Regulation of Selected MicroRNAs in Multiple Myeloma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3916-3916
Author(s):  
Reinhold Munker ◽  
Tetsuro Setoyama ◽  
Madeleine Duvic ◽  
Robert Z. Orlowski ◽  
George Calin
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Ionizing Radiation ◽  
Cell Lines ◽  
Expression Profiles ◽  
Early Time ◽  
Pegylated Interferon ◽  
Protective Mechanism ◽  
Time Points ◽  
High Risk Disease

Abstract Abstract 3916 Introduction: Multiple myeloma is clinically and biologically heterogeneous. Certain translocations and chromosomal losses (t{14;16}, t{14;20}, del 17p) and gene expression profiles define high-risk disease. Recently, several groups found microRNAs (miRs) dysregulated in multiple myeloma. A profile composed of 28 miRs was found to define high-risk disease. Among the dysregulated miRs, miR15a, miR16 were down-regulated, miR19b, miR20a, miR181b were increased according to most publications. MiR-21 was generally upregulated in high-risk disease and inducible by interleukin-6. We hypothesized that commonly administered treatments for multiple myeloma would alter the expression pattern of these miRs. Materials and Methods: For these in-vitro experiments, 4 established cell lines were used: RPMI8226, OPM-2 (t4;14), Kas-6 (IL-6-dependent) and MM1-S (t14;16). The cells were treated with ionizing radiation (3- 6 Gy), lenalidomide (10 μM), doxorubicin (50 ng/ml), bortezomib (2- 50 nM), SAHA (1–3x 10−6 M), pegylated interferon α (3–300 ng/ml) and nutlin-3 (10 μM) between 2 and 48 hours. RNA was extracted and quantitative real-time RT-PCR was performed for miR-15a, miR-16, miR19b, miR-20a, miR21, miR-181b and a control gene (U6). The expression was calculated and compared by the ΔΔ CT method. Results: Ionizing radiation increased miR15a in 1/2 cell lines at early time points, increased miR-19b at early time points in 2/2 cell lines (decreased later) and increased MiR20a in 2/2 cell lines at early time points. Lenalidomide induced miR15a in 2/4 cell lines, miR19b in 3/ 4 cell lines and miR-20a in 3/ 4 cell lines. Doxorubicin increased miR-16 in 2/3 cases and miR-20a in 2/3 cases (in 1 cell line decreased). Bortezomib overall induced few changes in miR-expression. SAHA induced miR-15a in 2/3 cell lines and decreased miR-16 in 1/3. MiR-19a was decreased with SAHA in 2/4 and increased in 1/4 cell lines. MiR-20 decreased in 1/4 and increased in 1/4. MiR-21 decreased in 1 and increased in 1/4 SAHA-treated cell lines. MiR-181b increased in 2/4 cell lines. Pegylated interferon decreased MiR-15a in 3/4 cell lines, decreased miR-16a in 3/4 cell lines, increased miR19b in 2/4 cell lines. MiR-20a was increased in 2/4 and decreased in 1/4 cell lines. MiR-181b was decreased in 2/ 4 cell lines. Nutlin-3 increased miR16 in 1/3 cell lines, increased miR-20a in 2/4 cell lines, increased miR-181b in 2/4, decreased miR-181b in 1/4 cell lines. Most changes observed are in the range of −50 – + 200%. Conclusions: Many miRs are induced at early time points under non-cytotoxic conditions. The variability observed in these experiments may be due to the genetic heterogeneity of the cell lines. Interferon mostly down-modulates the expression of the miRs studied. Previous experiments, for example using endothelial cells also showed an induction of certain miRs after cytotoxic or cytostatic treatments. This can be explained as a stress response or protective mechanism enhancing tumor cell survival. However, the functional relevance of our data was not investigated. The downregulation of miRs following interferon treatment is surprising and would argue for a combination of interferon with cytostatic treatments. If confirmed using CD138 selected samples from patients with multiple myeloma, our data may be used to develop a treatment profile which ultimately might prognosticate treatment response. Our results are also relevant for future miR-based treatments for multiple myeloma. Disclosures: Orlowski: Onyx Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Jumping translocations of 1q12 in multiple myeloma: a novel mechanism for deletion of 17p in cytogenetically defined high-risk disease

Blood ◽  
2014 ◽  
Vol 123 (16) ◽  
pp. 2504-2512 ◽  
Author(s):  
Jeffrey R. Sawyer ◽  
Erming Tian ◽  
Christoph J. Heuck ◽  
Joshua Epstein ◽  
Donald J. Johann ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Copy Number ◽  
High Risk Disease ◽  
Key Points ◽  
Gains And Losses

Key Points Jumping translocations of 1q12 (JT1q12) provide a mechanism for the deletion of 17p in cytogenetically defined high-risk myeloma. Sequential JT1q12s introduce unexpected copy number gains and losses in receptor chromosomes during subclonal evolution.

Real-World Experience with Minimal Residual Disease Testing with Next Generation Flow Cytometry and Functional Imaging in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
David Böckle ◽  
Paula Tabares Gaviria ◽  
Xiang Zhou ◽  
Janin Messerschmidt ◽  
Lukas Scheller ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
High Risk ◽  
Functional Imaging ◽  
Real World ◽  
Research Funding ◽  
Residual Disease ◽  
Focal Lesions ◽  
High Risk Disease ◽  
Minimal Residual

Background: Minimal residual disease (MRD) diagnostics in multiple myeloma (MM) are gaining increasing importance to determine response depth beyond complete remission (CR) since novel agents have shown to induce high rates of deep clinical responses. Moreover, recent reports indicated combining functional imaging with next generation flow cytometry (NGF) could be beneficial in predicting clinical outcome. This applies in particular to the subset of patients suffering from relapsed/refractory multiple myeloma (RRMM) who tend to show a higher incidence of residual focal lesions despite serological response. Here, we report our institutions experience with implementing both functional imaging and NGF-guided MRD diagnostics in clinical practice. Methods: Our study included patients with newly diagnosed multiple myeloma (NDMM) and RRMM achieving VGPR, CR or sCR. Bone marrow aspirates were obtained for MRD-testing according to IMWG 2016 criteria. Samples were collected between July 2019 and July 2020 and analyzed with NGF (according to EuroFlowTM guidelines) at a sensitivity level of 10-5. Results were compared to functional imaging obtained with positron emission tomography (PET) and diffusion-weighted magnetic resonance imaging (DW-MRI). High-risk disease was defined as presence of deletion 17p, translocation (14;16) or (4;14). Results: We included 66 patients with NDMM (n=39) and RRMM (n=27) who achieved VGPR or better. In patients with RRMM the median number of treatment lines was 2 (range 2-11). Fifteen patients suffered from high-risk disease. Median age at NGF diagnostics was 64 years (range 31-83). Among patients achieving VGPR (n=27), CR (n=10) and sCR (n=29) seventeen (26%) were MRD-negative by NGF testing. CR or better was significantly associated NGF MRD-negativity (p=0.04). Notably, rates of NGF MRD-negativity were similar among patients with NDMM (28%) and RRMM (26%). Even some heavily pretreated patients who underwent ≥ 4 lines of therapy achieved MRD-negativity on NGF (2 of 9). Functional imaging was performed in 46 (70%) patients with DW-MRI (n=22) and PET (n=26). Median time between NGF and imaging assessment was 2 days (range 0-147). Combining results from imaging and NGF, 12 out of 46 (26%) patients were MRD-negative with both methods (neg/neg). Three patients displayed disease activity as measured with both, imaging and NGF (pos/pos). Twenty-nine of the remaining patients were MRD-positive only according to NGF (pos/neg), while two patients were positive on imaging only (neg/pos). More patients demonstrated combined MRD-negativity on NGF and imaging (neg/neg) in the NDMM setting than in RRMM (32% versus 19%). We also observed that 30% of the patients with high-risk genetics showed MRD-negativity on both imaging and NGF. Of note, none of the patients with very advanced disease (≥4 previous lines) was MRD-negative on both techniques. Conclusion In the clinical routine, MRD diagnostics could be used to tailor maintenance and consolidation approaches for patients achieving deep responses by traditional IMWG criteria. Our real-world experience highlights that MRD-negativity can be achieved in patients suffering from high-risk disease and also in late treatment lines, supporting its value as endpoint for clinical trials. However, our data also support MRD diagnostics to be combined with functional imaging at least in the RRMM setting to rule out residual focal lesions. Future studies using MRD for clinical decision-making are highly warranted. Disclosures Einsele: Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau. Rasche:Celgene/BMS: Honoraria; GlaxoSmithKline: Honoraria; Oncopeptides: Honoraria; Skyline Dx: Research Funding; Janssen: Honoraria; Sanofi: Honoraria.

Bortezomib Inhibits mTOR Pathway in Multiple Myeloma Cell Lines Via Induced Expression of REDD1.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 242-242
Author(s):  
Olivier Decaux ◽  
Monique Clement ◽  
Florence Magrangeas ◽  
Laurence Lode ◽  
Catherine Charbonnel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Size ◽  
Cell Lines ◽  
Stress Responses ◽  
Cellular Proliferation ◽  
Expression Profiles ◽  
Mtor Pathway ◽  
Molecular Signature ◽  
Gene And Protein Expression

Abstract Pharmacogenomic profiles of genes involved in bortezomib - dexamethasone response may help to understand resistance and could provide new therapeutic targets as well as contributing to novel prognostic markers in multiple myeloma. We have used gene expression profiling to analyze the complex signaling pathways regulating the response to bortezomib - dexamethasone. Gene expression profiles were established in 9 cell lines, derived from 9 myeloma patients, incubated or not with a combination of bortezomib 10 nM and dexamethasone 1 μM. These concentrations correspond to the ones used for patients in the IFM 2005-01. Cells were collected after 6 hours of treatment. We focused our interest in early response genes, making the hypothesis that the comprehension of early effects would help to better understand the mechanisms of resistance that take place in at least two third of myeloma patients. Supervised analysis with permutations identified significantly up regulated genes involved in stress responses (heat shocks proteins, RTP801/dig2/REDD1/DDIT4), endoplasmic reticulum stress (HERP/HERPUD1, gadd145/CHOP/DDIT3), ubiquitin/proteasome pathway (proteasome 26S subunits PSMB7, PSMC4, PSMD3 and PSMD13), unfolded protein response (such as SQSTM1, ATF4) or redox equilibrium (PLRX, PRDX1). We assumed that these genes might represent a molecular signature of response to bortezomib and provide important insight into the complex mechanisms of action of these drugs. We focused on REDD1 a gene cloned in 2002 that is known to be rapidly induced by a wide variety of stress conditions (arsenic, hypoxia, dexamethasone, thapsigargin, tunimycin and heat shock) and DNA damages (ionizing radiation, ultraviolet radiation, DNA alkylant). We found that both REDD1 gene and protein expression were early and highly induced after bortezomib exposure alone or in combinaison with dexamethasone. This effect was dependent upon cell line: REDD1 was overexpressed within two hours in resistant cell lines in association with a cell size decrease while in sensitive cell lines, neither REDD1 induction nor morphological changes occured. REDD1 induction was associated with the dephosphorylation of S6K1, a key substrat of mTOR, a protein kinase which controls cell growth and cell size in response to various signals. SiRNA studies confirmed that bortezomib lead to a negative regulation of mRTor activity mediated by REDD1: disruption of REDD1 abrogates both S6K1 phosphorylation and early transitory cell size reduction. Our results are in accordance with data obtained in mouse showing an early regulation of mTOR pathway and cellular proliferation induced by REDD1 expression in response to stress. Our study suggests that mTOR regulation could be a resistance mechanism mediated by REDD1 expression. As we found that REDD1 was differentially induced in primary plasma cells from patients, this gene expression could help to predict response to bortezomib. Our objective is now to clarify the pathway that links bortezomib to REDD1 in multiple myeloma and to investigate REDD1 expression in patients enrolled in IFM 2005-01 clinical trial.

In Silico Prediction of Novel Drug Combinations to Combat Bortezomib-Resistant Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1344-1344
Author(s):  
Holly A. F. Stessman ◽  
Tian Xia ◽  
Aatif Mansoor ◽  
Raamesh Deshpande ◽  
Linda B. Baughn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
In Silico ◽  
Research Funding ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Mouse Cell ◽  
Resistant Mouse ◽  
Novel Therapeutic

Abstract Abstract 1344 Bortezomib/VELCADE® (Bz) is a proteasome inhibitor that has been used successfully in the treatment of multiple myeloma (MM) patients. However, acquired resistance to Bz is an emerging problem. Thus, there is a need for novel therapeutic combinations that enhance Bz sensitivity or re-sensitize Bz resistant MM cells to Bz. The Connectivity Map (CMAP; Broad Institute) database contains treatment-induced transcriptional signatures from 1,309 bioactive compounds in 4 human cancer cell lines. An input signature can be used to query the database for correlated drug signatures, a technique that has been used previously to identify drugs that combat chemoresistance in cancer (Wei, et al. Cancer Cell (2006) 10:331). In this study we used in silico bioinformatic screening of gene expression profiles from isogenic pairs of Bz sensitive and resistant mouse cell lines derived from the iMycCα/Bcl-xL mouse model of plasma cell malignancy to identify compounds that combat Bz resistance. We established Bz-induced kinetic gene expression profiles (GEPs) in 3 pairs of Bz sensitive and resistant mouse cell lines over the course of 24 hours. GEPs were collected in the absence of large-scale cell death. The 16 and 24 hour time points were averaged and compared between each Bz sensitive and resistant pair. Genes in the sensitive cell line with a fold change greater than 2, relative to the resistant line, were given the binary distinction of “up” or “down” depending on the direction of change. Genes that met these criteria were assembled into signatures, and then used as inputs for CMAP queries to identify compounds that induce similar transcriptional responses. In all pairs, treatment of the Bz sensitive line correlated with GEPs of drugs that target the proteasome, NF-κB, HSP90 and microtubules, as indicated by positive connectivity scores. However eight compounds, all classified as Topoisomerase (Topo) I and/or II inhibitors, were negatively correlated to our input signature. A negative connectivity score could have two interpretations: (1) this could indicate simply that Topos are upregulated by Bz treatment in Bz sensitive lines, which has been previously reported (Congdan, et al. Biochem. Pharmacol. (2008) 74: 883); or (2) this score could be interpreted as Topos are inhibited in Bz resistant cells upon Bz treatment. This led us to ask whether Topo inhibitors could target Bz resistant MM cells and re-sensitize them to Bz. Indeed, we found that multiple Topo inhibitors were significantly more active against Bz resistant cells as single agents and restored sensitivity to Bz when combined with Bz as a cocktail regimen. This work demonstrates the potential of this in silico bioinformatic approach for identifying novel therapeutic combinations that overcome Bz resistance in MM. Furthermore, it identifies Topo inhibitors – drugs that are already approved for clinical use – as agents that may have utility in combating Bz resistance in refractory MM patients. Disclosures: Stessman: Millennium: The Takeda Oncology Company: Research Funding. Van Ness:Millennium: The Takeda Oncology Company: Research Funding.

NEDD4-1 Modulates the Resistance of Multiple Myeloma to Bortezomib

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2068-2068
Author(s):  
Xi Huang ◽  
Enfan Zhang ◽  
Xing Guo ◽  
Jing Chen ◽  
Xuanru Lin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Expression Profiles ◽  
Acquired Resistance ◽  
Gene Array ◽  
Cell Counting ◽  
Control Group ◽  
Protein Levels ◽  
Significant Difference

Abstract Background: Multiple myeloma (MM) is among the most common hematologic malignancies. Proteasome inhibitor bortezomib (Bor) is one of the most effective drugs for treatment of MM. However, during long-term Bor treatment, MM cells may eventually develop acquired-resistance to Bor which results in recurrence and a poor prognosis of MM. Several researches show that E3 ubiquitin ligases (E3s) primarily determine the substrate specificity of ubiquitin proteasome system and play an essential role in Bor resistance of MM. NEDD4-1 E3s, a founding member of the Neural precursor cell-Expressed Developmentally Downregulated gene 4 (NEDD4) family, was proved to involve in the proliferation, migration, invasion of cancer cells and the sensitivity of anticancer therapies. Our current study aims to explore the role and underlying mechanism of NEDD4-1 in acquired resistance of Bor in MM. Methods: The mRNA and protein levels of NEDD4-1 and its substrates in MM cell lines (H929, LP-1, RPMI8226, OPM-2 and ARP-1) and MM patients were detected by Quantitative Realtime PCR and Western Blotting. Lentiviral plasmids containing shRNA against NEDD4-1 were transfected into MM cells. Cell viability, proliferation and apoptosis of MM cells were measured by Cell Counting kit8 (CCK8) and flow cytometry. Gene array was used to compare the gene expression profiles of a panel of Bor treated MM cells vs vehicle-treated MM cells. Results: Gene array showed NEDD4-1 was significantly increased in MM cells treated with Bor. MM cells (CD138+ plasma cells of the bone marrow) from refractory/recurrence patients expressed lower NEDD4-1 than primary patient myeloma cells. Also, MM cell lines H929, ARP-1, LP-1 highly expressed NEDD4-1 at mRNA and protein levels. RPMI8226 and OPM-2 were relatively low expressed. Cell growth assay displayed no significant difference in proliferation between the NEDD4-1 knockdown (KD) and the control group (P>0.05). After suppression of NEDD4-1 using shRNAs, the killing effect of Bor in MM was significantly weaker than the control group (P<0.05). We also found that PTEN was decreased in the NEDD4-1 KD H929 cell line. Otherwise, phospho-STAT3 (ser727) and oncoprotein c-Myc and Bcl-2 were upregulated. Conclusion: Collectively, our study reveals that inhibition of NEDD4-1 can reduce MM sensitivity to Bor via regulating PTEN, c-Myc and Bcl-2, may be related to JAK/STAT signaling pathway, which suggests that NEDD4-1 probably acts as a novel drug target and therapeutic paradigm in the battle against multiple myeloma. Disclosures No relevant conflicts of interest to declare.

Inhibition of the Epigenetic Modifier EZH2 Upregulates Cell Cycle Control Genes to Inhibit Myeloma Cell Growth and Overcome High-Risk Disease Features

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3289-3289 ◽  
Author(s):  
Charlotte Pawlyn ◽  
Michael Bright ◽  
Amy Buros ◽  
Caleb K. Stein ◽  
Zoe Walters ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
High Risk ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Cycle Control ◽  
Myeloma Cell ◽  
Expression Arrays ◽  
Molecular Features ◽  
High Risk Disease

Abstract Introduction High expression of the H3K27 histone methyltransferase EZH2 mRNA in myeloma (MM) patient samples is associated with molecular features of high risk disease, including increased proliferation, and adverse outcomes (1). Mutations or deletions in the H3K27 demethylase KDM6A are associated with similar findings (2) and would be expected to have the same epigenetic effect, increasing H3K27me3 levels, a mark associated with repression of gene expression. We, therefore, sought to identify the role EZH2 plays in controlling myeloma cell proliferation. Methods A panel of MM cell lines and primary patient samples (CD138 selected from bone marrow with consent) representing a variety of different MM molecular subgroups were used. Cell viability (WST-1), cell cycle (PI) and apoptosis (AnnexinV/PI, Caspase-Glo 3/7) assays were performed. Affymetrix gene expression arrays followed by validation with RT-PCR were used to identify patterns of gene expression change with EZH2i. Western blotting confirmed changes at the protein level and Chip-PCR was performed using a validated antibody and isotype control to identify H3K27me3 changes at the relevant gene promotors. Affymetrix gene expression data for 1213 patients enrolled in the Total Therapy studies were used to investigate the relevance of our findings in myeloma patient samples. Results We confirmed a reduction in viability following EZH2i using two chemically distinct, specific small molecule inhibitors (EPZ005687 and UNC1999) and the negative control compound UNC2400. There was a reduction in viability in 6/8 cell lines and 5/6 patient samples. Response to inhibition was not related to molecular subgroup or the presence of high-risk molecular features including del17p. Global levels of H3K27me3 measured by Western blot were reduced in all cell lines regardless of response to EZH2i. In responding cell lines EZH2i induced cell cycle arrest at G1/S followed by induction of apoptosis. Gene expression arrays performed using mRNA from KMS11 and KMM1 cell lines highlighted a change in expression of cell cycle control genes associated with EZH2i. This finding was validated using qRT-PCR, which demonstrated upregulation of the cyclin dependent kinase inhibitors CDKN2B, CDKN1A or both. These findings were confirmed at the protein level by Western blotting. Chip-PCR experiment using cell lysates from KMS11 cells following incubation with EZH2i over 6 days identified changes in H3K27me3 at the promoter and transcriptional start site (PROM/TSS) regions of the CDKN2B and CDKN1A genes. The most specific changes occurred at the CDKN1A PROM/TSS, which were more heavily marked with H3K27me3 at baseline compared to a region approx. 5KB upstream. Given these results, which suggest that CDKN1A expression may be controlled by changes in H3K27me3, we explored the effect of CDKN1A mRNA expression in our patient datasets. We found the expression of EZH2 and CDKN1A to be inversely correlated (R=-0.170, p<0.0001) and that low expression of CDKN1A was associated with a significantly shorter progression free and overall survival (p<0.001). In order to confirm whether these gene expression changes could be used as a potential biomarker of response we looked at our panel of cell lines with variable responses to EZH2i. We identified a consistent increase in expression of CDKN1A only in responding cell lines suggesting it could be used as a biomarker of efficacy in the clinic. Conclusions These data support the hypothesis that CDKN1A expression is suppressed by increased H3K27me3, due to high expression of EZH2 and that this can be reversed with pharmacological EZH2 inhibition leading to a reduction in proliferation of myeloma cells. We provide data which supports the investigation of EZH2i in clinical trials of myeloma patients, which has the potential to be an effective therapeutic strategy even for those with high-risk disease, for whom current treatment approaches are ineffective.Pawlyn et al, EZH2 Overexpression in Myeloma Patients Shortens Survival and in-vitro Data Supports a Potential New Targeted Treatment Strategy. AACR and IMW abstracts, 2015Pawlyn et al, The Spectrum and Clinical Impact of Epigenetic Modifier Mutations in Myeloma. Clinical Cancer Research, 2016 Disclosures Pawlyn: Celgene: Consultancy, Honoraria, Other: Travel Support; Takeda Oncology: Consultancy. Kaiser:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; BMS: Consultancy, Other: Travel Support; Takeda: Consultancy, Other: Travel Support; Chugai: Consultancy. Jones:Celgene: Honoraria, Research Funding. Jackson:Amgen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding, Speakers Bureau. Bergsagel:Novartis: Research Funding; Amgen, BMS, Novartis, Incyte: Consultancy. Morgan:Univ of AR for Medical Sciences: Employment; Janssen: Research Funding; Bristol Meyers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Davies:Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Examination Of Ionizing Radiation, Bortezomib And GSK-3 Inhibition In Several Multiple Myeloma Cell Lines

2011 ◽  
Vol 81 (2) ◽  
pp. S749
Author(s):  
J.S. Silverman ◽  
H.J. Cho ◽  
S.E. Millman ◽  
L. Busino ◽  
A. Mazumder ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Ionizing Radiation ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Multiple Myeloma Cell

scholarly journals Targeted sequencing using a 47 gene multiple myeloma mutation panel (M3P) in -17p high risk disease

2014 ◽  
Vol 168 (4) ◽  
pp. 507-510 ◽  
Author(s):  
Klaus M. Kortüm ◽  
Christian Langer ◽  
Jorge Monge ◽  
Laura Bruins ◽  
Jan B. Egan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Targeted Sequencing ◽  
High Risk Disease

scholarly journals NEK2 Inhibition Enhances the Efficacy of PD-1/PD-L1 Blockade in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2671-2671
Author(s):  
Yan Cheng ◽  
Fumou Sun ◽  
Huojun Cao ◽  
Dongzheng Gai ◽  
Bailu Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
B Cells ◽  
Cell Lines ◽  
Immune Checkpoint ◽  
Mononuclear Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Gene Set Enrichment Analysis ◽  
Myeloma Cells

Abstract Introduction The development of new treatments for high-risk multiple myeloma (HRMM) are needed. The PD-1/PD-L1 axis is one of the chief inhibitory immune checkpoints in antitumor immunity. Despite the success of PD-1 (PDCD1) / PD-L1 (CD274) blockade in some neoplasms, use of it as a monotherapy has failed to improve outcome in RRMM. We have previously demonstrated that the cell-cycle-regulated serine-threonine kinase, NEK2 is elevated in HRMM and that inhibition of NEK2 can overcome drug-resistance and prolong survival of xenografted MM cells. Here, we aimed to investigate the possible role of NEK2 in regulating the immune checkpoint response in MM and development of possible anti-PD1/PDL1 combination therapies. Methods Gene expression profiles and pathway enrichment analyses were conducted on oligonucleotide microarray gene expression profiles from over 1000 primary MM samples to evaluate the correlation of NEK2 and immune checkpoint expression levels. To elucidate the underlying mechanism, we used Nek2 -/- mice crossed with EμMyc mice to generate B cell tumor mouse model with NEK2 deficiency. RNA-sequencing analyses of premalignant B cells was compared between EμMyc/Nek2 WT and EμMyc/Nek2 -/- mice. The hub molecular regulators in the NEK2 correlated pathways were further determined by western blot using NEK2 overexpressing and knockdown cell lines and then verified by co-immunoprecipitation with a NEK2 antibody. Lastly, to establish its clinic significance, the efficacy of INH1 (small compound NEK2 inhibitor), (D)-PPA 1 (peptide-based PD-1/PD-L1 interaction inhibitor) or a PD-L1 (monoclonal antibody) was tested in bone marrow BM mononuclear cells from primary MM patients in-vitro as well as in MM xenografts. Tumor burden and T cell immune responses were monitored by M-spike and mass cytometry. Results Gene expression profiles demonstrated that CD274 expression was significantly higher in the non-proliferative hyperdiploid (HY) subtype of MM, representing between 25-35% of all MM. NEK2 was negatively correlated with CD274 gene expression across all 7 MM subtypes. Gene set enrichment analysis showed that the IFN-γ signaling pathway, which can induce CD274 expression, was significantly enriched in the HY subtype as well as premalignant B cells from EμMyc/Nek2 -/- mice. Elevated expression of EZH2, a histone methyltransferase gene, is also highly correlated wirth NEK2 levels in primary MM. We found that NEK2 inhibition increases CD274 expression as well as reduced EZH2 expression and H3K27me3 levels in MM cell lines. In contrarst, myeloma cells overexpressing NEK2 showed increased expression and activity of EZH2 and H3K27me3 levels. Thus, NEK2 appears to regulate CD274/PD-L1 expression through EZH2-mediated histone methylation. Next we demonstrated that NEK2 and EZH2 directly interact and that overexpression of NEK2 leads to increased methylation of the CD274/PD-L1 gene. We treated BM mononuclear cells from primary MM with PD-1/PD-L1 inhibitor with and without a NEK2 inhibitor. The combination was most effective at eliminating CD138 + myeloma cells while having no effects on T, B and myeloid cell populations. Conclusion Our study showed that expression of CD274/PD-L1 is suppressed in primary HRMM and that CD274/PD-L1 expression is negatively regulated by NEK2 via EZH2-mediated methylation. Inhibition of NEK2 sensitizes myeloma cells to PD-1/PD-L1 blockade, showing either a synergistic or an additive effect in MM cell cytotoxicity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Super-Enhancer Driven Regulation of CKS1B in Multiple Myeloma: Implications in Mediating Response to BET Inhibitor and Celmod Agent Combination

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2658-2658
Author(s):  
Aarif Ahsan ◽  
Ann Polonskaia ◽  
Chih-Chao Hsu ◽  
Chad C Bjorklund ◽  
Maria Ortiz Estevez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
High Risk ◽  
Cell Lines ◽  
Publicly Traded ◽  
Chip Sequencing ◽  
Chromosome 1Q ◽  
Cycle Arrest ◽  
Super Enhancer ◽  
Sequencing Studies

Abstract Introduction: The Myeloma Genome Project (MGP) characterized the genomic landscape of patients with newly diagnosed multiple myeloma (NDMM) (Walker BA, et al. Blood 2018; 132[6]:587-597). Using a multi-omics unsupervised clustering approach, 12 molecularly-defined disease segments were identified (Ortiz M, et al. Blood 2018; 132[suppl 1]:3165). Here, we performed experimental validation of CDC28 Protein Kinase Regulatory Subunit 1B (CSK1B) that was identified as a putative target from the disease segment with poorest clinical outcome. CKS1B was selected for in-depth validation due to their role in cell cycle pathways associated with high-risk disease, biological mechanisms of chromosome 1q amplification and druggability. Methods: Association of CKS1B with outcomes was analyzed in NDMM patients, across relapses and with clinical outcome datasets from MGP and Mayo clinic. Inducible shRNAs of CKS1B and bromodomain containing protein 4 (BRD4, a member of the BET [bromodomain and extra terminal domain] family) were generated in MM cell lines. BRD4 and Aiolos ChIP-seq datasets were analyzed for binding on CKS1B gene. BRD4 inhibitors JQ1 and CC-90010 were utilized for inhibition studies in MM cell lines. Results: Higher expression of CKS1B was associated with significantly poorer PFS, OS, disease severity and relapse. Knock-down of CKS1B in MM cells led to a significant decrease in proliferation (P&lt;0.001) and enhanced apoptosis in MM cell lines. BRD4-ChIP sequencing studies revealed that the expression of CKS1B was regulated by super-enhancer (SE) associated elements. As expected, two BRD4 inhibitors, JQ1 and CC-90010 and inducible BRD4 shRNAs downregulated the expression of CKS1B resulting in decreased proliferation, cell cycle arrest and apoptosis in MM cell lines. Furthermore, MM cell lines harboring chromosome 1q gain/amp showed higher sensitivity to BRD4 inhibition compared to cell lines with normal 1q copy number. Mechanistic studies revealed that BRD4inh and BRD4 shRNAs downregulated the expression of Aiolos and Ikaros in MM cell lines. Interestingly, Aiolos ChIP-sequencing studies demonstrated the binding of Aiolos at the transcriptional start sites of CKS1B with the transcriptional activation mark. The immunomodulatory agent (IMiD ®) pomalidomide (Pom) transcriptionally downregulated CKS1B in Pom-sensitive cells downstream of Aiolos, Ikaros degradation. Based on these mechanisms, IMiD agents, lenalidomide, Pom and the novel Cereblon E3 ligase modulating degrader (CELMoD ®) agent CC-92480 in combination with BRD4inh promoted a synergistic decrease in proliferation, cell cycle arrest and increase in apoptosis in both Pom-sensitive and -resistant cell lines. The combination of IMiD or novel CELMoD agent with BRD4inh also promoted deeper downregulation of CKS1B, Aiolos, Ikaros, c-Myc and survivin proteins with enhanced levels of apoptotic marker cleaved Caspase 3 as compared to single agents alone. Conclusions: In summary, we have identified CKS1B as a key target associated with poor outcome in MM patients. Translational studies suggest a profound downregulation of CKS1B and key pro-survival effector proteins following combination treatment with BRD4inh and IMiD agents/novel CELMoD agents resulting in synergistic anti-tumor effects. These data provide rationale for testing these agents in the clinic for high-risk and IMiD-relapsed patients. Figure: Changes in cell proliferation and protein levels of key signaling mediators were studied in K12PE cell line treated with increasing doses of Lenalidomide, Pomalidomide and CC-92480 in combination with JQ1. Figure 1 Figure 1. Disclosures Ahsan: BMS: Current Employment, Current equity holder in publicly-traded company. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hsu: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bahlis: Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

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