HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

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.

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Interleukin-16 Is An Important Growth-Promoting Factor and a Novel Diagnostic and Therapeutic Target for Multiple Myeloma

Blood ◽

10.1182/blood.v116.21.4056.4056 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4056-4056

Author(s):

Djordje Atanackovic ◽

York Hildebrandt ◽

Tim Luetkens ◽

Axel R. Zander ◽

Carsten Bokemeyer ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Cells ◽

Cell Lines ◽

Therapeutic Target ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Antibody Array ◽

Western Blots ◽

Myeloma Cells ◽

Growth Promoting

Abstract Abstract 4056 Background: Multiple myeloma (MM) is a malignancy characterized by the expansion of a plasma cell (PC) clone that localizes to the bone marrow (BM). Myeloma cells and BM stromal cells both produce soluble factors promoting the survival and progression of MM. Interleukin-(IL)-16 is involved in regulating migration and proliferation of normal leukocytes, however, it has been unclear whether IL-16 also plays a role in the pathophysiology of human cancers. Methods: Using an antibody array we screened supernatants of myeloma cell lines for the presence of a variety of cytokines/chemokines. We confirmed IL-16 expression in myeloma cell lines as well as in malignant PC and BM plasma from MM patients applying real-time PCR, western blots, ELISA, and flow cytometry. We applied inhibitory RNA to analyze IL-16 function and we used anti-IL-16 antibodies to evaluate possible therapeutic options for MM. Results: We found IL-16 to be strongly overexpressed in the BM of myeloma patients. Myeloma cell lines as well as primary tumor cells from MM patients constitutively expressed IL-16 RNA and protein and spontaneously secreted soluble IL-16. Functional analyses revealed that IL-16 supports the proliferation of myeloma cells. Accordingly, silencing of IL-16 expression had an anti-proliferative effect on the tumor cells. Most importantly, the application of a monoclonal antibody directed against IL-16had a strong growth-inhibiting influence on myeloma cells. Conclusions: These findings suggest that cytokine IL-16 is an important growth-promoting factor in MM and might represent a novel diagnostic and therapeutic target for this incurable human malignancy. Disclosures: No relevant conflicts of interest to declare.

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Good and Poor Response Gene Expression Signatures to Proteasome Inhibitors Using a Mouse Model of Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.1843.1843 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1843-1843

Author(s):

Holly Stessman ◽

Linda B. Baughn ◽

Aaron G. Sarver ◽

Aatif Mansoor ◽

Tzu G. Wu ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Cell Lines ◽

Copy Number ◽

Research Funding ◽

Proteasome Inhibitors ◽

Gene Signature ◽

Mouse Tumor ◽

Tumor Cell Lines

Abstract Abstract 1843 The proteasome inhibitor bortezomib (Bz) has been used extensively and with much success in the treatment of multiple myeloma (MM) patients; however, patients eventually relapse, many as non-responders to subsequent treatments with Bz making drug resistance a significant problem. Here we utilized cell lines created using a iMycCa/Bcl-xL transgenic mouse model of MM (Cheung, et al. J Clin Invest (2004) 113: 1763) to identify 1) gene expression signatures of Bz response, 2) differences in gene expression between sensitive and resistant cell lines, and 3) cytogenetic abnormalities associated with Bz sensitive and resistant phenotypes. The iMycCa/Bcl-xL transgenic mice develop plasma cell tumors with 100% penetrance and have shown strikingly strong similarities to human MM by extensive gene expression profiling (GEP), spectral karyotyping and histology (Boylan, et al. Cancer Res (2007) 67: 4069). Six cell lines created from these mice were dose escalated with Bz over approximately six months to create Bz resistant (BzR) cell lines with approximately 5–8 fold increase in IC50 to Bz compared to their sensitive counterparts. The BzR characteristics were stable, as lines grown in the absence of drug for as long as 6 months maintained drug resistance upon subsequent challenge. Notably, BzR lines showed cross resistance to other investigational proteasome inhibitors (MLN9708 and carfilzomib) while maintaining sensitivity to other chemotherapeutic agents (dexamethasone and melphalan), suggesting a common mechanism of emerging resistance to proteasome inhibitors. The results of GEP of these mouse tumor cell lines treated with Bz were compared with a recently published human drug trial where GEP was completed prior to and 48 hours after a “test dose” of Bz was administered to patients (Shaughnessy, et al. Blood (2011), ahead of print). In the mouse tumor cell lines, 116 genes were differentially expressed upon in vitro Bz treatment (p=0.001, ≥1.5 fold change). Between the mouse and human drug response data sets was an overlapping common 27-gene signature (p=1×10−25, Fishers exact test) of Bz-induced expression changes that has not previously been described. Time points were collected in these mouse cell line GEP experiments at 0, 2, 8, 16, and 24 hours after Bz treatment. A comparison of the Bz sensitive and derived BzR lines prior to drug treatment revealed a 50 gene signature (p=0.05, ≥2 fold change) that distinguishes three pairs of sensitive and resistant lines. Gene-set enrichment analyses have revealed significant pathways that are differentially regulated in the sensitive and resistant responses. Additional GEP differences were seen when time course expression patterns were examined from Bz sensitive compared to resistant tumor lines. Thus, GEP signatures that distinguish tumor lethality from resistance were identified both prior to Bz treatment, as well as in the early response to Bz. In addition, array comparative genomic hybridization on 4 pairs of mouse Bz sensitive and established BzR lines revealed not only gross differences in copy number between the differentially responding groups of cells but copy number abnormalities that may be unique to the emerging resistance. Taken together, these data indicate that this model is useful for the identification of good and poor Bz response signatures in MM. These signatures are currently being evaluated in human tumor cells from single agent bortezomib phase II and phase III clinical trials. Because the in vitro adapted tumor mouse lines can be genetically manipulated using lentiviral vectors, this model can be used as a preclinical platform to validate existing gene models with respect to Bz response, something that cannot be done using human patients. Subsequent transfer of manipulated lines into syngeneic, immunocompetent recipients can further test Bz response in vivo presenting a significant advantage of this robust mouse MM model system over other in vitro systems. Disclosures: Stessman: Millennium: The Takeda Oncology Company: Research Funding. Mansoor:Millennium: The Takeda Oncology Company: Research Funding. Janz:Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

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Identification of a Gene Signature Associated to Treatment Response to Palbociclib in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.5666.5666 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5666-5666

Author(s):

Angelique Bruyer ◽

Alboukadel Kassambara ◽

Paul Anziani ◽

Donia El Bahlagui ◽

Nicolas Robert ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Multiple Myeloma ◽

Rna Sequencing ◽

Cell Lines ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Gene Expression Signature ◽

G1 Arrest ◽

Expression Signature

Abstract Background: Inpatients with relaspsed/refractoryMultiple Myeloma (MM), outcomes are far from optimal, especially in patients refractory to current treatments Recent studies and clinical trials have highlighted the therapeutic potential of Palbociclib, a CDK4/6 inhibitor, in various cancers including MM. Deregulation of CDK4/6 is involved in the loss of cell cycle control in MM. Response to Palbociclib combined with bortezomib and dexamethasone was acquired in 20% of the relapsed/refractory MM patients, suggesting that biomarkers to identify patients that could benefit from this treatment are needed. Additional studies are required to understand the biological pathways associated with sensitivity or resistance of MM cells to Palbociclib. Methods: 14 human MM cell lines and 12 primary MM samples were tested for response to Palbociclib treatment. The concentration required to inhibit growth by 50% (IC50) was calculated. Gene expression signature associated with multiple myeloma response to Palbociclib, as well as, genes deregulated by the treatment have been analyzed using microarray and RNA-sequencing methods. Results: Palbociclib had an heterogeneous in vitro activity among the 14 human myeloma cell lines tested, which aggregated into three groups based on the distribution of the IC50 values: sensitive (n = 5, IC50: 0.2 - 0.3µM), intermediate (n = 3, IC50: 0.5 - 0.7µM) or more resistant group (n = 6, IC50: 0.9 - 2.4µM). The same holds true when testing the Palbociclib on primary multiple myeloma samples. The evaluation of the Palbociclib effect on cell cycle progression and the induction of the apoptosis, reveals that Palbociclib is essentially cytostatic, inducing prolonged G1 arrest in sensitive cell lines with a strong reduction of the percentage of cells in S phase. To better understand the molecular mechanisms associated with Palbociclib response, we identified a gene expression signature correlated with the response in both MM cell lines and primary myeloma cells from patients. Additionally, we have analyzed differentially expressed genes after Palbociclib treatment in human MM cell lines using RNA sequencing (n = 4). The physiological role of the downregulated genes after Palbociclib treatment is associated with cell cycle, mitosis and E2F mediated regulation of DNA replication. Significantly upregulated genes, after Palbociclib treatment, were enriched in genes encoding proteins involved in glutathione synthesis and recycling, and biological oxidations. Conclusion: Altogether, our data demonstrated a high heterogeneity in the response of MM cells to Palbociclib. We identified a gene expression signature associated with Palbociclib response in MM. These genes could help to identify MM patients that could benefit from Palbociclib treatment and provide novel targets for efficient combination therapy. Disclosures No relevant conflicts of interest to declare.

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Mucin-1 (MUC1) Oncoprotein in Multiple Myeloma Cells Inhibits the Th1 Responses By Down Regulating the Expression of Mir-200c and up-Regulating the PDL1 Expression

Blood ◽

10.1182/blood.v124.21.2072.2072 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2072-2072 ◽

Cited By ~ 1

Author(s):

Hasan Rajabi ◽

Maxwell Douglas Coll ◽

Jacalyn Rosenblatt ◽

Li Yin ◽

Dina Stroopinsky ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Cell Lines ◽

Immune Escape ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Micro Rna ◽

P Value ◽

Muc1 Expression ◽

Myeloma Cells

Abstract Introduction: The PDL1/PD-1 pathway is a critical mediator of immune escape in patients with multiple myeloma (MM). Regulation of this pathway has not been well characterized. MicroRNAs (miRNAs) are a conserved class of small (~22 nucleotides) RNAs that post-transcriptionally regulate gene expression by interacting with the 3′ untranslated region (3′ UTR) and, in some settings, coding regions of target mRNAs. MiRNAs suppress gene expression by promoting mRNA degradation or inhibiting translation. Of note, the 3’UTR of the PDL1 gene contains putative binding sites for miR-200 family of micro-RNA’s, suggesting a possible role of miR-200’s in regulation of PDL1 expression. We have previously demonstrated that miR-200c is suppressed by the MUC1 oncoprotein, and hypothesized that MUC1 expression on myeloma cells upregulates the expression of PDL1, via suppressing miR-200c. In the present study, we investigated the relationship between MUC1, miR-200c and PDL1 in multiple myeloma. Methods and Results: Lentivirus vectors expressing miR-200c or a control vector with green fluorescence protein (GFP) were transduced in two different MM cell lines (MM-RPMI, MM-U266). Cells were harvested sorted by Fluorescence-Activated Cell Sorting (FACS) after 72 hours of transduction, using a dual fluorescence for GFP and anti-PDL1 antibody to analyze the changes in PDL1 expression. MiR-200c transduction of U266 cells resulted in a decrease in mean expression of PDL1 from 69.55% to 1.4% (n=2). Similarly, RPMI cells demonstrated a reduction in mean expression of PDL1 from 62.5% to 1.9% (n=2) following miR-200c transduction. The abrogation of PDL1 expression in MM cells by ectopic expression of miR-200c was confirmed using western immunoblot analysis. Having previously demonstrated that miR-200c is suppressed by MUC1 in a solid tumor model, we evaluated the effect of silencing MUC1 in U266 and RPMI cell lines on miR-200c and PDL1 expression. MUC1 silenced stable cell lines of RPMI and U266 cells were generated using lentivirus shRNA vectors against MUC1 or a scrambled vector control. MUC1 silenced cells demonstrated an increase in miR-200c expression (> 2 fold, p value <0.05). Notably, PDL1 expression decreased from 52% to 3.7% and from 62.5% to 6.1% following silencing of MUC1 on U266 and RPMI cells respectively. Conclusions: Ectopic expression of micro-RNA miR-200c in RPMI-MM and U266-MM cell lines results in down regulation of PDL1 expression. Silencing MUC1 in RPMI-MM and U266-MM cell lines results in both increased expression of miR-200c and downregulation of PDL1 expression. These results support the hypothesis that MUC1 expression on myeloma cells contributes to tumor mediated immunosuppression, by suppressing miR-200c thereby enhancing PDL1 expression. Interfering with MUC1 mediated signaling represents a novel approach towards augmenting immune mediated targeting of myeloma. Disclosures No relevant conflicts of interest to declare.

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A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo

Blood ◽

10.1182/blood-2019-121480 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1821-1821

Author(s):

Mario I Vega ◽

Yijiang Shi ◽

Patrick Frost ◽

Sara Huerta-Yepez ◽

Alan Lichtenstein

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Therapeutic Target ◽

Cytotoxic Effect ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Inhibition Of Proliferation ◽

Induction Of Apoptosis

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831 Disclosures No relevant conflicts of interest to declare.

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Identification of functionally distinct and interacting cancer cell subpopulations from glioblastoma with intratumoral genetic heterogeneity

Neuro-Oncology Advances ◽

10.1093/noajnl/vdaa061 ◽

2020 ◽

Vol 2 (1) ◽

Author(s):

Min Guo ◽

Marjolein van Vliet ◽

Jian Zhao ◽

Teresita Díaz de Ståhl ◽

Mikael S Lindström ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Cancer Cell ◽

Copy Number ◽

Evolutionary Relationship ◽

Intratumoral Heterogeneity ◽

Phenotypic Traits ◽

Dna Copy Number ◽

Model Cell ◽

High Level

Abstract Background Glioblastomas display a high level of intratumoral heterogeneity with regard to both genetic and histological features. Within single tumors, subclones have been shown to communicate with each other to affect overall tumor growth. The aim of this study was to broaden the understanding of interclonal communication in glioblastoma. Methods We have used the U-343 model, consisting of U-343 MG, U-343 MGa, U-343 MGa 31L, and U-343 MGa Cl2:6, a set of distinct glioblastoma cell lines that have been derived from the same tumor. We characterized these with regard to temozolomide sensitivity, protein secretome, gene expression, DNA copy number, and cancer cell phenotypic traits. Furthermore, we performed coculture and conditioned media-based experiments to model cell-to-cell signaling in a setting of intratumoral heterogeneity. Results Temozolomide treatment of a coculture composed of all 4 U-343 cell lines presents a tumor relapse model where the least sensitive population, U-343 MGa 31L, outlives the others. Interestingly, the U-343 cell lines were shown to have distinct gene expression signatures and phenotypes although they were derived from a single tumor. The DNA copy number analysis revealed both common and unique alterations, indicating the evolutionary relationship between the cells. Moreover, these cells were found to communicate and affect each other’s proliferation, both via contact-dependent and -independent interactions, where NOTCH1, TGFBI, and ADAMTS1 signaling effects were involved, respectively. Conclusions These results provide insight into how complex the signaling events may prove to be in a setting of intratumoral heterogeneity in glioblastoma and provide a map for future studies.

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Genome-Wide shRNA Screen Identifies WEE1 As a Critical Mediator of Cell Fate and Novel Therapeutic Target in AML,

Blood ◽

10.1182/blood.v118.21.3503.3503 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3503-3503

Author(s):

Christopher C. Porter ◽

Jihye Kim ◽

Susan Fosmire ◽

Christy M. Gearheart ◽

Annemie van Linden ◽

...

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Cell Lines ◽

Therapeutic Target ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Cell Cycle Checkpoint ◽

Phase Ii Trials ◽

Genome Wide ◽

Cycle Checkpoint

Abstract Abstract 3503 Acute myeloid leukemia (AML) remains a therapeutic challenge despite increasing knowledge of the molecular origins of the disease, as the mechanisms of AML cell escape from chemotherapy remain poorly defined. We hypothesized that AML cells are addicted to specific molecular pathways in the context of chemotherapy and used complementary approaches to identify these addictions. Using novel molecular and computational strategies, we performed genome-wide shRNA screens to identify proteins which mediate AML cell fate in cytarabine in 2 AML cell lines (Molm13 and MV4-11). Over 55,000 shRNAs targeting over 11,000 genes were quantified by deep sequencing to identify shRNAs under-represented in the context of cytarabine as compared to no treatment. Complementary analyses identified 125 genes as mediators of AML cell fate in cytarabine. In addition we performed gene expression profiling of AML cells exposed to cytarabine to identify genes with induced expression in this context and examined existing gene expression data from primary patient samples. The integration of these independent analyses strongly implicates cell cycle checkpoint proteins, particularly WEE1, as critical mediators of AML cell fate in cytarabine. Knockdown of WEE1 in a secondary screen confirmed its role in AML cell survival in cytarabine. Pharmacologic inhibition of WEE1 in several, but not all, AML cell lines is synergistic with cytarabine, suggesting underlying molecular susceptibility to this combination of drugs. A WEE1 inhibitor is in Phase II trials in solid tumors, primarily as a means to abrogate the G2/M checkpoint in tumors with TP53 dysfunction. Further experiments demonstrate that inhibition of WEE1 prevents slowed S-phase progression induced by cytarabine in AML cells, broadening the functions of WEE1 that may be exploited therapeutically. Preliminary experiments indicate synergistic inhibition of AML cellular proliferation with daunorubicin in some AML cell lines. Experiments to determine whether WEE1 inhibition in combination with chemotherapy prolongs survival of mice with leukemia are underway. These data highlight the power of integrating functional and descriptive genomics, and identify WEE1 as potential therapeutic target in AML. Disclosures: No relevant conflicts of interest to declare.

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Generation of a Predictive Score for Ixazomib Response in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.5695.5695 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5695-5695

Author(s):

Amit Kumar Mitra ◽

Sanjoy Dey ◽

Andrew Hangsleben ◽

Michael Steinbach ◽

Vipin Kumar ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Cell Lines ◽

Conflicts Of Interest ◽

Single Agent ◽

The United States ◽

Myeloma Cell ◽

Chemotherapeutic Agents ◽

Predictive Score ◽

Molecular Characteristics

Abstract Multiple myeloma (MM) is the second-most common hematopoietic malignancy in the United States accounting for 1% of all cancers and 10% of all hematologic malignancies. Despite recent improvements in treatment strategies including the emergence of proteasome inhibitors (PIs) as effective chemotherapeutic agents, MM still remains difficult to cure with median survival rate of around 7 years, primarily due to wide inter-individual variation in response to treatment. We believe such heterogeneity in response to PIs is governed by the underlying molecular characteristics of the tumor including alterations in gene expression profile (GEP). In the current study, we used a panel of Human Myeloma Cell Lines (HMCLs) representing the gamut of biological and genetic heterogeneity in MM to evaluate the gene expression signatures associated with response to the second-generation PI Ixazomib and produced a predictive score (PI score) for Ix response. HMCLs (n=45) were treated with increasing concentrations of Ixazomib used as single agent and half maximal inhibitory concentration (IC50) values were determined using cell viability equation. Gene expression profiling data was obtained as publicly available data from the Keats lab website at TGen (http://www.keatslab.org/myeloma-cell-lines). Genes with high expression value and high standard deviation beyond the median values were pre-filtered and log expression values were normalized by subtracting mean expression of individual genes across all the samples and the housekeeping genes (GAPDH). Subsequently, analysis of correlation between Ix IC50 data and GEP data and the False Discovery Rate (FDR) based on 1000 random permutations were performed to identify true patterns of genes that are highly predictive of Ix response and to look for the top genes that could discriminate between the top sensitive and top resistant cell lines. Gene clusters were identified that correlated with response and will be presented. Our results will demonstrate in vitro modeling of response using GEP approaches that may provide predictive scoring algorithms of a defined set of genes that will be useful in clinical evaluation of drug choice in treating individual patients. Disclosures No relevant conflicts of interest to declare.

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The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽

10.1182/blood.v85.2.319.319 ◽

1995 ◽

Vol 85 (2) ◽

pp. 319-329 ◽

Cited By ~ 66

Author(s):

S Dziennis ◽

RA Van Etten ◽

HL Pahl ◽

DL Morris ◽

TL Rothstein ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Transgene Expression ◽

Heterologous Gene Expression ◽

Myeloid Cells ◽

Reporter Genes ◽

Regulatory Elements ◽

Specific Expression ◽

High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

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Methylation of the Cyclin A1 Promoter Correlates with Gene Silencing in Somatic Cell Lines, while Tissue-Specific Expression of Cyclin A1 Is Methylation Independent

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3316-3329.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3316-3329 ◽

Cited By ~ 55

Author(s):

Carsten Müller ◽

Carol Readhead ◽

Sven Diederichs ◽

Gregory Idos ◽

Rong Yang ◽

...

Keyword(s):

Gene Expression ◽

Cell Lines ◽

Promoter Methylation ◽

Cpg Island ◽

Trichostatin A ◽

Specific Gene ◽

Cyclin A1 ◽

Specific Expression ◽

Tissue Specific ◽

Tissue Specific Expression

ABSTRACT Gene expression in mammalian organisms is regulated at multiple levels, including DNA accessibility for transcription factors and chromatin structure. Methylation of CpG dinucleotides is thought to be involved in imprinting and in the pathogenesis of cancer. However, the relevance of methylation for directing tissue-specific gene expression is highly controversial. The cyclin A1 gene is expressed in very few tissues, with high levels restricted to spermatogenesis and leukemic blasts. Here, we show that methylation of the CpG island of the human cyclin A1 promoter was correlated with nonexpression in cell lines, and the methyl-CpG binding protein MeCP2 suppressed transcription from the methylated cyclin A1 promoter. Repression could be relieved by trichostatin A. Silencing of a cyclin A1 promoter-enhanced green fluorescent protein (EGFP) transgene in stable transfected MG63 osteosarcoma cells was also closely associated with de novo promoter methylation. Cyclin A1 could be strongly induced in nonexpressing cell lines by trichostatin A but not by 5-aza-cytidine. The cyclin A1 promoter-EGFP construct directed tissue-specific expression in male germ cells of transgenic mice. Expression in the testes of these mice was independent of promoter methylation, and even strong promoter methylation did not suppress promoter activity. MeCP2 expression was notably absent in EGFP-expressing cells. Transcription from the transgenic cyclin A1 promoter was repressed in most organs outside the testis, even when the promoter was not methylated. These data show the association of methylation with silencing of the cyclin A1 gene in cancer cell lines. However, appropriate tissue-specific repression of the cyclin A1 promoter occurs independently of CpG methylation.

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