Sulforaphane Synergistically Enhances the Cytotoxicity of Arsenic Trioxide in Multiple Myeloma Cell Lines Through Generation of ER Stress

Abstract Abstract 5140 Persistent paraprotein production in plasma cells necessitates a highly developed rough endoplasmic recticulum (ER) that is exquisitely sensitive to perturbations in protein synthesis. Targeting ER stress- related signaling has been clinically validated in the treatment of multiple myeloma (MM) as evidenced by the response to treatment with bortezomib (BTZ). Despite impressive response rates, BTZ carries the potential for serious side effects, and the development of resistance to BTZ is a clinical issue. We therefore sought to identify novel drug combinations that effectively generate ER stress. Here, we report that sulforaphane, a naturally occurring isothiocyanate found in cruciferous vegetables, synergistically enhances the cytotoxicity of arsenic trioxide (ATO), an agent that has shown clinical activity in MM, in a panel of MM cell lines. As single agents, both 1 μM sulforaphane and 0.5 μM ATO have a modest effect on cellular proliferation in a panel of MM lines. However, when the agents are administered in combination, cellular proliferation is dramatically reduced. For example, in PCNY-1 MM cells, 1 μM sulforaphane has no effect and 0.5μM ATO causes a 29% reduction in proliferation. However, when administered together, the agents enhance growth inhibition to 73%, with a CI of 0.632 indicative of synergy. Four out of 5 MM cell lines tested displayed sulforaphane and ATO synergy. Combination treatment resulted in enhanced apoptotic induction as demonstrated by cleavage of PARP. Enhanced induction of ER stress signaling and activation of the unfolded protein response (UPR) upon combination treatment was demonstrated by enhanced expression of the molecular chaperone HSP90 along with increased phosphorylation of PERK (an ER transmembrane kinase and proximal effector of the UPR) and eIF2 (translational initiation factor). Additionally, increased splicing of XBP1 (a transcription factor of UPR target genes) was apparent upon combination treatment as compared to treatment with either agent alone. Our results show that sulforaphane can synergistically sensitize MM cells to the cytotoxic effects of ATO through promotion of ER stress generating mechanisms. Based upon these promising results, further evaluation of this safe, natural product as an ATO sensitizer in a clinical trial of MM patients is warranted. Additionally, this approach holds the promise as a means to identify and clinically validate natural products effective in the treatment of MM and/or inhibition of progression of asymptomatic MM. Disclosures: No relevant conflicts of interest to declare.

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Membrane Lipid Biosynthesis As a Potential Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.1836.1836 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1836-1836

Author(s):

Carolyne Bardeleben ◽

Alan Lichtenstein

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Cell Lines ◽

Cell Apoptosis ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Membrane Lipid ◽

Dependent Manner ◽

Kennedy Pathway ◽

Phosphatidylcholine Synthesis

Abstract Abstract 1836 Phosphatidylcholine (PC) is the most prominent phospholipid in mammalian endoplasmic reticulum (ER) membranes. The rate-limiting step in PC synthesis through the Kennedy pathway is the conversion of phosphocholine + cytidine triphosphate (CTP) to cytidine diphosphocholine, (CDP)-choline, via the enzyme CTP:phosphocholine cytidylyltransferase (CCT) (see figure). Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. In contrast, similar concentrations of LPC in the same cell lines could not rescue apoptosis induced by bortezomib. An additional intervention to inhibit phosphatidylcholine synthesis, namely inducing pyrimidine starvation, also resulted in MM cell apoptosis and down-regulation of CDP-choline levels. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy. Disclosures: No relevant conflicts of interest to declare.

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Predictive Simulation Based Design and Validation Of Repurposed Novel Therapeutics With Multi-Target Mechanisms For Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3859.3859 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3859-3859

Author(s):

Nicole A Doudican ◽

Shireen Vali ◽

Shweta Kapoor ◽

Anay Talawdekar ◽

Zeba Sultana ◽

...

Keyword(s):

Multiple Myeloma ◽

Growth Inhibition ◽

Cell Lines ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Single Agent ◽

Biological Mechanisms ◽

Minimal Growth ◽

Predictive Simulation ◽

Simulation Based

Abstract Introduction Development of resistance to single agent therapy is a significant clinical obstacle in the treatment of multiple myeloma (MM). Genetic mutations and the bone marrow micro-environment are major determinants of MM resistance mechanisms. Given the complexity of MM, the need for combinatorial therapeutic regimens targeting multiple biological mechanisms of action is pressing. Repurposing has the advantage of using drugs with known clinical history. Methodology We used a predictive simulation-based approach that models MM disease physiology in plasma cells by integrating and aggregating signaling and metabolic networks across all disease phenotypes. We tested the efficacy of over 50 repurposed molecularly targeted agents both individually and in combination across simulation avatars of the MM cell lines OPM2 and U266. OPM2 harbors mutations in KRAS, CDKN2A/2C, PTEN, RASSF1A and P53, whereas U266’s mutational components include BRAF, CDKN2A, P53, P73, RASSF1A and RB1. These cell lines were used as models because they possess mutations in genes classically known to be involved in myeloma. The predicted activity of novel combinations of existing drug agents was validated in vitro using standard molecular assays. MTT and flow cytometry were used to assess cellular proliferation. Western blotting was used to monitor the combinatorial effects on apoptotic and cellular signaling pathways. Synergy was analyzed using isobologram plots and the Bliss independence model. Results Through simulation modeling, we identified two novel therapeutic regimens for MM using repurposed drugs: (1) AT101 (Bcl2 antagonist) and tesaglitazar (PPAR α/γ agonist) and (2) Ursolic acid (UA, inhibitor of NFκβ) and SP600125 (pan-JNK inhibitor). Simulation predictions showed that combining the IC30 concentrations with respect to viability of AT101 and tesaglitazar reduced proliferation by 40% and viability by 50%. Similarly simulation predictions showed that the combination of the IC30 concentrations of UA and SP600125 reduced proliferation by 50% and viability by 40%. Corroborating our predictive simulation assays, 10 µM tesaglitazar and 2 µM AT101 caused minimal growth inhibition as single agents in OPM2 and U266 MM cell lines. Growth inhibition in these cell lines is synergistically enhanced when the drugs are used in combination, reducing cellular viability by 88% and 77% in OPM2 and U266 cells, respectively. Similarly, proliferation was reduced by 34% with 7.5 μM UA and 25% with 10 μM SP600125 in OPM2 cells. When used in combination, cellular proliferation was synergistically reduced by 64%. In addition, isobologram analysis predicted synergy of lowered doses of the drugs in combination. Both combinations synergistically inhibited proliferation and induced apoptosis as evidenced by an increase in the percentage sub-G1 phase cells and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Conclusions These results highlight and validate the use of our predictive simulation approach to design therapeutic regimens with novel biological mechanisms using drugs with known chemistries. This allows for design of personalized treatments for patients using their tumor genomic signature beyond the “one-gene, one-drug” paradigm. The reuse of existing drugs with clinical data facilitates a rapid translational path into clinic and avoids the uncertainties associated with new chemistry. The corroboration of these results with patient derived cell lines will be pursued and discussed. Disclosures: No relevant conflicts of interest to declare.

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Adenylate Kinase 2 Is a Selective Dependency in NSD2-High Multiple Myeloma

Blood ◽

10.1182/blood-2020-141266 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-31

Author(s):

Amin Sobh ◽

Charlotte Kaestner ◽

Jianping Li ◽

Alberto Riva ◽

Richard Lynn Bennett ◽

...

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Candidate Genes ◽

Cell Lines ◽

Adenylate Kinase ◽

Conflicts Of Interest ◽

Essential Gene ◽

Gene Knockout ◽

A Genome

Background: Multiple myeloma (MM)-associated t (4;14) chromosomal translocation leads to overexpression of NSD2, the histone H3 lysine 36 specific methyltransferase. t(4;14) MM patients have a high risk of relapse and NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. The lack of a specific and potent NSD2 inhibitors mandates finding alternative strategies for treating NSD2-high MM. Aim: This study aims to test the hypothesis that NSD2 overexpression in MM cells generates cellular vulnerabilities that can be therapeutically exploited for treatment of t (4;14) MM. Methods: We conducted a genome wide CRISPR-based loss-of-function genetic screen using the human Brunello library in isogenic NSD2-high (NTKO) and NSD2-low (TKO) KMS-11 derived MM cells to define genes whose loss is selectively detrimental to cells with NSD2 overexpression. The cellular dependency of each identified candidate was then investigated across hundreds of human cell lines using the Cancer Dependency Map portal (www.Depmap.org). Candidate genes were validated using CRISPR-Cas9 gene knockout and shRNA knockdown of individual target genes followed by in vitro competitive growth assays and cell viability assays. Results: Our study revealed multiple candidate genes with increased dependency in NSD2-high cells including the adenine nucleotide regulator Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible conversion of ADP to AMP and ATP and can thus modulates energy balance within the cell. Dependency map analysis showed that AK2 is not a commonly essential gene. The top enriched lineages with AK2 dependency included MM with notable representation of t(4;14)-positive MM cell lines. The increased dependency of NTKO and other t (4;14) MM cells on AK2 was confirmed by in vitro competition assays. Disruption of AK2 in TKO cells had a minimal effect on cellular fitness but the dependency on AK2 was restored upon engineered overexpression of NSD2 in these cells. In addition, NSD2-high cells displayed higher sensitivity to the proteasome inhibitor bortezomib than NSD2-low cells suggesting elevated levels of endoplasmic reticulum (ER) stress in cells overexpressing NSD2. Elevated ER stress necessitates increased levels of ATP to refold proteins and could underlie the increased dependency of NSD2-high cells on AK2. Notably, suppression of AK2 increased bortezomib sensitivity in t (4;14) MM cell lines. Conclusions: Our findings indicate that NSD2 high t(4;14) MM may have a vulnerability due to increased proteostatic stress. Accordingly, AK2 inhibition could be used in combination with proteasome inhibitors to treat MM patients with t (4;14) translocations by inducing the accumulation of lethal levels of unfolded proteins. Disclosures No relevant conflicts of interest to declare.

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The Cell Cycle Regulator CDC25A Is a Target for JAK2V617F Oncogene

Blood ◽

10.1182/blood.v118.21.1385.1385 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1385-1385

Author(s):

Emilie-Fleur Gautier ◽

Christian Recher ◽

Camille Laurent ◽

Caroline Marty ◽

Jean-Luc Villeval ◽

...

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Primary Myelofibrosis ◽

Initiation Factor ◽

Proliferative Potential ◽

Erythroid Progenitors ◽

Translational Initiation

Abstract Abstract 1385 The JAK2V617F mutation is present in the majority of polycythemia vera (PV) patients and half of those with essential thrombocythemia and primary myelofibrosis. JAK2V617F is a gain-of-function mutation resulting in constitutive JAK2 signalling involved in the pathogenesis of these diseases. JAK2V617F has been shown to promote S phase entry. Here, we demonstrate that the CDC25A phosphatase, a key regulator of the G1/S cell cycle transition, is constitutively over-expressed in JAK2V617F -positive cell lines, JAK2-mutated patient CD36+ progenitors and in vitro differentiated proerythroblasts. Accordingly, CDC25A is also overexpressed in bone marrow and spleen of Jak2V617F knock-in mice compared to wild-type (WT) littermates. Using murine FDC-P1-EPOR and human HEL and SET-2 cell lines, we found that JAK2V617F-induced CDC25A up-regulation was not caused by increased transcription or CDC25A stability or the involvement of its known upstream regulators AKT and MAPK. Instead, our results suggest that CDC25A is originaly regulated at the translational level through the implication of the transcription factor STAT5 and the translational initiation factor eIF2α. CDC25A inhibition reduces the clonogenic and proliferative potential of JAK2V617F-expressing erythroid progenitors, while moderately affecting normal erythroid differentiation. These results suggest that CDC25A deregulation may be involved in hematopoietic cells expansion in JAK2V617F patients, making of this protein an attracting potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.

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Identification of the Black Tea Polyphenol Theaflavin-3, 3'-Digallate From Screen of Natural Product Inducers of Endoplasmic Reticulum Stress

Blood ◽

10.1182/blood.v120.21.5021.5021 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5021-5021

Author(s):

Nicole A Doudican ◽

Shih Ya Wen ◽

Amitabha Mazumder ◽

Seth J Orlow

Keyword(s):

Multiple Myeloma ◽

Endoplasmic Reticulum ◽

Natural Products ◽

Er Stress ◽

Natural Product ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Black Tea ◽

Parp Cleavage ◽

Tea Polyphenol

Abstract Abstract 5021 Background: A distinguishing characteristic of myeloma plasma cells is the large quantity of paraprotein that they synthesize and secrete, rendering them especially sensitive to the effects of endoplasmic reticulum (ER) stress. Consistent with this notion, the proteasome inhibitor bortezomib disrupts protein equilibrium in the ER by preventing misfolded proteins from being properly degraded. Given the clinically validated importance of targeting ER stress mediated pathways in the treatment of multiple myeloma (MM), we sought to identify natural products that modulate pathways known to be an effective therapeutic target for MM for potential use to inhibit progression of asymptomatic MM to symptomatic MM without the limiting side effects of current targeted therapies. Methods: Using decreased protein processing in the secretory pathway as a measurable hallmark of ER stress, our screen employed the naturally secreted Gaussia luciferase (Gluc) as a reporter that can be easily monitored through extracellular release of luciferase activity in real time. KMS11 and ARP-1 MM cells expressing Gluc were exposed to compounds in our natural products library in order to identify those which potentially induce ER stress as measured by inhibition of Gluc secretion. The growth inhibitory activity of theaflavin-3, 3'–digallate (TF3) was further characterized by MTS assay. Mechanistic studies of ER stress related pathways including the unfolded protein response (UPR) and apoptotic cascades were analyzed by standard Western blotting techniques. Results: Our screen identified the black tea polyphenol TF3 as a significant inhibitor of GLUC secretion in ARP-1 and KMS-11 cells. TF3 at 0. 5 μM inhibits GLUC secretion by 73 and 68% in ARP-1 and KMS-11 cells, respectively. This inhibition observed is on par with that observed for bortezomib and tunacamycin (a well known inducer of ER stress). TF-3 effectively inhibits cellular proliferation and induces apoptosis in a panel of MM cell lines at physiologically achievable concentrations. Apoptotic induction is at least partially mediated by ER stress mediated pathways as upregulation of the protein chaperone HSP90 and phosphorylation of eIF2-a, a key mediator of the UPR pathway, occurs prior to caspase and PARP cleavage. Conclusions: Our results suggest that TF-3 inhibits protein secretion and MM cell growth through promotion of ER stress generating mechanisms. Based upon these promising results, further mechanistic evaluation and characterization of this safe, natural product as a prophylactic agent in the treatment of asymptomatic conditions like monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) is warranted. Disclosures: No relevant conflicts of interest to declare.

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Super-Enhancer Profiling Identifies Novel Oncogenes and Therapeutic Targets in Multiple Myeloma

Blood ◽

10.1182/blood-2019-124252 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 362-362

Author(s):

Jianbiao Zhou ◽

Yunlu Jia ◽

Tze King Tan ◽

Tae-Hoon Chung ◽

Takaomi Sanda ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Plasma Cell ◽

Conflicts Of Interest ◽

Cell Cancer ◽

Ectopic Expression ◽

Therapeutic Targets ◽

Physical Interaction ◽

Rna Seq ◽

Normal Plasma

Background: Multiple myeloma (MM) is an aggressive neoplastic plasma cell cancer characterized by diversely cytogenetic abnormalities. MM can be divided into subtypes with immunoglobulin heavy chain (IGH) gene translocations involving CCND1-3, FGFR3/MMSET, MAFs and hyperdiploid myeloma containing trisomies of several odd numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and 21. Although several new drugs have been introduced into clinic, treatment for MM patients remains challenge and refractory/resistant to therapy is often seen. Thus, a better understanding of the molecular pathogenesis of MM can lead to generate new prognostic classification and identify new therapeutic targets. Super-enhancers (SEs) are defined as large clusters of cis-acting enhancers, marked by high level bindings of acetylation of histone H3 lysine 27 (H3K27ac) and mediator complex. SEs have been shown to control genes for maintaining cellular identity and also key tumor drivers in various malignancies. Methods: H3K27Ac ChIP-seq and RNA-seq were performed on primary MM patient samples, MM cell lines. Normal plasma cells and lymphoma cell lines were served as controls. We systematically compared SEs and their associated genes of normal and cancerous tissue. THZ1, a CDK7 inhibitor, was used to efficiently down-regulate SE-associated genes. Combinatory analysis of THZ1-sensitive and SE-associated gene revealed a number of promising MM oncogenes. CRISPR/Cas9 technology and ectopic expression experiments in conjunction with cellular functional assays were performed to determine the effects of candidate SE-genes on MM cells. Circularized chromatin conformation capture followed by sequencing (4C-seq) was applied to explore the direct contact of SE and promoter. Results: SE analysis uncovered some cell lineage-specific transcription factors (TFs) and known oncogenes in MM. Several key TFs, including IRF4, PRDM1, MYC and XBP1, were identified in most MM samples, confirming the origin of MM cells. These data reinforce the concept that SE establishment is a key component of MM biology. The acquisition of SEs around oncogene drivers is widely observed during tumorigenesis. ST3GAL6 and ADM were two known oncogenic drivers in myeloma cells, which were associated with super-enhancers in all MM samples but not in normal plasma cell and lymphoma cells. We also found SE constituents for multiple subtype-specific key oncogenes such as CCND1 in t(11;14) cells, C-MAF in t(14;16) cells, and NSD2 and FGFR3 in t(4;14) cells. Furthermore, THZ1 showed prominent anti-neoplastic effect against MM cells. SE-associated genes were more sensitive to THZ1 compared with those genes associated with typical enhancers (TEs). By overlapping THZ1-sensitve gene with SE-associated genes, we identified a number of novel MM oncogenes, including MAGI2, EDEM3, HJURP, LAMP5, MBD1 and UCK2 as a potential druggable kinase. The expression level of MAGI2 and HJURP confers poor prognosis in several MM datasets. MAGI2 silencing in MM cells decreased cell proliferation and induced apoptosis. qRT-PCR and Western blot analysis confirmed the overexpression of HJURP in t(4;14) cells relative to non-t(4;14) MM cells. Furthermore, 4C-seq analysis revealed the physical interaction between HJURP-SE and promoter and THZ1 treatment diminished this interaction. Motif search at SE constituents revealed a highly significant enrichment of NSD2 recognition. Significant reduction of NSD2 binding at HJURP-SE region was observed in KMS11 infected with NSD2-specific shRNAs. Interestingly, blocking SE sites by CRISPR/Cas9i or silencing HJURP by shRNA led to decreased HJURP expression and cell apoptosis, whereas overexpression of this gene promoted cell growth. Taken together, our data demonstrated that HJURP is a novel SE-associated oncogene in t(4;14) MM. Conclusions: Our integrative approaches by combing H3K27Ac ChIP-seq, RNA-seq and THZ1-sensitive transcript defined the landscape of SE and identified SE-associated novel oncogenes, as well as lineage-specific TFs in MM. Furthermore, we also revealed subtype-specific SE-driving oncogenic program in MM. Taken together, these results not provide novel insight into the MM pathology, but also offer novel, potential therapeutic targets, such as MAGI2, and HJURP for the treatment of MM patients. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v110.11.242.242 ◽

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.

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Delineation of Canonical and Non-Canonical NF-κB Pathways in Multiple Myeloma: Therapeutic Implications.

Blood ◽

10.1182/blood.v110.11.670.670 ◽

2007 ◽

Vol 110 (11) ◽

pp. 670-670

Author(s):

Teru Hideshima ◽

Noopur Raje ◽

Ruben Carrasco ◽

Hiroshi Ikeda ◽

Yutaka Okawa ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Combination Treatment ◽

Inhibitory Effect ◽

Significant Inhibition ◽

Canonical Pathway ◽

Mobility Shift ◽

Therapeutic Implications ◽

Real Time Quantitative Pcr ◽

Mobility Shift Assay

Abstract NF-κB pathway plays a crucial role in the pathogenesis in cancer cells including multiple myeloma (MM). The NF-κB complex is dimer in different combinations of Rel family proteins, including p65 (RelA), RelB, c-Rel, p50 (NF-κB1), and p52 (NF-κB2). Recent studies have revealed that NF-κB activity is mediated via two distinct pathways. In the canonical pathway, NF-kB is typically a heterodimer composed of p50 and p65 subunits. In the non-canonical pathway, NF-kB is typically a heterodimer composed of RelB and p100 subunits. We have shown anti-MM activities of IKKβ inhibitors (PS-1145, MLN120B); however, effects of these agents were modest. Our studies therefore suggest that baseline NF-kB activity in MM cells is not totally dependent on the canonical pathway, and that inhibition of only canonical NF-κB pathway may not be sufficient to block total NF-kB activity. In this study, we therefore hypothesized whether non-canonical inhibitors significantly enhanced NF-κB inhibition induced by canonical inhibitors in MM cells. We first examined baseline NF-κB activity using electrophoretic mobility shift assay (EMSA). NF-κB activity varied between cell lines; for example MM.1S, MM.1R and H929 cells have higher level of NF-κB activity than in RPMI8226, INA6 and OPM2 cells. To define the role of canonical and non-canonical pathway, we next examined protein expression of p50, p65 and p52 NF-κB in these cell lines: p65 was highly expressed in all MM cell lines; however, expression of p50 and 52 is variable. Surprisingly, no detectable or weak expression of p50 was observed in U266, RPMI8226, LR5, H929 and OPM2 cell lines, suggesting that baseline NF-kB activity in these cell lines is not maintained only by the canonical pathway. We then attempted to block non-canonical NF-κB pathway in MM cell lines. Specifically since IKKα and IKKβ are client proteins of heat shock protein (Hsp) 90, we examined whether 17AAG could inhibit expression and/or function of IKKα and IKKβ in MM cells. Importantly, both IKKα and IKKβ were significantly downregulated by 17AAG in MM cell lines. To determine whether downregulation of these IKK proteins by 17AAG was due to inhibition of transcription, we next performed real-time quantitative PCR and no significant inhibition of relative expression of IKKβ was observed by 17AAG treatment, suggesting that downregulation of these proteins was a post transcription event. We further examined whether 17AAG enhanced the effect of IKKβ inhibitor MLN120B on NF-κB activity. Although the inhibitory effect by either MLN120B or 17AAG alone on phosphorylation (p) of IκBα triggered by TNFα was marginal, combination treatment of MLN120B with 17AAG almost completely blocked IκBα, suggesting that this combination synergistically inhibit canonical NF-κB activity in MM cells. Importantly, the combination of MLN120B with 17AAG also significantly blocked baseline and TNFα-triggered NF-κB activity, assessed by EMSA, in MM cells. Finally, 17AAG augmented the growth inhibitory effect of MLN120B in the context of bone marrow stromal cells. Taken together, these results showed that baseline and TNFα-triggered NF-κB activities were completely blocked by this combination treatment, and provide the rationale for its clinical evaluation to induce maximum inhibition of NF-κB activity and improve patient outcome in MM.

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RFP2 Knockdown Disrupts Cell Growth and Induces Apoptosis in Multiple Myeloma

Blood ◽

10.1182/blood.v112.11.5327.5327 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5327-5327

Author(s):

Moshe E. Gatt ◽

Mala Mani ◽

Jui Dutta ◽

Daniel E. Carrasco ◽

John D Shaughnessy ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Tumor Suppressor ◽

Er Stress ◽

Cell Lines ◽

Growth Retardation ◽

Copy Number ◽

Suppressor Gene ◽

Phase Arrest ◽

G2 Phase

Abstract Background: Multiple Myeloma (MM) is characterized by a clonal proliferation of antibody producing malignant plasma cells. Complete or partial monoallelic deletion of chromosome 13, is commonly observed in tumor cells of patients with monoclonal gammopathy of unknown significance and in over 50% of MM patients, as well as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Recurrent loss of a minimal common region (MCR) of 10 megabases at 13q14, in MM and CLL suggests the MCR harbors a tumor suppressor gene(s) (TSG) with biological and clinical relevance. Within this MCR resides the Ret Finger Protein 2 (RFP2) encoding gene, which produce an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). Because of its copy number-dependent expression, its strong and unique promoter, and its associated inferior survival with reduced expression in MM, RFP2 represents a candidate TSG. Nevertheless, its role and targets have not yet been established. Here we describe a functional analysis of RFP2 in MM cells. Methods: The MMS1 MM cell line lacks chromosome 13 deletion. To study the effects of loss of RFP2 in this line we used the PLKO-GFP lentiviral vector to stably transduce a RFP2 shRNA. Flow cytometer selected cell lines exhibit significantly reduced expression of RFP2 relative transduced shRNA controls or to the parental line. Cell growth rate was measured using trypan blue counting, soft agar colony formation and thymidine incorporation. Cell cycle analysis and apoptosis were measured by flow cytometry after staining with PI or Annexin-V PE and 7AAD, respectively. Intracellular signal modulation was demonstrated by Western blotting. Results: At day six post transduction, 75–95% of MMS1 cells were GFP positive. RFP2 downregulation induced an impairment of cell growth with a G2 phase arrest and a profound apoptosis (over 50% at day six as compared with less than 15% of controls). This effect was mediated through ER stress evidenced by upregulation of p-eIF2α and Bip, and the induction of Caspase-8, 9 and 3 cleavage. These effects could be abrogated by the ZVAD-FMK pancaspase inhibitor and by overcoming the G2 phase arrest with caffeine. Similar results were observed in MM cell lines RPMI-8226, NCI-H929, MM1S, and SACHI, and were independent of presence of a monoallelic 13q deletion. RFP2 complementation did not produce by itself a significant growth promoting effect, but was able to rescue the knockdown-induced growth retardation. In order to identify potential RFP2 target proteins, RFP2 was immunoprecipitated from MM cell lines. RFP2 protein complexes are currently being analyzed by mass-spectometry and results of these studies will be presented. Conclusions: RFP2 is a copy number sensitive gene mapping to a deletion hotspot at 13q14 and reduced RNA expression is associated with poor survival in MM. Functional studies revealed that shRNA mediated knockdown of RFP2 in MM causes growth retardation and apoptosis, mediated by ER stress and a G2 arrest. Although RFP2 did not prove itself to be a tumor suppressor gene in our studies, disrupting RFP2 function may represent a novel therapeutic target in MM and other lymphoid malignancies.

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HOXA9 Is a Novel Therapeutic Target in Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.832.832 ◽

2009 ◽

Vol 114 (22) ◽

pp. 832-832 ◽

Cited By ~ 1

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.

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