreover, a 48-h incubation with dasatinib significantly reduced the transcription of cd20 gene, as assessed with RT-PCR (Fig.2B). To further elucidate the mechanism of transcriptional regulation of CD20 we performed qRT-PCR studies. A strongly reduced transcription of cd20 gene was observed in Raji cells over a dose range of dasatinib (20–200 nM) after 24- (Fig.2C) and 48h- incubation (Fig.2D). Additionally, the CD20 promoter activity measured with reporter Firefly luciferase assay has been reduced as early as 1 hour after dasatinib treatment (Fig.2E). To elucidate in more detail binding of transcription factors to the promoter of cd20 gene, a chromatin immunoprecipitation assay was performed. Our early results indicate that dasatinib impairs binding of PU.1 transcription factor to its consensus site within cd20 promoter in Raji cells. [Display omitted] [Display omitted] Conclusions:Our studies indicate for the first time that SFTKs are involved in the transcriptional regulation of CD20 levels in lymphoma cells. Elucidation of the exact mechanism of this phenomena needs further studies. Results of these experiments will help to understand the biology and regulation of CD20 levels in lymphoma cells.The research was supported by Polish Ministry of Science and Higher Education [N N402 352938 (MW), IP2010/046570 (MW), IP2010/028670 (DN)]. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Regulation of CD20 Protein Expression in B-Cell Lymphoma Cells after Rituximab-Containing Chemotherapy.

For the past 10 years, rituximab has been used with some forms of conventional chemotherapy to treat CD20(+) B-cell malignancies as a new molecular targeting drug. Although significant improvements in disease-free survival and cure rate have been obtained, resistance to rituximab has been recognized as a considerable problem. Here we report that CD20(−) tumor transformation is one important mechanism of resistance to rituximab. We also demonstrate a possibility that CD20 protein expression is downregulated by some epigenetic mechanisms, and interestingly, the protein expression can be restored by treatment with certain epigenetic drugs. During the 5 years beginning in 2001, 124 patients with B-cell malignancies were treated with rituximab-containing induction, consolidation, or salvage chemotherapies in our hospital. Relapse or recurrence of disease were confirmed in 36 patients (29.0%), and re-biopsy of tumor tissues was performed in 19 patients. Five cases (DLBCL, 3 cases, and DLBCL transformed from FL, 2 cases) of those 19 patients (26.3%) indicated CD20 protein-negative transformation in flow cytometry (FCM) and/or immunohistochemistry (IH) analyses. We established a CD20(−) lymphoma cell line RRBL1 from a DLBCL patient with CD20(−) transformation from CD20(+) FL after repeated treatment with rituximab, and analyzed molecular mechanisms of the CD20 protein-negative phenotype. CD20 was not expressed by IH, FCM, and immunoblotting analyses, and resistance to rituximab was observed after cell culture with rituximab treatment. Expression of wild-type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that the CD20 mRNA expression level was almost 100 times lower than that of CD20(+) B-cell lymphoma cells typically obtained from DLBCL patients. These data suggest that aberrant down regulation of CD20 mRNA may be closely related to the negative CD20 surface antigen expression. Interestingly, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression using specific epigenetic drugs including histone deacetylase inhibitor Trichostatin A (TSA). In addition, after TSA treatment, sensitivity for rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of histone deacetylase complexes to CD20 promoter may be related to the down regulation of CD20 expression. Our data may provide the basis for a new therapeutic strategy, epigenetic therapy combined with molecular targeting therapy using rituximab, for rituximab-resistant CD20(−) transformed B-cell malignancies.