RNAi Screen of the Druggable Genome Identifies Modulators of Proteasome Inhibitor Sensitivity in Myeloma Including CDK5.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 602-602
Author(s):  
Yuan Xiao Zhu ◽  
Rodger E. Tiedemann ◽  
Chang-Xin Shi ◽  
Jessica Schmidt ◽  
Laura Bruins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Shrna Expression ◽  
Primary Screening ◽  
A Genome ◽  
Proteasome Subunits ◽  
Druggable Genome ◽  
Sensitization Effect

Abstract Abstract 602 The molecular target(s) which co-operate with proteasome inhibition in inducing drug sensitivity or resistance in Multiple Myeloma (MM) remain unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in KMS11 MM cells in the presence or absence of bortezomib without regard to pre-conceived mechanistic notions. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising 13,984 siRNA targeting 6,992 genes and comprising two RNAi per gene. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. Bortezomib was added (at the IC10, 25, 70 and 90) 24 hours post transfection. After 96 hours (72 hours after bortezomib), viability was measured by ATP-dependent luminescence. In primary screening 320 candidate bortezomib sensitizing genes were identified and rescreened with four siRNA oligos each gene. 57 of the top sensitizer hits were selected for which at least two distinct siRNA decreased the EC50 by 2 standard deviations from cells treated with control siRNAs. By cross referencing gene expression profile (GEP) data of KMS11, we furthered pared the list to 37 plausibly expressed targets (0.5% of those genes originally screened) as bortezomib sensitizers. After silencing, 50% of these 37 genes also sensitized the lung cancer cell line A549 to bortezomib. After further parsing of genes which also modulated the sensitivity of MM to Melphalan (non specific chemosensitizers), 34 genes remained: The strongest sensitizers to bortezomib were the proteasome subunits PSMA5, PSMB2, PSMB3, PSMB7 but included less obvious targets such as BAZ1B, CDK5, CDC42SE2, MDM4, NME7, TFE3, TNFAIP3, TNK1, TOP1, VAMP2 and YY1 were also identified. Of these, the most potent synergetic effects were observed with siRNAs against the proteasome subunits and against cyclin dependent kinase 5 (CDK5), which caused the greatest shift in EC50. CDK5 is of particular interest as a therapeutic target as it is expressed at high levels in MM and neural tissues but has low expression in other organs. Using viral shRNA expression, silencing CDK5 consistently increased the sensitivity of genetically variable MM cell lines (n=5) to all of the proteasome inhibitors tested: bortezomib, carfilzomib and PR047, and the effect could be at least partially rescued by overexpression of an RNAi resistant CDK5. To explore therapeutic relevance the small molecule CDK5 inhibitor, Roscovitin, was shown to be synergistic or additive with bortezomib in both MM cell lines and primary patient samples. Gene expression profiling was then performed to seek an explanation for the CDK5 sensitization effect and regulation of a proteasome subunit PSMB5 by CDK5 was identified as a probable route to sensitization. In summary inhibition of the existing proteasome either directly by suppression of proteasome subunits, or indirectly by suppression of modulators such as CDK5 appears to confer the greatest sensitization effect suggesting that combinations of bortezomib with other unique proteasome inhibitor drugs or combinations with inhibitors of CDK5 is a logical avenue for clinical exploration. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Contribution of Proteasome Subunits to Myeloma Cell Viability and Proteasome Inhibitor Sensitivity

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4337-4337
Author(s):  
Chang-Xin Shi ◽  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Cecilia Bonolo De Campos ◽  
William Stewart ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Viability ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Myeloma Cell ◽  
Resistance Function ◽  
Proteasome Subunits ◽  
Underlying Mechanisms

Background Bortezomib (BTZ) is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. The underlying mechanisms of such proteasome inhibitor resistance are still incompletely understood. Methods To further understand its resistant mechanism, we generated eight multiple myeloma (MM) cell lines resistant to bortezomib (BTZ) by exposure to increasing drug concentration: five of them acquired novel PSMB5 mutations. Given the rarity of similar mutations in over 1,500 analyzed MM patients, we explored in depth the role of the proteasome on MM cell viability and BTZ sensitivity by systematically deleting the major proteasome targets of BTZ by CRISPR. Results We demonstrated that MM cell lines without PSMB5 were surprisingly viable (mutation corresponding yeast gene pre2 is lethal). PSMB5 mutated, BTZ resistant, MM cell lines were re-sensitized to BTZ when PSMB5 was experimentally deleted, implying that this mutation is activating in its drug resistance function. In contrast PSMB6 knockout was lethal to MM cell lines, which were efficiently rescued by re-introduction of wild type PSMB6. Interestingly, reduction in PSMB6 levels also prevented the splicing of the major catalytic subunits PSMB5, PSMB7, PSMB8 and PSMB10. PSMB6 engineered with no splicing function or catalytic activity, also restored viability, inferring that the contribution of PSMB6 to proteasome structure is more important than functional activity. Supporting this observation, BTZ sensitivity was restored in resistant MM cells line by introducing low level expression of mutated PSMB6 lacking splicing function. As with PSMB6, PSMB7 knockout was lethal to MM cell lines. In contrast, loss of immunoproteasome subunits PSMB8 and PSMB9 was neither lethal nor restored sensitivity to BTZ. Our results demonstrate that expression of the three constitutive proteasome subunits PSMB5, PSMB6 and PSMB7 is highly co-dependent. This dependence is relying on the structure, but not the function, of PSMB5 and PSMB6. Conclusions In summary, PSMB5 and PSMB6, but not PSMB8 and PSMB9, are highly relevant for BTZ sensitivity in MM. Absence of PSMB6 or PSMB7, but not PSMB5, was lethal in MM cell lines. Expression of PSMB5, PSMB6 and PSMB7 was highly co-dependent. Together these findings suggest that the modulation of expression rather than function of PSMB5, PSMB6 or PSMB7 may be a new therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

A Genome-Wide Approach Identifies Variations in the Aspartate Metabolism Pathway That Are Associated with Asparaginase Sensitivity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 213-213
Author(s):  
Shih-Hsiang Chen ◽  
Wenjian Yang ◽  
Yiping Fang ◽  
Gabriele Stocco ◽  
Kristine R. Crews ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Interindividual Variation ◽  
European Ancestry ◽  
Mixed Effect ◽  
Metabolism Pathway ◽  
Genome Wide ◽  
Individual Snps ◽  
A Genome ◽  
Aspartate Pathway

Abstract Asparaginase is an important drug for acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in asparaginase sensitivity, we employed a genome-wide association approach using the HapMap lymphoblastoid cell lines from 87 individuals of European ancestry (CEU) and diagnostic ALL blasts from 42 newly diagnosed, genomically-determined white patients. In vitro sensitivity was based on IC50 values measured following 48 hour exposures to native E. coli asparaginase (0.003–100 IU/ml) in CEU cell lines and 96 hour exposures (0.003–10 IU/ml) in patient samples using the methylthiazol tetrazolium assay. For CEU cell lines, single nucleotide polymorphism (SNP) genotypes were downloaded from the International HapMap database (www.hapmap.org) and gene expression data (Affymetrix GeneChip Human Exon 1.0 ST Array) were downloaded from http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE7761. For patients with ALL, we used the 500K SNP arrays to interrogate germline DNA and Affymetrix U133A GeneChip Array to assess gene expression in ALL blasts. We tested whether 2,390,203 SNP genotypes were associated with asparaginase IC50 using a linear mixed effect model in CEU cell lines, setting a p value threshold of p &lt; 0.001 for individual SNPs and p &lt; 0.05 at the gene level. This approach yielded 329 SNPs representing 94 genes. Combining these germline SNPs with those representing genes whose expression was also associated with IC50 at the p &lt; 0.05 level (1,706 genes), there were 6 SNPs representing 5 genes, two of which (rs8135371 and rs17001863, both in the ADSL gene) contributed to asparaginase sensitivity (p = 6.9 × 10−4 and 9.1 × 10− 4, respectively) through their effects on ADSL gene expression. The top ranked KEGG pathway overrepresented by the 94 top-ranked genes (329 SNPs) was that of aspartate metabolism, which may be directly linked to the mechanism of action of asparaginase. The two most highly ranked genes (ADSL and DARS) in this pathway encompassed 7 SNPs (rs8135371, rs17001863, rs3768998, rs2278683, rs11893318, rs2322725, and rs7587285), all with p &lt; .001. Using multiple linear regression analysis, 32% of the variability in asparaginase IC50 among the CEU cell lines could be accounted for by these 7 SNPs (p = 5.9 × 10−7). To examine the overall contribution of the aspartate metabolism pathway to asparaginase IC50, we compared all SNPs (935 in cell lines, 717 in patients) representing the aspartate pathway with those SNPs representing other pathways, using a random forest model. We found that the SNP genotypes in the aspartate pathway explained significantly more variation in asparaginase IC50 in cell lines (11.4%, p = 6.9 × 10−4) and in ALL patient samples (11.2%, p = 0.02) than other pathways. The expression of ADSL differed among ALL subtypes, with more sensitive subtypes (hyperdiploid and TEL-AML1 ALL) having lower ADSL expression than more resistant subtypes (T-ALL) (p = 1.1 × 10−5 and 2.9 × 10−9, respectively). Genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited and acquired genomic interindividual variation in a plausible candidate pathway contribute to asparaginase sensitivity.

HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 832-832 ◽  
Author(s):  
Michael A Chapman ◽  
Jean-Philippe Brunet ◽  
Jonathan J Keats ◽  
Angela Baker ◽  
Mazhar Adli ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Histone Modification ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Specific Expression ◽  
Aberrant Expression ◽  
High Level

Abstract Abstract 832 We hypothesized that new therapeutic targets for multiple myeloma (MM) could be discovered through the integrative computational analysis of genomic data. Accordingly, we generated gene expression profiling and copy number data on 250 clinically-annotated MM patient samples. Utilizing an outlier statistical approach, we identified HOXA9 as the top candidate gene for further investigation. HOXA9 expression was particularly high in patients lacking canonical MM chromosomal translocations, and allele-specific expression analysis suggested that this overexpression was mono-allelic. Indeed, focal copy number amplifications at the HOXA locus were observed in some patients. Outlier HOXA9 expression was further validated in both a collection of 52 MM cell lines and 414 primary patient samples previously described. To further verify the aberrant expression of HOXA9 in MM, we performed quantitative RT-PCR, which confirmed expression in all MM patients and cell lines tested, with high-level expression in a subset. To further investigate the mechanism of aberrant HOXA9 expression, we interrogated the pattern of histone modification at the HOXA locus because HOXA gene expression is particularly regulated by such chromatin marks. Accordingly, immunoprecipitation studies showed an aberrantly low level of histone 3 lysine 27 trimethylation marks (H3K27me3) at the HOXA9 locus. H3K27me3 modification is normally associated with silencing of HOXA9 in normal B-cell development. As such, it appears likely that the aberrant expression of HOXA9 in MM is due at least in part to defects in histone modification at this locus. To determine the functional consequences of HOXA9 expression in MM, we performed RNAi-mediated knock-down experiments in MM cell lines. Seven independent HOXA9 shRNAs that diminished HOXA9 expression resulted in growth inhibition of 12/14 MM cell lines tested. Taken together, these experiments indicate that HOXA9 is essential for survival of MM cells, and that the mechanism of HOXA9 expression relates to aberrant histone modification at the HOXA9 locus. The data thus suggest that HOXA9 is an attractive new therapeutic target for MM. Disclosures: No relevant conflicts of interest to declare.

CBX8, a Polycomb-Group Protein, Is Essential for MLL-AF9-Induced Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4174-4174
Author(s):  
Jiaying Tan ◽  
Jay L. Hess
Keyword(s):  
Gene Expression ◽  
Cell Growth ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Polycomb Group ◽  
Human Leukemia ◽  
Interaction Sites

Abstract Abstract 4174 Trithorax and Polycomb-group (Trx-G and Pc-G) proteins are antagonistic regulators of homeobox-containing (Hox) gene expression that play a major role in regulation of hematopoiesis and leukemogenesis. Mixed lineage leukemia (MLL), a mammalian Trx-G protein, is a histone methyltransferase crucial for embryonic development and hematopoiesis that is commonly altered by translocation in acute leukemia. Recent evidence suggests that transformation by MLL fusion proteins is dependent on multiple interaction complexes, including the polymerase associated factor complex (PAFc) and the elongation activating protein complex (EAPc) or a closely related AF4 family/ENL family/P-TEFb complex (AEPc). CBX8 is a human PcG protein, functioning as a transcription repressor in the polycomb repressive complex 1 (PRC1). Previous studies have shown that CBX8 also interacts with the EAPc components AF9 and ENL; however, its role in leukemogenesis is unknown. To elucidate the significance of this interaction between these two proteins thought to have antagonistic function, we generated a large series of point mutations in AF9 and identified two amino acids that are essential for CBX8 interaction but preserve the interaction with other EAP components. Mutation of the two sites reduced the transcriptional activation of the MLL-AF9 target promoters by nearly 50% and completely inhibits the ability of MLL-AF9 to immortalize bone marrow (BM) as assessed by methylcellulose replating assays. This finding suggests that CBX8 interaction is essential for MLL-AF9-induced leukemogenesis. Several lines of evidence further support this finding. First, CBX8 knockdown by siRNAs decreased MLL-AF9-induced transcriptional activation by approximately 50%. Second, the ability of MLL-AF9 to transform primary BM was markedly reduced by retroviral shCbx8 transduction. Notably, this inhibitory effect is specific for MLL-AF9 because the BM transformation ability of E2A-HLF was unaffected by Cbx8 suppression. Third, Cbx8 suppression by shCbx8 in MLL-AF9 and MLL-ENL, but not E2A-HLF transformed AML cell lines, significantly inhibited the expression of MLL-dependent target genes, as well as cell growth and colony forming ability. Fourth, inducing CBX8 knockdown in human leukemia cell lines expressing MLL-AF9 led to a marked decrease in the localization of basic transcription machinery at the Hoxa9 locus and a corresponding reduction in Hoxa9 transcription. Importantly, the observed effects of CBX8 on MLL-rearranged leukemia cells are PRC1-independent: no effects on MLL target gene expression, cell growth, or BM transformation ability were observed by suppressing other core components of PRC1. Taken together, our results indicate that CBX8, independent of its transcription repression role in PRC1, interacts with and synergizes with MLL fusion proteins to promote leukemogenesis. Defining the interaction sites between AF9/ENL and CBX8 and the dependence of other AML subtypes and normal hematopoiesis on CBX8 will be important for the further development of agents that target this mechanism in MLL-rearranged and potentially other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

The Alteration of SEPT5 Gene Expression in BCR-ABL Positive Cells and Its Possible Correlation with the Development and / or Progression of Chronic Myeloid Leukemia (CML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4415-4415
Author(s):  
Cintia Do Couto Mascarenhas ◽  
Anderson Ferreira Cunha ◽  
Ana Flavia Brugnerotto ◽  
Sheley Gambero ◽  
Joao Machado-Neto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Blood Donors ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Blood Platelets ◽  
Gene Expression Pattern ◽  
P Value

Abstract Abstract 4415 The CML is a clonal disease of stem cells and its main feature is the unregulated production of a tyrosine kinase protein called BCR-ABL, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. Because of this, the use of tools for the study of gene expression could bring new insights in the understanding of these mechanisms in the CML. In a recent study using SSH libraries, we compared the gene expression pattern between granulocytes of health control and CML patients, and we identified the gene SEPT5 expressed only in CML patients. Although the studies in the literature, there is not a clear relationship between the expression of this gene and the development or progression of CML. SEPT5 is a member of nucleotide binding proteins called septins that were firstly described in yeast as cell division cycle regulatory proteins. This gene was reported in patients with AML translocated with MLL gene, in adult human brain and heart; it is also associated with alpha granules of human blood platelets. The aims of this study are to carry a functional analysis of SEPT5 in differents cells line and to study the relationship of this gene and the development and/or progression of CML. The gene expression evaluation was made in granulocytes, mononuclear cells and total leukocytes of CML patients and healthy blood donors in peripheral blood. It was also evaluated in bone marrow donors, in human cell lines (K562, HL60 and NB4) and in mice cell lines (BaF3/BCR-ABLp210 and BaF3T315I), performed by real-time PCR for the following genes: SEPT5, β-actin and GAPDH. Experiments were also performed to verify the difference between the chemotaxis of granulocytic cells from controls and patients by ELISA. Data were analysed statistically using the ANOVA followed by Dunnett’s test – P value of less than 0.05 was considered to be significant. The study was approved by the Research Ethic Committee of the Faculty of Medical Sciences of University of Campinas. The gene expression of SEPT5 was evaluated by real time PCR using the same samples used in the library construction to validate the results found in the SSH library. The data confirmed our previous results, showing that the SEPT5 expression is increased in all cells of patients compared to controls. The same results were observed when we studied the expression comparing individually patients and health blood donors, suggesting that this protein could be increased in all human cells that present the translocation BCR-ABL. The level of expression of this gene in HL60 and NB4 was significantly lower than in K562 cell line. The experiments with mice cell lines showed a higher expression of this gene in BaF3T315I when compared to BaF3BCR-ABLp210. We obtained a significant expression difference in all experiments (p <0.05). The spontaneous and stimulated with IL-8 chemotaxis assays used granulocytes and were assessed using chamber containing 96 wells. However, although the results suggest an increased chemotactic activity in patients, there were no significant differences (p<0.05) between controls and patients – regardless of whether the chemotaxis was spontaneous or stimulated with IL-8. In mammals the SEPT5 gene is associated with cellular processes such as exocytosis, apoptosis, leukemogenesis, carcinogenesis and neurodegeneration. Therefore, molecules capable of interacting with the septins, either at biochemical or molecular level, can bring information about their functions in cytokinesis. Studies indicate that the human septins can interact among themselves and with other components of the cytoskeleton – this may be a relevant observation regarding the function of this gene in cancer. The SEPT5 can be activated by different pathways – this may increase expression in translocated cells. Despite major advances in the treatment of CML, the treatments available are not capable of inactivating all the signaling pathways activated by BCR/ABL. Our results demonstrate that SEPT5 may be involved in the pathophysiology of CML. Also, it is clear the importance of the study of pathways that could culminate in its high expression or the triggering of other unknown pathways involved in the development of CML. The increased expression of this gene may be related to disease progression, and finally, the identification of several important genes may lead to a better understanding of CML and helping to identify new therapeutic targets. FAPESP/INCT. Disclosures: No relevant conflicts of interest to declare.

Characterization of lincRNA BALIR-6 in MLL rearranged B-lymphoblastic leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3730-3730
Author(s):  
Norma Iris Rodriguez-Malave ◽  
Weihong Yan ◽  
Giuseppe Basso ◽  
Martina Pigazzi ◽  
Dinesh S. Rao
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Microarray Data ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Regulate Gene Expression ◽  
In Cis ◽  
Regulate Gene

Abstract A new class of non-coding RNA, known as long intergenic non-coding RNAs (lincRNAs), has only recently been described. These lincRNAs have been found to play a role in various molecular processes within the cell including gene regulation, acting as sinks for microRNAs, and regulating splicing, implicating them in development and oncogenic processes. B lymphoblastic leukemia (B acute lymphoblastic leukemia; B-ALL), a malignancy of precursor B-cells, harbors mutations and translocations that result in a dysregulated gene expression. Interestingly, dysregulated expression of lincRNAs has been found in various cancers, but has not yet been described in B-ALL. Recently, we completed a gene expression profiling study in human B-ALL samples, which showed differential lincRNA expression in samples with particular cytogenetic abnormalities. This led us to hypothesize that lincRNAs may be related to disease pathogenesis. Here, we describe a promising lincRNA from our microarray data designated B-ALL associated long intergenic RNA 6 (BALIR-6). Expression of BALIR-6 is highest in patient samples carrying the MLL rearrangement (n=16; when compared to patients with TEL-AML1-translocated, n=39; E2A-PBX1-translocated, n=8; BCR-ABL-translocated, n=3; and cytogenetically normal cases, n=56; 1-way ANOVA p<0.0001) and showed significant variance in the expression level based on the immunophenotype (1-way ANOVA p=0.0004). BALIR-6 is located on chromosome 3p24.3 in humans, and exists in a syntenic gene block in with neighboring genes SATB1 and TBC1D5, and is conserved in mammals. Rapid Amplification of cDNA Ends (RACE) uncovered multiple transcript isoforms; from these, three were cloned out and sequenced, corresponding to the genomic locus as predicted. In B-ALL cell lines, BALIR-6 expression was highest in RS411 cells, which carry the MLL rearrangement, when compared to other B-ALL cell lines. This suggests that the cell lines may show a similar expression pattern to human B-ALL samples. To study the functional role of BALIR-6 we utilized siRNA in a mmu-miR-155 expression cassette to knockdown the transcript. In RS411 cells we observed a reduction in proliferation by MTS assay. Additionally, we observed an increase Sub-G0 cells and a decrease in G2-M phase cells by propidium iodide staining, suggesting an increase in apoptosis. Conversely, overexpression of BALIR-6 in a mouse pre-B cell line (70Z/3), leads to an increase in proliferation. Interestingly, during normal B cell development, BALIR-6 is dynamically expressed, with high expression in pre-B cells and subsequent downregulation, suggesting that a normal role during development is being hijacked in patients with B-ALL. Mechanistically, a few recent studies have described that lincRNAs can regulate gene expression in cis. To explore whether BALIR 6 regulates surrounding genes in cis, we analyzed microarray data of MLL rearranged B-ALL samples, finding that expression of BALIR-6 correlates with expression of surrounding genes SATB1 and TBC1D5. Interestingly for SATB1, this correlation is also seen in human B cell developmental stages. Altering BALIR-6 expression by siRNA mediated knockdown or overexpression causes an effect on the expression of surrounding genes SATB1 and TBC1D5. Previous findings have shown that dysregulated SATB1 has been seen in a variety of malignancies, suggesting a mechanism for how BALIR-6 may produce the changes in cell growth and apoptosis described above. Altogether, these results identify a novel and interesting RNA transcript with the potential to regulate gene expression and pathogenesis in B-ALL with MLL rearrangement, suggesting novel diagnostic, prognostic, and therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

Antitumor Activity of a Novel Proteasome Inhibitor BSc2118 Against Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5725-5725
Author(s):  
Meirong Zang ◽  
Lanting Liu ◽  
Xin Li ◽  
Wei Li ◽  
Ulrike Kuckelkorn ◽  
...  
Keyword(s):  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Molecular Mechanisms ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Proteasome Activity ◽  
Autophagy Flux ◽  
Ubiquitinated Proteins ◽  
Rpmi 8226

Abstract Background: Although the first generation of proteasome inhibitor bortezomib is very effective, the development of resistance limits it long-term utility. In this study, we explored the efficiency and molecular mechanisms of the novel, irreversible proteasome inhibitor BSc2118, particularly, the reversal of bortezomib resistance. Materials and Methods: Human MM cell lines (MM.1S, MM.1R, RPMI-8226, U266, NCI-H929) were treated with BSc2118 at various concentrations for 48h, and assessment for cell viability by CCK-8 assay. MM.1S and MM.1R cells were treated with BSc2118 for 24 hours, and cell cycle and apoptosis analysis were conducted by flow cytometry. Associated molecules were detected by qRT-PCR and western blot. Chymotrypsin-like proteasome activity assay was performed by using the 20S proteasome assay kit. Ubiquitinated proteins were isolated and determined with ubiquitin enrichment kit. Results: Our results revealed that treatment of MM cell lines with BSc2118 inhibits the chymotrypsin-like proteasome activity and induces accumulation of ubiquitinated proteins. BSc2118 inhibits MM cell growth and induces MM apoptosis via induction of G2/M phase arrest, activation of cleaved caspase-3, caspase-8 and caspase-9 and PARP, increasing p53, p21 and E2F1, and inhibition of autophagy in MM.1S, MM.1R and RPMI-8226 cell lines. In addition, BSc2118 dramatically inhibits cytokines mRNA, such as IL-6, VEGF and bFGF in both myeloma cells line and primary bone marrow stromal cells from myeloma patients. More importantly, BSc2118 could overcome bortezomib resistance in vitro by using primary CD138 positive plasma cells from bortezomib-resistant myeloma patients and bortezomib resistance cell line ANBL-6 (ANBL-6.BR), most likely as the consequence of inhibition of autophagy flux which is responsible for bortezomib resistance. Conclusion: Our study revealed BSc2118, a novel irreversible proteasome inhibitor, exerts anti-MM effect, mainly through activation of caspase pathway and inhibition of basal autophagy. It is of great importance that BSc2118 could overcome bortezomib resistance via inhibition of autophagy flux. A head to head of BSc2118 versus Bortezomib is performing in human plasmacytoma xenograft tumor model to evaluate drug safety, anti-tumor efficiency, in particular, reversal of bortezomib resistance. Our preclinical study supports clinical evaluation of BSc2118, particularly, overcomes bortezomib resistance, as a potential MM therapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5658-5658
Author(s):  
Mariana Bleker de Oliveira ◽  
Angela Isabel Eugenio ◽  
Veruska Lia Fook Alves ◽  
Daniela Zanatta ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Immunodeficient Mice ◽  
Late Apoptosis

Abstract Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12). Disclosures No relevant conflicts of interest to declare.

scholarly journals Induction of Resistance to Proteasome Inhibition Preferentially Switches Survival Dependence from Bcl-2 to XIAP in Preclinical Models of Waldenstrom Macroglobulinemia: Pre-Clinical Rationale for Early Clinical Sequencing of ABT199

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4839-4839
Author(s):  
Sharoon Akhtar ◽  
Aneel Paulus ◽  
Kelara Samuel ◽  
Hassan Yousaf ◽  
Davitte Cogen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Specific Inhibitor ◽  
Cross Resistance ◽  
Preclinical Models ◽  
Waldenström Macroglobulinemia ◽  
Waldenstrom Macroglobulinemia ◽  
Resistant Cells

Abstract Background: The proteasome is an established and druggable target for the treatment of plasma cell-related malignancies including Waldenstrom macroglobulinemia (WM). WM cells as a consequence of high immunoglobulin production and increased B-cell receptor (BCR) mediated proliferation upregulate proteasome activity. Additionally, microenvironmental influence mediated through BCR signaling directly influences Bcl-2 and its BH3 family members, supporting tumor cell survival. Indeed, WM patients derive significant clinical benefit from proteasome-inhibitor (PI) based therapy with agents such as bortezomib and carfilzomib. However, resistance to PI develops over time and for these patients the optimal choice and sequence of therapy has yet to be determined. Using our WM models of PI-resistance we interrogated molecular events within the BCR and Bcl-2 pathways to determine therapeutic potential of targeting these crucial pathway in PI-resistance. Materials & Methods: WM cell lines (BCWM.1 and MWCL-1) and carfilzomib-resistant (CR) subclones (BCWM.1/CR and MWCL-1/CR) were used in experiments. Gene-expression and long-noncoding (LNC) RNA analysis was performed (Arraystar) and validated by real-time PCR. Bortezomib, carfilzomib, ABT199 and ibrutinib were purchased from Sellekchem. Results: To determine the functional impact of BCR and Bcl-2 signaling in PI-resistance, as well as therapeutic sensitivity of PI-resistant cells to their inhibitors (ibrutinib, ABT-199, respectively), we established and characterized WM cell lines resistant to carfilzomib. BCWM.1/CR cells showed approximately 20-fold resistance to carfilzomib (IC50 = 92.75nM) and MWCL-1/CR cells approximately 10-fold resistance (Fig. 1A). Both CR clones also displayed some cross-resistance to bortezomib. Gene expression and LNC-RNA profiling demonstrated several changes between carfilzomib-resistant vs. sensitive WM cells. Analysis of proteasome-related mRNA revealed downregulation of PSMB5, PSMB1, PSMB2 and PSMB8. Similarly, profiling of BCR-associated genes demonstrated decreased expression of several components, including BTK and SPI1. This observation functionally manifested as reduced sensitivity to the BTK-inhibitor ibrutinib, wherein CR cells displayed 1.5 - 2 fold growth resistance to ibrutinib on 72hr MTS assay. Next we examined the expression of Bcl-2 family members in CR cells. Intriguingly, we observed that Bcl-2 and Mcl-1 were significantly downregulated but XIAP (inhibitor of apoptosis) was significantly increased in CR cells vs. wildtype WM cells- both at transcriptional and translational levels. This suggested that upon acquisition of CR, a transcriptional shift towards XIAP occurs to accommodate sustained therapeutic stress from carfilzomib and maintain steady antiapoptotic composure. To test if the PI-resistant cells have moved away from their survival dependence on Bcl-2, we treated CR cells to increasing concentrations of the Bcl-2-specific inhibitor, ABT199, and as anticipated, CR cells displayed reduced apoptosis in presence of ABT199 compared to wildtype WM cells (32% annexin-V staining vs. 50%, respectively) (Fig. 1B). Conclusions: Our study sheds insight into the differential drivers of PI-resistance particularly towards carfilzomib, in WM cells. We demonstrate that acquisition of CR is associated with downregulation of Bcl-2 and Mcl-1, which is countered by upregulation of XIAP- an event that renders CR cells resistant to ABT199 (as it targets only Bcl-2). Likewise, downregulation of BCR-related components in CR cells was associated with reduced sensitivity towards ibrutinib. These observations suggest that acquisition of resistance to PI such as carfilzomib can impact future treatment with agents such as ibrutinib or ABT199. Our preclinical models provide rationale or early sequencing of ibrutinib or ABT199 in therapeutic planning of WM patients prior to induction of PI resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Amplification of 6p Associated with Familial CLL

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2432-2432
Author(s):  
Jennifer R Brown ◽  
Bethany Tesar ◽  
Megan Hanna ◽  
Megan Ash ◽  
Stacey M Fernandes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Second Generation ◽  
Somatic Mutations ◽  
Risk Allele ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
High Density ◽  
A Genome

Abstract Abstract 2432 Chronic lymphocytic leukemia (CLL) is one of the most familial of all cancers but the genetic basis of this heritability remains poorly characterized. Families with very strong inheritance of CLL have been described in the literature, and recently the occurrence of CLL in one such family was associated with a polymorphism in the DAPK gene. Here we report the genomic characterization of a family in which CLL appears to be inherited in a Mendelian autosomal dominant manner. Within this family, five of eleven siblings of the first generation were affected, and one of those affected siblings had five children, of whom three were also affected (the second generation). The children of the second generation are currently aged 20–30 and hence too young to know whether they will develop CLL. We performed high-density single-nucleotide polymorphism (SNP) array analysis and gene expression profiling on tumor and germline DNA from four of the offspring of the second generation, as well as six of their children. Analysis of the SNP array data revealed a significant germline amplification of 6p, spanning 0–720 Mb and encompassing a known copy number variant (CNV) region but significantly larger than the CNV region. This amplification was found in both affected individuals with samples available from the second generation, and was transmitted by each of them to one of their two children in the third generation. This amplification was absent from the two unaffected members of the second generation, their children, or any of the other 189 individuals with CLL who were analyzed in our high-density SNP array dataset. None of the unaffected individuals with or without the amplification had evidence of monoclonal B cell lymphocytosis (MBL) by highly sensitive flow cytometry. These unaffected individuals also lacked any PCR-detectable oligoclonal or monoclonal immunoglobulin heavy chain gene rearrangement suggestive of MBL. The region of amplification contains four protein-coding genes: EXOC2, DUSP22, HUS1B and IRF4. We sequenced the coding regions of these four genes and the 5` and 3` UTRs of IRF4 in all family members, but found no somatic mutations in this family. All four genes were also sequenced in 92 other familial CLLs, identifying no somatic mutations. We then analyzed our gene expression profiling data to assess whether any genes in this region were altered in the affected individuals with the amplification. This analysis revealed a significant 1.74X increase in IRF4 expression in the CLLs with the amplification compared to those without (q value < 0.001). By Western blotting, we confirmed that IRF4 protein was increased approximately two-fold in amplified compared to non-amplified samples. These data suggest that the amplification may target IRF4, which has been previously implicated in CLL by a genome wide association study that identified a tag SNP in its 3` UTR as a CLL risk allele. Further analysis of our SNP data demonstrated allele specific amplification in this region, and mass-spectrometric genotyping confirmed enrichment of the CLL risk allele in the individuals with amplification. We conclude that amplification of IRF4 carrying the risk allele for CLL appears likely to be the culprit predisposing to CLL in this family. Disclosures: No relevant conflicts of interest to declare.

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