Mafb Protein Confers Primary Resistance of Myeloma to Proteasome Inhibitors

Abstract Multiple myeloma (MM) is a malignancy of terminally differentiated clonal plasma cells displaying significant molecular heterogeneity with 7 subgroups defined by gene expression profiling (GEP). Our previous work showed that both the MS and MF subgroups associated with inferior survival (Zhan et al, Blood 2006). Furthermore, clinical studies have demonstrated that the addition of the proteasome inhibitor (PI) bortezomib (Bzb) to high dose melphalan based regimens provided a major advantage to patients in MS subgroup while patients in the MF subgroups (including C-MAF and MAFb) did not benefit from Bzb (Nair, Blood 2010). We have previously demonstrated that Bzb prevents c-MAF protein degradation leading to primary drug resistance (Abstract # 281, ASH 2013). In the present study, we assessed the ability of MAFb, another MAF family member, to influence the innate resistance to proteasome inhibitors (PI) and identify the molecular mechanism underlying the resistance of proteasome inhibitors in high MAFb-expressing patients. To investigate the association of the limited therapeutic effect of proteasome inhibitors in the different molecular subgroup of myeloma, we compared the IC50 of Bzb and carfilzomib (CFZ) in 29 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentrations of Bzb were higher (>25 nM) in all 4 MAFb MMCL and >60 nM in one MAFb MMCL, which expressed the highest level of MAFb protein, as determined by immunoblot analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. For CFZ, IC50 concentrations were higher (>30nM) in all 11 c-MAF cell lines, while the IC50 levels were lower (2-20 nM) for the MMCL belonging to other molecular subgroup. One MMCL harboring t (14; 20) with high IC50 of Bzb (35 nM) showed low IC50 (10 nM) for CFZ. These results indicate that high MAFb expression in myeloma cells may contribute to primary resistance to Bzb and MM cells that express high MAFb protein although resistant to Bzb are sensitive to CFZ. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAFb protein levels in a dose-dependent manner, suggesting that Bzb prevents the degradation of MAFb protein in myeloma cells. To further confirm that drug-induced stabilization of MAFb protein confers resistance to Bzb and partially to CFZ, we generated loss of functional MAFb cells by silencing MAFb expression in a t (14;20) positive myeloma cell line using lentiviral shRNA specific to MAFb mRNA (shMAFb). shMAFb infected myeloma cells had 85% lower levels of MAFb mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, significantly decreased ITGB (9.1 fold), E-cadherin (2.5-fold), CCND2 (5.5-fold), and CCR1 (25-fold) levels were observed in these cells, compared with the cells infected with control viral vector. Silencing MAFb expression significantly decreased proliferation of myeloma cells (65.1% decrease, p=2.1E-6). Moreover, Bzb treatment of the cells infected with shMAFb led to 50.2%, inhibition (P=2.9E-8) of proliferation compared with control cells. Similarly, CFZ treatment of cells with silenced MAFb resulted in 54.3% (P=3.5E-8) inhibition of proliferation compared with control cells. Taken together, our results indicate that high expression of MAFb protein, similar to c-MAF, confers primary resistance to Bzb as well as to CFZ. In addition Bzb induces stabilization of MAFb protein, further increasing resistance to Bzb. These data provide the molecular rational for adopting an alternative therapeutic strategy for high-MAFb expressing myeloma patients. Disclosures Morgan: Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Myeloma UK: Membership on an entity's Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity's Board of Directors or advisory committees; The Binding Site: Membership on an entity's Board of Directors or advisory committees; MMRF: Membership on an entity's Board of Directors or advisory committees.

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XRCC5 Plays an Important Role in Homologous Recombination, Genome Stability and Survival of Myeloma Cells

Blood ◽

10.1182/blood.v126.23.1218.1218 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1218-1218 ◽

Cited By ~ 1

Author(s):

Edward Laane ◽

Purushothama Nanjappa ◽

Subodh Kumar ◽

Florence Magrangeas ◽

Stephane Minvielle ◽

...

Keyword(s):

Cell Cycle ◽

Board Of Directors ◽

Genomic Instability ◽

Cell Lines ◽

Copy Number ◽

Genome Stability ◽

Myeloma Cell ◽

Advisory Committees ◽

Myeloma Cells ◽

Elevated Expression

Abstract Understanding mechanisms underlying genomic instability is critical in delineating pathogenesis and development of new treatments for prevention and treatment of cancer. We have previously shown that dysregulated homologous recombination (HR) significantly contributes to genomic instability and progression in multiple myeloma (MM). To identify the regulators of HR and genome stability in MM, we conducted a functional shRNA screen and identified XRCC5 (Ku80) as a novel regulator of HR in MM cells. XRCC5 has been known to work as part of DNA ligase IV-XRCC4 complex in the repair of DNA breaks by non-homologous end joining (NHEJ) and the completion of V(D)J recombination events. Evaluation by Western blotting showed that all myeloma cell lines tested (RPMI, MM1S, OPM2, MM1R, U266, ARP, H929) had elevated expression of XRCC5, ranging from 3- to 10-fold elevation relative to average expression in two normal PBMC samples. Expression profiling showed a wide range of XRCC5 expression in myeloma patients, with a subset of patients with very high expression. To investigate the role of XRCC5 in ongoing acquisition of genomic changes, we investigated the association of XRCC5 with genomic instability using two different patient datasets (gse26863, n=246 and IFM 170 pt dataset) in which both the gene expression and genomic copy number information for each patient was available. Copy events were defined as changes observed in ≥ 3 and/or 5 consecutive SNPs. Higher XRCC5 expression significantly correlated with increase in the number of copy number change events in both the 170 dataset (p ≤ 0.005 for amplifications and p = 0.0001 for deletions) as well as in gse26863 dataset (p ≤ 0.004 for amplifications and p ≤ 0.00003 for deletions). To understand mechanisms by which XRCC5 regulates HR in myeloma cells, we investigatedprotein-protein interactions using a custom protein array coated with antibodies against major DNA repair and cell cycle proteins. Array was sequentially incubated with MM cell lysate and HRP-conjugated anti-XRCC5 antibody, and interacting partners were then identified by their address on the array. Investigation in two different cell lines (RPMI and U266) showed that XRCC5 in myeloma interacts with XRCC4 (an NHEJ protein), a panel of major HR regulators (RAD51, RAD52, BRCA2, BRCA1, BARD1, P73, P53, C-ABL) and with components of cell cycle including CDC42, CDK1 (which controls entry from G2 to mitosis), CDK4, CDK6, CHK, CDC36, CDC34, and cyclins E and H. Consistent with these data, knockdown (KD) of XRCC5 was associated with reduced HR as well as reduced proliferation rate followed by a complete cell death over a period of two to three weeks in different experiments, in all 3 myeloma cell lines tested. Moreover, the investigation in U266 cells showed that XRCC5-KD is associated with 3-fold increase in the fraction of cells in G2 phase of cell cycle. Importantly, the elevated expression of XRCC5 was associated with shorter event free (p < 0.013) as well as poor overall survival (p < 0.008) in 170 patient dataset. We evaluted the expression and clinical correlation of XRCC5 in RNA-seq data from 311 newly-diagnosed MM patients and observed that the elevated expression of XRCC5 also correlated with event free survival (p = 0.03). In summary, we report that XRCC5, besides its known role in NHEJ, has important roles in HR, cell cycle and may be involved in the crosstalk among these DNA repair pathways. Elevated XRCC5 expression is associated with dysregulation of HR with consequent impact on survival of myeloma patients. Elevated XRCC5 is, therefore, a promising new target to inhibit/reduce genomic evolution as well as MM cell growth. Disclosures Avet-Loiseau: celgene: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees; jansen: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; millenium: Membership on an entity's Board of Directors or advisory committees.

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Sensitizing shRNA Screen for Molecular Targets in CDK4/CDK6-Based Combination Therapy in Multiple Myeloma

Blood ◽

10.1182/blood.v124.21.3440.3440 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3440-3440 ◽

Cited By ~ 1

Author(s):

Xiangao Huang ◽

Maurizio Di Liberto ◽

David Chiron ◽

Ruben Niesvizky ◽

Anna C. Schinzel ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Clinical Trial ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Proteasome Inhibitors ◽

Advisory Committees ◽

Myeloma Cells ◽

Shrna Screen

Abstract CDK4 and CDK6 are rarely mutated but are overexpressed or hyperactivated at a very high frequency in human cancers. By inhibiting CDK4/CDK6 with an exceptionally selective and reversible inhibitor, palbociclib (PD 0332991), we have developed a novel strategy to reprogram cancer cells for cytotoxic killing through induction of prolonged early G1 arrest (pG1). We have demonstrated that pG1 sensitizes cancer cells expressing Rb, the substrate of CDK4 and CDK6, to cytotoxic killing by forcing an imbalance in gene expression because only genes scheduled for early G1 are expressed. This sensitization is exacerbated after palbociclib withdrawal due to incomplete restoration of gene expression despite S phase synchronization (pG1-S). This study aims to identify genes that mediate pG1-S sensitization to two clinically-relevant agents for myeloma, the proteasome inhibitors carfilzomib and bortezomib, in model cell lines by a sensitizing pool genome-wide shRNA screen, and by validating the hits in a clinical trial of palbociclib in combination with bortezomib and dexamethasone. We ranked the hits based on the enrichment of target shRNAs, and representation in replica of each cell lines and among different human myeloma cell lines (HMCLs) as well as functional analyses. In myeloma cells, cell cycle control by palbociclib was intact in all hits, demonstrating that CDK4 and CDK6 are indispensable for myeloma replication. Among the top ranking 20 candidates, we found that NEDD4L was essential for proteasome inhibitor killing, FTH1 modulated the threshold of killing by diverse agents especially in pG1-S, and IL10RAappeared to be required for pG1-S sensitization to proteasome inhibitors. Moreover, RNA-sequencing analysis of primary myeloma cells from a phase II clinical trial targeting CDK4/CDK6 with palbociclib in combination with bortezomib in myeloma revealed that a higher level of FTH1 expression in myeloma cells in vivo correlated with sensitivity to this therapy, suggesting a role for FTH1 in differential sensitivity to this CDK4/CDK6-based therapy in myeloma. Selective inhibition of CDK4/CDK6 with palbociclib, or another specific inhibitor such as LY2835219 or LEE011, in combination therapy has now achieved unprecedented clinical efficacy in diverse human cancers. Most notably, palbociclib more than doubled the progression free survival of metastatic breast cancer patients when it was combined with letrozole, and has been designated a “breakthrough therapy” by the FDA for breast cancer. Our work provides the first insight into genes that mediate cell cycle sensitization to cytotoxic killing through selective CDK4/CDK6 inhibition. It provides an exciting potential for further investigation in a clinical context, such as the ongoing phase I clinical trial combining palbociclib with the immunomodulatory drug lenalidomide in patients with relapsed/refractory myeloma. Disclosures Huang: Celgene: Research Funding. Off Label Use: PD 0332991 (palbociclib) is a specific CDK4/CDK6 inhibitor used to stop the cell cycle.. Niesvizky:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Chen-Kiang:Celgene: Consultancy, Research Funding.

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Mapping of the Multiple Myeloma Transcriptional Core Regulatory Circuitry Reveals TCF3 As a Novel Dependency and an Oncogenic Collaborator of MYC

Blood ◽

10.1182/blood.v130.suppl_1.64.64 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 64-64

Author(s):

Charles Y Lin ◽

Mariateresa Fulciniti ◽

Michael A Lopez ◽

Mehmet Kemal Samur ◽

Raphael Szalat ◽

...

Keyword(s):

Multiple Myeloma ◽

Transcription Factor ◽

Transcription Factors ◽

Board Of Directors ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Advisory Committees ◽

Myeloma Cells ◽

Over Time

Abstract Multiple Myeloma (MM) is a complex plasma cell malignancy driven by numerous genetic and epigenetic alterations that are acquired over time. The events controlling and modifying transcriptomic changes that drive MM cell growth and progression remains undefined. To reveal the epigenetic circuitry governing myeloma cells, we performed a comprehensive analysis integrating data obtained from Multiplexed Indexed T7 Chromatin IP (Mint-ChIP), Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), and RNA-seq in 10 primary MM cells as well as 3 MM cell lines to identify genome-wide the master transcription factors (TFs), the enhancer elements they occupy, and the genes they regulate. Using these data, we have identified myeloma-specific core regulatory circuitry which includes several well-established regulators of MM such as IKZF, E2F, MYC and IRF family of genes. For example, our data show elevated MYC at numerous tissue specific enhancers in myeloma cells, including those that regulate lineage specifying transcription factors such as IRF4 and TCF3 (aka E2A). When translocated to the immunoglobulin enhancer, MYC in turn is regulated by these lineage transcription factors thus integrating MYC into the interconnected transcriptional core regulatory circuitry of MM (Figure 1a,b). We propose that this oncogenic "re-wiring" accounts for the observed addiction of MM cells to lineage factors such as IRF4 and in this work, we implicate the B-cell factor TCF3 as a novel multiple myeloma dependency. Using myeloma cell lines and primary samples, we observed elevated enhancer activity at TCF3 in primary CD138+ cells from myeloma patients compared to normal plasma cells (NPCs) (Figure 1c). As a result, TCF3 expression is significantly upregulated in our large cohort of MM patients (n=370) compared to normal bone marrow plasma cells (n=18). As MYC proteins can only bind pre-established and acetylated regions of active chromatin, we hypothesize that enhancer specifying lineage transcription factors such as TCF3 may cooperate with MYC to alter tissue specific gene expression programs. We show that TCF3 is regulated by a large proximal enhancer that is bound by MYC, and is highly sensitive to chemical perturbation of enhancer co-activators such as BRD4. As a helix-loop-helix transcription factor that similar to MYC binds short (CANNTG) E-box sequences, we computationally predict co-occupancy of MYC and TCF3 at ~80% of all enhancers that form the multiple myeloma transcriptional core regulatory circuitry. To evaluate the functional role of TCF3 in myeloma cells, we established TCF3 knock down myeloma cell lines and followed the cell growth over time. Stable knockdown of TCF3 preferentially blocks proliferation of IgH MYC translocated cell lines (such as MM1.S cells) versus non-translocated lines (such as U266 cells). Finally, high expression of TCF3 correlates with poor clinical outcome in myeloma patients. Together these data suggest TCF3 acts as an oncogenic collaborator with deregulated MYC and implicates transcriptional control of lineage as a dependency in multiple myeloma. Figure 1: Transcriptional core regulatory circuitry of multiple myeloma: A) ChIP-Seq tracks of IRF4, MYC, BRD4, and H3K27ac occupancy at the IRF4, IgH enhancer, and TCF3 loci respectively. B) Schematic of transcription factor to enhancer connectivity of the partial multiple myeloma transcriptional core regulatory circuitry highlighting interactions between IRF4, MYC, and TCF3 (computationally predicted based on TCF3 motif data). C) ChIP-Seq tracks of H3K27ac occupancy at the TCF3 locus in patient multiple myeloma (top, n=3) or normal plasma cells (bottom, n=2). Figure 1 Figure 1. Disclosures Bradner: Acetylon: Other: Scientific Founder; Novartis: Employment. Anderson: Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees.

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Targeting PI3K-AKT-mTOR Signaling in Multiple Myeloma Mesenchymal Stem Cells Mediates Antiproliferative Effect on Myeloma Cells

Blood ◽

10.1182/blood-2021-150413 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1600-1600

Author(s):

Luca Heinmann ◽

Helal Mohammed Mohammed Ahmed Noman ◽

Klara Möllers ◽

Subbaiah Chary Nimmagadda ◽

Kaiyan Sun ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Therapeutic Strategies ◽

Advisory Committees ◽

Myeloma Cells ◽

Bm Niche ◽

Novel Therapeutic Strategies ◽

Novel Therapeutic

Abstract Introduction: Multiple myeloma (MM) is a B-cell malignancy characterized by an abnormal proliferation and infiltration of malignant plasma cells in the bone marrow (BM). Mesenchymal stromal cells (MSCs) represent a crucial component of the BM niche and mediate essential signalling via cytokines and cell-cell interactions. The interplay of MM cells and BM-MSC is complex and relies on multiple signaling pathways leading to MM progression and therapeutic resistance. Objectives: MM remains an incurable disease so far. Distinctive for this disease is a long-lasting polarization of the BM niche influencing MM progression and prognosis. We, therefore, focussed on MSCs to identify enrichment for different hallmark gene sets and their aberrant signaling contributing to the pathogenesis of the disease, therapy response and to further identify novel therapeutic strategies. Methods: BM-MSCs were isolated from patients with MM at diagnosis (MM-D-MSC) and in remission (MM-R-MSC) as well as from donors with other malignant diseases (CTR-MSC). RNA sequencing and Western Blot were used for examination of enriched pathways. Various functional assays for proliferation, apoptosis and cell cycle were performed either using a mono-culture or co-culture protocol of MSC and the MM-cell lines MM.1S and SKMM2 treating the cells with the pan-PI3K-inhibitor GDC-0941. Results: MM-D-MSCs supported the growth of myeloma cell lines better (3 fold, p<0.01) than MM-R- and CTR-MSCs. Our results demonstrate that MM-D-MSCs have a distinct gene expression profile compared to CTR-MSC indicating potential nodes of crosstalk and therapeutic importance. Amongst others, the PI3K-AKT-mTOR hallmark gene set was significantly enriched in MM-D-MSCs as compared to CTR-MSCs (p<0.001). We confirmed these findings on a proteomic level. We found evidence for the upregulation of PI3Kα, AKT, pAKT and mTOR in MM-D-MSC comparing to the other MM-R- and CTR-MSCs (p<0.05). We treated these MSC and the MM-cell lines MM.1S and SKMM2 with the PI3-Kinase inhibitor GDC-0941. The treatment reduced the signaling PI3Kα, AKT and mTOR in both, MSC and MM-cells. As stated MM-D-MSC supported the growth of myeloma cells better than other MSC types. However, upon GDC-0941 treatment, the proliferation of MM-D-MSCs was significantly reduced compared to the other MSC-types. In addition, the inhibition of proliferation of myeloma cell lines MM1S and SKMM2 was much more pronounced when they were cocultured with MM-D-MSC (32 and 34 %, p=0.04) compared to the growth of myeloma cells in coculture with MSC types, either in remission or other malignancies. Conclusion: We here identified functionally distinct differences in MM-D-MSCs compared to MM-R-MSCs or CTR-MSCs. Our data further provides a deeper insight into the molecular signature of MM-MSCs, a predictive of patient prognosis and treatment outcome. Targeting MSCs as a crucial part of the MM-BM niche by using PI3K-inhibitors could contribute to novel therapeutic strategies to effectively block MM-MSC interaction improving overall patient survival. Disclosures Raab: Roche: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Khandanpour: BMS/Celgene: Honoraria; Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria; GSK: Honoraria.

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MAF Protein Elicits Innate Resistance To Bortezomib In Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.281.281 ◽

2013 ◽

Vol 122 (21) ◽

pp. 281-281 ◽

Cited By ~ 1

Author(s):

Shiqiao Ye ◽

Wei Qiang ◽

Yu Chen ◽

Bo Hu ◽

Qing Zhang ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Research Funding ◽

Molecular Heterogeneity ◽

Molecular Subgroups ◽

Immunoblotting Analysis ◽

Primary Resistance ◽

Myeloma Cells ◽

Innate Resistance ◽

E Cadherin

Abstract Multiple myeloma (MM) is a malignancy of terminally differentiated clonal plasma cells displaying significant molecular heterogeneity with 7 subgroups defined by gene expression profiling (GEP). Our previous work showed that MS and MF subgroups have been associated with inferior survival (Zhan et al, blood 2006). Furthermore, clinical studies have demonstrated that the addition of the proteasome inhibitor (PI) bortezomib (Bzb) to high dose melphalan based regimens provided a major advantage to patients in MS subgroup (Barlogie NJE 2006, Blood 2008; Pineda-Roman et al BJH 2008), while patients in the MF subgroups did not benefit from Bzb (Nair, Blood 2010). These findings led us to hypothesize that overexpression of MAF protein confers innate resistance to Bzb. In the present study, we assessed the ability of MAF to influence the innate resistance to Bzb and identify the molecular mechanism underlying the resistance of Bzb in high MAF-expressing patients. To investigate association of the limited therapeutic effect of Bzb with molecular subgroup of myeloma, we established the IC50 of Bzb in 24 myeloma cell lines (MMCL) belonging to different GEP-based molecular subgroups. IC50 concentration were higher (>25nM) in 7 of 9 MAF and in all 5 MAFb MMCL, and >40 nM in 5 MAF and one MAFb MMCL, which expressed the highest levels of MAF protein, as determined by immunoblotting analysis. In contrast, Bzb IC50 levels were lower (7.5-20 nM) for the MMCL belonging to the other molecular subgroups. These results indicate that high MAF expression in myeloma cells may contribute to primary resistance to Bzb. Mechanistically, immunoblotting analysis demonstrated that exposure to Bzb resulted in increased MAF protein levels. These results suggested that Bzb prevents the degradation of MAF protein in myeloma cells. To further confirm that Bzb-induced stabilization of MAF protein confers resistance to Bzb, we overexpressed MAF cDNA in myeloma cells lacking MAF expression, and silenced MAF expression in myeloma cells expressing high level of MAF mRNA and protein. MM cells were infected with Lentiviral vector containing MAF cDNA or with empty vector, and stable clones selected with puromycin, designated as MMmaf and MMEV, respectively. qPCR and immunoblotting analysis showed that expression of MAF mRNA and protein in MMmaf cells were significantly higher (1.8x105-fold) than in MMEV cells. The functionality of ectopic MAF protein was confirmed by qRT-PCR analysis of downstream target genes; the mRNA level of E-cadherin was higher in MMmaf cells than those of MMEV (1.42-fold, p<0.01). MTT assay showed that the proliferation rate of MMmaf cells was 53% higher than that of MMEV cells (p <0.001). Similar results were observed in other two MM cell lines that transiently ectopic expressed MAF. Moreover, MMmaf showed higher IC50 of Bzb than that of MMEV , indicating that increase MAF protein in myeloma cells reduces sensitivity to Bzb. We further generated loss of functional MAF by silencing MAF expression in MAF positive myeloma cells using shRNA specific to maf mRNA (shMAF) by lentiviral expressing system. shMAF infected myeloma cells had 75% lower levels of MAF mRNA and protein level compared with the cells infected with scrambled shRNA. Additionally, significantly decreased integrin E-cadherin (9.1-fold), cyclin D2 (4.99x105-fold), and CCR1 (4.9-fold) levels were observed in these cells, compared with the cells infected with control viral vector. Silencing MAF expression significantly decreased proliferation of myeloma cells (81.9% decrease, p=2.5E-6). Moreover, Bzb treatment of the cells infected with shMAF lead to 53.1%, inhibition (P=3.3E-8) of proliferation compared with control cells. Taken together, our results indicate that high expression of MAF protein confers myeloma primary resistance to Bzb, and Bzb induces stabilization of MAF protein further increases resistance to Bzb, providing the molecular rational for therapeutic strategy for high-MAF expressing myeloma patients. Disclosures: van Rhee: Jansen & Jansen: Research Funding. Barlogie:Celgene: Consultancy, Honoraria, Research Funding; Internation Myeloma Foundation: Consultancy, Honoraria; Millennium: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; National Cancer Institute: Research Funding; Johnson & Johnson: Research Funding; Centocor: Research Funding; Onyx: Research Funding; Icon: Research Funding; Myeloma Health, LLC: Patents & Royalties.

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Expression Of Truncated Protein Tyrosine Phosphatase, Receptor Type, O (PTPROt) Influences Multiple Myeloma Sensitivity To Bortezomib Through Akt Signaling, and Is a Favorable Clinical Prognostic Factor

Blood ◽

10.1182/blood.v122.21.2520.2520 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2520-2520

Author(s):

Hua Wang ◽

Veerabhadran Baladandayuthapani ◽

Zhiqiang Wang ◽

Jiexin Zhang ◽

Heather Yan Lin ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Advisory Committees ◽

Over Expression ◽

Resistant Disease ◽

Drug Naïve ◽

Rpmi 8226

Abstract Background Proteasome inhibitors such as bortezomib and carfilzomib are an important part of our current chemotherapeutic armamentarium against multiple myeloma, and have improved outcomes in the up-front, relapsed, and relapsed/refractory settings. Their efficacy has been demonstrated both as single agents, and as part of rationally designed combination regimens, but they are at this time used empirically, since biomarkers to identify patients who would most or least benefit from their application have not been clinically validated. Moreover, the vast majority of patients eventually develop drug-resistant disease which precludes further proteasome inhibitor use through mechanisms that have not been fully elucidated. Methods We compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. The list of genes whose expression was changed by at least 2-fold was compared with independent RNA interference studies whose goal was to identify genes whose suppression conferred drug resistance. Further validation of genes of interest was pursued in a panel of myeloma cell lines, and in clinically annotated GEP databases. Results Suppression of PTPROt expression was noted in bortezomib-resistant RPMI 8226 and ANBL-6 myeloma cells compared to isogenic, drug-naïve controls, and this was confirmed by quantitative PCR. Overexpression of PTRPOt in RPMI 8226, ANBL-6 and other myeloma cell lines was by itself sufficient to increase the level of apoptotic, sub-G0/G1 cells compared to vector controls, or cells expressing a phosphatase-dead PTPROt mutant. Moreover, PTPROt enhanced the ability of bortezomib to reduce myeloma cell viability, in association with increased activation of caspases 8 and 9. Exogenous over-expression of PTPROt was found to reduce the activation status of Akt, a known anti-apoptotic pathway that reduces bortezomib activity, based on Western blotting with antibodies to phospho-Akt (Ser473), and Akt kinase activity assays. Notably, we also found that exogenous over-expression of PTPROt resulted in increased expression levels of p27Kip1. Interestingly, array CGH data from studies of myeloma cell lines and primary cells showed that the PTPROt gene was located in a genomic region with a high propensity for loss. Analysis of the Total Therapy databases of GEP and patient outcomes available on the Multiple Myeloma Genomics Portal showed that higher than median expression of PTPROt was associated with better long-term survival (P=0.0175). Finally, analysis of the Millennium Pharmaceuticals database of studies of bortezomib in the relapsed and relapsed/refractory setting showed high PTRPOt expression was more frequently seen in patients who achieved complete remission (P<0.01), and was associated with a better median overall survival (P=0.0003). Conclusions Taken together, the data support the possibility that high expression of PTPROt is a good prognostic factor for response to bortezomib-containing therapies, and that this may occur through modulation by PTPROt of the Akt pathway. Moreover, they suggest that strategies to enhance the expression of PTPROt should be investigated to restore bortezomib sensitivity in patients with proteasome inhibitor-resistant disease. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

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RNA Polymerase I Inhibition with CX-5461 As a Novel Strategy to Target MYC in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3010.3010 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3010-3010 ◽

Cited By ~ 1

Author(s):

Hans Lee ◽

Hua Wang ◽

Heather Lin ◽

Veera Baladandayuthapani ◽

Jin He ◽

...

Keyword(s):

Western Blot ◽

Board Of Directors ◽

Cell Lines ◽

Ribosomal Proteins ◽

Research Funding ◽

Myeloma Cell ◽

Ribosomal Biogenesis ◽

Advisory Committees ◽

Pol I

Abstract Background: The role of dysregulation of the proto-oncogene MYC in both early and late myeloma progression events is well established. Among key MYC -downstream targets is upregulation of ribosomal biogenesis, resulting in increased protein translational capacity and biomass accumulation that is characteristic of neoplastic cells. Thus, given the relationship between myeloma pathobiology, MYC dysregulation, and ribosomal biogenesis, we hypothesized that selective targeting of ribosomal RNA (rRNA) transcription with the small molecule RNA polymerase (pol) I inhibitor CX-5461 (Senhwa Biosciences) may represent a novel therapeutic strategy in myeloma. Methods: Studies with CX-5461 were performed in human myeloma cell lines, isogenic p53 wild-type (wt) and knock-out (KO) p53 cells generated using sequence-specific zinc-finger nucleases, drug-resistant cell lines, primary patient samples, and myeloma murine xenograft models using NOD-SCID IL2Rgnull mice. Results: CX-5461 treatment of p53 wt (MM1.S, MOLP-8) and p53 mutant (U266, RPMI-8226) myeloma cell lines demonstrated a time- and dose-dependent decrease in cell proliferation with a median inhibitory concentration (IC50) at nM levels after 72 hours. A corresponding increase in cleaved-PARP, cleaved caspase-9, and cleaved caspase-3 expression was seen on Western blot as well as increased Annexin V staining on flow cytometry analysis, although this was more pronounced in p53 wt versus mutant cell lines. CX-5461 also retained activity in a panel of cell lines resistant to standard myeloma therapeutic agents (bortezomib, carfilzomib, lenalidomide, and doxorubicin) and in primary patient samples, including a heavily pretreated relapsed/refractory patient and a de novo plasma cell leukemia patient with del 17p. In vivo studies using a systemic isogenic MM1.S p53 wt and KO myeloma murine xenograft model demonstrated significant improvement in median overall survival in the CX-5461-treated p53 wt cohort (41 days vs. not reached, P .05), although outcomes were more modest in the p53 KO cohort with only a trend towards improved survival (P.1) in the drug-treated mice. To probe the p53-independent effects of CX-5461, gene expression profiling and gene set enrichment analysis was performed on isogenic MM1.S and MOLP-8 p53 wt and KO myeloma cell lines treated with CX-5461 or vehicle. These results suggested downregulation of MYC downstream targets as one p53-independent effect of RNA pol I inhibition. qPCR and Western blot studies revealed rapid downregulation of MYC at the transcript level within 1-hour of CX-5461 treatment followed by decreases in MYC protein levels. Previous studies have suggested ribosomal biogenesis is tightly controlled by an auto-regulatory feedback mechanism in which ribosomal proteins such as RPL5 and RPL11 can bind to the 3'UTR of MYC mRNA and facilitate its degradation through the RNA-induced silencing complex (RISC). Because RNA pol I inhibition is known to induce a nucleolar stress response and increase the availability of free ribosomal proteins, RISC-mediated degradation of MYC mRNA was explored as one possible mechanism of CX-5461-mediated MYC downregulation. Indeed, treatment with CX-5461 led to increased pull-down of RPL5 when immunoprecipitated with the RISC subunit TAR (HIV-1) RNA Binding Protein 2 (TARBP2) compared to vehicle-treated controls, and RNA immunoprecipitation assays with the catalytic RISC subunit, Argonaute 2 (AGO2), demonstrated enrichment of MYC mRNA with CX-5461 treatment. These results suggest that CX-5461 may induce degradation of MYC through the cooperative binding of ribosomal proteins, RISC subunits, and MYC mRNA. Finally, to evaluate the role of MYC expression and ribosomal biogenesis in relation to CX-5461 sensitivity, MYC was overexpressed in the H1112 myeloma cell line, which at baseline does not harbor a MYC translocation. MYC overexpression in H1112pCDH-myc cells led to increased basal pre-rRNA transcript levels compared to H1112pCDH cells, and furthermore, led to enhanced sensitivity to CX-5461. Conclusion: RNA pol I inhibition by CX-5461 is a promising target in myeloma therapy, with downregulation of MYC representing one mechanism of action. Moreover, increased MYC expression enhances sensitivity to CX-5461, providing rationale for the clinical translation of CX-5461 for the treatment of myeloma and other MYC-driven cancers. Disclosures O'Brien: Senhwa Biosciences, Inc.: Employment. Keats:Translational Genomic Research Institute: Employment. Orlowski:Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding.

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Expression and in Vitro Binding of Protease Activated Receptor1 (PAR1) with Novel Anti-PAR1 Molecules: Data on Fresh Myeloma Marrow Plasma Cells and Human Myeloma Cell Lines

Blood ◽

10.1182/blood.v126.23.4440.4440 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4440-4440

Author(s):

Meral Beksac ◽

Pinar Ataca ◽

Berna Atesagaoglu ◽

Klara Dalva ◽

Andry Nur Hidayat ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Myeloma Cell ◽

Lower Percentage ◽

Advisory Committees ◽

In Silico Docking ◽

Human Myeloma Cell

Abstract Introduction and Aim: Myeloma plasma cells are dependent on stromal support which is mediated through cell adhesion. Heparanase activity has been shown to be associated with aggressive behavior or Bortezomib resistance and can lead to increased levels of proteases as well as shedding of heparan sulfate proteoglycan syndecan-1(CD138) from myeloma cells. We have recently published in vivo anti-myeloma effects of low molecular weight heparin (Beksac et al Acta Haematol 2015). Protease activated Receptor (PAR1) is a thrombin receptor. PAR1 gene and antigen expression on myeloma patient samples and cell lines (HMCL) has been recently reported by University of Arkansas (UAMS) group (Tian et al ASH 2011). They were able to find HMCLs H929, U266, JJN3 to express PAR1. Also expression was found to be highest among patients with 5q amplification where the PAR1 gene is located. Patients and Methods: We analyzed PAR1 expression (WEDE15 PE, Beckman Coulter) by flow cytometry, on CD38+CD138+/-CD27+/- cells obtained from fresh patient bone marrow samples obtained either at diagnosis (n: 84)(NDMM) or relapse (n: 54)(RRMM) and were compared with marrow samples taken from patients without MM (n: 43). Our group in Ankara University had previously synthesized and published novel benzamide and phenyl acetamide derivatives. We performed an in silico docking analysis on these molecules, and eleven (TD10,TD12,TD12A,TD12B,TD13,TD14,TD14B,XT2,XT2B,XT5,XT11) were found to bind to PAR1. These molecules were screened using 72 hour MTT assay on primary and refractory cell lines (U266BR ,U266, JJN3BR, JJN3, H929R, OPM2, OPM2R, KMS28PE). Results: PAR1 expression was highest on platelets followed by myeloma plasma cells (0-81.9%) and did not correlate with ISS. PAR1 expression (Threshold: >2.5 % or >5%) could be detected in NDMM (35 % or 14%) and RRMM (31% or 19%) of patients (Table1). PAR1+CD38+138+ cells were more frequent among patients with lower percentage of plasma cells in RRMM group (2,98 ± 4,5 vs 1,93 ± 3,96, P=0.028) but not NDMM. PAR1 was similarly highly expressed on HCML. Two of the novel PAR1 binding molecules (XT5 and XT2B) were found to have the lowest IC50. The IC50 were similar for all HMCLs, primary and refractory, with XT5. With XT2B the IC50 was less (U266) or higher (JJN3) or similar (OPM2) for refractory compared to the primary HMCL. PAR1 expression and anti-myeloma IC50 values of cell lines are summarized in Table 2. Conclusion: PAR1 expression is detectable at very low or very high percentages on CD138+plasma cells. Expression is higher on cells with CD27 expression (patient samples) or lacking CD27 (HMCL). Inverse correlation between PAR1 expression and plasma cell percentage among myeloma patients is detected among RRMM but not on NDMM samples. This finding may point to expression of PAR1 on quiescent plasma progenitors as suggested by Tian et al previously. The intensity or frequency of PAR1 expression on HMCL did not influence the anti-myeloma effects of these novel molecules. PAR1 binding molecules, in particular XT5, are promising as they are effective even on Bortezomib refractory HCML. However their mechanism of action and the role of PAR1 require further investigations. This study has been supported by a research grant from Turkish Academy of Sciences. Table 1. Frequency of PAR1 expression (> 2.5 %) on total plasma cells (CD38+138+) and on quiescent plasma cells (CD38+138+27+) Control (n=43) NDMM (n=84) RRMM (n=54) P CD38+138+ (%) 0,56± 0,66 4,48 ± 7,67 5,44 ± 12,13 0,007 PAR1+ among CD38+138 (%) 6,18 ± 13,14 4,14 ± 11,00 3,42 ± 8,81 0,394 PAR1+ among CD38+138+27+(%) 5,44 ± 12,13 3,42 ± 8,81 3,58 ± 8,57 0,207 Table 1. Comparison of Flow Cytometric PAR1 expression and IC50 (in uM after 72 hours)of the two novel molecules on three Human Myeloma Cell Lines. H929 RPMI8221 U266 IC50 XT2B 33.9 >100 34.3 IC50 XT5 8.12 5.45 9.77 CD38+138+ (total%) 85 % 75 % 80 % PAR1% and (MFI) within CD38+138+ 83 %(13,6) 90 % (2,1) 85 % (2,1) Disclosures Beksac: Celgene: Consultancy, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Off Label Use: Elotuzumab is an investigational agent being studied for the treatment of multiple myeloma.. Usmani:Millennium: Honoraria, Speakers Bureau; Sanofi: Honoraria, Research Funding; Onyx: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Array BioPharma: Honoraria, Research Funding; Pharmacyclics: Research Funding; Janssen Oncology: Honoraria, Research Funding. Tian:University of Arkansas for Medical Sciecnes: Employment.

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Genomic Characterization of in Vitro Acquired-Resistance to Proteasome Inhibitors

Blood ◽

10.1182/blood-2021-148584 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2651-2651

Author(s):

Hugues De Boussac ◽

Alboukadel Kassambara ◽

Amelie Machura ◽

Djamila Chemlal ◽

Claire Gourzones ◽

...

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Board Of Directors ◽

Cell Lines ◽

Expression Profiling ◽

Proteasome Inhibitors ◽

Resistance Mechanisms ◽

P Value ◽

Glycolytic Metabolism ◽

Advisory Committees

Abstract Multiple myeloma (MM) is the second most common hematological malignancy characterized by an abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novels agents that have significantly improved clinical outcomes, MM patients invariably relapse. A better understanding of the drug resistance mechanisms and development of biomarkers remain of major interest to improve the treatment of patients. In order to further investigate the mechanisms involved in resistance to proteasome inhibitors (PI), we have derived and characterized PI-resistant human myeloma cells lines (HMCLs) from different molecular subgroups including XG2BR t(12;14), XG7BR t(4;14), XG19BR t(14;16), and XG1BR t(11;14). These cell lines were cultured continuously with escalating concentrations of Bortezomib (Btz) during 12 months and showed a significant resistance to Bortezomib compared to their parental cell lines (mean IC50: Btz-resistant HMCLs =5.5nM vs parental HMCLs=2.5nM, p<0.05). Of interest, we demonstrated that Btz-resistant HMCLs are also significantly more resistant to Carfilzomib (Cfz) and Ixazomib (Ixa) PIs (mean IC50: Btz-resistant HMCLs =6nM for Cfz and =70nM for Ixa vs parental HMCLs=3nM, for Cfz, p<0.05 ; and =21nM for Ixa, p<0.05. No significant cross-resistance was observed with other therapeutic agents including melphalan, dexamethasone and IMIDs indicating that the observed drug resistance mechanisms are specifically related to PIs. In order to understand the PIs resistance mechanisms acquired by MM cells, we used a combination of genomic approaches including whole genome sequencing, and comparative transcriptomic analysis. Among the 40 mutations identified in Btz-resistant HMCLs compared to the parental ones, a mutation residing in the Btz-binding pocket in the proteasome beta5-subunit (PSMB5) gene was identified. This mutation has already been found in other models of PIs-resistant MM cell lines and in relapsed MM patients, and is associated to PIs- resistance by reducing the PI binding capacity and impairing the chemotrypsin-like catalytic activity of the 20S proteasome. When we compared the gene expression profiling of Btz-resistant HMCLs with the parental ones, we identified a gene expression signature significantly enriched in nucleotide excision repair (NER) pathway with an increased expression of ERCC1, ERCC5, LIG1, POLD1 in the Btz-resistant HMCLs (FC >1.5). Since, protein ubiquitination is essential in regulation and coordination of various pathways of DNA damage recognition, signaling and repair, proteasome inhibitors affect DNA repair, overexpression of DNA repair pathways may participate in drug resistance mechanisms. Using gene expression profiling data, we also identified a significant downregulation of 8 solute carrier protein (SLC) intake transporters (SLC6A6, SLC16A1, SLC16A14, SLC16A10, SLC25A13, SLC5A6, SLCO3A1, SLCO4A1) together with a significant upregulation of xenobiotic receptors (RXRA, RXRB) in Btz-resistant HMCLs compared to parental HMCLs (FC> or < 1.5; p value <0.05). In addition, several genes involved in antioxidant response (NQO1), and in glutathione regulation (MGST1, MGST2, GSTO1) were also overexpressed in Btz-resistant HMCLs (FC>1.5 p value <0.05). Investigating the deregulated genes involved on energy metabolism that is often associated with resistance, we identified an upregulation of glycolytic enzymes directly involved in glycolytic metabolism (ALDOC, ENO3, HK1, PDK1, PDK3, PFKB3, PFKB4, PFKL, SLC2A1 (FC>1.5 p value <0.05) in the Btz-resistant HMCLs. Altogether our data underline a significant deregulation of genes involved in cell metabolism and drug clearance system that allow the PI resistant-MM cells to maintain metabolic homeostasis and survival in stringent redox conditions. This is in accordance with described mechanisms linking drug resistance and glycolytic metabolism in cancers cells. Metabolomic analyzes are currently ongoing for functional validation. Altogether, drug-resistant cell lines represent an attractive preclinical model to test molecules targeting these pathways in order to identify new therapeutic strategies to overcome PI resistance in MM. Disclosures De Boussac: Diag2Tec: Current Employment. Kassambara: Diag2Tec: Consultancy. Machura: Diag2Tec: Current Employment. Chemlal: Diag2Tec: Current Employment. Vincent: Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Herbaux: Abbvie: Honoraria, Research Funding; Roche: Honoraria; Janssen: Honoraria; Takeda: Honoraria, Research Funding. Bruyer: Diag2Tec: Current Employment. Moreaux: Diag2Tec: Consultancy.

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Targeting Glycolysis in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant in Hypoxic Conditions

Blood ◽

10.1182/blood-2019-129818 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4344-4344

Author(s):

Seiichi Okabe ◽

Yuko Tanaka ◽

Mitsuru Moriyama ◽

Akihiko Gotoh

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Proteasome Inhibitors ◽

Myeloma Cell ◽

New Agents ◽

Myeloma Cells ◽

Hypoxic Conditions ◽

Inhibitor Treatment

Introduction: Multiple myeloma (MM) is one of the hematological malignancy and characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Hypoxia is an important component of the bone marrow microenvironment. Hypoxia may increase myeloma cell survival. Because cells shift primarily to a glycolytic mode for generation of energy in hypoxic conditions, glycolytic activities can be targeted therapeutically in MM patients. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate which is a regulator of glycolysis. Materials and Methods: In this study, we investigated whether PFKFB was involved in myeloma cells in hypoxia condition. We also investigated whether PFKFB inhibitors could suppress myeloma cells and enhance the sensitivity of myeloma cells to proteasome inhibition. Results: We first investigated the expression of PFKBP in the myeloma cell lines in hypoxia condition. PFKFB family contains four tissue-specific isoenzymes encoded by four different genes. We found expression of PFKBP3 and PFKBP4 were increased in hypoxia condition. We found gene expression of PFKBP3 and PFKBP4 were involved in myeloma cell lines and myeloma patient samples in hypoxia condition from the public microarray datasets (GSE80140 and GSE80545). In hypoxia condition, expression of hypoxia-inducible factor 1α (HIF1α) was increased and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was activated in myeloma cell lines. Expression of PFKBP3 and PFKBP4 were inhibited by HIF1α inhibitor and p38 MAPK inhibitor treatment. In the hypoxia condition, activity of proteasome inhibitors were reduced compared to normoxia condition. We next investigated whether PFKBP3 inhibitor, PFK158 and PFKBP4 inhibitor, 5MPN could inhibit the proliferation of myeloma cells. We found PFK158 and 5MPN treatment inhibited the growth of myeloma cells in a dose dependent manner in hypoxia condition. Combined treatment of myeloma cells with carfilzomib and PFK158 or 5MPN caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We found proteasomal activity was also reduced by carfilzomib and PFK158 or 5MPN treatment. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after carfilzomib and PFK158 or 5MPN treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by carfilzomib and PFK158 or 5MPN on myeloma cells. In the hypoxia condition and inhibitor treatment, glycolytic activities (e.g. glucose and lactate) were changed in myeloma cells. Conclusion: The PFKBP3 and PFKBP4 are enhanced in hypoxia condition and involved in proteasome inhibitor sensitivity. Our data also suggested that administration of PFKBP3 and PFKBP4 inhibitors may be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

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