scholarly journals An IgG3 switch variant of rituximab mediates enhanced complement-dependent cytotoxicity against tumour cells with low CD20 expression levels

2013 ◽  
Vol 161 (2) ◽  
pp. 282-286 ◽  
Author(s):  
Thies Rösner ◽  
Stefanie Derer ◽  
Christian Kellner ◽  
Michael Dechant ◽  
Stefan Lohse ◽  
...  
Keyword(s):  
Tumour Cells ◽  
Expression Levels ◽  
Complement Dependent Cytotoxicity ◽  
Cd20 Expression ◽  
Switch Variant

An IgG3 switch variant of rituximab mediates enhanced complement-dependent cytotoxicity against tumor cells with low CD20 expression levels

Immunobiology ◽  
2012 ◽  
Vol 217 (11) ◽  
pp. 1138
Author(s):  
Stefanie Derer ◽  
Thies Rössner ◽  
Michael Dechant ◽  
Stefan Lohse ◽  
Christian Kellner ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Expression Levels ◽  
Complement Dependent Cytotoxicity ◽  
Cd20 Expression ◽  
Switch Variant

Upregulation of CD20 on Human Acute Lymphoblastic Leukemia Cells by IL-4 and CpG Motif Containing Oligonucleotides Increases Susceptibility to Rituximab.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1879-1879
Author(s):  
Bart Nijmeijer ◽  
Marianke L.J. Van Schie ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Surface Expression ◽  
Chimeric Antibody ◽  
Transcriptional Level ◽  
Apparent Lack ◽  
Expression Levels ◽  
Cd20 Expression ◽  
Cpg Motif

Abstract Monoclonal antibodies are emerging modalities in the treatment of hematologic malignancies. Rituximab (RTX), a chimeric antibody that recognizes CD20, shows therapeutic efficacy in non-Hodgkin lymphoma (NHL). Precursor-B ALL (pB-ALL) may also express CD20. However, little is known on the activity of RTX in this disease. We determined CD20 surface expression levels on primary pB-ALL cells by flow cytometry following a standardized protocol in which cells are stained with RTX and secondary antibody. CD20 on primary ALL cells was generally absent or expressed at lower levels than on primary NHL cells (median mean fluorescence intensity (MFI): 35.1, range 5–423 in 17 pB-ALL samples, and median MFI 1082, range 440–1818 in 6 NHL samples). The ALL cells that expressed the highest levels of CD20 were similarly susceptible to RTX mediated complement dependent cytotoxicity (CDC) as NHL cells (median 17.8% lysis, range 9.5–98% and 41% lysis, range 13–72%, respectively). However, in pB-ALL cells, RTX activity was strongly limited by CD20 expression levels as lysis strictly correlated with CD20 surface expression (P=0.89), and CD20 negative pB-ALL cells were not killed. Despite apparent lack of surface expression, quantitative RT-PCR (qPCR) demonstrated that all samples expressed CD20 mRNA. Transcriptional expression levels correlated with surface CD20 expression (P=0.72). Therefore we explored upregulation of CD20 as a strategy to augment activity of RTX in ALL. We previously established an in vitro culturing system that supports long-term proliferation of ALL cells. Using the LeidenALL cell lines that were generated in this system, we evaluated the effect of various agents on expression of CD20. Ten LeidenALL pB-ALL cell lines were cultured in the presence or absence of IL-2, IL-3, IL-5, IL-7, IL-15, a CpG motif containing oligonucleotide (CpG), TNFa or IFNg. Culture of LeidenALL cells in the presence of IL-4 resulted in upregulation of CD20 in 4 out of 10 samples, 24 hours after incubation. Culture of LeidenALL cells in the presence of CpG resulted in upregulation of CD20 in another 4 out of 10 samples, 48 hours after incubation. None of the other cytokines affected expression of CD20. IL-4 and CpG displayed synergistic action as co-incubation of LeidenALL cells with IL-4 and CpG resulted in further increased expression of CD20 in 9 out of the 10 samples after 48 hours of co-incubation. Mean CD20 expression increased from MFI 406 (range 14–2668) to MFI 1572 (range 70–4394). qPCR revealed that CD20 was upregulated on a transcriptional level in all of the 10 cell lines. Upregulation of CD20 augmented RTX-mediated CDC (from 41% lysis, range 8–95% to 78% lysis, range 3–93%). Three CD20-negative LeidenALL cell lines that initially were not susceptible to RTX-mediated CDC were lysed after upregulation. In an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay, using peripheral blood mononuclear cells from healthy donors, rituximab-mediated ADCC was increased in 7 out of the 10 samples (from 18% lysis, range 4–48% to 25% lysis, range 6–51%). In conclusion, these results demonstrate that RTX may possess activity in pB-ALL. Activity of RTX in ALL can be potentiated by modulation of the leukemic cells. These insights may allow successful application of rituximab in ALL.

Predominant Complement Mediated Lysis of B-CLL Cells by Therapeutic MAbs Rituximab and Campath-1H in Whole Blood Assays.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3445-3445 ◽  
Author(s):  
Josee Golay ◽  
Luca Bologna ◽  
Elisa Gotti ◽  
Alessandro Rambaldi ◽  
Renato Bassan ◽  
...  
Keyword(s):  
Target Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Human Igg ◽  
Blood Samples ◽  
Expression Levels ◽  
Cd20 Expression

Abstract Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit (<0.1%) in most cases analysed, suggesting that ADCC in the circulation is not a major mechanism of lysis in this disease subtype. Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.

Prenyl Transferases Are Involved in the Regulation of CD20 Levels and Influence Anti-CD20 Monoclonal Antibody-Mediated Activation of Complement-Dependent Cytotoxicity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3722-3722
Author(s):  
Dominika Nowis ◽  
Magdalena Winiarska ◽  
Jacek Bil ◽  
Angelika Muchowicz ◽  
Malgorzata Wanczyk ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Antitumor Effects ◽  
Raji Cells ◽  
Farnesyl Transferase ◽  
Complement Dependent Cytotoxicity ◽  
Cd20 Expression ◽  
Geranylgeranyl Transferase ◽  
Anti Cd20 ◽  
Hodgkin's Lymphomas

Abstract Abstract 3722 Anti-CD20 monoclonal antibodies (mAbs) (rituximab or ofatumumab) are being successfully used in the treatment of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). They exert antitumor effects by triggering indirect effector mechanisms of the immune system, such as activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, upon crosslinking with secondary antibodies, anti-CD20 mAbs can induce cell death. It is frequently underscored that CD20 expression levels in various B-cell tumors is relatively constant. However, accumulating evidence indicates that CD20 can be modulated at transcriptional, posttranscriptional, and even posttranslational levels. Moreover, it has been clearly shown in vitro that CDC efficacy of anti-CD20 mAbs clearly depends on CD20 expression. We have previously observed that statins impair detection of CD20 in non-Hodgkin lymphoma cells and impair rituximab-mediated CDC and ADCC (Winiarska et al. PLoS Med 2008). Statins are inhibitors of cholesterol synthesis and decrease production of prenyl groups (farnesyl and geranylgeranyl PPi), which are necessary for posttranslational modification of approximately 1% of cellular proteins. In experiments aimed at elucidation of the molecular mechanisms of statin-mediated modulation of CD20 we observed that neither geranylgeranyl transferase (GGT) nor farnesyl transferase (FNT) inhibitors could mimic statins effects. On the contrary, prenyltransferase inhibitors improved rituximab-mediated CDC. Therefore, we decided to investigate in more detail the interaction of prenyltransferase inhibitors and anti-CD20 mAbs. In the initial experiments we evaluated the effects of three different farnesyl transferase inhibitors as well as three different geranylgeranyl transferase inhibitors. Among all FNT and GGT inhibitors, L-744,832 seemed to produce the most significant influence on both rituximab-mediated CDC and CD20 levels (Figure). Moreover, L-744,832 significantly increased rituximab-mediated CDC in 3 out of 5 primary tumor cell cultures isolated from patients with NHL or CLL. Therefore, L-744,832 was selected for further more systematic studies. Interestingly, in Raji cells L-744,832 did not improve rituximab-mediated ADCC and only at the highest non-toxic concentrations it sensitized to rituximab+crosslinking antibody-mediated cytotoxicity. In 10 out of 17 (58.8%) primary lymphoma/leukemia cells L-744,832 increased CD20 expression by at least 20% as measured with flow cytometry. Moreover, we observed that upon L-744,832 treatment CD20 is up-regulated in Raji cells at both mRNA as well as protein level. Experiments aimed at investigation of FTI influence on proteasome activity as well as CD20 endocytosis and shedding revealed that L-744,832 influences CD20 levels independently from its posttranslational regulation. To verify whether modulation of CD20 levels by L-744,832 results from specific inhibition of farnesyltransferase or is an off-target effect of this compound we performed FNT B subunit (FNTB) knock-down experiments that resulted in increased CD20 levels by almost 60%. Incubation of Raji cells with a transcription inhibitor cycloheximide completely prevented L-744,832-mediated increase of CD20 levels in WB. Therefore, a chromatin immunoprecipitation assay was performed to determine whether inhibition of FNT activity is associated with binding of transcription factors to the promoter of cd20 gene. These studies revealed that L-744,832 promotes binding of PU.1 and Oct2, but not TFE3 to target DNA sequences within cd20 promoter in Raji cells. To conclude, our studies indicate for the first time that CD20 expression can be modulated by prenyltransferase inhibitors. While inhibition of FNT activity significantly up-regulates expression of CD20, the influence of GGT inhibitors on this protein is more complex, and requires further studies. Furthermore, pre-incubation of NHL and CLL cells with L-744,832, a FNT inhibitor, potentiates anti-CD20 mAb-mediated activation of the complement-mediated cytotoxicity. Therefore, the combination seems to be promising and its efficacy should be determined in patients with NHL or CLL. Disclosures: Winiarska: Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.

scholarly journals Mutations at 3' Untranslated Region (3'UTR) of NOTCH1 Are Associated with Low CD20 Expression Levels in Chronic Lymphocytic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 306-306
Author(s):  
Tamara Bittolo ◽  
Federico Pozzo ◽  
Riccardo Bomben ◽  
Tiziana D'Agaro ◽  
Vanessa Bravin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Untranslated Region ◽  
Lymphocytic Leukemia ◽  
Transcript Levels ◽  
Expression Levels ◽  
Cd20 Expression ◽  
Trisomy 12 ◽  
Anti Cd20 ◽  
Notch1 Mutations

Abstract Background. In chronic lymphocytic leukemia (CLL), NOTCH1 mutations associate with clinical resistance to anti-CD20 immunotherapy in FCR combination (Stilgenbauer et al., Blood, 2014, Dal Bo et al., AHO, 2014), that can be ascribed to a NOTCH1 mutation-driven repression of CD20 levels by HDACs (Pozzo et al., Leukemia, 2016). Recently, novel recurrent mutations have been identified in the 3'untranslated region of NOTCH1 (3'UTR NOTCH1 mutations), determining a novel splicing event within the last NOTCH1 exon (Puente et al., Nature, 2015), leading to an impaired degradation of NOTCH1 protein. Aim. To determine if 3'UTR NOTCH1 mutations associate with low CD20 levels. Methods. NOTCH1 mutations were screened by next-generation sequencing (NGS) in exon 34 and part of 3'UTR with at least 1000X coverage. NOTCH1 (transmembrane, TM, or intracytoplasmic domain, NICD) protein levels were investigated by western blot (WB). CD20 expression was investigated by flow cytometry with a FITC-conjugated anti-CD20 antibody (L27 clone). MS4A1 (encoding for CD20 protein) transcript levels were investigated by qRT-PCR. Susceptibility to anti-CD20 rituximab and ofatumumab was investigated by complement-dependent cytotoxicity (CDC) assay. NOTCH1 signaling was inhibited by gamma-secretase inhibitor (GSI). Results. i) NOTCH1 mutational screening. In 649 CLL, NOTCH1 mutations were detected in 115 cases (17.72%), overall accounting for 127 mutations (73 c.7541-7542delCT, 11 other frameshift, 17 nonsense, 1 missense, and 25 3'UTR mutations). For analysis purposes, the 115 mutated cases were subdivided in cases with NOTCH1 coding mutations (coding NOTCH1-mut, 90 cases) and cases with 3'UTR NOTCH1 mutations (3'UTR NOTCH1-mut, 25 cases). Four cases with concomitant 3'UTR NOTCH1 mutation and coding NOTCH1 mutation were assigned to the 3'UTR group according to the substantially higher mutational burden detected for the 3'UTR mutation. ii)NOTCH1protein expression. In keeping with alternative splicing events causing an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases showed higher levels than NOTCH1 wild-type (NOTCH1-wt) cases of both NOTCH1 TM and NICD by WB, and similar to coding NOTCH1-mut cases (Fig. 1a). iii) CD20 expression levels. According to FISH classification, variable CD20 levels were found by flow cytometry, with the highest levels in trisomy 12 CLL. Of note, 3'UTR NOTCH1-mut cases expressed lower CD20 levels than NOTCH1-wt cases in both trisomy 12 CLL (mean MFI in 9 3'UTR NOTCH1-mut cases = 2446.11 vs mean MFI in 66 NOTCH1-wt cases = 8254.20; p<0.0001) and non-trisomy 12 CLL (mean MFI in 16 3'UTR NOTCH1-mut cases = 2033.50 vs. mean MFI in 468 NOTCH1-wt cases = 3294.07; p=0.0001), and comparable to coding NOTCH1-mut cases (trisomy 12 CLL, mean MFI in 34 coding NOTCH1-mut cases = 2570.73; p=0.8530; non-trisomy 12 CLL, mean MFI in 56 coding NOTCH1-mut cases = 2538.13; p=0.1500) (Fig. 1b). Similarly, in both trisomy 12 CLL and non-trisomy 12 CLL categories, MS4A1 transcript levels were lower in 3'UTR NOTCH1-mut cases than in NOTCH1-wt cases (p=0.0274 and p=0.0072, respectively), again with expression levels comparable with coding NOTCH1-mut cases (p=0.2874 and p=0.9610, respectively, Fig. 1c). iv) Susceptibility to anti-CD20 in vitro. In keeping with CD20 levels, 3'UTR NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab, in CDC assay, than NOTCH1-wt cells (7 3'UTR NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.67% vs. 25.56%; p=0.0349; mean relative lysis upon ofatumumab: 39.26% vs. 60.64%; p=0.0286). In the same manner, coding NOTCH1-mut cases showed lower relative lysis induced by rituximab or ofatumumab than NOTCH1-wt cases (9 coding NOTCH1-mut cases, 9 NOTCH1-wt cases; mean relative lysis upon rituximab: 2.53% vs. 25.56%; p=0.0339; mean relative lysis upon ofatumumab: 30.60% vs. 60.64%; p=0.0114; Fig. 1d). v) Modulation of CD20 expression by NOTCH1 inhibition. GSI treatment increased both CD20 protein and transcript levels in 3'UTR NOTCH1-mut cases, as previously reported for coding NOTCH1-mut cases (p=0.0376 and p=0.0326, respectively; Fig. 1e,Pozzo et al., Leukemia, 2016). Conclusions.In keeping with an impaired NOTCH1 protein degradation, 3'UTR NOTCH1-mut cases had low CD20 expression levels, suggesting the introduction of 3'UTR NOTCH1 mutation evaluation in CLL patients undergoing anti-CD20 immuno-chemotherapy. Disclosures No relevant conflicts of interest to declare.

Effect of CD20 Expression Levels On B-Cell Lymphoma Patients' Background and Prognosis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5000-5000
Author(s):  
Yuhko Suzuki ◽  
Tsutomu Yoshida ◽  
Naoya Nakamura ◽  
Tomiteru Togano ◽  
Koji Miyazaki ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Overall Survival ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
World Health ◽  
Expression Levels ◽  
Significant Difference ◽  
Cd20 Expression ◽  
Anti Cd20

Abstract Abstract 5000 AIM The anti-CD20 antibody Rituximab has improved the disease-free survival and overall survival of patients with several types of B-cell lymphoma, in which CD20 is expressed on the cell surface and in the cytoplasm. Flow cytometry indicated that CD20 expression varies from null, weak to strong. Despite its clinical importance, the influence of CD20 expression levels on lymphoma therapy has not been well elucidated. Patients, Material and Methods 214 cases were analyzed, all newly diagnosed with B-cell lymphoma at our institute from January in 2002 to April in 2009. All were biopsied and analyzed by flow cytometry. The biopsy specimens were fixed in formalin and stained with Hematoxylin-Eosin; they were also immunostained. Histological subtypes were defined according to the World Health Organization Classification Ver. 3. The mean florescence intensities of CD20 and CD19 were determined by flow cytometry, and cytoplasmic expression of CD20 was determined by immunohistochemistry. 1) The cases were categorized as follows: A: CD20-negative, B: CD20/19 less than 20%, C: CD20/19 20-50% and D: CD20/19 more than 50%. Patients' backgrounds, pathological diagnoses, primary lesions, etc. were also analyzed. 2) Among DLBCL cases, 76 treated with R-chemo were selected and analyzed with respect to treatment response and overall survival. Results 1) Diagnoses: DLBCL 128, Follicular lymphoma 58, MALT 7, CLL 4 and so on. Among the DLBCL cases, immunohistochemistry indicated six (4%) were CD20-negative and three were CD20-positive; the ages in the CD20-negative DLBCL group tended to be high (74.28 vs 64.36, p=0.06, t-test). Weak CD20 expression was observed in 15 cases (B: 4, C:11). No statistically significant differences were seen with respect to gender, IPI, clinical stage, biopsy lesion, CD5 expression or Karyotype. No FL cases were CD20-negative, but two ( 3.4%) showed low CD20 expression. 2) Kaplan-Meier analysis of 76 cases of DLBCL treated with R-Chemo showed a significantly lower overall survival for Group A, (CD20-negative) while there was no significant difference in overall survival of Groups B+C(CD20-weak) and D(CD20-normal). Our results indicate that even patients with lower levels of CD20 expression in B-cell lymphoma may respond favorably to anti-CD20 therapy. Further studies will be necessary to explore this possibility. Disclosures No relevant conflicts of interest to declare.

Understanding CD20 Regulation By Loss-Of-Function RNAi Screen

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4221-4221
Author(s):  
Mikolaj Slabicki ◽  
Leopold Sellner ◽  
Alexander Jethwa ◽  
Tatjana Stolz ◽  
Srinivas Mamidi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Rnai Screen ◽  
Lymphoma Cell ◽  
Raji Cells ◽  
Expression Levels ◽  
Cd20 Expression ◽  
Anti Cd20 ◽  
Druggable Targets

Abstract Introduction CD20 is a cell surface glycoprotein, encoded by the MS4A1 gene, which is highly expressed on most B-cells. Although CD20 is suggested to be involved in calcium signaling, its exact function is unknown. The monoclonal antibody rituximab, which targets CD20, has revolutionized the treatment of B-cell lymphoma. Despite the widespread use of CD20 antibodies across lymphoma subtypes, little is known about the endogenous regulation of CD20. The tight correlation between CD20 expression and responsiveness to CD20 antibody suggests that increasing CD20 expression levels could improve rituximab sensitivity. This could be particularly useful for B-cell lymphoma with low CD20 surface expression (e.g. chronic lymphocytic leukemia). The main goal of the project is to dissect and understand the CD20 regulation using an unbiased RNAi approach and to exploit this knowledge to improve the efficacy of anti-CD20 therapy. Methods In order to discover novel regulators of CD20 expression, we performed a genome-wide RNAi screen. The shRNA library targets over 5000 mRNAs, with 5 to 6 shRNAs per target. The CD20 regulatome was studied in a Burkitt lymphoma cell line (Raji) characterized by medium expression of CD20. Results MS4A1 was the most significant screen hit that decreased CD20 surface levels with three shRNAs and served as positive control. Based on stringent selection criteria, we identified hits involved in diverse molecular mechanisms such as signaling, trafficking, chromatin remodeling and cell membrane composition. In total, 21 genes targeted with two non-overlapping shRNAs were selected for validation: 12 decreasing and 9 increasing CD20 levels. For every gene, two shRNAs were synthesized and cloned into shRNA expression vectors. Every shRNA was investigated in separate experiments for its effect on CD20 expression levels on Raji cells. We were able to validate and confirm the influence of those genes on CD20 phenotype on Raji cells, as well as on other B-cell lymphoma cell lines (SuDHL5, BL2 and BL60). The group of genes decreasing CD20 expression can provide an insight into physiological regulation and the machinery involved in CD20 trafficking and processing. More relevant from a therapeutic viewpoint are genes where silencing increases the expression of CD20. In order to investigate a potential therapeutic synergy of our hits with Rituximab, we investigated complement-mediated lysis triggered by Rituximab in vitro after silencing of several hits. In this assay we observed direct correlation between CD20 surface levels with the sensitivity towards Rituximab for our hits: increased CD20 levels led to more Rituximab induced lysis (up to two fold increase), and vice versa. One of the main aims of the screen was the discovery of druggable targets that can be potentially used in combination therapy with Rituximab. Indeed, a chemical inhibitor targeting one of our screen hits was able to increase CD20 expression levels on Raji cells. Conclusion With an unbiased shRNA screen we identified potent regulators of CD20 expression levels. Our approach led to the discovery of novel druggable targets, which modulate CD20 levels. This may provide the rationale for novel combinatory therapies, which might improve the efficiency of anti-CD20 therapy in B-cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

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