Rituximab-mediated Complement-dependent Cytotoxicity Research Articles

Abstract 4990: Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis

Abstract Monoclonal antibodies such as rituximab exert targeted tumor cell lysis via complement dependent cytotoxicity (CDC). These antibodies are often combined with chemotherapy in clinical treatment protocols. However, the impact of chemotherapy on cell-intrinsic pathways that regulate antibody-mediated CDC is not known. Through a phage display screen, we identified that genotoxic stress caused upregulation of a membrane complement regulatory protein (mCRP), CD46, prompting further investigation into its implications and mechanism. Using cell lines from diffuse large B cell lymphoma (DLBCL) as our model, we confirmed that genotoxic chemotherapy upregulates CD46, as well as other mCRPs such as CD55 and CD59. We hypothesized that upregulated mCRPs could impede Rituximab-mediated CDC. We assessed this in-vitro using rituximab and complement human serum. Aligned with our hypothesis, chemotherapy treatment of DLBCL cells significantly impeded rituximab-induced CDC. Using blocking antibodies to each mCRP, reversal of the effect of chemotherapy on CDC only occurred after co-treatment with a CD59 blocking antibody, suggesting that CD59 is a key player for chemotherapy-invoked resistance to CDC. To further elucidate the underlying cellular pathway, we performed a high-throughput small molecule screen using rituximab-induced CDC as a readout, with or without cotreatment of a genotoxic chemotherapeutic (etoposide). We identified Chk1 inhibitors as consistent top rescuers. qPCR and flow cytometry revealed that Chk1 inhibition prevented the upregulation of mCRPs transcriptionally. Sp1 is a known transcription factor driving constitutive CD59 expression. Inhibiting Sp1 similarly prevented chemotherapy-induced upregulation of CD59 and resistance to CDC. However, CHIP-qPCR showed no increased binding of total Sp1 protein to the CD59 promoter after chemotherapy. Instead, positive co-immunoprecipitation of pATM and p300 with Sp1 only with etoposide and not with co-treatment of Chk1 inhibitor suggests Chk1-mediated activation of Sp1. Mechanistically, we have found that chemotherapy-activated Chk1 modulates Sp1-mediated CD59 transcription and that the induced upregulation of CD59 is able to prevent antibody-mediated CDC. Overall, these results indicate novel immune regulation by DNA damage response, particularly in the context of complement regulatory proteins. Our results advocate consideration when using simultaneous administration of chemotherapy with CDC-inducing monoclonal antibodies, and suggest Chk1 blockade as a possible therapeutic addition. Citation Format: Allison S. Chan, Patrick W. Jaynes, Nurulhuda B. Mustafa, Akshaya A. Anbuselvan, Charmaine Z. Ong, Anand D. Jeyasekharan. Genotoxic chemotherapy impedes antibody-mediated complement dependent cytotoxicity (CDC), via a Chk1-CD59 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4990.

Herbal NF-κB Inhibitors Sensitize Rituximab-Resistant B Lymphoma Cells to Complement-Mediated Cytolysis.

BackgroundDrug resistance remains a serious challenge to rituximab therapy in B-NHL (B cell non-Hodgkin’s lymphoma). CDC (complement-dependent cytotoxicity) has been proposed as a major antitumor mechanism of rituximab, and direct abrogation of CD59 function partially restores rituximab sensitivity with high efficacy. However, universal blockade of CD59 may have deleterious effects on normal cells. Sp1 regulates constitutive CD59 expression, whereas NF-κB and CREB regulate inducible CD59 expression.MethodsImmunohistochemistry (IHC) assay was used to detect the expression levels of CD59 and other related molecules. Quantitative Real-time PCR (RT-PCR) analysis was used to explore the levels of transcripts in the original and resistant cells. We chose LY8 cells to test the effects of NF-κB and CBP/p300 inhibition on CD59 expression using flow cytometry (FACS). Immunoblotting analysis was employed to detect the effects of curcumin and POH. The in vitro and in vivo experiments were used to evaluate the toxicity and combined inhibitory effect on tumor cells of curcumin and POH.ResultsWe demonstrated that herbal (curcumin and perillyl alcohol) blockade of NF-κB specifically suppresses the expression of inducible CD59 but not CD20, thus sensitizing resistant cells to rituximab-mediated CDC. Moreover, activation of NF-κB and CREB is highly correlated with CD59 expression in B-NHL tissues.ConclusionsOur findings suggest the potential of CD59 expression as a predictor of therapeutic efficacy of NF-κB inhibitors in clinical application as well as the rationality of a NF-κB inhibitor-rituximab regimen in B-NHL therapy.

Tumor-Targeting Liposomes with Transient Holes Allowing Intact Rituximab Internally.

In this study, the strategy of transient generation of holes in the liposome surface has been shown to enable safe encapsulation of a high-molecular weight antibody (rituximab, Mw ∼140 kDa) within liposomes. These transient holes generated using our magnetoporation method allowed rituximab to safely enter the liposomes, and then the holes were plugged using hyaluronic acid grafted with 3-diethylaminopropylamine (DEAP). In the tumor microenvironment, the resulting liposomal rituximab was destabilized because of the ionization of the DEAP moiety at the acidic pH 6.5, resulting in extensive release of rituximab. Consequently, the rituximab released from the liposomes accumulated at high levels in tumors and bound to the CD20 receptors overexpressed on Burkitt lymphoma Ramos cells. This event led to significant enhancement in tumor cell ablation through rituximab-mediated complement-dependent cytotoxicity and Bcl-2 signaling inhibition-induced cell apoptosis.

Rituximab-Mediated Complement-Dependent Cytotoxicity Enhanced by Gemcitabine in Older Patients with Previously Rituximab-Treated Diffuse Large B-Cell Lymphoma: Study Protocol.

High-dose chemotherapy and autologous stem cell transplantation is too toxic for elderly patients with relapsed or refractory (DLBCL). Therefore, tolerable and efficient salvage regimens for elderly patients are greatly needed. In this study, therapy with rituximab, gemcitabine, dexamethasone, and cisplatin (R-GDP) will be performed every 4 weeks, and an interim evaluation will be performed after the completion of the 3rd course. If a complete response (CR) is achieved at the time of interim evaluation, 1 course of R-GDP therapy and 2 courses of monotherapy with rituximab will be additionally performed. If a partial response (PR) is achieved, 3 courses of R-GDP therapy will be additionally conducted. In patients without a PR or CR by the time of the interim evaluation, treatment will be discontinued. Treatment will also be discontinued at any point if disease progression is observed during protocol treatment. After the completion of the final course of R-GDP therapy, final effects of the regimen will be evaluated. A primary endpoint is the efficacy of R-GDP therapy (CR and response rates). This is the first multicenter phase II clinical study of R-GDP therapy to examine post-treatment activities of daily living in addition to the safety and efficacy of treatment in elderly patients with relapsed or refractory transplantineligible DLBCL.

Abstract B27: B-cell lymphoma response to anti-CD20 antibodies based therapies is tightly modulated by FOXO1-mediated MS4A1 gene transcription

Abstract No remission or early relapse remains a major clinical concern in diffuse large B cell lymphoma (DLBCL) with a third of patients failing to respond to current regimens or relapsing with resistant disease. Genome and transcriptome sequencing studies have identified FOXO1 as one of the recurrent target for somatic mutations in DLBCL associated with patient short survival. These mutations enhance FOXO1 nuclear localization and activity on target genes. Herein, we determine the role FOXO1 (wild type and mutated) on the expression of the rituximabtarget, CD20. The effect of active mutated FOXO1 on CD20 expression was determined upon inhibition of AKT, exogenous expression of active myristolated AKT, and mutated FOXO1 at its N-terminal region. FOXO1 binding activity to CD20 promoter was assessed using various assays combined with an in silico analysis of a publicly available ChipSeq data. Our results show that the activation of FOXO1 using AKT inhibitors (MK2206 and GDC0068) led to a significant decrease of CD20 transcript and protein levels, which resulted in a significant resistance to rituximab mediated complement dependent cytotoxicity (CDC). Consistently, the overexpression of either wild-type or mutated FOXO1 repressed CD20 expression. Unlike the activity of exogenous wild type FOXO1 that can be inhibited by AKT1, the activity of mutated FOXO1AAA (Thr24, Ser256 and Ser319 mutated to Ala and that cannot be phosphorylated) markedly inhibits CD20 expression. Furthermore, CD20 levels remained high and unchanged by the DNA binding defective mutant of FOXO1H215R, suggesting a FOXO1 mediated regulation of CD20 transcription through FOXO1 binding to CD20 promoter, as confirmed by ChIP, EMSA and the analysis of ChIPSeq data using antiFOXO1 antibody. These results show that FOXO1 mutants that are insensitive to AKT1 kinase activity, effectively suppress CD20 expression in comparison to wild type FOXO1, through a binding to CD20 promoter. In summary, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab induced cytotoxicity. In addition, our findings indicate that the genetic status of FOXO1 together with its transcriptional activity need careful attention while designing rituximab-based regimens for the therapy of specific B-lymphomas. Supported by: National Science Center, Poland (NCN#2013/11/B/NZ5/03240), STREAM project (Horizon 2020,#692180). Citation Format: Abdessamad Zerrouqi, Beata Pyrzynska, Michal Dwojak, Nina Miazek, Piotr Zapala, Jakub Golab, Magdalena Winiarska. B-cell lymphoma response to anti-CD20 antibodies based therapies is tightly modulated by FOXO1-mediated MS4A1 gene transcription [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B27.

Abstract B51: FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy

Abstract Lack of remission or early relapse remains a major clinical concern in diffuse large B-cell lymphoma (DLBCL), with a third of patients failing to respond to current regimens or relapsing with resistant disease. Genome and transcriptome sequencing studies have identified FOXO1 as one of the recurrent targets for somatic mutations in DLBCL associated with patient short survival. These mutations enhance FOXO1 nuclear localization and activity on target genes. Herein, we determine the role FOXO1 (wild-type and mutated) on the expression of the rituximab-target, CD20. The effect of active mutated FOXO1 on CD20 expression was determined upon inhibition of AKT, exogenous expression of active myristolated AKT, and mutated FOXO1 at its N-terminal region. FOXO1 binding activity to CD20 promoter was assessed using various assays combined with an in silico analysis of a publicly available Chip-Seq data. Our results show that the activation of FOXO1 using AKT inhibitors (MK-2206 and GDC-0068) led to a significant decrease of CD20 transcript and protein levels, which resulted in a significant resistance to rituximab-mediated complement-dependent cytotoxicity (CDC). Consistently, the overexpression of either wild-type or mutated FOXO1 repressed CD20 expression. Unlike the activity of exogenous wild-type FOXO1 that can be inhibited by AKT1, the activity of mutated FOXO1-AAA (Thr24, Ser256 and Ser319 mutated to Ala and that cannot be phosphorylated) markedly inhibits CD20 expression. Furthermore, CD20 levels remained high and unchanged by the DNA-binding defective mutant of FOXO1-H215R, suggesting a FOXO1-mediated regulation of CD20 transcription through FOXO1 binding to CD20 promoter, as confirmed by ChIP, EMSA, and the analysis of ChIP-Seq data using anti-FOXO1 antibody. These results show that FOXO1 mutants that are insensitive to AKT1 kinase activity effectively suppress CD20 expression in comparison to wild-type FOXO1, through a binding to CD20 promoter. In addition, the disruption of FOXO1 with CRISPR/Cas9 genome editing resulted in CD20 upregulation and improved the cytotoxicity induced by rituximab and survival of mice with sgFOXO1 tumors. Consistently, in primary samples pharmacologic inhibition of FOXO1 activity upregulated surface CD20 levels. In summary, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab-induced cytotoxicity. In addition, our findings indicate that the genetic status of FOXO1 together with its transcriptional activity needs further attention while designing rituximab-based regimens in the therapy of selected lymphomas. Supported by National Science Center, Poland (NCN#2013/11/B/NZ5/03240), STREAM project (Horizon 2020,#692180). Citation Format: Abdessamad Zerrouqi, Beata Pyrzynska, Michal Dwoyak, giulia Morlino, Piotr Zapala, Nina Miazek, Jakub Golab, Magdalena Winiarska. FOXO1 determines non-Hodgkin lymphomas response to anti-CD20-based therapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B51.

Gemcitabine enhances rituximab-mediated complement-dependent cytotoxicity to B cell lymphoma by CD20 upregulation.

Although rituximab, a chimeric monoclonal antibody that specifically binds to CD20, has significantly improved the prognosis for diffuse large B cell lymphoma (DLBCL), one‐third of DLBCL patients demonstrate resistance to rituximab or relapse after rituximab treatment. Thus, a novel approach to rituximab‐based treatment is likely to be required to improve the efficacy of DLBCL treatment. As complement dependent cytotoxicity (CDC) is a key mechanism mediating rituximab's tumoricidal activity, rituximab binding to CD20 on tumor cells is a critical factor for effective rituximab‐based treatments against DLBCL. We found that gemcitabine (GEM), but not lenalidomide (LEN) or azacitidine (AZA), can upregulate CD20 expression in TK and KML‐1 cells, two human DLBCL cell lines. Treatment of TK and KML‐1 cells with GEM enhanced CD20 expression at both the mRNA and protein levels. CD20 upregulation by GEM treatment was accompanied by increased rituximab binding to CD20. In TK cells, GEM treatment synergistically increased rituximab‐mediated CDC activity in a dose‐dependent manner. In KML cells, GEM treatment also induced upregulation of complement regulatory proteins, possibly leading to resistance to CDC. Treatment with LEN, a drug that did not upregulate CD20, did not enhance rituximab‐mediated CDC activity. GEM treatment activated nuclear factor‐kappa B (NF‐kB) signaling in these cells. Furthermore, a specific inhibitor to NF‐kB suppressed GEM‐induced CD20 upregulation, indicating that GEM‐induced NF‐kB activation is closely associated with CD20 upregulation. These results suggest that when used in combination, GEM might enhance the antitumor efficacy of rituximab against DLBCL due to its unique ability to upregulate CD20.

Erratum: Reduction of complement factor H binding to CLL cells improves the induction of rituximab-mediated complement-dependent cytotoxicity

Correction to: Leukemia (2013) 27, 2200–2208; doi:10.1038/leu.2013.169; published online 16 July 2013 Since the publication of this article, the authors have identified two errors in Figure 2, namely that in both panels a and b, ‘1200’ has been incorrectly listed instead of ‘120’. The correct figureis shown below.

Reduction of complement factor H binding to CLL cells improves the induction of rituximab-mediated complement-dependent cytotoxicity

A main effector mechanism of rituximab (RTX) is the induction of complement-dependent cytotoxicity (CDC). However, this effector function is limited, because CLL cells are protected from complement-induced damage by regulators of complement activation (RCAs). A prominent RCA in fluid phase is factor H (fH), which has not been investigated in this context yet. Here, we show that fH binds to CLL cells and that human recombinant fH-derived short-consensus repeat 18–20 (hSCR18–20) interferes with this binding. In complement-based lysis assays, CLL cells from therapy-naive patients were differently susceptible to RTX-induced CDC and were defined as CDC responder or CDC non-responder, respectively. In CDC responders, but notably also in non-responders, hSCR18–20 significantly boosted RTX-induced CDC. Killing of the cells was specific for CD20+ cells, whereas CD20− cells were poorly affected. CDC resistance was independent of expression of the membrane-anchored RCAs CD55 and CD59, although blocking of these RCAs further boosted CDC. Thus, inhibition of fH binding by hSCR18–20 sensitizes CLL cells to CDC and may provide a novel strategy for improving RTX-containing immunochemotherapy of CLL patients.

Silencing of Human Phosphatidylethanolamine-Binding Protein 4 Enhances Rituximab-Induced Death and Chemosensitization in B-Cell Lymphoma

Rituximab is the first line drug to treat non Hodgkin’s lymphoma (B-NHL) alone or in combination with chemotherapy. However, 30–40% of B-NHL patients are unresponsive to rituximab or resistant after therapy. Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of PEBP family and functions as an anti-apoptotic molecule. In this study, we found hPEBP4 to be expressed in up to 90% of B-cell lymphoma patients, but in only 16.7% of normal lymph nodes. Interestingly, hPEBP4 overexpression inhibited rituximab-mediated complement dependent cytotoxicity (R-CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in B-NHL cells while downregulation of hPEBP4 augmented the therapeutic efficacy of rituximab both in vitro and in vivo. Furthermore, hPEBP4 silencing sensitized the primary B-acute lymphocytic leukemia (B-ALL) cells to R-CDC. During rituximab-mediated complement dependent cytotoxicity, hPEBP4 was recruited to the cell membrane in a PE-binding domain dependent manner and inhibited R-CDC induced calcium flux and reactive oxygen species (ROS) generation. These events contributed to the decrease of cell death induced by R-CDC in B-cell lymphomas. Meanwhile, hPEBP4 knockdown potentiated the chemosensitization of the rituximab in B-cell lymphoma cells by regulating the expression of Bcl-xl, Cycline E, p21waf/cip1 and p53 and the activation of caspase-3 and caspase-9. Considering that hPEBP4 conferred cellular resistance to rituximab treatment and was preferentially expressed in lymphoma tissue, it could be a potential valuable target for adjuvant therapy for B-cell lymphoma.

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors

BackgroundIn clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence.MethodsHere we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC).ResultsCharacterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC.ConclusionsThese results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.

A rheumatoid factor paradox: inhibition of rituximab effector function

IntroductionRituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).MethodsWe developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.ResultsUsing human sera, addition of RTX resulted in rapid and profound (> 50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.ConclusionsContrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.

Prenyltransferases Regulate CD20 Protein Levels and Influence Anti-CD20 Monoclonal Antibody-mediated Activation of Complement-dependent Cytotoxicity

Anti-CD20 monoclonal antibodies (mAbs) are successfully used in the management of non-Hodgkin lymphomas and chronic lymphocytic leukemia. We have reported previously that statins induce conformational changes in CD20 molecules and impair rituximab-mediated complement-dependent cytotoxicity. Here we investigated in more detail the influence of farnesyltransferase inhibitors (FTIs) on CD20 expression and antitumor activity of anti-CD20 mAbs. Among all FTIs studied, L-744,832 had the most significant influence on CD20 levels. It significantly increased rituximab-mediated complement-dependent cytotoxicity against primary tumor cells isolated from patients with non-Hodgkin lymphomas or chronic lymphocytic leukemia and increased CD20 expression in the majority of primary lymphoma/leukemia cells. Incubation of Raji cells with L-744,832 led to up-regulation of CD20 at mRNA and protein levels. Chromatin immunoprecipitation assay revealed that inhibition of farnesyltransferase activity was associated with increased binding of PU.1 and Oct-2 to the CD20 promoter sequences. These studies indicate that CD20 expression can be modulated by FTIs. The combination of FTIs with anti-CD20 mAbs is a promising therapeutic approach, and its efficacy should be examined in patients with B-cell tumors.

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

Abstract 3722Anti-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. [Display omitted] Disclosures:Winiarska:Genmab A/S: Research Funding. Golab:Genmab A/S: Research Funding.

Sorafenib Affects Membrane Complement Inhibitors and Improves Antitumor Activity of Rituximab,

Abstract 3723Monoclonal antibodies (mAbs) targeting CD20 antigen are now routinely used in the treatment of various types of non-Hodgkin's lymphomas (NHL) and B-cell chronic lymphocytic leukemia (B-CLL). Their antitumor action results from the ability to trigger complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or immunophagocytosis. Moreover, direct cell death can be induced upon crosslinking with secondary antibodies. Previous studies have demonstrated a sigmoidal correlation between CD20 expression level and rituximab-mediated CDC but not ADCC. Next to CD20 expression level also other potential mechanisms of anti-CD20-mediated cytotoxicity have been observed. Among them an increased expression of complement regulatory proteins (CRP), such as CD46, CD55 or CD59 has been shown to contribute to resistance to mAb-mediated CDC. Antibodies blocking CRP or siRNA that knocks-down CRP expression facilitate rituximab-mediated cytotoxicity. Also in xenograft in vivo models it was shown that antibodies blocking the activity of CD55 and CD59 or a recombinant adenoviral fiber knob protein that cross-links CD46 molecules enhance therapeutic effects of rituximab. Fludarabine, the nucleoside analogue clinically active against CLL and indolent NHL has been shown to act synergistically with rituximab and to down-regulate the membrane expression of CD55 without significantly altering CD20 levels.The aim of this study was to investigate the influence of sorafenib, a multikinase inhibitor on the expression of CRPs in human CD20+ B-cell lymphomas and to evaluate its ability to trigger CDC in combination with rituximab. A 48-h incubation of four different human non-Hodgkin lymphoma cells DoHH2 (Fig. 1A), Daudi, Raji and Ramos with sorafenib resulted in a marked concentration-dependent drop in the surface expression of CD46, CD55 and CD59, but did not affect the levels of CD20. Analysis of primary cells isolated from 5 consecutive patients with CD20+ CLL also revealed a significant drop in the levels of CD46 (5 of 5 patients), CD55 (4 of 5 patients), and CD55 (5 of 5 patients) after a 48 h incubation of tumor cells with sorafenib. Accordingly, with previous reports, sorafenib significantly decreased phosphorylation of STAT3 transcription factors that are involved in the regulation of CRP expression. Incubation of tumor cells with sorafenib lowered the levels of mRNA for all three CRPs, but not that of CD20 indicating that sorafenib-mediated downregulation of CRPs occurs at transcriptional level. Pre-incubation of all four lymphoma cell lines cells with sorafenib at concentrations that down-modulate CRPs levels also significantly sensitized tumor cells to rituximab-mediated CDC (Fig. 1B). Moreover, sorafenib led to increased incorporation of membrane-attack complex (C5b-9) into the membranes of DoHH2 cells. On the other hand, sorafenib did not affect rituximab-mediated ADCC nor apoptosis in DoHH2 cells. Sunitinib, another multi-kinase inhibitor, did not affect rituximab-mediated complement-dependent cytolysis. [Display omitted] Altogether, these results indicate that sorafenib, which undergoes clinical trials in patients with NHL and B-CLL might be effectively used in combination with anti-CD20 mAbs. Disclosures:No relevant conflicts of interest to declare.

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.

RILYd4, a Human CD59 Inhibitor, Enhances Complement-Dependent Cytotoxicity of Ofatumumab against Rituximab-Resistant B-cell Lymphoma Cells and Chronic Lymphocytic Leukemia

Ofatumumab is an anti-CD20 antibody recently approved for treatment of fludarabine and alemtuzumab refractory chronic lymphocytic leukemia (CLL); it mediates much stronger complement-dependent cytotoxicity (CDC) than rituximab. Human CD59, a key membrane complement regulator that inhibits CDC, is highly expressed in B-cell malignancies and its upregulation is an important determinant of the sensitivity of B-cell malignancies to rituximab treatment. Previously, we have shown that the potent CD59 inhibitor rILYd4 sensitizes rituximab-resistant lymphoma cells to rituximab-mediated CDC. Here, we further investigated whether rILYd4 can sensitize B-cell malignancies to ofatumumab-mediated CDC and whether either ofatumumab-mediated CDC or rILYd4-enhanced ofatumumab-mediated CDC correlates with CD20 or CD59 expression, known biomarkers involved in rituximab activity. Rituximab-resistant cell lines and primary CLL cells were used to investigate the antitumor efficacy of the combination of rILYd4 with ofatumumab or rituximab. Propidium iodide staining or alamarBlue assay were used to evaluate the CDC effect. The levels of CD20 and CD59 on the cell membrane were analyzed by flow cytometry. rILYd4 enhanced CDC effects mediated by ofatumumab or rituximab on rituximab-resistant lymphoma cells and primary CLL cells in vitro. The sensitivity to CDC effects mediated by ofatumumab positively correlated with the ratio of CD20/CD59 and negatively correlated with CD59 levels on CLL cells. The degree to which rILYd4 enhanced CDC correlated positively with the CD59 levels on CLL cells. These data suggest that rILYd4 may enhance the anticancer activity of ofatumumab and rituximab in B-cell malignancies that have relapsed after prior antibody-based therapies.

Human CD59 inhibitor sensitizes rituximab-resistant lymphoma cells to complement-mediated cytolysis.

Rituximab efficacy in cancer therapy depends in part on induction of complement-dependent cytotoxicity (CDC). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex, thereby inhibiting induction of CDC. hCD59 is highly expressed in B-cell non-Hodgkin's lymphoma (NHL), and upregulation of hCD59 is an important determinant of the sensitivity of NHL cells to rituximab treatment. Here, we report that the potent hCD59 inhibitor rILYd4 enhances CDC in vitro and in vivo, thereby sensitizing rituximab-resistant lymphoma cells and primary chronic lymphocytic leukemia cells (CLL) to rituximab treatment. By defining pharmcokinetic/pharmacodynamic profiles of rILYd4 in mice, we showed that by itself rILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes. Increasing expression levels of the complement regulators CD59 and CD55 in rituximab-resistant cells occur due to selection of preexisting clones rather than de novo induction of these proteins. Moreover, lymphoma cells overexpressing CD59 were directly responsible for the resistance to rituximab-mediated CDC therapy. Our results rationalize the use of rILYd4 as a therapeutic adjuvant for rituximab treatment of rituximab-resistant lymphoma and CLL. Furthermore, they suggest that preemptive elimination of CD59-overexpressing subpopulations along with rituximab treatment may be a useful approach to ablate or conquer rituximab resistance.

Bortezomib modulates surface CD20 in B-cell malignancies and affects rituximab-mediated complement-dependent cytotoxicity

AbstractUnresponsiveness to rituximab treatment develops in many patients prompting elucidation of underlying molecular pathways. It was recently observed that rituximab-resistant lymphoma cells exhibit up-regulation of components of the ubiquitin-proteasome system (UPS). Therefore, we investigated in more detail the role of this system in the regulation of CD20 levels and the influence of proteasome inhibitors on rituximab-mediated complement-dependent cytotoxicity (R-CDC). We observed that incubation of Raji cells with rituximab leads to increased levels of ubiquitinated CD20. However, inhibition of the UPS was not associated with up-regulation of surface CD20 levels, although it significantly increased its ubiquitination. Short-term (24 hours) incubation of Raji cells with 10 or 20nM bortezomib did not change surface CD20 levels, but sensitized CD20+ lymphoma cells to R-CDC. Prolonged (48 hours) incubation with 20nM bortezomib, or incubation with 50nM bortezomib for 24 hours led to a significant decrease in surface CD20 levels as well as R-CDC. These effects were partly reversed by bafilomycin A1, an inhibitor of lysosomal/autophagosomal pathway of protein degradation. These studies indicate that CD20 levels are regulated by 2 proteolytic systems and that the use of proteasome inhibitors may be associated with unexpected negative influence on R-CDC.

A recombinant adenovirus type 35 fiber knob protein sensitizes lymphoma cells to rituximab therapy

AbstractMany tumors, including lymphomas, up-regulate expression of CD46 to escape destruction by complement. Tumor cells are therefore relatively resistant to therapy by monoclonal antibodies, which act through complement-dependent cytotoxicity (CDC). From an Escherichia coli expression library of adenovirus type 35 fiber knob mutants, we selected a variant (Ad35K++) that had a higher affinity to CD46 than did the natural Ad35 fiber knob. We demonstrated that incubation of lymphoma cells with recombinant Ad35K++ protein resulted in transient removal of CD46 from the cell surface. Preincubation of lymphoma cells with Ad35K++ sensitized cells to CDC, triggered by the CD20-specific monoclonal antibody rituximab. In xenograft models with human lymphoma cells, preinjection of Ad35K++ dramatically increased the therapeutic effect of rituximab. Blood cell counts and organ histology were normal after intravenous injection of Ad35K++ into mice that express human CD46. The presence of polyclonal anti-Ad35K++ antibodies did not affect the ability of Ad35K++ to enhance rituximab-mediated CDC in in vitro assays. The Ad35K++-based approach has potential implications in monoclonal antibody therapy of malignancies beyond the combination with rituximab.