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

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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Functional CRISPR Screening Identifies Ptprg As a Driver of Migration and Adhesion in NSD2-E1099K ALL

Blood ◽

10.1182/blood-2021-154009 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1149-1149

Author(s):

Charlotte L Kaestner ◽

Amin Sobh ◽

Jianping Li ◽

Alberto Riva ◽

Richard Lynn Bennett ◽

...

Keyword(s):

Candidate Genes ◽

Cell Lines ◽

Essential Gene ◽

Gene Knockout ◽

Lymphoblastic Leukemia ◽

Tyrosine Phosphatase ◽

Brain Parenchyma ◽

High Dose ◽

Loss Of Function

Abstract Background: Acute Lymphoblastic Leukemia (ALL) is the most common childhood cancer and frequently infiltrates the central nervous system (CNS). CNS-directed therapy is currently limited to intrathecal and systemic high-dose methotrexate, or less commonly craniospinal irradiation, both of which are associated with substantial neurotoxicity. A lack of mechanistic understanding of the mechanisms of CNS infiltration presents an obstacle for the development of more specific and less toxic therapeutic approaches. We previously showed that ALL cells with a specific mutation (E1099K) in the histone methyltransferase NSD2 have aggressive CNS tropism by not only infiltrating the leptomeninges but also the brain parenchyma in murine xenografts models. Analysis of cBioPortal data shows that NSD2-E1099K is associated with a higher rate of testicular involvement in ALL also suggesting more aggressive infiltration behavior of the tumor. Accordingly, using gene editing to revert mutant NSD2 back to wild-type, we also showed that NSD2-E1099K cells have an enhanced ability to migrate and adhere in vitro. RNA-seq data on four NSD2-E1099K cell lines revealed genes that may play a role in ALL brain infiltration. However, it remains unknown which of those upregulated genes could be potential therapeutic targets against CNS leukemia. Aim: This study aims to Identify therapeutically targetable genes that are important for migration of NSD2-E1099K ALL cells Methods: Using a focused CRISPR-gene-knockout library of 5600 sgRNAs directed against 500 genes upregulated in NSD2-E1099K cells, we ascertained the necessity of the selected genes for migration in the RCH-ACV cell line. Candidate genes were evaluated for cellular dependency using a CRISPR-loss of function screen and the cancer dependency map portal. Overexpression of the candidate genes in NSD2-E1099K cell lines was confirmed with qPCR analysis. Candidate genes were validated by individual shRNA knockdown followed by migration and adhesion assays. Results: Our study identified genes whose knockout led to enhancement of migration and others whose knockout resulted in inhibition of migration. Protein Tyrosine Phosphatase Receptor Type G (PTPRG) was one of the top candidate genes whose knockout resulted in inhibition of migration. Dependency map analysis showed that PTPRG is not a commonly essential gene and a CRISPR-based-loss-of function screen performed in parallel to the migration screen confirmed that ALL cell survival is not dependent on PTPRG. We also found that PTPRG is overexpressed in multiple NSD2-E1099K ALL cell lines. Individual Knockdown of PTPRG in NSD2-E1099K ALL cell lines not only inhibited migration, but also led to a loss of adhesion ability to endothelial cells of the Blood Brain Barrier. Conclusions: Our findings implicate PTPRG as an important modulator of migration and adhesion in ALL cells and a potential therapeutic target for preventing ALL brain infiltration, especially in NSD2-E1099K ALL. Disclosures Licht: Epizyme: Research Funding.

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Investigational Agent MLN2238/MLN9708, a Specific, Orally Available, Small Molecule Proteasome Inhibitor, Shows Promising In Vitro Activity Against Multiple Myeloma Cell Lines

Blood ◽

10.1182/blood.v116.21.3014.3014 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3014-3014

Author(s):

Giada Bianchi ◽

Vijay G. Ramakrishnan ◽

Teresa Kimlinger ◽

Jessica Haug ◽

S. Vincent Rajkumar ◽

...

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Cell Lines ◽

Small Molecule ◽

Proteasome Inhibitor ◽

Research Funding ◽

Proteasome Inhibitors ◽

Biologically Active ◽

Investigational Agent

Abstract Abstract 3014 Background: Proteasome inhibitors have proven particularly effective in treatment of multiple myeloma, the second most frequent hematologic malignancy in the western world. Bortezomib, the first in class proteasome inhibitor in clinical use, was first approved in 2003 via fast FDA track, given the remarkable activity shown during phase II clinical trials. Nevertheless, more than 50% of multiple myeloma patients did not respond to single agent bortezomib when administered as second line agent. Moreover, bortezomib is only available for intravenous administration, representing a cumbersome therapy for patients, and its use is limited by significant toxicities (especially peripheral neuropathy). MLN9708 (Millennium Pharmaceuticals, Inc.), an investigational orally available, small molecule, is a potent, specific and reversible inhibitor of the 20S proteasome. It is currently under clinical investigation for the treatment of hematologic and non-hematologic malignancies. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form, and MLN2238 was used for all of the preclinical studies reported here. In vitro biochemistry studies have shown that MLN2238 has a faster dissociation rate from the proteasome compared to bortezomib, and in vivo studies of MLN2238 have shown antitumor activity in a broader range of tumor xenografts when compared to bortezomib. Given these encouraging preclinical results, we set to investigate the anti-myeloma activity of MLN2238 in vitro. Results: MLN2238 proved to have anti-proliferative and pro-apoptotic activity against a broad range of MM cell lines with EC50 at 24 hours ranging between 10 and 50 nM, even in relatively resistant MM cell lines (OPM2, DOX6, RPMI, etc.). In MM.1S cells, induction of apoptosis was time and dose dependent and related to activation of both caspase 8 and 9. When compared to MM.1S treated for 24 hours with EC50 dose of bortezomib, treatment with EC50 dose of MLN2238 resulted in the same extent of caspases cleavage occurring at an earlier time point (8-12 hours), possibly suggesting more rapid onset and/or irreversibility of apoptosis in cells treated with MLN2238. Treatment with MLN2238 was associated with early, but persistent induction of endoplasmic reticulum (ER) stress with BiP being induced 2–4 hours after treatment with EC50 dose and gradually increasing over time. While bortezomib has been associated with early induction and late decrease in proteins involved in ER stress, MLN2238 appears to induce a persistent rise in these factors, suggesting either more sustained proteasome blockade with stabilization of proteasome substrates or de-novo induction of unfolded protein response (UPR) genes. MLN2238 also proved effective in reducing phosphorylation of ERK1-2 with no overall alteration in the total ERK level, thus accounting for the observed reduction in proliferation upon treatment. Preliminary data indicate potential for additive and synergistic combination with widely used drugs, including doxorubicin and dexamethasone. Conclusion: While further clinical data are needed to establish the effectiveness of MLN2238 in the treatment of multiple myeloma, these preliminary nonclinical data, together with the favorable biochemical and pharmacokinetic properties, including oral bioavailability, make the investigational agent MLN9708 an appealing candidate for treatment of multiple myeloma. Further in vitro data could help establish whether a difference in the apoptotic mechanisms exist between MLN2238 and other proteasome inhibitors, primarily bortezomib, and could also help inform combination treatment approaches aimed at increasing effectiveness, overcoming bortezomib resistance and decreasing toxicity. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Research Funding; Novartis: Research Funding; Genzyme: Consultancy, Research Funding; Cephalon: Research Funding.

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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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An Investigational Proteasome Inhibitor MLN9708 (MLN2238) Induces Apoptosis In Human Multiple Myeloma Cells In Vitro

Blood ◽

10.1182/blood.v116.21.3066.3066 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3066-3066

Author(s):

Aisha Masood ◽

Kasyapa Chitta ◽

Kiersten M Miles ◽

Nazmul H Khan ◽

Remi Adelaiye ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Conflicts Of Interest ◽

Active Form ◽

Biologically Active ◽

Mitochondrial Outer Membrane ◽

Dependent Manner ◽

In Vivo Models

Abstract Abstract 3066 Targeting the proteasome has proven to be one of the most effective therapeutic strategies in the treatment of multiple myeloma (MM), and the proteasome inhibitor bortezomib is approved for treatment of MM. However its clinical efficacy is compromised by the acquired resistance in patients, necessitating the development of new therapeutics. Several new proteasome inhibitors are under investigation for their therapeutic efficacy in MM. MLN9708 (Millennium Pharmaceuticals, Inc., Cambridge, MA) is a proteasome inhibitor which shows refined pharmacokinetic and pharmacodynamic properties in preclinical studies and is currently in Phase I clinical development. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to MLN2238, the biologically active form. MLN2238 was used for all of the studies reported here, in which we report the efficacy of MLN2238 on three established MM cell lines-KMS11, OPM2 and U266. MLN2238 was found to inhibit the chymotrypsin-like proteasomal activity of all MM cell lines in a dose dependent manner. Investigation of the IC50 of MLN2238 on these cell lines demonstrated that KMS11 is the most sensitive (IC50 of 15.9 nM) while U266 was found to be the least sensitive cell line (IC50 of 511 nM). OPM2 cells also showed intermediate sensitivity with an IC50 of 58.6 nM. MLN2238 induced apoptosis in KMS11 cells as evidenced by annexin V staining and PARP-1 cleavage. Cleavage of caspases 9 and 3 suggested activation of the intrinsic apoptotic pathway by MLN2238. Furthermore, MLN2238 treatment was shown to increase the mitochondrial outer membrane permeability (MOMP) and decrease BCL-2 levels. Evaluation of the expression of PSMB5, the preferred proteasomal subunit target for both bortezomib and MLN2238, revealed that it is expressed at approximately 3 fold more in KMS11 cells as compared to U266, suggesting a possible mechanism for higher sensitivity of KMS11 to the proteasomal inhibitor, MLN2238. This preclinical evaluation confirms the anti-myeloma effects of MLN2238, warranting further in-depth evaluation in both in vitro and in vivo models of MM. Disclosures: No relevant conflicts of interest to declare.

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HIF 1 Alpha: A Suitable Target for Multiple Myeloma

Blood ◽

10.1182/blood.v118.21.2901.2901 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2901-2901 ◽

Cited By ~ 2

Author(s):

Giulia Perrone ◽

Enrica Borsi ◽

Carolina Terragna ◽

Sandra Durante ◽

Marina Martello ◽

...

Keyword(s):

Multiple Myeloma ◽

Solid Tumors ◽

Cell Lines ◽

Conflicts Of Interest ◽

Critical Role ◽

Flow Cytometric Analysis ◽

Parp Cleavage ◽

Survival Assay ◽

The Impact

Abstract Abstract 2901 Hypoxia-inducible factor-1 alpha (HIF1 α) is a transcription factor that plays a critical role in survival and angiogenesis. In solid tumors, elevated expression of HIF-1 α, in response to hypoxia or activation of growth factor pathways, is associated with tumor proliferation, metastasis, and drug resistance and correlated with poor prognosis. In contrast to solid tumors, the role of HIF1 α in hematological malignancies is not completely known. In particular in multiple myeloma (MM) HIF1 α has been suggested to be constitutively expressed and HIF1 α knockdown cell lines have shown higher sensitivity to standard chemotherapy, suggesting a role in the pathophysiology of MM. In the present study, we explored the effect of EZN2968, an antisense oligonucleotide against HIF1 α, as a molecular target in MM. We showed, using real time PCR, and Western blotting analysis, that the expression of HIF1 α in several MM cell lines (MM1S, U266, OPM2, RPMI8226) is detectable under conditions of normoxia or hypoxia and is increased in the presence of growth stimuli (IL-6 and stroma cells). The immunofluorescence analysis suggested that the protein is ubiquitously present in both the cytosol and nucleus. To evaluate the specificity of the oligonucleotide for the target, we tested whether EZN2968 was able to induce a selective and stable down-modulation of HIF1 α mRNA and protein expression. We confirmed that the downmodulation was lasting in a long term culture experiment (up to 96 hours) either in normoxic or hypoxic conditions, and did not affect the expression of other family members of hypoxia inducible transcription factors (HIF2 α). We next explored the effects of EZN-2968 on the growth and survival of MM cells. Using an MTT colorimetric survival assay, we showed that, after 48 hours of culture in the presence of the HIF1 α inhibitor (20μM), MM1.S and U266 cell lines exhibited a reduction of 30% of viability compared to untreated cells, while RPMI8226 of 15%. AnnexinV/PI staining revealed that EZN-2968 (20μM) increased, after 48 hours of culture, the percentage of PI+ cells compared to the control, suggesting a disruption on membrane permeability. In addition, immunoblotting revealed PARP cleavage as early as 24 hours. Evaluation of cell cycle profile, by flow cytometric analysis, showed an increase of the sub-G0/G1 population from 3.5% to 30 %, after 48 hour of exposure to EZN-2968. To evaluate if the impact on cell viability was irreversible, we performed a cell death commitment assays. MM1S cells were incubated with EZN2968 (20 μM) for 24 to 96 hours, following incubation in drug-free medium for additional 24 to 72 hours. MTT colorimetric survival assay showed that EZN-2968 treatment for as early as 24h resulted in commitment to death in all cell lines tested. To evaluate the effect of microenvironment, MM cells treated with EZN2968 were exposed to IL-6 and stroma cells for additional 24 hours. EZN2968 overcame the proliferative effect induced by cytokines. We next evaluated the impact of EZN-2968 on purified CD138+ cells from MM patients with advanced MM. MTT colorimetric survival assay showed a reduction of cells viability of 30% after 24 hours of incubation. In addition we observed a low sensitivity of PBMCs and CD34+cells, derived from healthy donors, to EZN-2968 treatment suggesting that EZN-2968 has selective in vitro activity against MM cells. Evaluation of gene expression profiling modulation induced by EZN 2968 is on going. In summary, our results suggests that the inhibition of HIF1 α activity can be used as an attractive therapeutic target for MM patients and provide the rationale for clinical evaluation of HIF inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Adenylate Kinase 2 Is a Selective Multiple Myeloma Cell Dependency That Is Preferentially Essential in NSD2-Overexpressing Cells

Blood ◽

10.1182/blood-2021-153284 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1586-1586

Author(s):

Amin Sobh ◽

Charlotte L Kaestner ◽

Jianping Li ◽

Alberto Riva ◽

Richard Lynn Bennett ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Adenylate Kinase ◽

Chromosomal Translocation ◽

Purine Metabolism ◽

Essential Genes ◽

Competitive Growth ◽

High Confidence ◽

Increased Sensitivity

Abstract Background: Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable. Advances in MM therapy have come about due to therapies that target vulnerabilities of the plasma cell such as high protein load (proteasome inhibitors; PIs), dependence on specific transcription factors such as IKZF1 and IKZF3 which are degraded by immunomodulatory drugs (IMiDs), the susceptibility of B cells to glucocorticoids and the presence of specific B cell markers that can serve as targets for monoclonal antibodies and CAR-T cells. Gene editing screens offer a way to identify novel MM therapeutic targets. Objectives: The molecular heterogeneity of MM imposes challenges to discovering generalized therapeutic targets. Therefore, identification of selective dependencies associated with a particular recurrent genetic lesion is a promising strategy to personalize therapy. Here, we aim to identify vulnerabilities linked to the chromosomal translocation t(4;14), a recurrent rearrangement in MM characterized by overexpression of the histone methyltransferase NSD2. Methods: Genome-wide CRISPR-based loss-of-function screens were performed in NSD2-high and low isogenic cells derived from the t(4;14) MM cell line KMS11 to define selective dependencies associated with NSD2 overexpression. High-confidence hits were corroborated by in vitro competitive growth assays where individual candidates are genetically knocked out or suppressed or chemically inhibited. Detailed investigation was performed for selected candidates using various molecular and biochemical assays to elucidate mechanisms by which these genes contribute to MM cell fitness. Results: A fitness screen in NSD2-high and low isogenic MM cells identified 1118 essential genes which are common between the cell pair. We further revealed 282 genes whose loss is more detrimental to cells overexpressing NSD2 and 139 genes that are preferentially essential when NSD2 levels are low. Pathway analysis of NSD2-high selectively essential genes indicated that these cells are more dependent on mitochondrial processes including oxidative phosphorylation. Although proteasomal degradation is essential for all MM cells, our screens indicated that NSD2-high cells are more dependent on the proteasome, which was validated by increased sensitivity to the PI bortezomib. One of the high-confidence selective NSD2-high hits was the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). Analyzing the dependence of hundreds of human cell lines on AK2 using the cancer dependency map portal (depmap.org/portal/), we found that AK2 is not a common essential gene. The top enriched linages with AK2 dependency included MM with notable representation of t(4;14)-positive cell lines. Analysis of the multiple myeloma research foundation (MMRF)-CoMMPass data demonstrated that MM patients with high NSD2 expression, despite poor prognosis, display enhanced overall survival when AK2 levels are low. In vitro competitive growth assays in NSD2-high and low MM cells confirmed the increased dependence of NSD2-overexpressing cells on AK2. In addition, NSD2-high MM cells displayed elevated sensitivity to AK2 inhibitors. Moreover, AK2 knockdown in t(4;14) MM cell lines increased sensitivity to the PI bortezomib. Mechanistically, we showed that AK2 disruption activates apoptotic unfolded protein response (UPR) signaling in MM cells. Metabolomic profiling in NSD2-high and low MM cells revealed accumulation of purine metabolites and reduction of pyrimidine metabolites upon NSD2 overexpression. Intriguingly, purine supplementation rescued MM cell depletion due to AK2 loss. These observations suggested that MM cells, especially those with NSD2 overexpression, are addicted to elevated purine levels and that lethality of MM cells upon AK2 loss is due to perturbed purine metabolism. How impaired purine metabolism activates UPR signaling is currently under investigation. Conclusions: Our work indicated that NSD2 overexpression resulting from chromosomal translocation t(4;14), despite its oncogenic role, generates metabolic dependencies in MM cells. Our findings further suggest that inhibition of AK2, a mitochondrial enzyme involved in purine metabolism, can induce UPR-mediated apoptosis in MM cells and could be used in combination with PI therapy to treat MM patients with t(4;14) translocations. Disclosures Licht: Epizyme: Research Funding.

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

Blood ◽

10.1182/blood.v118.21.5140.5140 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5140-5140

Author(s):

Nicole A Doudican ◽

Amitabha Mazumder ◽

Seth J Orlow

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Arsenic Trioxide ◽

Cell Lines ◽

Combination Treatment ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Initiation Factor ◽

Clinical Issue ◽

Translational Initiation

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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Frequent Involvement of PVT1 in Multiple Myeloma Carrying 8q24 Rearrangement and Identification of Novel PVT1-NBEA Chimeric Gene,

Blood ◽

10.1182/blood.v118.21.3917.3917 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3917-3917

Author(s):

Hisao Nagoshi ◽

Tomohiko Taki ◽

Ichiro Hanamura ◽

Masakazu Nitta ◽

Takemi Otsuki ◽

...

Keyword(s):

Multiple Myeloma ◽

Candidate Genes ◽

Cell Lines ◽

Conflicts Of Interest ◽

Chromosomal Translocation ◽

Fusion Transcript ◽

Chimeric Gene ◽

Oligonucleotide Array ◽

Immunoglobulin Gene ◽

Molecular Features

Abstract Abstract 3917 The 8q24 rearrangement has been identified in 3.5–5.0 % of multiple myeloma (MM) patients with conventional cytogenetic analyses, such as G-banding, and in 9.5–20% of MM patients with fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) techniques. Of note, 8q24 rearrangements accompany with advanced MM patients and MM cell lines more frequently. Chromosomal translocation of the immunoglobulin gene (Ig), such as t(8;14)(q24;q32) and t(8;22)(q24;q11), occur in approximately 25% in MM with 8q24 rearrangements, while non-Ig chromosomal loci, including 1p13, 1p21–22, 6p21, 6q12–15, 13q14, and 16q22, have been also identified as translocation partners where no candidate genes have been delineated so far. In this study, we precisely investigate molecular features of chromosomal 8q24 rearrangements to access pathophysiology of MM. FISH, SKY, and RT-PCR analyses were performed as described previously. DNA gain and loss assay based on oligonucleotide array (GeneChip Human Mapping 50K, 250K, or 6.0 array, Affymetrix) was performed on the genomic DNA extracted from MM cells. Oligonucleotide array data was analyzed using the CNAG3.0 or 3.3 programs. Various types of 8q24 rearrangements were detected in 12 (22.2 %) of 54 MM patients and 8 (72.7 %) of 11 MM cell lines by means of FISH procedure. A breakpoint cluster of approximately 360 kb region containing myelocytomatosis oncogene (MYC) and plasmacytoma variant translocation 1 (PVT1) genes at 8q24 was divided into three subclusters according to rearranged region, that is, PVT1 region, region which is centromerically adjacent to PVT1 (including MYC), and region which is 120kb centromeric to MYC. Seven of 12 patient-derived cells and 5 of 8 cell lines with 8q24 abnormalities showed PVT1 rearrangements with various partner loci, such as 4p16, 4q13, 13q13, 14q32, or 16q23–24 in SKY combined with FISH analyses (SKY-FISH). Oligonucleotide array combined with SKY analysis delineated the candidate genes within partner loci of 8q24 rearrangements by mapping boundary breakpoints of copy number gains and losses, identifying MMSET, EPHA5, NBEA, and WWOX as candidate genes at 4p16, 4q13, 13q13, and 16q23–24, respectively. We identified the novel chimeric gene PVT1-NBEA in AMU-MM1 cell line with t(8;13)(q24;q13). The PVT1-NBEA fusion transcript in which PVT1 exon 1 was fused to NBEA exon 3 was accompanied with overexpression of abnormal NBEA lacking its part of N-terminus. In conclusion, PVT1 rearrangement may play pivotal roles in pathophysiology of MM harboring 8q24 abnormalities. Disclosures: No relevant conflicts of interest to declare.

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Mir-29a Displays in Vitro and in Vivo Anti-Tumor Activity in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.2090.2090 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2090-2090

Author(s):

Manujendra N Saha ◽

Yan Chen ◽

Jahangir Abdi ◽

Hong Chang

Keyword(s):

Multiple Myeloma ◽

Tumor Suppressor ◽

Cell Lines ◽

Conflicts Of Interest ◽

Therapeutic Potential ◽

Critical Role ◽

Treatment Strategies ◽

Loss Of Function

Abstract Despite advances in recent therapeutic approaches including targeted therapies, multiple myeloma (MM) remains still incurable necessitating the development of novel treatment strategies. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate post-transcriptional gene expression and play a critical role in tumor pathogenesis. Tumor suppressor miRNAs are generally down-regulated in cancer cells compared to their normal counterpart, and their enforced expression indeed represents a promising strategy for cancer treatment. In this study, we sought to characterize the role of miR-29a as a tumor suppressor as well as evaluated its therapeutic potential in MM. miR-29a expression levels were found down-regulated in a panel of 5 MM cell lines, 6 newly diagnosed MM patient samples compared to its expression in normal hematopoietic cells collected from 10 normal healthy individuals suggesting that high expression of miR-29a might be involved in MM pathogenesis. We further assessed the functional significance of miR-29a by both gain- and loss-of-function studies. A significant decrease in cell viability (22-32%, p<0.05), along with induction of apoptosis (30-35%, p<0.05) was observed at 48 hrs in MM cell lines, MM.1S and 8226 transfected with miR-29a compared to cells transfected with scrambled miRNA. In contrast, cell lines transfected with miR-29a antagonist prevented the loss of viability in such cells indicating the specificity of miR-29a. At the molecular level, we have identified c-Myc, an important oncogenic transcription factor known to stimulate MM cell proliferation, as a target of miR-29a. Binding site of miR-29a was first identified by computer algorithm and further confirmed by the use of a 3’UTR of c-Myc reporter (luciferase renilla/firefly) constructs containing, miR-29a target site. Moreover, treatment with PRIMA-Met, a small molecule anti-tumor agent in phase I/II clinical trials, significantly increased the expression of miR-29a (2 to 6-fold) and decreased expression of c-Myc in MM cell lines and primay MM patient samples suggesting an important role of miR-29a in inhibiting proliferation of MM cells. On the other hand, overexpression of c-Myc in 8226 and MM.1S cells at least partially reverted the functional effect of miR-29a or PRIMA-1Metsuggesting a specific role of c-Myc in mediating its anti-proliferative activity. To examine therapeutic potential of our studies, we took advantage of novel lipid based delivery method of miRNA. Intratumor delivery of the miR-29a by intraperitoneal injection route against MM xenografts in SCID mice resulted in a significant inhibition of tumor growth (~60%) at 12 days of treatment and prolongation of survival (median survival increased from 22 days to 35 days, p<0.038) compared to the mice receiving scrambled miRNA. Retrieved tumors from treated mice showed efficient increase in miR-29a (5.5-fold, p=0.025), and decrease in c-Myc protein as well as reduced expression of Ki67 and increase of Tunel expression. Similar phenomenon was observed by systematic delivery of miR-29a (by intraveneous injection) in mice with no significant side effects or toxicity in mice. Our study reveals an important role of miR-29a as a tumor suppressor in mediating anti-tumor activities in MM cells by targeting c-Myc. Our findings provide a proof-of-principle that formulated synthetic miR-29a exerts therapeutic activity in preclinical models, and support a framework for development of miR-29a based treatment strategies in MM patients. Disclosures No relevant conflicts of interest to declare.

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The Role of Chaperone-Mediated Autophagy in Bortezomib Resistant Multiple Myeloma

Cells ◽

10.3390/cells10123464 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3464

Author(s):

Nicholas Nikesitch ◽

Patricia Rebeiro ◽

Lye Lin Ho ◽

Srinivasa Pothula ◽

Xin Maggie Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Er Stress ◽

Cell Line ◽

Cell Lines ◽

Western Blotting ◽

Resistant Cell ◽

Novel Agents ◽

Cell Line Model ◽

Chaperone Mediated Autophagy

Background: Multiple myeloma (MM) remains incurable despite high-dose chemotherapy, autologous stem cell transplants and novel agents. Even with the improved survival of MM patients treated with novel agents, including bortezomib (Bz), the therapeutic options in relapsed/refractory MM remain limited. The majority of MM patients eventually develop resistance to Bz, although the mechanisms of the resistance are poorly understood. Methods: Lysosomal associated membrane protein 2A (LAMP2A) mRNA and protein expression levels were assessed in ex vivo patient samples and a Bz-resistant MM cell line model by in real-rime PCR, western blotting and immunohistochemistry. In vitro modelling of chaperone-mediated autophagy (CMA) activity in response to ER stress were assessed by western blotting and confocal microscopy. The effects of CMA inhibition on MM cell viability and Bz sensitivity in MM cells were assessed by Annexin V/7AAD apoptosis assays using flow cytometry. Results: In this study, there is evidence that CMA, a chaperone-mediated protein degradation pathway, is upregulated in Bz-resistant MM and the inhibition of CMA sensitises resistant cells to Bz. The protein levels of LAMP2A, the rate-limiting factor of the CMA pathway, are significantly increased in MM patients resistant to Bz and within our Bz-resistant cell line model. Bz-resistant cell lines also possessed higher basal CMA activity than the Bz-sensitive parent cell line. In MM cell lines, CMA activity was upregulated in response to ER stress induced by Bz. The inhibition of CMA sensitises Bz-resistant cells to Bz and the combination of CMA inhibition and Bz in vitro had a more cytotoxic effect on myeloma cells than Bz alone. Conclusion: In summary, the upregulation of CMA is a potential mechanism of resistance to Bz and a novel target to overcome Bz-resistant MM.

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