MS4A1 (CD20) Gene Expression Is Down-Regulated by Recruiting the Histone Deacetylase Protein Complex to the Promoter in the CD20-Negative B-Lymphoma Cells After Treatment with Rituximab.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1286-1286
Author(s):  
Takumi Sugimoto ◽  
Akihiro Tomita ◽  
Junji Hiraga ◽  
Kazuyuki Shimada ◽  
Hitoshi Kiyoi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Protein Expression ◽  
B Cell ◽  
Protein Complex ◽  
Research Funding ◽  
Gene Promoter ◽  
B Lymphoma ◽  
B Lymphoma Cells ◽  
Cd20 Expression

Abstract Abstract 1286 Poster Board I-308 Background Rituximab, a mouse/human chimeric-monoclonal antibody, is now one of the critical molecular targeting drugs for treatment of CD20-positive B-cell lymphomas. Although the survival benefit of rituximab has been proved for several types of CD20-positive B-cell malignancies, resistance to rituximab has also become a considerable problem. Very recently, we reported that down-modulation of CD20 protein expression in CD20-positive B-cell lymphoma patients after treatment with rituximab-containing combination chemotherapies had been observed in 26.3% of re-biopsied patients under relapsed/progress disease (RD/PD) condition (Hiraga et al., 2009, Blood). Interestingly, CD20 expression and the rituximab sensitivity were partially restored by some epigenetic drugs in vitro, suggesting that aberrant down-regulation of MS4A1 gene expression by epigenetic mechanisms may be related to the loss of CD20 protein expression. Aims Analyses of the molecular mechanisms of MS4A1 gene down-regulation after treatment with rituximab-containing chemotherapies. Results Primary B-lymphoma cells and RRBL1 cells (Hiraga et al., 2009, Blood; Tomita et al., 2007, Int J Hematol.), that showed CD20-negative phenotype after using rituximab, were analyzed in these assays. CD20 mRNA and protein expression was partially stimulated by decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, and the expression was enhanced by trichostation A (TSA), a histone deacetylase (HDAC) inhibitor. Immunoblot analysis indicated that DNMT1 expression was once down-regulated one day after treatment with DAC, and reversed within 3 days. On the other hand, IRF4/Pu.1, the transcription regulators of MS4A1 gene expression, were consistently present with or without DAC. Bisulfite sequencing was performed to check the CpG methylation status of MS4A1 gene promoter, with the result that no significant methylation was confirmed in CD20-negative transformed cells without DAC. Chromatin immunoprecipitation (ChIP) assay indicated that Sin3-HDAC1 co-repressor complex was recruited to MS4A1 gene promoter without DAC/TSA. In the presence of those drugs, Sin3-HDAC1 recruitment was dissociated from the promoter and the histone acetylation of the promoter was confirmed. Under these conditions with/without DAC/TSA, IRF4 and Pu.1 were constantly recruited to the promoter. Immunoprecipitation using whole cell lysate of RRBL1 cells indicated that endogenous Sin3-HDAC1 forms a protein complex, but IRF4 and/or Pu.1 interaction with the complex was not confirmed under this condition. To explore the critical factors for CD20 transcription regulation, expression-profiling assay using cDNA micro array was performed. mRNA from RRBL1 cell with/without DAC/TSA was harvested, and expression profiles were compared. In the presence of DAC or DAC+TSA, 0.7% and 7.0%, respectively, of genes were significantly activated. We are now analyzing some candidates that are critical for the transcription regulation of MS4A1 gene expression. Conclusions Our data indicate that the transcription repression of MS4A1 gene in the CD20-negative phenotypic change after treatment with rituximab is, in part, introduced by the recruitment of Sin3-HDAC1 co-repressor protein complex, not by CpG methylation of the promoter. However, the direct interaction of the complex with IRF4/Pu.1 transcription factors was not confirmed in our assay, and the existence of other transcription factors that interact with Sin3-HDAC1 complex was suggested. To confirm the key regulators of CD20 expression is quite useful for exploring some strategies to overcome the rituximab resistance through CD20-negative transformation after using rituximab. Disclosures Kiyoi: Novartis Pharma Co. Ltd.: Research Funding; Kyowa Hakko Kirin Co. Ltd.: Consultancy. Naoe:Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Wyeth K.K.: Research Funding.

ROR1 Protein Expression Is Regulated By UHRF1 in t(1;19) Pre B-Cell ALL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3648-3648
Author(s):  
Marilynn Chow ◽  
Jason D. MacManiman ◽  
Vincent T. Bicocca ◽  
Lina Gao ◽  
Joshi A. Alumkal ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Transcription Factors ◽  
Protein Expression ◽  
B Cell ◽  
Chromatin Remodeling ◽  
Research Funding ◽  
Actinomycin D ◽  
Regulatory Mechanism ◽  
Protein Levels ◽  
Epigenetic Regulators

Abstract Introduction: Acute lymphoblastic leukemia is the most common form of pediatric cancer, and a translocation between chromosomes 1 and 19 represents one of the most frequent cytogenetic abnormalities, accounting for ~5% of ALL cases. The 1;19 rearrangement results in a fusion between the E2A and PBX1 genes, and pre B-ALL cells carrying the E2A-PBX1 fusion are consistently arrested at an intermediate stage of B cell lineage development in which the cells express a functional pre B-cell receptor (pre-BCR). We have previously shown that receptor orphan tyrosine kinase receptor 1 (ROR1) is highly expressed on t(1;19) pre B-ALL blasts and required for their viability, even though ROR1 is not directly regulated by either the E2A-PBX1 fusion or the pre-BCR. Moreover, ROR1 exhibits compensatory signaling cross-talk with the pre-BCR such that maximal killing of t(1;19) pre B-ALL cells is achieved by combining silencing of ROR1 with siRNA and inhibition of the pre-BCR with kinase inhibitors such as dasatinib. However, clinical employment of this strategy is hampered as ROR1 is a pseudokinase whose biology is poorly understood and no ROR1 small-molecule inhibitors currently exist. Therefore, it is critical to advance our understanding of the upstream regulatory mechanisms responsible for ROR1 expression to uncover new therapeutic strategies for t(1;19) pre B-cell ALL. Methods: To identify potential regulators of ROR1 expression, we designed two siRNA panels targeting transcription factors predicted to bind the ROR1 locus or an array of chromatin remodeling factors and epigenetic regulators. Pre B-ALL cell lines that are positive or negative for the 1;19 rearrangement were screened with these siRNA panels and any gene targets required for the viability of t(1;19) pre B-cell ALL cells but not control cells lines were considered candidate regulators of ROR1 expression. Candidates were validated for the capacity to regulate ROR1 expression by qRT-PCR and immunoblot. Results: Screening of the t(1;19) pre B-cell ALL cell line, RCH-ACV, with our siRNA panels targeting transcription factors and chromatin remodeling/epigenetic regulators identified four potential regulators of ROR1 that were required for viability of RCH-ACV cells but not our negative control cell line, REH (t(12;21)-positive). Among these four candidates was UHRF1, which interacts with histone and DNA methyltransferases. Interestingly, silencing of UHRF1 by siRNA led to reduced ROR1 protein levels, but had no significant impact on ROR1 mRNA expression. Since one primary function of UHRF1 is to regulate DNA methylation, we hypothesized that UHRF1 regulates ROR1 protein expression through an intermediate gene whose transcription is impacted by UHRF1-mediated DNA methylation. To test this hypothesis, RCH-ACV cells were treated with the RNA synthesis inhibitor actinomycin D 48 hours after silencing UHRF1. This resulted in a transient rescue of ROR1 protein levels, indicating that UHRF1 regulation of ROR1 protein depends on the transcription of an intermediate gene whose gene product subsequently regulates ROR1 protein levels. Conclusions: Data from siRNA screens and subsequent validation experiments suggest that UHRF1 functions to regulate ROR1 protein levels, but not mRNA expression. This is intriguing due to the primary role of UHRF1 as a modulator of DNA and histone methylation. The observed rescue of ROR1 protein with actinomycin D suggests that UHRF1 regulation of ROR1 protein occurs through an undefined intermediary whose locus is methylated in a UHRF1-dependent manner and whose gene product modulates ROR1 protein levels. Inhibitors of additional stages of gene transcription, translation, and stabilization are currently being tested to continue characterizing UHRF1-dependent regulation of ROR1. Cumulatively, this work has identified a novel regulatory mechanism of ROR1 protein expression by UHRF1 in t(1;19) pre B-cell ALL. In addition to advancing our understanding of ROR1 biology, this regulatory mechanism suggests a new therapeutic strategy to target ROR1-expressing cancers. Disclosures Tyner: Incyte: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding.

Distinct Gene Expression Signatures in Patients with Richter's Syndrome and Chronic Lymphocytic Leukemia with Prior Exposure to Ibrutinib

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-31
Author(s):  
Hanyin Wang ◽  
Shulan Tian ◽  
Qing Zhao ◽  
Wendy Blumenschein ◽  
Jennifer H. Yearley ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Activation ◽  
Expression Profiles ◽  
Lymphocytic Leukemia ◽  
B Cell Activation ◽  
Advisory Committees ◽  
Upregulated Genes

Introduction: Richter's syndrome (RS) represents transformation of chronic lymphocytic leukemia (CLL) into a highly aggressive lymphoma with dismal prognosis. Transcriptomic alterations have been described in CLL but most studies focused on peripheral blood samples with minimal data on RS-involved tissue. Moreover, transcriptomic features of RS have not been well defined in the era of CLL novel therapies. In this study we investigated transcriptomic profiles of CLL/RS-involved nodal tissue using samples from a clinical trial cohort of refractory CLL and RS patients treated with Pembrolizumab (NCT02332980). Methods: Nodal samples from 9 RS and 4 CLL patients in MC1485 trial cohort were reviewed and classified as previously published (Ding et al, Blood 2017). All samples were collected prior to Pembrolizumab treatment. Targeted gene expression profiling of 789 immune-related genes were performed on FFPE nodal samples using Nanostring nCounter® Analysis System (NanoString Technologies, Seattle, WA). Differential expression analysis was performed using NanoStringDiff. Genes with 2 fold-change in expression with a false-discovery rate less than 5% were considered differentially expressed. Results: The details for the therapy history of this cohort were illustrated in Figure 1a. All patients exposed to prior ibrutinib before the tissue biopsy had developed clinical progression while receiving ibrutinib. Unsupervised hierarchical clustering using the 300 most variable genes in expression revealed two clusters: C1 and C2 (Figure 1b). C1 included 4 RS and 3 CLL treated with prior chemotherapy without prior ibrutinib, and 1 RS treated with prior ibrutinib. C2 included 1 CLL and 3 RS received prior ibrutinib, and 1 RS treated with chemotherapy. The segregation of gene expression profiles in samples was largely driven by recent exposure to ibrutinib. In C1 cluster (majority had no prior ibrutinb), RS and CLL samples were clearly separated into two subgroups (Figure 1b). In C2 cluster, CLL 8 treated with ibrutinib showed more similarity in gene expression to RS, than to other CLL samples treated with chemotherapy. In comparison of C2 to C1, we identified 71 differentially expressed genes, of which 34 genes were downregulated and 37 were upregulated in C2. Among the upregulated genes in C2 (majority had prior ibrutinib) are known immune modulating genes including LILRA6, FCGR3A, IL-10, CD163, CD14, IL-2RB (figure 1c). Downregulated genes in C2 are involved in B cell activation including CD40LG, CD22, CD79A, MS4A1 (CD20), and LTB, reflecting the expected biological effect of ibrutinib in reducing B cell activation. Among the 9 RS samples, we compared gene profiles between the two groups of RS with or without prior ibrutinib therapy. 38 downregulated genes and 10 upregulated genes were found in the 4 RS treated with ibrutinib in comparison with 5 RS treated with chemotherapy. The top upregulated genes in the ibrutinib-exposed group included PTHLH, S100A8, IGSF3, TERT, and PRKCB, while the downregulated genes in these samples included MS4A1, LTB and CD38 (figure 1d). In order to delineate the differences of RS vs CLL, we compared gene expression profiles between 5 RS samples and 3 CLL samples that were treated with only chemotherapy. RS samples showed significant upregulation of 129 genes and downregulation of 7 genes. Among the most significantly upregulated genes are multiple genes involved in monocyte and myeloid lineage regulation including TNFSF13, S100A9, FCN1, LGALS2, CD14, FCGR2A, SERPINA1, and LILRB3. Conclusion: Our study indicates that ibrutinib-resistant, RS-involved tissues are characterized by downregulation of genes in B cell activation, but with PRKCB and TERT upregulation. Furthermore, RS-involved nodal tissues display the increased expression of genes involved in myeloid/monocytic regulation in comparison with CLL-involved nodal tissues. These findings implicate that differential therapies for RS and CLL patients need to be adopted based on their prior therapy and gene expression signatures. Studies using large sample size will be needed to verify this hypothesis. Figure Disclosures Zhao: Merck: Current Employment. Blumenschein:Merck: Current Employment. Yearley:Merck: Current Employment. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Parikh:Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Kenderian:Sunesis: Research Funding; MorphoSys: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Gilead: Research Funding; BMS: Research Funding; Tolero: Research Funding; Lentigen: Research Funding; Juno: Research Funding; Mettaforge: Patents & Royalties; Torque: Consultancy; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding. Kay:Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:DTRM: Research Funding; Astra Zeneca: Research Funding; Abbvie: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Characterization of Novel Subtypes in B Progenitor Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 565-565
Author(s):  
Zhaohui Gu ◽  
Michelle L. Churchman ◽  
Kathryn G. Roberts ◽  
Ian Moore ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Transcriptome Sequencing ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Genetic Alterations ◽  
Advisory Board ◽  
The Other ◽  
Cell Maturation ◽  
B Cell Maturation

Abstract Introduction B progenitor acute lymphoblastic leukemia (B-ALL) is a leading cause of childhood cancer death. Many chimeric genes have been identified and led to a refined classification of B-ALL and tailored therapies. Still, up to 30% of B-ALL cannot be classified into established subtypes, and the outcome for many is poor. Methods To identify novel subtypes of B-ALL, we performed integrative genomic analysis including transcriptome sequencing (RNA-seq) of 1,988 cases from St. Jude, Children's Oncology Group and adult cooperative group studies and analyzed chromosomal rearrangements, gene-expression profiles (GEP), somatic mutations and chromosome-level copy-number alterations. Cases lacking known or putative subtype-defining alterations underwent whole genome sequencing. Effects on proliferation and transformation of novel lesions were assessed by retroviral expression in cell lines and point-mutation knock-in mice using CRISPR/Cas9 genome editing. Results Using integrated genetic alterations and gene expression profiling, we classified 23 B-ALL subtypes (Table and Figure). Three groups included cases with similar GEP as canonical subtypes (ETV6-RUNX1, KMT2A-rearranged, and ZNF384-rearranged), but lacking the expected drivers (e.g., ETV6-RUNX1-like, n=42). Eighteen cases (0.9%) had rearrangements of BCL2, MYC and/or BCL6 showing a distinct GEP; they were mostly adults (n=16) with very poor outcome. These alterations, rarely seen in ALL, are identical to those observed in "double/triple hit" lymphoma, and are of pre-B immunophenotype. Eight cases with tightly clustered GEP comprised a novel subtype defined by IKZF1 N159Y missense mutation. N159Y is in the DNA-binding domain of IKZF1, and is known to perturb IKZF1 function, with distinct nuclear mislocalization and induction of aberrant intercellular adhesion. We identified two subtypes with distinct GEP characterized by PAX5 alterations. One, herein termed PAX5 altered (PAX5alt), comprised 148 (7.4%) cases, was characterized by diverse PAX5 alterations including rearrangements (n=57), sequence mutations (n=46) and/or focal intragenic amplifications (n=8). These PAX5 alterations were found in 73.6% of PAX5alt cases and different alteration types were mutually exclusive. Other PAX5 alterations, including deletions and large-scale amplifications were also assessed using SNP array, but were not enriched in the PAX5alt group. Clinically, PAX5alt pediatric and adult patients had favorable (96.8±3.2%) and intermediate (42.1±10.2%) 5-year overall survival (OS), respectively. The other GEP distinct subtype comprised 44 cases, all with PAX5 P80R missense mutations. In 30 of these cases, PAX5 P80R was homozygous due to deletion of the wild-type (WT) PAX5 allele or copy-neutral loss of heterozygosity. Of the other 14 cases with heterozygous PAX5 P80R mutations, 13 had a second frameshift (n=7), nonsense (n=2) or deleterious missense (n=4) PAX5 mutation. Four of the remaining 1,944 cases also had the PAX5 P80R mutation, but all were heterozygous with preservation of a WT PAX5 allele, consistent with the notion that homozygous or compound heterozygous PAX5 P80R mutation is the hallmark of this subtype. Adult PAX5 P80R cases (n=14) showed better 5-year OS (61.9±13.4%) than those in PAX5alt subtype (42.1±10.2%). To examine the effects of PAX5 P80R on B-cell maturation, WT PAX5, PAX5 P80R, V26G and P34Q were expressed in Pax5-/- lineage-depleted bone marrow cells. Expression of WT PAX5, PAX5 V26G and P34Q resulted in near complete rescue of B cell differentiation; however, expression of PAX5 P80R blocked the differentiation at the pre-pro-B stage of B-cell maturation. Further, Pax5 P80R heterozygous or homozygous mice developed pre-B-ALL with a median latency of 166 and 87 days, respectively, with heterozygous mice acquiring alterations on the second allele. In contrast, Pax5+/- mice, and those harboring G183S mutation observed in familial leukemia, do not spontaneously develop B-ALL. Conclusions These results show the utility of transcriptome sequencing in defining subtypes and founding genetic alterations in B-ALL, provide a revised taxonomy of the disease across the age spectrum, and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis. Disclosures McKay: ImmunoGen Inc.: Employment. Tallman:Orsenix: Other: Advisory board; AROG: Research Funding; BioSight: Other: Advisory board; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding. Stock:Jazz Pharmaceuticals: Consultancy. Konopleva:Stemline Therapeutics: Research Funding. Relling:Shire Pharmaceuticals: Research Funding. Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

scholarly journals Roles of the Tumor Suppressor p53 and the Cyclin-dependent Kinase Inhibitor p21WAF1/CIP1 in Receptor-mediated Apoptosis of WEHI 231 B Lymphoma Cells

1998 ◽  
Vol 187 (10) ◽  
pp. 1671-1679 ◽  
Author(s):  
Min Wu ◽  
Robert E. Bellas ◽  
Jian Shen ◽  
Gail E. Sonenshein
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Cyclin Dependent Kinase ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
Cyclin Dependent Kinase Inhibitor ◽  
B Lymphoma Cells ◽  
Wehi 231

Treatment of WEHI 231 immature B lymphoma cells with an antibody against their surface immunoglobulin M (anti-IgM) induces apoptosis and has been studied extensively as a model of self-induced B cell tolerance. Since the tumor suppressor protein p53 has been implicated in apoptosis in a large number of cell types and has been found to be mutated in a variety of B cell tumors, here we sought to determine whether p53 and the p53 target gene cyclin-dependent kinase inhibitor p21WAF1/CIP1 were involved in anti-IgM–induced cell death. Anti-IgM treatment of WEHI 231 cells increased expression of p53 and p21 protein levels. Ectopic expression of wild-type p53 in WEHI 231 cells induced both p21 expression and apoptosis. Ectopic expression of p21 similarly induced apoptosis. Rescue of WEHI 231 cells from apoptosis by costimulation with CD40 ligand ablated the increase in p21 expression. Lastly, a significant decrease in anti-IgM–mediated apoptosis was seen upon downregulation of endogenous p53 activity by expression of a dominant-negative p53 protein or upon microinjection of an antisense p21 expression vector or antibody. Taken together, the above data demonstrate important roles for p53 and p21 proteins in receptor-mediated apoptosis of WEHI 231 B cells.

IKZF3 Overexpression Phenocopies Gain-of-Function Mutation in Chronic Lymphocytic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Shanye Yin ◽  
Gregory Lazarian ◽  
Elisa Ten Hacken ◽  
Tomasz Sewastianik ◽  
Satyen Gohil ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Binding ◽  
B Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Advisory Committees ◽  
Bcr Signaling ◽  
Experimental Group

A hotspot mutation within the DNA-binding domain of IKZF3 (IKZF3-L162R) has been identified as a putative driver in chronic lymphocytic leukemia (CLL); however, its functional effects are unknown. We recently confirmed its role as a CLL driver in a B cell-restricted conditional knock-in model. IKZF3 mutation altered mature B cell development and signaling capacity, and induced CLL-like disease in elderly mice (~40% penetrance). Moreover, we found IKZF3-L162R acts as a gain-of-function mutation, altering DNA binding specificity and target selection of IKZF3, and resulting in overexpression of multiple B-cell receptor (BCR) genes. Consistent with the murine data, RNA-sequencing analysis showed that human CLL cells with mut-IKZF3 [n=4] have an enhanced signature of BCR-signaling gene expression compared to WT-IKZF3 [n=6, all IGHV unmutated] (p<0.001), and also exhibited general upregulation of key BCR-signaling regulators. These results confirm the role of IKZF3 as a master regulator of BCR-signaling gene expression, with the mutation contributing to overexpression of these genes. While mutation in IKZF3 has a clear functional impact on a cardinal CLL-associated pathway, such as BCR signaling, we note that this driver occurs only at low frequency in patients (~3%). Because somatic mutation represents but one mechanism by which a driver can alter a cellular pathway, we examined whether aberrant expression of IKZF3 could also yield differences in BCR-signaling gene expression. We have observed expression of the IKZF3 gene to be variably dysregulated amongst CLL patients through re-analysis of transcriptomic data from two independent cohorts of human CLL (DFCI, Landau et al., 2014; ICGC, Ferreira et al., 2014). We thus examined IKZF3 expression and BCR-signaling gene expression, or the 'BCR score' (calculated as the mean expression of 75 BCR signaling-associate genes) in those cohorts (DFCI cohort, n=107; ICGC cohort, n=274). Strikingly, CLL cells with higher IKZF3 expression (defined as greater than median expression) had higher BCR scores than those with lower IKZF3 expression (<median) (p=0.0015 and p<0.0001, respectively). These findings were consistent with the notion that IKZF3 may act as a broad regulator of BCR signaling genes, and that IKZF3 overexpression, like IKZF3 mutation, may provide fitness advantage. In support of this notion, our re-analysis of a gene expression dataset of 107 CLL samples (Herold Leukemia 2011) revealed that higher IKZF3 expression associated with poorer prognosis and worse overall survival (P=0.035). We previously reported that CLL cells with IKZF3 mutation appeared to increase in cancer cell fraction (CCF) with resistance to fludarabine-based chemotherapy (Landau Nature 2015). Instances of increase in mut-IKZF3 CCF upon treatment with the BCR-signaling inhibitor ibrutinib have been reported (Ahn ASH 2019). These studies together suggest an association of IKZF3 mutation with increased cellular survival following either chemotherapy or targeted treatment. To examine whether higher expression of IKZF3 was associated with altered sensitivity to ibrutinib, we performed scRNA-seq analysis (10x Genomics) of two previously treatment-naïve patients undergoing ibrutinib therapy (paired samples, baseline vs. Day 220). We analyzed an average of 11,080 cells per patient (2000 genes/cell). Of note, following ibrutinib treatment, remaining CLL cells expressed higher levels of IKZF3 transcript compared to pretreatment baseline (both p<0.0001), whereas no such change was observed in matched T cells (n ranging between 62 to 652 per experimental group, p>0.05), suggesting that cells with high expression of IKZF3 were selected by ibrutinib treatment. Moreover, we showed that ibrutinib treatment resulted in consistent upregulation of BCR-signaling genes (e.g., CD79B, LYN, GRB2, FOS, RAC1, PRKCB and NFKBIA) (n ranging between 362 to 1374 per experimental group, all p<0.0001), which were likewise activated by mutant IKZF3. Altogether, these data imply that IKZF3 mutation or overexpression may influence upregulation of BCR-signaling genes and enhance cellular fitness even during treatment with BCR-signaling inhibitors. We highlight our observation that IKZF3 mutation appears to be phenocopied by elevated IKZF3 expression, and suggest that alterations in mRNA or protein level that mimic genetic mutations could be widespread in human cancers. Disclosures Kipps: Pharmacyclics/ AbbVie, Breast Cancer Research Foundation, MD Anderson Cancer Center, Oncternal Therapeutics, Inc., Specialized Center of Research (SCOR) - The Leukemia and Lymphoma Society (LLS), California Institute for Regenerative Medicine (CIRM): Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech/Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VelosBio: Research Funding; Oncternal Therapeutics, Inc.: Other: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory, Research Funding; Ascerta/AstraZeneca, Celgene, Genentech/F. Hoffmann-La Roche, Gilead, Janssen, Loxo Oncology, Octernal Therapeutics, Pharmacyclics/AbbVie, TG Therapeutics, VelosBio, and Verastem: Membership on an entity's Board of Directors or advisory committees. Wu:BionTech: Current equity holder in publicly-traded company; Pharmacyclics: Research Funding.

Pax5 Haploinsufficiency Cooperates with BCR-ABL1 to Induce Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 293-293 ◽  
Author(s):  
ChristoPher B Miller ◽  
Charles G Mullighan ◽  
Xiaoping Su ◽  
Jing Ma ◽  
Michael Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Point Mutation ◽  
Copy Number ◽  
Lymphoblastic Leukemia ◽  
Wild Type ◽  
Lymphoid Development ◽  
B Lymphoid

Abstract Genes regulating B lymphoid development are somatically mutated in over 40% of B-progenitor acute lymphoblastic leukemia (ALL) cases, with the most common targets being the transcription factors PAX5, IKZF1 (encoding Ikaros), and EBF1. Notably, BCR-ABL1 ALL is characterized by a high frequency of mutations of IKZF1 (85%), PAX5 (55%), and CDKN2A/B (encoding INK4/ARF, 55%), suggesting that these lesions cooperate with BCR-ABL1 in lymphoid leukemogenesis. To examine cooperativity between Pax5 haploinsufficiency and BCR-ABL1, we transplanted Pax5+/+ and Pax5+/− bone marrow cells transduced with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 transduced Pax5+/− marrow developed B progenitor cell ALL with significantly higher penetrance and decreased latency when compared to animals transplanted with BCR-ABL1 transduced Pax5+/+ marrow (median survival 36 vs. 60 days, P=0.0003). The latency of tumor onset was further decreased in the presence of Arf haploinsufficiency (Pax5+/+Arf+/+ 60 days, Pax5+/−Arf+/+ 36 days, Pax5+/−Arf+/− 21 days, P<0.0001). All leukemias were of B cell lineage and were transplantable to secondary recipients. In addition, Southern blot analysis revealed the Pax5+/−Arf+/+ leukemias to be monoclonal, where as the Pax5+/−Arf+/− leukemias were oligoclonal. Importantly, the Pax5+/− leukemias exhibited a more immature B cell immunophenotype than Pax5 wild type leukemias. Moreover, a proportion of the Pax5+/− leukemias (19%) exhibited a very immature early pro B cell immunophenotype (Cd19−, Bp1−), suggesting the possibility of acquired lesions in other key regulators of normal B cell differentiation. To explore this possibility and to identify the total complement of genetic lesions required to generate overt leukemia, we performed genome-wide copy number analysis on 30 murine leukemias (15 Pax5+/+, 15 Pax5+/−) using a custom CGH microarray (Agilent) that interrogated 477,000 autosomal loci, including 18,000 probes covering 20 genes encoding B lymphoid transcription factors and genes targeted by recurring copy number abnormalities (CNAs) in human BCR-ABL1 ALL (Bcl11a, Cdkn2a, Ebf1, Ikzf1, Ikzf2, Ikzf3, Il7r, Lef1, Mdm2, Mef2c, Myb, Pax5, Pten, Rb1, Sfpi1, Sox4, Stat5a, Tcf3, Tcf4, and Trp53). This analysis identified focal recurring CNAs in multiple genes including Cdkn2a/b, Ebf1, Ikzf1, Ikzf2, Ikzf3, and Pax5, each of which is a target of mutation in human B-ALL. Overall, there were on average 3.5 CNAs in Pax5+/+ leukemias versus 0.7 CNAs in Pax5+/− leukemias. Genomic resequencing was also performed on Pax5 and revealed three missense mutations in the DNA binding paired domain (R38H, P80R and G85R), one of which (P80R) is the most common PAX5 point mutation in human B-ALL. All three point mutations are predicted to impair DNA binding of Pax5. Interestingly, the majority of the pro-B cell leukemias that arose in the Pax5+/−Arf+/+ animals were found to harbor mutations (CNAs or point mutation) of the retained Pax5 allele, consistent with the immature immunophenotype. To further explore the relationship between our murine model and human BCR-ABL1 ALL, we performed gene expression profiling of Pax5+/+ and Pax5+/− leukemias and compared their signatures to those of human BCR-ABL1 ALL and stage-specific murine B lymphoid developmental signatures using gene set enrichment analysis (GSEA). This analysis identified significant similarity between murine and human BCR-ABL1 leukemias, thus providing further evidence that this model closely recapitulates human BCR-ABL1 ALL. Notably, Pax5+/− leukemias, or Pax5+/+ leukemias that acquired additional mutations of B-lymphoid regulators exhibited a less mature gene expression profile than leukemias lacking B-lymphoid regulatory mutations. These data indicate that loss of Pax5 contributes to leukemogenesis, that additional genomic alterations in genes regulating B lymphoid development and cell cycle regulators/tumor suppressors (Arf) are frequent events in BCR-ABL1 acute lymphoblastic leukemia, and that these lesions result in impaired B-lymphoid maturation in B-ALL. The genetic complexity of BCR-ABL1 ALL is likely to have important therapeutic implications for this poor prognosis subtype of leukemia.

MYC Translocations and Expression Are Clinically Important in R-CHOP Treated Patients with De Novo DLBCL.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1100-1100
Author(s):  
Nathalie A. Johnson ◽  
Susanna Ben-Neriah ◽  
Kerry J. Savage ◽  
Tang Lee ◽  
Douglas E. Horsman ◽  
...  
Keyword(s):  
Protein Expression ◽  
B Cell ◽  
Research Funding ◽  
De Novo ◽  
Cell Lymphoma ◽  
Molecular Subtype ◽  
B Cell Lymphoma ◽  
Prognostic Impact ◽  
Statistical Software ◽  
Bcl2 Protein

Abstract Abstract 1100 Poster Board I-122 Background MYC, an oncogene associated with cellular proliferation, is deregulated as a result of chromosomal translocation in Burkitt lymphoma (BL), and in 8-12% of diffuse large B cell lymphomas (DLBCL). In 2006, 2 studies defined the molecular features of BL and DLBCL by gene expression profiling (GEP) (Dave, NEJM 2006; Hummel, NEJM 2006) and identified a subset of cases that resembled DLBCL by morphology, but by GEP, expressed MYC and MYC target genes similar to classical BL, i.e. molecular BL (mBL) signature. The clinical outcome of these cases is poorly defined. More recently, MYC expression and MYC translocations (MYC tr+) have been associated with an inferior overall survival (OS) in de novo DLBCL patients (pts) treated with R-CHOP (Rimsza, Blood 2008; Savage, Blood 2009) but the prognostic impact of BCL2 protein expression and concurrent BCL2 translocations (BCL2 tr+) is poorly understood. We investigated the prognostic impact of the presence of a mBL signature by GEP, high MYC mRNA expression, and the presence of a MYC tr+ with or without a concurrent BCL2 tr+ in DLBCL pts treated uniformly with R-CHOP. Methods 315 samples were reviewed by a panel of expert hematopathologists and classified according to the WHO 2008 criteria. Pts with high grade B cell lymphoma otherwise unclassifiable, BL, primary mediastinal B cell lymphoma (PMBCL), T-cell-rich B cell lymphoma and pts that were not treated with R-CHOP were excluded from this analysis. The remaining 259 DLBCL samples were subjected to GEP as previously described (Lenz, NEJM 2008). Tissue microarrays (TMA) were constructed in cases with available paraffin material. 184 had successful GEP arrays, 186 were included on a TMA and 151 cases were assessed on both platforms. Presence of a mBL signature was determined according to the method described by Dave (NEJM 2006). MYC expression was determined using log normalized expression values from Affymetrix U133 Plus 2.0 probe set id 202431_s_at and dichotomized into high versus low expression using a cut off threshold determined by the statistical software X-Tile (http://www.tissuearray.org/rimmlab/). The presence of MYC tr+ or BCL2 tr+ was determined by fluorescence in situ hybridization (FISH) using MYC and BCL2 breakapart probes (Abbott) on TMAs. BCL2 protein expression was determined by immunohistochemistry (IHC) using clone 124 (Dako). Correlation between variables and association with OS was performed by Pearson Chi-Square, Kaplan-Meier and Cox regression analysis using SPSS statistical software. Results A mBL signature was found in 4/184 samples (2%). One case was MYC tr+, one was MYC tr-, and the MYC translocation status was unknown in the remaining 2 cases. All 4 pts with a mBL had a complete response to R-CHOP lasting >2 years after diagnosis. MYC tr+, BCL2 tr+ or concurrent MYC tr+/ BCL2 tr+ were present in 12%, 20% and 4% of 186 DLBCL cases, respectively. BCL2 tr+ were predominately found in the germinal center B cell (GCB) molecular subtype (36%) compared to the activated B cell (ABC) or unclassifiable (U) subtypes (4% and 19%, p=0.0001) but were not associated with an inferior OS. In contrast, MYC tr+ were not associated with a specific molecular subtype (GCB 15%, ABC 8%, U 19%, p=0.2) but were associated with an inferior OS (p=0.0078). When dichotomizing patients with MYC tr+ according to the BCL2 status, pts with concurrent MYC tr+/ BCL2 tr+ (4%) and pts with MYC tr+ and BCL2 protein-positive biopsies (7%) had a markedly inferior OS compared to pts with MYC tr+ and BCL2 protein-negative biopsies or pts with no MYC tr (median OS 7 months vs. not reached, both p < 0.00001). The presence of MYC tr+ correlated with high MYC expression in 6/16 (38%) MYC tr+ cases whereas high MYC expression was present in 5/111 (5%) MYC tr- cases (p=0.0001). High MYC expression alone was also associated with an inferior OS (p<0.00001). In multivariate analysis, high MYC mRNA expression, concurrent MYC tr+/ BCL2 tr+, and the IPI were independent predictors of OS (p=0.04, p=0.05, p=0.007, respectively). Conclusion MYC expression, as prognostic marker in DLBCL, should be investigated in routine clinical practice. Cytogenetic analysis to determine MYC and BCL2 translocation status by FISH and/or karyotype of de novo DLBCL samples, and BCL2 protein expression by IHC are clinically indicated to identify a group of high-risk pts that may benefit from up-front intensified therapy. Disclosures Connors: Roche Canada: Research Funding. Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Inhibition of Phosphatidylinositol 3-Kinase Is Effective to Suppress the Growth of CD20-Negative Refractory Diffuse Large B-Cell Lymphoma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2421-2421
Author(s):  
Tsuyoshi Nakamaki ◽  
Kunihiko Fukuchi ◽  
Hidetoshi Nakashima ◽  
Hirotsugu Ariizumi ◽  
Takashi Maeda ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Expression ◽  
B Cell ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Copy Number ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Copy Number Loss ◽  
Cd20 Expression

Abstract Abstract 2421 CD20 is not only a therapeutic target but also its expression has prognostic importance in B cell lymphoma. Decreased CD20 expression is often associated with refractory phenotypes, especially in lymphoma treated with therapy including rituximab, an anti-CD20 antibody. To address the molecular mechanism(s) of down-regulation of its expression and to find an alternative therapeutic target(s) in resistant lymphoma, we analyzed a CD20-negative B-cell lymphoma cell line (SD07). SD07 was established from lymphoma cells without expressing CD20, which appeared in pleural effusion in a patient with diffuse large B-cell lymphoma (DLBCL), after 2-years of repeated anti-lymphoma therapy including rituximab, While initial diagnosis was CD20 positive activated B cell-like DLBCL (ABCDLBCL). SD07 selectively lacks CD20 protein expression, but not other B cell antigens, such as CD19, CD21 and CD22 by flowcytometry(FCM). In SCID mice, subcutaneously transplanted SD07 developed tumors which are positive for B cell antigen such as CD79a, but not CD20 (L26) and EBER. Array CGH revealed 0.7 Mb region with copy number loss less than −1.0 of log2 ratios on chromosome 11q12 in SD07. Within this region, 30 kb segment, which showed further loss (less than −2.0),contained two genes, MS4A1(CD20) and MS4A5. Southern blot analysis using CD20 exon 1 as a probe showed homozygous deletion of CD20 gene in SD07. As expected, incubation with rituximab in culture failed to suppress the cell growth of SD07 up to 20 μg/ml(cell No. rituximab/control=0.98±0.04, P=NS). This suggests deletion of CD20 gene with genomic copy number loss in 11q12 produced the loss of CD20 expression and resulted in resistant to rituximab. It is currently studied whether the loss of CD20 expression is directly involved in the tumorigenicity of SD07. Array CGH also showed several genomic regions with copy number loss which are possibly involved in refractory phenotype of SD07. Those includes 10q23 (PTEN), 12q23 (APAF1) and 16q21 (NFATC3). Immunoblot analysis showed the absence of PTEN protein expression and constitutive AKT Ser473 phosphorylation in SD07. SDO7 also showed constitutive phosphorylation of both SykTyr525/526 and Btk Tyr223,however, those did not differ much in those in three germinal center B-like(GCB) cell lines, two Burkitt lymphoma cell lines, Daudi and N8, and a DLBCL cell line, TK, derived from follicular lymphoma. In SD07, somatic mutations of ITAM of either CD79A or CD79B was not detected. This suggests inactivation of PTEN, rather than chronic active BCR signaling, is likely to promote constitutive PI3K-AKT signaling in SD07. Nuclear NF-kB DNA binding by EMSA, SD07 showed constitutively higher binding signals of NF-kB, compared with either Daudi, N8 or TK (SD07=3.6±0.1,GCB cell lines(Daudi,N8,TK)=1.2±0.2, p<0.01, arbitrary density units). Supershift analysis showed increased NF-kB DNA binding in SD07 mainly consist of p50 and c-Rel. By immunoblot, SD07 showed steady-state increased accumulation of p50 protein, but not p65, in nuclear fraction, compared with either in Duadi,N8 or TK. Protein expression of BClxL, a NF-kB target gene, increased in SD07 (SD07=1.9±0.1, GCB cell lines=0.7±0.2, p<0.01, signal intensity, SI). Those suggest that oncogenic PI3K-AKT activation and/or constitutive activation of NF-kB pathway contribute pro-survival signaling in SD07 and those are possible therapeutic target in SD07. Incubation with LY294002(LY), selective PI3K inhibitor, at more than 0.1μM for 4 days, dose-dependently inhibited the cell growth of SD07(10μMLY/DMSO=0.35±0.01, P<0.01), without significant effects on cell viability. FCM analysis with PI showed that incubation with LY produced G1 accumulation and decrease of cells in the S (LY/DMSO,G1=1.5±0.1,S=0.2±0.1,G2M=0.4±0.1, P<0.05). 10 μM LY inhibited both AKT Ser473 phosphorylation (LY/DMSO=0.18±0.01,SI,P<0.01) and protein expression of BClxL (LY/DMSO=0.43±0.02,P<0.05) in SD07. LY increased p27 protein expression (LY/DMSO=1.83±0.04, P<0.05), without affecting NF-kB DNA binding in SD07. It may suggest PI3K are required for expression of some NF-kB target gene without suppressing nuclear NF-kB DNA binding. In summary, although a limited in a cell line, we clarify a novel molecular mechanism of acquired loss of expression of CD20 in DLBCL. In addition, we show that de-regulated PI3K-AKT pathway is a possible therapeutic target for CD20-negative refractory ABCDLBCL. 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.

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