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.

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.

scholarly journals Targeting Maternal Embryonic Leucine Zipper Kinase with OTSSP167 Displays Anti-Tumor Activities in Chronic Lymphocytic Leukemia through Down-Regulation of FoxM1

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 801-801
Author(s):  
Ya Zhang ◽  
Xiangxiang Zhou ◽  
Ying Li ◽  
Yangyang Xu ◽  
Xin Wang
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Leucine Zipper ◽  
Lymphocytic Leukemia ◽  
Functional Enrichment ◽  
Down Regulation ◽  
Cell Chemotaxis

Abstract Introduction Maternal embryonic leucine zipper kinase (MELK) is a novel oncogene, which exerts pivotal roles in maintenance of cancer stem cells. OTSSP167, an orally bioavailable inhibitor targeting MELK, is currently in a phase I/II clinical trial in patients with acute myeloid leukemia and breast cancer. Yet, no literature has been reported regarding the effects of MELK and OTSSP167 in chronic lymphocytic leukemia (CLL). Intriguingly, previous studies identified that OTSSP167 contributed to tumorigenesis via p53 pathway, highlighting its potential efficacy in relapsed/ refractory CLL. Hence, the aim of this study was to determine the anti-tumor potency and mechanism of OTSSP167 targeting MELK in CLL. Methods Peripheral blood samples from 55 de novo CLL patients were collected with informed consents at the Department of Hematology in Shandong Provincial Hospital Affiliated to Shandong University (SPHASU). CD19+ B cells were isolated with informed consents from healthy donors. Expression levels of MELK mRNA and protein in CLL cells were determined by quantitative RT-PCR and western blotting. Kaplan-Meier survival curves with log-rank test of overall survival (OS) were analyzed. Microarray datasets GES22762 and GSE25571 were obtained from Gene Expression Omnibus. Functional enrichment analyses of gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) and gene set enrichment analysis (GSEA) in gene expression profiles were performed. Lentiviral vectors transfected CLL cells to stably silence MELK and adenovirus delivery were constructed to overexpress MELK. Besides, cell viability, apoptosis and cell cycle were assessed by cell counting kit-8, annexin V-PE/7AAD and PI/RNase staining, respectively. Cell migration was evaluated by CXCL12-induced chemotaxis. Results Aberrantly increased expression of MELK was detected in CLL primary cells and cell lines (MEC1 and EHEB) at mRNA and protein level compared with normal B cells (Figure 1A-D). We observed MELK over-expression in significant correlation with higher WBC count( p=0.022), advanced Binet stage (p=0.023), high-risk Rai stage (p=0.022), elevated LDH level (p=0.002) and increased β2-MG levels (p=0.009), unmutated IGHV (p=0.037), positive ZAP-70 (p=0.037) and deletion of 17p13 (p=0.009). Besides, MELK high expression was revealed in significant association with reduced OS of enrolled patients, which was further confirmed in GSE22762 (Figure 1E). Annotations of bioinformatics analyses indicated that MELK was functional enriched in cell proliferation, regulation of G2/M cell cycle, response to drug and p53 signaling pathway in cancer. In consistent with functional enrichment analyses, CLL cells with silence or inhibition of MELK exhibited attenuated cell proliferation, increased fast-onset apoptosis, induced G2/M phase arrest, impaired cell chemotaxis and enhanced sensitivity to fludarabine and ibrutinib (Figure 2A-E). Whereas, gain-of-function assay showed promoted cell proliferation and cell chemotaxis (Figure 2E-F). Additionally, serial dilution of OTSSP167 decreased phosphorylation of AKT and ERK1/2 in CLL cells (Figure 3A). Besides, expression of phosphorylated FoxM1, FoxM1, cyclinB1 and CDK1 decreased with treatment of OTSSP167 in CLL cells. p53 modestly changed with MELK suppression in MEC1 cells, yet visibly elevated in EHEB cells. Besides, activation of p21 was observed (Figure 3B). Furthermore, confocal immunofluorescent images revealed that MELK and FoxM1 co-localized in CLL cells and were down-regulated by OTSSP167 in a dose-dependent manner (Figure 3C). Collectively, the interactive network of MELK and FoxM1-signaling cascade in genomic expression profile was established, illuminating the potential mechanism of MELK inhibition by OTSSP167 in CLL (Figure 3D). Conclusion Our investigations identified for the first time the oncogenic role of MELK in CLL tumorigenesis and regulatory mechanism of OTSSP167 in CLL cells by bioinformatics analysis and ex vivo evaluation. Expression of MELK was upregulated, and associated with adverse outcome of CLL patients. OTSSP167 exerted therapeutic potential at nanomolar concentrations in abrogating cell survival and migration, inducing cell cycle arrest through down-regulation of FoxM1. Further clinical investigation is warranted to substantiate OTSSP167 as a novel therapeutic strategy in progressed CLL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Expression of MicroRNA (miR) miR-15a/miR-16-1 Downregulates Expression of BCL-2 Protein in Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2796-2796 ◽  
Author(s):  
Liguang Chen ◽  
Li Tang ◽  
George Calin ◽  
Carlo M. Croce ◽  
Thomas J. Kipps
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Human Cancer ◽  
Human Tumor ◽  
Immunoblot Analysis ◽  
Lymphocytic Leukemia ◽  
Control Vector ◽  
Rt Pcr ◽  
Target Mrna

Abstract MicroRNAs (miRNAs) comprise a family of small RNA, each member of which can potentially regulate post-transcription gene expression in a temporal and tissue-specific manner. Each miRNA encodes a transcript of about 22 nucleotides that can modulate expression of specific target mRNA. We identified that the chronic lymphocytic leukemia (CLL) cells of a large proportion of patients have aberrant, low-level expression of two clustered miRNAs located at 13q14, designated miR-15a and miR-16–1, providing the first example of dysregulated miRNA expression in a human cancer (PNAS USA99:15524, 2002). These miRNAs each have sequences that potentially could target mRNA encoding the anti-apoptotic protein bcl-2, which is expressed at high levels in CLL. Transfection of a lymphoma B cell line lacking expression of miR-15a and miR-16–1 with an expression vector encoding miR-15a and miR-16–1, designated pmiR-15/16, attenuated the expression-level of bcl-2 protein and enhanced the susceptibility of such lymphoma B cells to apoptosis in vitro. However, it was not certain whether miR-15a and miR-16–1 could modulate expression of bcl-2 in primary leukemia cells that were not adapted for propagation in vitro. We selected primary CLL cell samples (n = 3) that harbored deletions at 13q14 and that lacked expression of miR-15a and miR-16–1, as assessed via microRNA array and quantitative RT-PCR analyses. We transfected these cells with pmiR-15/16 or a control vector via electroporation and, in parallel studies, also transfected these CLL cells with sense and antisense control oligo-RNAs via transmessenger transfection. Before and after such manipulations we monitored for expression of miR-15a and miR-16–1 by RT-PCR, for relative expression of BCL-2-family member transcripts using a multiplex ligation-dependent probe amplification (MLPA) assay, and for expression of bcl-2 protein using immunoblot analysis and flow cytometry. We found that CLL cells transfected with either pmiR15/16 or with miR-15a and miR-16–1 sense oligo-RNAs had increased expression-levels of miR-15a and miR-16–1 within 24 hours after transfection, whereas CLL transfected with the control vector or antisense oligo-RNA did not. Depite high-level expression of miR-15a and miR-16–1, the relative levels of BCL-2 transcript did not change over the 48 hours after transfection that we examined. However, in this time period we observed that CLL cells made to express miR-15a and miR-16–1, experienced significant reductions in the levels of bcl-2 protein, which were not observed in control transfected CLL cells. This is the first demonstration that miRNAs can effect post-transcriptional regulation of protein expression in a primary human tumor and suggest that miRNAs may have potential therapeutic utility in the modulation of pathogenic gene-expression in CLL and other cancers.

scholarly journals Lenalidomide and Dexamethasone Combination in Patients with Chronic Lymphocytic Leukemia (CLL) Relapsing or Resistant to Treatment (LENDEX-LLC-09): A Gene Expression Profiling Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4675-4675
Author(s):  
Anna Puiggros ◽  
Pau Abrisqueta ◽  
Lara Nonell ◽  
Marta Bodalo ◽  
Eulalia Puigdecanet ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Lymphocytic Leukemia ◽  
Down Regulation ◽  
Tnf Α

Abstract Background. Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which immune evasion of tumoral cells, as well as, an impaired CD4 and CD8 T-cell function have been described. Immunomodulatory drugs, such as lenalidomide, alone or in combination with other treatments are promising strategies for those patients with refractory disease. The combination of lenalidomide with dexamethasone has been investigated in multiple myeloma and has revealed as a highly efficient treatment. Nonetheless, the efficacy and mechanisms of action of this combination in CLL have not been elucidated. Aim. To assess the effect of lenalidomide and dexamethasone combination in gene expression of CLL B cells, as well as CD4+ and CD8+ T cells from CLL patients enrolled in LENDEX-LLC-09 trial. Methods. Four patients included in the LENDEX-LLC-09 trial (NCT01246557) were studied (2M/2F, med age 72). All presented with advanced CLL (2 B and 2 C Binet stages), and were previously treated by a minimum of two chemo-immunotherapy regimens. Peripheral blood samples were taken at the recruitment and the 7th day of the first cycle of lenalidomide (2.5mg/day) and dexamethasone (20mg/day, 4 days). Total RNA was extracted from CLL B cells (CD5+ CD19+) and T cells (CD4+ or CD8+) positively selected by immunomagnetic methods (Miltenyi Biotec). Good quality RNA (RIN>7) was hybridized to Human Gene 2.0 ST array (Affymetrix). Differences between gene expression of pretreated and treated samples were assessed for each cell type using linear models for microarrays. Genes with a |logFC|>1 were considered as potentially relevant. Functional analysis was performed using Ingenuity Pathway Analysis (IPA). Results and discussion. The major effect in the gene expression due to treatment was observed in CD4+ T cells, which presented 290 up-regulated genes and 103 down-regulated. CLL cells showed up-regulation of 189 and down-regulation of 53 genes, while increase and decrease in the expression of 112 and 37 genes, respectively, were found in CD8+ T cells. Globally, the most important involved networks were related to cell-to-cell signaling, cellular growth and proliferation, cell death and survival, as well as inflammatory response and immune cell trafficking. Regarding CLL B cells, TNF-α was the most up-regulated gene, as previously described in lenalidomide treated B cells. Contrarily, we did not observe significant differences in genes involved in the immunologic synapse, as CD80, CD86, CD200, PD-L1, CD276 and CD270, which have been reported as key regulators in lenalidomide mechanism of action. Of note, a general increase of genes associated with binding to cells (CD68, CTLA4, ADAM28, ITGAX, LY96) was detected. In contrast to previous studies that demonstrated a growth arrest and induction of apoptosis by lenalidomide or dexamethasone in monotherapy (Baptista et al, 2012; Fecteau et al, 2014), a global inhibition of the apoptosis (up-regulation of BTK and CD79B and inhibition of SMAD7, among others) were observed when both drugs were combined. Considering CD8+ T cells gene expression, an up-regulation of genes involved in leukocyte activation and cell-to-cell binding was detected. The most remarkable changes were found in TNF-α and IFN-γ induction, as well as in ADAM28, LY96 and CD68. In contrast to CD8+ T cells, an inhibition of CD4+ T cell proliferation was observed after the combined treatment (up-regulation of VSIG4, LILRB4 and down-regulation of ICOS). This observation suggests that dexamethasone administration inhibits the CD4+ activation promoted by lenalidomide, as has been described in multiple myeloma (Hsu et al, 2011). Regarding response to treatment, two patients initially presented a complete response with positive minimal residual disease. However, all patients finally progressed after treatment and one died due to disease progression. No significant differences in gene expression patterns were found among patients. Conclusions. Our results suggest that lenalidomide and dexamethasone combination leads to an anti-tumoral activity displayed by an activation of CD8+ T cells against the tumor, rather than an increase of apoptosis in CLL cells. More studies are needed to confirm these preliminary findings of the combined effect of lenalidomide and dexamethasone in refractory CLL patients. Acknowledgments. This work was funded by Celgene, and supported by PI11/1621, 14SGR585 and Fundació LaCaixa. Disclosures Off Label Use: Lenalidomide and dexamethasone combination in CLL.

scholarly journals Nonconventional Gene Expression within the NF-κb Signaling Pathway Induced By Poly(propylene)Imine Glycodendrimers in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5595-5595
Author(s):  
Ida Franiak-Pietryga ◽  
Kinga Ostrowska ◽  
Dietmar Appelhans ◽  
Henryk Maciejewski ◽  
Maria Bryszewska ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathway ◽  
Research Funding ◽  
Target Genes ◽  
Early Stage ◽  
Lymphocytic Leukemia ◽  
P Value

Abstract Introduction The nuclear factor kappa-light-chain enhancer of activated B-cells (NF-κB) signaling pathway is constitutively active in a variety of cancers, including chronic lymphocytic leukemia (CLL). The importance of this signaling pathway identifies it as a prime therapeutic target, however the complexity and potential side effects of inhibiting NF-κB have thus far made the clinical use of NF-κB inhibitors a relatively unexplored resource in this disease. There are a few combined therapies available for the treatment of CLL includes chemotherapy with agents such as chlorambucil, cyclophosphamide, fludarabine and bendamustine, along with immunotherapy including rituximab and alemtuzumab. None available therapy for CLL is curative. Nanotechnology, a new and promising field of scientific research, may be of use in medicine and the pharmaceutical industry. Dendrimers, nanoparticles of dendritic architecture, can interact effectively and specifically with cell components. We have already proved the influence of PPI-G4-OS-M3 dendrimers in cultures in vitro on CLL cells apoptosis.Herein, the objective was to evaluate how MEC-1 cells survival in vitro is affected by influence on NF-κB pathway by PPI-G4-OS-M3 dendrimer comparing to FA. Material and methods Dendrimer, in which approximately 35% of peripheral amino groups, was coated with maltotriose have been defined as PPI-G4-OS-M3 and was used in concentration of 8 mg/ml (the IC50 value for this dendrimer). 'OS' abbreviation stands for the open shell structure of carbohydrate-modified dendrimers. The molar mass of this PPI dendrimer was 31000 g/mol. Fludarabine (FA, Genzyme) in concentration of 1.6 µM, based on previous studies, was used. MEC-1 (DSMZ no. ACC 497) was used as a homogenous cell line with del(17p)(11q). In cultures the percentage of apoptotic cells was verified using AnnV and PI by means of flow cytometer. Cells predominated in the early stage of apoptosis.A microarray gene expression (Agilent SurePrint Technologies) was performed. Samples were hybridized to a whole human genome microarray 8x60K. Arrays were scanned on Agilent DNA Microarray Scanner. Data were deposited at Gene Expression Omnibus (GEO) (accession number GSE68094).Analysis of differential expression of genes was done with the limma method (Smyth, G. K., 2004) as implemented in R/Bioconductor software. We used the FDR multiple testing adjustment. We declared as differentially expressed the genes with FDR-adjusted p-value <0.1, which means that 10% of genes declared as DE are expected to be false positives. Results Dendrimer induced expression of REL, RELB and NFKBIB genes. In contrast, FA monotherapy resulted in significant differences in gene expression of cellular pathway-dependent transcription factor NF-kB. The most significant differences in the function of the FA and dendrimer are reflected in different levels of expression of three genes: NFKBIA, BCL3 and CHUK. Conclusion Constitutive NF-κB signaling contributes to cell growth, proliferation and survival. CLL cells have high basal levels of NF-κB compared with normal B cells. The activity is variable in CLL patients, correlates with in vitro cell survival, and importantly, increased levels of NF-κB activity enhanced resistance to the purine analogues FA (del17p). Therefore, disruption of NF-κB signaling and downstream target genes either promoted or repressed is an important strategy to pursue to disrupt drug resistance in CLL. The study indicates that the use of PPI dendrimers modified maltotriose may be the key to developing therapies deliberates CLL. The study was partially supported by Grant No. DEC-2011/01/B/NZ5/01371from the National Science Centre, Poland. Disclosures Robak: Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding.

scholarly journals Long Term Outcomes of Venetoclax Therapy for Complex Karyotype Relapsed/Refractory Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4431-4431 ◽  
Author(s):  
Thomas Lew ◽  
Mary Ann Anderson ◽  
Constantine S. Tam ◽  
Sasanka Handunnetti ◽  
Dennis Carney ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Board Of Directors ◽  
Research Funding ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Complex Karyotype ◽  
Advisory Committees ◽  
Pre Treatment

Abstract Background The selective BCL2 inhibitor venetoclax (Ven) achieves an overall response rate of approximately 75 - 80% as a single agent in patients (pts) with relapsed and refractory chronic lymphocytic leukemia / small lymphocytic lymphoma (RR-CLL/SLL)1. Ven is associated with 1 year estimates of progression free survival of ~75% for the approved single agent dose of 400 mg daily1,2 with promising efficacy among pts previously treated with ibrutinib3. Early reports of pre-treatment factors impacting durability of clinical benefit have identified bulky adenopathy, >3 lines of prior therapy and del(17p) as putative adverse factors in univariate analyses1. In analyses of data from ibrutinib and idelalisib naïve patients, the dominant predictors were fludarabine refractory disease and complex karyotype (CK), with del(17p) and/or TP53 mutations not reaching significance4. However, a systematic longer term follow up of pts with RR-CLL/SLL harboring CK treated with Ven has not been reported. We report the long term follow up of 31 pts with RR-CLL/SLL with a known karyotype treated with Ven ≥400 mg/d. Methods We retrospectively reviewed 67 pts with RR-CLL/SLL enrolled on early phase clinical studies of Ven at our two hospitals between June 2011 and July 2018 as of July 2018. The pts were treated in one of three ongoing clinical trials: Phase 1 Ven monotherapy (NCT01328626) (n=41), Phase 1b Ven plus rituximab (NCT01682616) (n=16), or Phase 2 Ven monotherapy in del(17p) CLL/SLL (NCT01889186) (n=10). Our analysis was restricted to pts who received ≥400 mg/daily2 and whose pre-treatment karyotype was known (n=31). CK is defined by the presence of ≥3 chromosomal aberrations using conventional cytogenetic analysis5. Time to progression (TTP) was estimated by the method of Kaplan and Meier, and comparisons among groups used the log-rank test (Mantel-Cox) and Fisher's exact test for categorical variables. Results 31 pts (median age 68 [range 45-83]) had known karyotype prior to Ven. They had received a median of 3 (1 - 8) prior therapies, but none had prior BTK inhibitor or idelalisib exposure. 12 pts had RR-CLL/SLL with known CK (median number of aberrations 4, range 3 - 8) and 19 were known non CK. Disease was fludarabine refractory in 15 pts and 16 had evidence of TP53 dysfunction (TP53 mutation and / or del(17p)) (Table 1). The median follow up was 33 (range 1 - 67) months. Twenty-eight (90%) pts responded and 18 (58%) have developed progressive disease; 7 with Richter transformation (RT) and 11 with CLL. RT occurred significantly earlier than CLL progression at a median of 7 (range 1 - 22) months v 33 (22 - 48) months, respectively (p = 0.0004). CK did not impact likelihood of overall response or complete response (CR) (p = 0.46), but was associated with a lower rate of attainment of minimal residual disease negative (MRD-neg) BM status (8% vs 47% in pts with non CK; p = 0.046). Four pts with CK achieved CR; one was fludarabine refractory, three harbored aberrations in TP53 and one lacked both risk factors. RT was largely confined to pts with CK CLL who had a 50% incidence of RT (6 of 7 RT events; p = 0.007). Two pts with CK RR-CLL/SLL received concomitant rituximab with Ven therapy: one developed Hodgkin variant RT at ~1 month, the other achieved a PR without clearance of PB or BM MRD, with progressive CLL at 25 months. Of the 5 pts with non CK RR-CLL/SLL who received Ven and rituximab, 4 (80%) achieved MRD-neg CR with no progressions at a median follow up of 57 (range 33 - 60) months and one achieved PR, with progressive CLL at 58 months. Compared to pts with a known non CK, patients with CK had significantly shorter TTP (median 22 [95% CI 4 - 48] months v 67 [95% CI 33 - undefined] months; p = 0.0011) (Figure 1). Conclusions Patients with CK RR-CLL/SLL treated with Ven have inferior outcomes relative to those with non-CK, predominantly due to early emergence of presumably unrecognized subclinical RT, consistent with patterns previously observed for ibrutinib and chemo-immunotherapy. Careful screening of these patients for nascent RT is important. However, deep remissions are possible in some patients and confer durable disease control. Ven combination therapies merit exploration with the aim of improving depth of response and outcomes in RR-CLL/SLL harboring CK.Roberts; N Engl J Med; 2016;374:311-22.Stilgenbauer; Lancet Oncol; 2016;17:768-78.Jones; Lancet Oncol; 2018;19:65-75.Anderson; Blood; 2017;129:3362-70.Rigolin; Blood; 2012;119:2310-3. Disclosures Lew: Walter and Eliza Hall: Employment, Patents & Royalties. Anderson:Genentech: Research Funding; AbbVie, Inc: Research Funding; Walter and Eliza Hall: Employment, Patents & Royalties. Tam:Pharmacyclics: Honoraria, Travel funding; Janssen: Honoraria, Research Funding; Beigene: Honoraria, Other: Travel funding; Pharmacyclics: Honoraria; Gilead: Honoraria; Roche: Honoraria; Roche: Honoraria; Beigene: Honoraria, Other: Travel funding; AbbVie: Honoraria, Research Funding; Gilead: Honoraria; AbbVie: Honoraria, Research Funding. Roberts:Janssen: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; Walter and Eliza Hall: Employment, Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone and royalty payments related to venetoclax. Seymour:Celgene: Consultancy; AbbVie: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding.

scholarly journals The expression BIRC6 gene in patients with chronic lymphocytic leukemia – a preliminary study

2014 ◽  
Vol 27 (3) ◽  
pp. 179-182 ◽  
Author(s):  
Piotr Chomik ◽  
Paulina Gil-Kulik ◽  
Malgorzata Filas ◽  
Agnieszka Wojcieszek ◽  
Mateusz Wilinski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood Lymphocytes ◽  
Lymphocytic Leukemia ◽  
Over Expression ◽  
Apoptosis Protein ◽  
Preliminary Study ◽  
Apoptotic Activity ◽  
Cancer Tissues ◽  
Resistance To Chemotherapy

ABSTRACT The BIRC6 gene encodes the Bruce (Apollon) protein. This belongs to the III class of Inhibitors of the Apoptosis Protein (IAP) and demonstrates anti-apoptotic activity (binding, inhibiting and degrading the caspases). Moreover, the Bruce protein shows multilevel activities and additional functions. The Bruce protein is involved in the maintenance of cell viability, and it is also suggested that it plays an important role in cell proliferation and diversification. Many researchers have noticed elevated BIRC6 gene expression in cell lines of brain cancer and ovarian carcinoma, leukemia, breast cancer and even in colorectal cancer tissues. Resistance to chemotherapy-inducted apoptosis in cancers characterized by BIRC6 gene over-expression was also reported. The aim of the study was to assess the BIRC6 gene expression in peripheral blood lymphocytes of patients diagnosed with chronic lymphocytic leukemia.

Deregulation of miRNAs by Epigenetic Silencing Disrupts Suppression of the Oncogene PLAG1 in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3463-3463
Author(s):  
Christian P Pallasch ◽  
Michaela Patz ◽  
Yoon Jung Park ◽  
Susanne Hagist ◽  
Daniela Eggle ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Specific Interaction ◽  
Target Prediction ◽  
Lymphocytic Leukemia ◽  
Luciferase Reporter ◽  
Binding Prediction ◽  
Down Regulation ◽  
Over Expression

Abstract Abstract 3463 Poster Board III-351 MicroRNAs play a key role in cellular regulation and if deregulated in the development of neoplastic disorders including chronic lymphocytic leukemia (CLL). Both deregulations of miRNAs as well as the identification of their functional relevant targets and regulatory circuits in CLL pathogenesis are only partly understood and remain to be elucidated. RNAs from primary cells of 50 treatment-naïve CLL patients and peripheral B-cells of 14 healthy donors were applied to miRNA-expression profiling using bead chip technology. The majority of patients presented with Binet stage A disease and showed a favorable risk profile as assessed by clinical and molecular features. Comparing the total number of miRNA being expressed a significantly lower number of miRNA was detected in CLL compared to normal B cells. The predominance of down-regulated miRNAs in CLL cells was accompanied by highly significantly lower total number of miRNAs expressed above the detection threshold in CLL patients (19.8% vs 23.5%; p<10-6). In CLL cells a set of 7 up- and 19 down-regulated miRNAs was identified. We could not identify significant differentially expressed miRNA in cytogenetic defined subgroups, in particular we could not detect significant deregulation of miRNAs in patients harboring del13q14. Moreover, we could not identify significant down-regulation of miR-15 and miR-16 except in one patient harboring a homozygous deletion of chromosome 13q14. However, the previous up-regulation of miR-155, a key regulator of B-cell ontogenesis, appeared to be the most prominent up-regulated miRNA in our cohort. Interestingly, we identified so far unknown down-regulation of a set of miRNAs in CLL such as miR-107, -424, -125a, -126 and -326. Among the miRNAs being downregulated in CLL cells, 6 out of 10 miRNA promoters (miR-126, miR-139, miR-181a2/b2, miR-582, miR-107, miR-449) being examined showed gain of methylation as compared to normal B cell controls. Subsequent target prediction of deregulated miRNAs revealed a highly significant binding prediction at the 3′UTR of the pleomorphic adenoma gene 1 (PLAG1) oncogene. Luciferase reporter assays including site directed mutagenesis of binding sites revealed a significant regulation of PLAG1 by miR-181a, miR-181b, miR-107 and miR-424. While expression of PLAG1 mRNA was not affected, PLAG1 protein expression was shown to be significantly elevated in CLL cells as compared to the levels in healthy donor B cells. In conclusion we demonstrate (I) predominant down-regulation of miRNAs in CLL, (II) identified novel deregulated miRNAs in CLL, (III) unraveled underlying epigenetic changes in loci of deregulated miRNA, (IV) applied in silico target prediction of miRNA interactions for identification of novel pathogenetic factors, and (V) identified specific interaction of deregulated miRNA with PLAG1 3'UTRs resulting in over-expression of this oncogene in CLL. Therefore, PLAG1 over-expression in CLL cells represents a novel oncogenic mechanism in CLL pathogenesis on the background of deregulation in miRNA-mediated control mechanisms. Disclosures No relevant conflicts of interest to declare.

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.

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