Restoring the Functional Immunogenicity of Chronic Lymphocytic Leukemia Using Epigenetic Modifiers.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5116-5116
Author(s):  
Jason A Dubovsky ◽  
John J. Powers ◽  
Daniel Wang ◽  
Eduardo M. Sotomayor ◽  
Javier Pinilla
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Hla Dr ◽  
Antigen Presenting

Abstract Abstract 5116 Background Chronic lymphocytic leukemia (CLL) is a malignancy arising from immune cells (B-lymphocytes) endowed with intrinsic antigen-presenting capabilities. Such a function however is lost during malignant transformation and CLL cells are well known for their inability to process and present antigens to the T-cell arm of the immune system. Instead, malignant CLL cells elicit a vast array of immune regulatory mechanisms conducive to T-cell dysfunction and immunosupression. Recent evidence suggests that DNA methylation inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) can have lasting cancer-specific effects on the expression of highly immunogenic cancer-testis antigens (CTAs) as well as modulating costimulatory and major histocompatability molecule (MHC) surface expression. Methods To investigate the potential efficacy of a combined therapy of DNMTi (5-aza-2'-deoxycytidine) and HDACi (LAQ) in human CLL we performed a gene expression microarray profile and further confirmed the expression of CTAs. Furthermore we investigated phenotypic alterations due to changes in costimulatory molecules (CD40, 80, and 86), and MHC molecules (HLA-A, B, C, and HLA-DR) in both cell lines and primary cells from patients with CLL. To confirm that alterations in molecule surface expression correlated to more potent immune stimulation we characterized the formation of the immune-synapse between the CLL antigen presenting cell and healthy T-lymphocytes in treated and untreated samples using confocal microscopy and flow conjugation assay. To demonstrate functional significance we subjected treated and untreated CLL cell lines to mixed lymphocyte proliferation assays with CD4, CD8, and NK cell subsets. Results As expected we demonstrate by gene expression profile and RT-PCR that many highly immunogenic CTAs (SSX family, MAGE family, NY-ESO-1, GAGE-2 and NXF2) are significantly upregulated after treatment with an HDACi and DNMTi in combination. Interestingly, preliminary data showed interferon gamma mRNA upregulation. Consistent with this information, our data also indicates that a more proinflammatory signaling pathway is developed between the CLL B-cell and the T-lymphocyte after combination treatment demonstrated by upregulated surface expression of CD86, 80, 40, HLA-A,B,C, and HLA-DR as well as the formation of significantly more, robust, numerous, and organized actin-mediated immune-synapses, changes which were not duplicated using either drug alone. Functionally, only the combined HDACi/DNMTi treatment of CLL cells led to increased allogenic T and NK cell proliferation and cytotoxicity. Conclusions Taken together these data indicate that combined HDACi/DNMTi may benefit current antigen specific vaccine strategies for CLL by inducing the expression of a highly antigenic CTAs, increasing CLL costimulatory capacity, repairing immunological synapse, and functionally increasing the proinflammatory status of the CLL APC. Disclosures Pinilla: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.

scholarly journals P09.02 Epigenetic modulation of neuroblastoma enhances T- and NK cell immunogenicity via induction of surface expression of MHC class I and MICA/MICB

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A28.1-A28
Author(s):  
AM Cornel ◽  
DAMH van den Beemt ◽  
E Dunnebach ◽  
MP van Dierselhuis ◽  
S Nierkens
Keyword(s):  
T Cell ◽  
Cell Lines ◽  
Healthy Donor ◽  
Nk Cell ◽  
Surface Expression ◽  
Immunomodulatory Effects ◽  
Mhc I ◽  
Adaptive Immune ◽  
Antigen Presenting

BackgroundNeuroblastoma (NBL) is the most common pediatric solid tumor and responsible for about 15% of all pediatric cancer deaths. The majority of high-risk (HR) patients suffers from relapse after intense therapy regimens, resulting in a 5-year survival rate of only 40%. Even though the potential of immune interference in HR-NBL is shown by the additive effect of anti-GD2 monoclonal antibody therapy to the treatment protocol, long-term follow-up studies reveal that the beneficial effect of immunotherapy diminishes over time. We hypothesize that this is a result of inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL and its microenvironment. One of the most remarkable immunomodulatory strategies of NBL tumors is the absence of MHC-I surface expression, thereby preventing cytotoxic T cell recognition and killing. MHC-I lacking cells are known to be subjected to NK cell mediated cytotoxicity, however, we have shown that NBL is able to evade this by temporary upregulating surface expression of MHC-I, thereby becoming temporarily more prone to T cell mediated cytotoxicity. The aim of this project is to identify pharmacological strategies to enhance adaptive immune activation and therewith immunogenicity of HR-NBL.Materials and MethodsFDA-approved drug libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cell lines using high-throughput flow cytometry analyses optimized for adherent NBL cells. The effect of positive hits was subsequently confirmed in a panel of NBL patient-derived tumeroids. Alterations in the transcriptome and translatome upon incubation with compounds of interest were further studied to identify potential additional immunomodulatory effects in NBL. Ultimately, compound treated NBL cell lines and tumeroids were co-cultured with PRAME reactive tumor-specific T cells and healthy-donor NK cells to determine the in vitro effect on T- and NK cell cytotoxicity.ResultsDrug library screening revealed MHC-I upregulation upon treatment of NBL cell lines and patient-derived tumeroids with multiple histon deacetylase inhibitors (HDACi). Further investigation of immunomodulatory effects of HDACi in NBL revealed enhanced expression of several additional players of the antigen presenting machinery, immunoproteasome expression, and MICA/MICB upregulation in NBL cells. We show that in untreated NBL cells, plasticity of MHC-I expression causes evasion of both NK- and T cell mediated cytotoxicity. Intriguingly, co-culture of NBL cells with tumor-specific T cells and healthy-donor NK cells upon treatment with the HDACi Entinostat resulted in enhanced in vitro T- and NK cell activation and cytotoxicity.ConclusionsWe show pharmacological upregulation of MHC-I, other antigen presenting machinery players, and the NKG2D ligands MICA/MICB upon HDACi in HR-NBL. Pre-treatment of NBL with HDACi resulted in enhanced in vitro T- and NK cell mediated cytotoxicity, substantiating HDACi as a potential strategy to improve adaptive immune engagement and therewith immunogenicity to aid NBL treatment.This work was supported by the Villa Joep Foundation [IWOV-Actief.51391.180034].Disclosure InformationA.M. Cornel: None. D.A.M.H. van den Beemt: None. E. Dunnebach: None. M.P. van Dierselhuis: None. S. Nierkens: None.

scholarly journals Reversible DNA Hypermethylation of the Interleukin-15 (IL-15) Promoter Induces IL-15 Expression, Drives the Pathogenesis of T-Cell Large Granular Lymphocytic Leukemia and Provides a Potential Therapeutic Approach Using 5-Azacitidine

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3776-3776
Author(s):  
Jonathan E Brammer ◽  
Amy E Boles ◽  
Anthony Mansour ◽  
Aharon G. Freud ◽  
Monique Mathé-Allainmat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
T Cell ◽  
Research Funding ◽  
Marked Decrease ◽  
Lymphocytic Leukemia ◽  
Dna Hypermethylation ◽  
Reduced Representation ◽  
Primary Driver ◽  
Interleukin 15

Background and Rationale: T-cell large granular lymphocytic leukemia (T-LGLL) is an incurable clonal proliferation of CD8+ memory T-cells that leads to profound neutropenia and anemia with limited treatment options. The primary driver of T-LGLL is overexpression of interleukin-15 (IL-15), a gamma-chain cytokine. Previously, we have demonstrated that mice overexpressing IL-15 develop DNA hypermethylation and chromosomal instability that leads to the spontaneous development of LGLL (Mishra et al. Cancer Cell 2012). Further, the IL-15 promoter is known to be hypermethylated in cutaneous T-cell lymphoma (CTCL), another IL-15 driven malignancy (Mishra et al. Cancer Discovery 2016). In CTCL patients, the counterintuitive increase in IL-15 mRNA was due to hypermethylation of its promoter at the repressor binding sequences in the IL-15 gene. However, the methylation status of the IL-15 promoter in T-LGLL patients remains unknown. Concept: We hypothesize that the IL-15 promoter is hypermethylated in patients with T-LGLL, leading to aberrant overexpression of IL-15 and that this hypermethylation is a critical event in the leukemogenesis of T-LGLL. If true, demethylation of the IL-15 promoter with a resultant decrease in IL-15 transcripts should lead to apoptosis of T-LGLL cells. Hypomethylation of the IL-15 promoter, therefore, may provide a novel therapeutic approach to inhibiting IL-15, the primary driver of T-LGLL. Results: CD3+/CD8+/CD5-/dim T-cells were purified from peripheral blood of LGLL patient (n=3) and normal donor (ND) (n=3) by flow cytometry sorting. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing. With bioinformatics analysis, we determined differential methylation (1-way ANOVA P= 0.0178) and expression (1-way ANOVA P =0.0059). These data sets revealed significant differential hypermethylation of gene promoters in leukemic samples, compared to controls (Figure 1A). Reduced representation bisulfite sequencing that can identify differentially methylated regions at single base-pair resolutions demonstrated an increase in DNA methylation of the IL-15 promoter in patient samples over controls. To determine the functional significance of this finding, we treated the MOTN-1 T-LGLL cell line in vitro with the hypomethylating agent, 5-azacytidine (5-aza) at concentrations of 0.5 uM, 1 uM, 2.5 uM, and 5 uM. At 24 and 48 hours, a marked decrease in the viability of T-LGLL cells was observed, from 100% to 49.50%, p=0.037; particularly at higher concentrations of 5-aza (100% to 27% +11.30%, p=0.0030). Next, we sought to determine whether 5-aza induced hypomethylation of the IL-15 promoter. IL-15 gene expression in MOTN-1 T-LGLL cells treated with 5-aza was measured in comparison to control treated MOTN-1 cells. A marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (Figure 1B, p=0.0001). These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. Finally, to determine whether a decrease in IL-15 alone was the cause of increased apoptosis of T-LGLL cells, we exposed MOTN-1 cells to a novel IL-15 inhibitor, IBI-15, and compared cell viability against MOTN-1 cells exposed to an inactive control, IBI-40. Even more profound decrease in cell viability was observed utilizing IBI-15 that targets the binding of IL-15 to its receptor (Figure 1C). Together, these data suggest that hypermethylation of the IL-15 promoter is critical to the pathogenesis of T-LGLL, and that treatment with 5-aza is sufficient to induce hypomethylation of the IL-15 promoter, decrease IL-15 transcription, and induce apoptosis in T-LGLL cells. Conclusions: Hypermethylation of the IL-15 promoter, with subsequent increase in IL-15, is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter hypermethylation by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce apoptosis of T-LGLL cells. These data suggest that 5-aza induced hypomethylation may be a novel method to induce IL-15 inhibition and a potentially efficacious clinical strategy against T-LGLL. Disclosures Brammer: Bioniz Therapeutics, Inc.: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Verastem, Inc: Research Funding. Porcu:Daiichi: Research Funding; BeiGene: Other: Scientific Board, Research Funding; Spectrum: Consultancy; Viracta: Honoraria, Other: Scientific Board, Research Funding; Innate Pharma: Honoraria, Other: Scientific Board, Research Funding; Kyowa: Honoraria, Other: Scientific Board, Research Funding; ADCT: Research Funding; Incyte: Research Funding. OffLabel Disclosure: IBI-15 IBI-40 IL-15 inhibitor

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.

Prior Treatment with Alemtuzumab Interferes with T-Cell Engraftment After Allogeneic Stem Cell Transplantation in Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3351-3351
Author(s):  
Johannes Schetelig ◽  
Christian Thiede ◽  
Alexander Kiani ◽  
Uwe Platzbecker ◽  
Uta Oelschlaegel ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Washout Period ◽  
Research Funding ◽  
Limit Of Detection ◽  
Lymphocytic Leukemia ◽  
Cell Engraftment ◽  
Median Level ◽  
Antibody Levels ◽  
Donor Lymphocyte Infusions

Abstract Abstract 3351 Poster Board III-239 Objectives: The majority of patients with chronic lymphocytic leukemia (CLL) who receive allogeneic hematopoietic cell transplantation (HCT) have fludarabine-refractory disease. The most active single agent in this disease stage is alemtuzumab. Alemtuzumab has a long half-life and induces profound T-cell depletion (TCD). Since TCD may mitigate graft-versus leukemia effects we evaluated „pre-conditioning“ with alemtuzumab followed by a washout period in order to minimize in vivo T-cell depletion of the graft in a phase II study (NCT 00337519). Methods: Patients received cytoreductive treatment with 3 × 30 mg alemtuzumab weekly prior to HCT. The scheduled interval between last dose of alemtuzumab and HCT was increased from two weeks to one month during the study. The antibody level at the day of HCT was measured with an ELISA with a lower limit of detection of 31.25 ng/mL (BioAnaLab lim., Oxford, UK). The conditioning regimen contained fludarabine (150 mg/m2) and busulfan (8 mg/kg). Cyclosporine (CSA) and methotrexate (MTX) were applied as GVHD-prophylaxis. Medically fit patients with relapsed CLL were elible. Results: 62 patients with a median age of 57 years were included between April, 2004 and October, 2008. A median of 3 prior regimens had been given. 55% of the patients had fludarabine-resistant disease. Two patients failed to reach HCT due to progressive disease during alemtuzumab therapy. Donors were matched siblings for 26 and matched unrelated donors for 34 patients. The median level of alemtuzumab in peripheral blood after a washout period of two weeks was 62 ng/mL (interquartile range, 45 to 196 ng/mL; minimum below the limit of detection; maximum 490 ng/mL) compared to a median level below the limit of detection after a delay of four weeks (interquartile range, between the limit of detection and 77 ng/mL, maximum 256 ng/mL) (p=0.005). Despite one month time between the last dose of alemtuzumab and HCT 4 out of 30 patients (13%) had alemtuzumab levels greater than 200 ng/mL. No primary or secondary graft failure occurred. A linear relationship between the alemtuzumab level at HCT and the time to complete CD4-T-cell chimerism (TCC) was observed (p=0.003). At day +100 a CD4 positive T-cell-chimerism (TCC) >95% had been achieved by 84% of patients with alemtuzumab levels <100 ng/mL, 83% of patients with antibody levels between 100 and 200 ng/mL and 25% of patients with antibody levels >200 ng/mL (p=0.006). All patients had a complete neutrophil-chimerism at day +100. After early taper of immunosuppression (N=2) or the application of donor lymphocyte infusions in incremental doses (N=5) mixed TCC has been converted to complete TCC in all patients. The median follow-up is 17 months (1 to 61 months). Day +100 non-relapse mortality was 2%. At two years non-relapse mortality and relapse incidence were 21% and 29%, respectively. Two-year overall survival and progression-free survival were 67% (95% CI, 51% to 83%) and 50% (95% CI, 31% to 69%). Conclusions: In patients who received alemtuzumab prior to HCT, residual drug levels may interfere with T-cell engraftment. Lineage specific T-cell chimerism should therefore be assessed prospectively in this group of patients. Persistent mixed T-cell chimerism can be converted by an early taper of immunosuppression and incremental doses of donor lymphocyte infusions. Disclosures: Schetelig: Bayer Schering: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Treatment of Chronic Lymphocytic Leukemia Patients with Lenalidomide Induces Down-Regulation of miR342-3p Associated with Over-Expression of Tumor Suppressor RASSF4,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3885-3885
Author(s):  
Emanuela M. Ghia ◽  
Lillian Werner ◽  
Danelle F. James ◽  
Donna Neuberg ◽  
Laura G Corral ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Family Member ◽  
Research Funding ◽  
Human Tumor ◽  
Lymphocytic Leukemia ◽  
Down Regulation ◽  
Antagonistic Effects ◽  
Over Expression ◽  
Pre Treatment

Abstract Abstract 3885 Lenalidomide has promising clinical activity in patients with chronic lymphocytic leukemia (CLL). Unlike other anti-leukemia drugs, lenalidomide is not cytotoxic for CLL cells in vitro. Similar to CD154, lenalidomide can enhance CLL-cell expression of immune co-stimulatory molecules, formation of immunologic synapse, activation of NK-cells, and generation of anti-tumor immunity. Furthermore, lenalidomide repeatedly can enhance expression of CD154, which we had observed was functionally deficient in patients with CLL. However the exact mechanism of action of lenalidomide is still under investigation. Herein, we studied the gene expression profile and microRNA (miR) of CLL cells collected from 20 patients before and at day 8 and day 15 of treatment with 2.5–5 mg of lenalidomide in the CRC014 trial. We observed significant changes in expression level of 54 genes at day 8 versus pre-treatment samples. We identified significant changes in expression level of 189 genes at day 15 versus pre-treatment samples. This included 44 of the 54 (81%) genes noted at day 8. Forty genes were expressed at significantly higher levels at day 8 and day 15 of lenalidomide treatment. We noted that 7 (17%) of these genes were related to Ras pathway and its downstream signaling pathways (i.e. NF-KappaB pathway): Ras association (RalGDS/AF-6) domain family member 4 (RASSF4), a member of RAS oncogene family (RAB13), Ras protein-specific guanine nucleotide releasing factor 1 (RASGRF1), GTPase IMAP family member 6 (GIMAP6), GTP-binding protein ras homolog gene family member S (RND1), kinase suppressor of Ras 2 (KSR2) and toll-like receptor adaptor molecule 2 (TICAM2). Ras signaling affects many cellular functions, which includes cell proliferation, apoptosis, migration, fate specification, and differentiation. In the resting cells, Ras is tightly bound to GDP (Guanosine Diphosphate), which is exchanged for GTP (Guanosine Triphosphate) upon binding to activated cell membrane receptors. In the GTP-bound form, Ras interacts with a broad range of effector proteins to induce a diverse array of biological consequences. Although typically associated with enhanced growth and transformation, activated Ras also may induce growth antagonistic effects such as senescence or apoptosis. Some of the growth-inhibitory properties of Ras are mediated via the RASSF family of Ras effector/tumor suppressors. RASSF4 is the fifth member of this family and it binds directly to activated K-Ras in a GTP-dependent manner via the effector domain, thus exhibiting the basic properties of a Ras effector. Overexpression of RASSF4 induces Ras-dependent apoptosis in 293-T cells and inhibits the growth of human tumor cell lines. Although broadly expressed in normal tissue, RASSF4 is frequently down-regulated by promoter methylation in human tumor cells and primary tumors. However changes in miR expression also may affect the level of gene expression. Therefore we analyzed miRs expression by microarray in pre treatment, day 8, and day 15 CLL samples. We observed significant changes in expression levels of 33 miRs between day 8 and pre treatment samples. We identified significant changes in expression levels of 11 miRs between day 15 and pre treatment samples. Of the 33 miRs differentially expressed at day 8, only 5 were up-regulated whereas the remaining 28 were down-regulated. Interestingly, among these 28 down-regulated miRs, 5 miRs (miR-103, miR-16, miR-30a, miR-30b and miR-342-3p) target RASSF4. Noteworthy, miR-342-3p was one of the 3 miRs (miR-26a, miR-138 and miR-342-3p) down-regulated both at day 8 and at day 15, suggesting that the down-regulation of such miR has a key role in the overexpression of RASSF4 leading to Ras-dependent apoptosis. Further studies are ongoing to elucidate lenalidomide action on CLL cells via RASSF4 overexpression. This study demonstrates that treatment with lenalidomide can induce down-regulation of miRs associated with changes in gene expression by CLL cells, leading to over-expression of RASSF4 and other Ras or GTPase related proteins that can induce growth antagonistic effects and account in part for the activity of this drug in CLL. Disclosures: James: Celgene: Research Funding. Neuberg:Celgene: Research Funding. Corral:Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

A Novel HIV Envelope Bi-Specific Killer Engager Enhances Natural Killer Cell Mediated ADCC Responses Against HIV-Infected Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2517-2517 ◽  
Author(s):  
Zachary B. Davis ◽  
Todd Lenvik ◽  
Louis Hansen ◽  
Martin Felices ◽  
Sarah Cooley ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Mhc I ◽  
Variable Heavy ◽  
Infected Cells ◽  
T Cell Lines ◽  
Hiv Envelope

Abstract Natural Killer (NK) cells, a critical component of the immune response to viral infection, recognize and destroy cells with diminished expression of major histocompatibility class-I (MHC-I) molecules and expression of ligands for activating NK receptors such as NKG2D. Down-modulation of MHC-I is a hallmark of viral infection, as it allows infected cells to evade a CD8 T-cell response. Stalling of the cell cycle to enhance viral replication induces NK activation ligands such as the NKG2D ligands unique long binding proteins (ULBP)-1 and -2 which could trigger NK destruction of infected cells. Unfortunately, incomplete down-modulation of MHC-I by HIV leaves HLA-C on the cell surface, which inhibits the majority of NK cells from killing infected targets. CD16, the low affinity Fc receptor, is the most potent NK cell activating receptor. It mediates antibody dependent cell-mediated cytotoxicity (ADCC), and can override inhibition by MHC-I. We designed a series of bi-specific killer-engager (BiKE) constructs to direct NK cell ADCC against an HIV-infected target. We linked the Fab portions of broadly neutralizing (bn)Abs to a novel llama-derived nanobody EF91 that binds CD16 at high affinity and signals strong activation. We chose to use EF91 as its structure is unique compared to the use of a single chain variable fragment (scFv). Rather than being composed of a variable heavy (VH) and variable light (VL) chain, the nanobody is composed of a single variable heavy (VHH) domain. A distinct advantage to using a CD16 nanobody over a scFv is in the purity of the generated product. During protein folding it is not uncommon for the wrong VH to associate with the wrong VL; the result of which is a nonfunctional product. Since the nanobody is single VHH, and does not require association with another domain, there is less risk of a misfolded product. Nanobodies are also known to have similar, if not increased, affinity for their target molecules. In the case of EF91, this may result in more robust activation of NK cells than with a traditional scFv. We tested a BiKE constructed with the bnAb, VRC01, which recognizes the CD4 binding domain of HIV-Env. The specificity of our novel anti-CD16 nanobody was demonstrated by binding of our BiKE construct to CD16+ NK cells (Figure 1A). Function of our BiKE construct was tested by incubating it with chronically infected T-cell lines (HIV-IIIB and ACH-2) or with their respective uninfected counterparts (H9 and CEM). We only observed binding to infected cells (Figure 1B), demonstrating HIV-Env binding specificity to the HIV strains ACH-2 (LAI strain) and HIV-IIIB. The ability of the anti-Env BiKE construct to mediate ADCC and IFNγ production was tested against two uninfected CD4 T-cell lines or their infected counterparts. While NK cells degranulated when incubated with the infected cell lines (50% against HIV-IIIB and 20% against LAI), this response was markedly enhanced when co-incubated with the HIV-Env specific BiKE (80% against HIV-IIIB and 60% against LAI) (Figure 1C). Furthermore, the HIV-Env BiKE enhanced IFNγ production against HIV-infected T-cell lines compared to responses in the absence of BiKE (28% against HIV-IIIB compared to 36% with BiKE; 15% against ACH-2 compared to 37% with BiKE) (Figure 1D). Our data demonstrate that a BiKE construct containing the Fab of an HIV bnAb and an anti-CD16 component can eliminate HIV-infected targets that express the HIV-envelope on their surface. The reservoir of latently infected CD4 T cells lack expression of any recognizable virus protein on the cell surface, we plan to combine our BiKE strategy with cellular activation using IL-15. Alternatively, we can construct a tri-specific engager (TriKE) with an IL-15 segment that may activate CD4 T cells while enhancing NK cell killing. Disclosures Cooley: Fate Therapeutics: Research Funding. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Miller:Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

scholarly journals Subcutaneous Epcoritamab in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia: Preliminary Results from the Epcore CLL-1 Trial

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2627-2627
Author(s):  
Arnon P. Kater ◽  
Jacob Haaber Christensen ◽  
Hans Herluf Bentzen ◽  
Carsten Utoft Niemann ◽  
Martin Hutchings ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Dose Escalation ◽  
Research Funding ◽  
Injection Site Reaction ◽  
Steering Committee ◽  
Lymphocytic Leukemia ◽  
Site Reaction ◽  
Phase 1B ◽  
Dose Levels

Abstract Background: Small molecules such as Bruton tyrosine kinase (BTK) and B-cell lymphoma 2 (BCL2) inhibitors have transformed the management of chronic lymphocytic leukemia (CLL). However, such treatments are not curative, and patients (pts) with relapsed or refractory (R/R) CLL following multiple targeted treatments present an emergent challenge with very limited therapeutic options. In addition, success rates of autologous T-cell-based therapies in CLL have been disappointing. In vitro data in CLL cells suggest potentially high efficacy of bispecific T-cell engagers (Martens et al, J Immunother Cancer 2020), but clinical data are extremely limited. Epcoritamab (GEN3013; DuoBody ®-CD3×CD20) is a bispecific antibody that can induce potent activation and cytotoxic activity of CD4+ and CD8+ T cells to specifically eliminate CD20-expressing cells (van der Horst et al, Blood Cancer J 2021). In the first-in-human trial in R/R B-cell non-Hodgkin lymphoma (B-NHL; EPCORE NHL-1; NCT03625037), epcoritamab showed manageable safety and meaningful antitumor activity across a range of aggressive and indolent B-NHLs. The most common treatment-emergent adverse events (AEs) were pyrexia (69%), cytokine release syndrome (CRS; 59%), and injection-site reaction (47%); CRS events were all grade 1-2 and most occurred in cycle 1 (Clausen et al, J Clin Oncol 2021). However, as CLL is characterized by presence of (high numbers of) circulating tumor cells, acquired T-cell dysfunction, and variable expression of CD20, data obtained in B-NHL are difficult to extrapolate to CLL. Herein we present the first results from the dose-escalation part of a phase 1b/2 trial evaluating epcoritamab in pts with R/R CLL. Methods: In this open-label, multicenter, phase 1b/2 trial, toxicity and efficacy of epcoritamab are investigated in adults with R/R CLL (EPCORE CLL-1; NCT04623541). Eligible pts previously received ≥2 lines of systemic antineoplastic therapy, including treatment with (or intolerance to) a BTK inhibitor. Epcoritamab is subcutaneously administered via 1-mL injections in 4-week cycles as follows: once weekly in cycles 1-3, every 2 weeks in cycles 4-9, and monthly in cycles ≥10 until progression or unacceptable toxicity. Step-up dosing during cycle 1 (ie, priming dose followed by an intermediate dose, then full doses) is used in combination with steroid prophylaxis to reduce the risk of CRS. In the dose-escalation part, pts with R/R CLL received epcoritamab at 2 full-dose levels (24 and 48 mg) according to a modified 3+3 design. Primary end points of the dose-escalation part included dose-limiting toxicities (DLTs) during the first 28-day treatment cycle and the incidence and severity of AEs, CRS, immune effector cell-associated neurotoxicity syndrome (ICANS), and tumor lysis syndrome (TLS). Results: The first pt was enrolled on November 30, 2020. As of July 12, 2021, 7 pts with R/R CLL received epcoritamab subcutaneously administered at 2 full-dose levels: 24 mg (n=3) and 48 mg (n=4). Six pts completed the DLT evaluation period, and 5 pts had a full response assessment. Pts had received a median of 4 lines of prior therapy (range, 2-5). Six of 7 pts had poor-risk features of del(17p), TP53 mutations, or both. Three of 7 pts had bulky disease. No DLTs occurred at 24 or 48 mg. The most common treatment-emergent AEs (&gt;30%) were CRS (100%), fatigue (71%), injection-site reaction (43%), and nausea (43%). All pts experienced CRS in the first cycle, but no CRS events were higher than grade 2. No cases of ICANS were observed. TLS was not observed. Antileukemic activity has been observed at both dose levels, with partial responses in 3 of 5 pts. Updated clinical and pharmacokinetic data, including data for additional pts treated with epcoritamab, will be presented. Conclusions: These data suggest that epcoritamab is well tolerated in pts with R/R CLL at dose levels up to 48 mg and has clinical activity in pts with high-risk features. The expansion part of this study in pts with R/R CLL and Richter syndrome will open later this year. Disclosures Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding. Niemann: CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding; Novo Nordisk Foundation: Research Funding. Hutchings: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Incyte: Research Funding; Janssen: Honoraria, Research Funding; Genmab: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Genentech: Honoraria, Research Funding. Chen: Genmab: Current Employment. Rios: Genmab: Current Employment. Palenski: AbbVie: Current Employment. Li: Genmab: Current Employment. Mato: AbbVie: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; MSKCC: Current Employment; Genmab: Research Funding; Johnson and Johnson: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; LOXO: Consultancy, Research Funding; Nurix: Research Funding; DTRM BioPharma: Consultancy, Research Funding; TG Therapeutics: Consultancy, Other: DSMB, Research Funding; BeiGene: Consultancy, Research Funding; Acerta/AstraZeneca: Consultancy, Research Funding; AstraZeneca: Consultancy; Sunesis: Consultancy, Research Funding.

scholarly journals Chronic Lymphocytic Leukemia Cells with Mutated Nfkbie Are Positively Selected By Microenvironmental Signals and Display Reduced Sensitivity to Ibrutinib Treatment

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 248-248
Author(s):  
Alice Bonato ◽  
Riccardo Bomben ◽  
Supriya Chakraborty ◽  
Giulia Felician ◽  
Claudio Martines ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Pi3k Inhibitor ◽  
Leukemia Cells ◽  
Wild Type ◽  
Clonal Composition

Abstract Inactivating mutations in NF-kB pathway genes, such as the NF-kB inhibitor NFKBIE, are among the more frequent genetic lesions in chronic lymphocytic leukemia (CLL). However, the role of these genetic lesions in CLL pathogenesis and treatment resistance is still largely unknown and requires further study in in vivo models of the disease. To this end, we generated transplantable murine leukemias with inactivating NFKBIE mutations and investigated their impact on leukemia growth and response to ibrutinib (IBR) treatment. The NFKBIE mutations were introduced by CRISPR/Cas9 editing in two recently established autoreactive leukemia lines derived from the Eμ-TCL1 murine CLL model. These cell lines proliferate spontaneously in vitro in a BCR-dependent manner, but also respond with increased proliferation to certain microenvironmental signals, such as those generated by Toll-like receptor (TLR) stimulation (Chakraborty S et al, Blood 2021). To investigate whether NFKBIE mutations can affect the proliferation of these cell lines in vitro, we performed competition experiments with mixed cultures of cells with wild type and mutated NFKBIE. Analysis of the clonal composition after 2 weeks showed no change in the mutant allele frequency (MAF), suggesting that NFKBIE mutations do not affect the spontaneous in vitro growth of the immortalized leukemia cells. However, repeated TLR or BCR stimulation of these cells with CpG-DNA, LPS, anti-IgM or autoantigen resulted in a 2-3 fold increase in MAF, suggesting that NFKBIE mutations provide a growth advantage when the cells are exposed to certain microenvironmental signals (n=3 experiments/condition, P&lt;0.05 for each condition). To investigate the impact of NFKBIE mutations on leukemia growth in vivo, the same cells were transplanted by intraperitoneal injection in wild type mouse recipients (n=8) and the clonal composition was determined 3 weeks later by MAF analysis of cells isolated from peritoneal cavity (PC), blood and spleen. A significant increase in MAF was observed only in leukemia cells isolated from the spleen (P&lt;0.05), suggesting that microenvironmental signals that positively select NFKBIE-mutated cells are available only in certain tissue compartments. Because mutations in other NF-kB pathway genes have been associated with resistance to IBR in mantle cell lymphoma, we next investigated whether NFKBIE mutations can also affect the response to IBR treatment. In vitro BrdU-incorporation experiments showed that IBR inhibits the proliferation of cells with mutated NFKBIE to a significantly lesser extent compared to cells with wild type NFKBIE (% proliferating cells with wild type and mutated NFKBIE, respectively, cultured without IBR: 90% vs 88%, P=n.s., with 0.2 μM IBR: 57% vs 73%, P&lt;0.001, with 1.0 μM IBR: 28% vs 53%, P&lt;0.001). Consistent with this finding, positive selection of NFKBIE-mutated cells was observed in the presence of IBR after 14 days in mixed culture competition experiments (mean MAF without IBR 47%, with 0.2 μM IBR 61%, p=0.032, with 1.0 μM IBR 64%, p=0.034). The greater resistance of NFKBIE-mutated cells to IBR was further validated by in vivo competition experiments showing a significantly greater increase in MAF in mice treated with IBR compared to controls in all three investigated compartments (n=4 mice/group, PC: P=0.029, blood P=0.029, spleen: P=0.001). To validate these findings in the clinical setting, we investigated the presence of NFKBIE mutations in a cohort of 84 IBR-treated CLL patients. Mutations of NFKBIE were detected at pre-treatment in 10/84 patients, 7/10 with &gt;10% VAF values. Kaplan Meier analysis showed a trend towards reduced progression-free and overall survival from the beginning of IBR treatment for NFKBIE-mutated cases (Figure 1A). Analysis of an extended cohort of over 200 cases is ongoing and will be presented at the meeting. Finally, to investigate whether leukemic cells with mutated NFKBIE remain sensitive to other BCR inhibitors, we tested their growth in the presence of the PI3K inhibitor idelalisib or SYK inhibitor fostamatinib (Figure 1B). In contrast to IBR, both drugs inhibited the proliferation of NFKBIE-mutated cells in vitro, with a greater effect observed with idelalisib. Collectively, these data demonstrate that NFKBIE mutations can reduce the response to IBR treatment and suggest that such cases may benefit more from treatment with a PI3K inhibitor. Figure 1 Figure 1. Disclosures Marasca: Janssen: Honoraria, Other: Travel grants; AstraZeneca: Honoraria; AbbVie: Honoraria, Other: Travel grants. Tafuri: Roche: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Laurenti: Janssen: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria; BeiGene: Honoraria. Gattei: abbVie: Research Funding; Janssen: Research Funding; Menarini: Research Funding.

scholarly journals RPS15 mutations Repress mRNA Translation in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1843-1843
Author(s):  
Stavroula Ntoufa ◽  
Stamatia Laidou ◽  
Fotis Psomopoulos ◽  
Marina Gerousi ◽  
Larry Mansouri ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Transcription Level ◽  
Triplet Periodicity

Abstract We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5' UTRs, CDS, and 3' UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity. To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, p<0.05), while 742 genes were modulated only at the translation level (log2FC RPF>I1I, p<0.05). We identified 322 genes with differential TE (log2FC TE<I1I, p<0.05) between PRS15-wt vs RPS15-mut CLL cases; 262/322 (81%) showed reduced TE in RPS15-mut versus RPS15-wt cases. Similar analysis for the stable MEC1 cell lines revealed 749 genes displaying differences only at the transcription level (log2FC mRNA>I1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE<I1I, adjusted p<0.1) between PRS15-wt vs RPS15-mut MEC1 cell lines; 48% of the genes showed reduced TE in mut vs wt cell lines and the remaining 52% augmented TE. The slightly different results compared to those obtained from primary CLL cells, may be attributed to the following reasons: (i) MEC1 cells are TP53-aberrant; (ii) the PRS15-wt cell line overexpresses the RPS15 gene compared to primary CLL cells; and,( iii) the RPS15-mut cell line expresses both the wt and mut RPS15 mRNAs (22% of the mapped reads correspond to the mut RPS15 and 78% to the wt gene, respectively). Gene ontology analysis (Enrichr) of the genes showing differential TE, revealed that in both primary CLL cells and MEC1 cell lines a large fraction of the deregulated transcripts is implicated in RNA binding processes (adj-p=0.0001; adj-p=1.98X10^-13, respectively) which are known to induce translational repression. Interestingly, in primary CLL cells, amongst genes with reduced TE we identified genes implicated in tRNA biosynthesis, protein processing in the endoplasmatic reticulum and the Hippo signaling pathway (p<0.01). Additionally, enrichment analysis revealed that a proportion of genes with reduced TE were targets of the MYC transcription factor (adj-p=0.0005). RP genes, despite unchanged mRNA levels, showed changes in RPF levels and differential TE, suggesting that RPs are also deregulated at the translational level. In conclusion, we show that RPS15 mutations rewire the translation program of CLL cells by reducing the TE of critical molecules, including translation initiation factors and other regulatory elements. Disclosures Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

Marrow Stromal Cells Induce TCL1 Expression in Chronic Lymphocytic Leukemia B Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2347-2347
Author(s):  
Mariela Sivina ◽  
Elena Hartmann ◽  
Michael Keating ◽  
William G Wierda ◽  
Andreas Rosenwald ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Stromal Cells ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Immunoglobulin Gene ◽  
Prolymphocytic Leukemia

Abstract Abstract 2347 Poster Board II-324 The human T cell leukemia/lymphoma 1 (TCL1) oncogene was initially identified as a target of chromosomal translocations and inversions at the 14q32.1 chromosome breakpoint region in T-cell prolymphocytic leukemia (T-PLL). Increased TCL1 expression is seen in follicular lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia (CLL). Transgenic mice over-expressing TCL1 under control of the mu immunoglobulin gene enhancer develop a CD5+ B cell lymphoproliferative disorder that mimics human CLL, indicating that TCL1 plays a central and/or causal role in the pathogenesis of CLL. However, chromosome aberrations that constitutively activate TCL1 have not (yet) been identified in the vast majority of CLL patients, and therefore the oncogenic mechanism(s) of TCL1 activation in CLL remain unclear. There is growing evidence that external signals from the microenvironment control and regulate the survival and proliferation of CLL cells. Marrow stromal cells (MSC) are highly effective in protecting CLL cells from spontaneous and drug-induced apoptosis, and are used as a model system to study the marrow microenvironment. In order to explore the molecular cross talk between CLL cells and MSC, we co-cultured CLL cells with different MSC and analyzed gene expression changes induced by co-cultures with MSC, an approach similar to our recent study with nurselike cells (Blood 113:3050-8, 2009). For this, RNA was extracted from 19-purified CLL cells from 10 different patients (baseline expression, day 0). Also, the same patients' samples were co-cultured on stroma cells (KUSA-H1, NK-Tert) for 2 and 7 days. At these time points, RNA again was isolated after CD19-purification. Then, gene expression was determined using HG U133 plus 2.0 oligonucleotide arrays from Affymetrix. Gene expression changes were analyzed in individual patients' samples, comparing baseline samples' gene expression to samples after 2 and 7 of co-culture on MSC. We observed relatively homogeneous gene expression changes in CLL cells after co-culture with MSC. We found that TCL1 was among the top 5 genes that were most highly up-regulated by MSC, based on at least 3-fold up-regulation in at least 6 of the paired samples. We also found an up-regulation of TCL1 at the protein level when assessed by immunoblotting and flow cytometry in CLL samples after co-culture with MSC. These findings indicate that MSC can induce and regulate TCL1 expression in CLL, suggesting that the microenvironment plays an even greater role in the pathogenesis of this disease than previously recognized. Disclosures: No relevant conflicts of interest to declare.

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