Inflammasome-Primed Myeloid Cells Maintain a Pro-Tumor Microenvironment in Multiple Myeloma

Abstract Background: Multiple myeloma (MM) disease progression is influenced by signals from the bone marrow (BM) microenvironment. Recently, we showed that the MM BM is characterized by inflammatory mesenchymal stromal cells (iMSCs) that transcribe MM survival factors and are predicted to recruit proliferating myeloma cells via CCL2-CCR2 interactions (de Jong et al. Nat Immunol. 2021). iMSCs also transcribed high levels of chemokines that can bind to CXCR1 and 2. Myeloid cells are known to express CXCR1/2, and have been implicated in both pro- and anti-tumor responses in various malignancies. Therefore, we hypothesized that iMSCs attract and influence myeloid populations in the MM BM. Results: Using flow cytometry, we verified expression of CXCR1/2 on myeloid cell populations in the BM of 5 newly diagnosed MM (NDMM) patients. CD15 + neutrophils were the most dominant population expressing these receptors, as 22.4% (± 9.8%) of cells expressed CXCR2 alone, and 72.6% (± 8.0%) expressed both CXCR1 and CXCR2. CD14 + monocytes only expressed CXCR2 (86.9% ± 15.8%). Importantly, less than 1% of myeloma cells expressed these receptors (n = 17 NDMM). As these findings suggested neutrophils and monocytes as potential targets of iMSC-mediated chemotaxis, we set out to identify MM-associated alterations in this population by performing single cell RNA sequencing of the full neutrophilic and monocytic lineages (n = 5 NDMM and 2 controls). In line with our flow cytometric data, CXCR1 transcripts were absent in monocytes, while CXCR2 was transcribed by classical monocytes of both myeloma patients and controls. Interestingly, CXCR1 and CXCR2 transcription was increased in mature neutrophils of MM patients compared to controls. Additionally, both mature classical monocytes as well as mature neutrophils of MM patients had an activated transcriptome as defined by increased transcription of C3AR1, SLPI, and IL6R, the plasma cell supportive factor TNFSF13B (encoding BAFF), and the inflammatory cytokines IL1B and IL18. Transcription of IL1B and IL18 can be regulated by pattern-recognition receptors (PRRs) binding damage-associated molecular patterns (DAMPs) resulting from e.g. matrix breakdown. Transcription of PRRs as TLR1, 2 and 4 was increased in mature neutrophils and classical monocytes of MM patients compared to controls. Secretion of IL-1β and IL-18 relies on the cleavage of pro-forms of these cytokines by the inflammasome, a multiprotein complex that is assembled in response to alarmins. Transcription of inflammasome components PYCARD, NLRP3 and CASP1 was increased in mature neutrophils and classical monocytes of patients with MM. Additionally, protein levels of both IL-18 and IL-1β are increased in BM plasma from MM patients, implicating activated neutrophils and monocytes as a potential sources of these cytokines. Conclusion: In MM, mature neutrophils and classical monocytes are activated and might interact with iMSCs via CXCR1 and/or 2. Moreover, these myeloid cells are inflammasome-primed and are likely to be sources of the increased IL-1β levels in the MM BM. Therefore, myeloid cells and iMSCs may form a feed-forward loop in which myeloid cells contribute to a pro-MM environment by maintaining iMSC and by directly providing BAFF to tumor cells. Disclosures Broyl: Celgene/BMS: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Sonneveld: Janssen: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; SkylineDx: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene/BMS: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

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Inhibition of protein geranylgeranylation induces apoptosis in myeloma plasma cells by reducing Mcl-1 protein levels

Blood ◽

10.1182/blood-2003-03-0970 ◽

2003 ◽

Vol 102 (9) ◽

pp. 3354-3362 ◽

Cited By ~ 85

Author(s):

Niels W. C. J. van de Donk ◽

Marloes M. J. Kamphuis ◽

Berris van Kessel ◽

Henk M. Lokhorst ◽

Andries C. Bloem

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Mevalonate Pathway ◽

Cytochrome C Release ◽

Hmg Coa Reductase ◽

Myeloma Cells ◽

Protein Levels ◽

Geranylgeranyl Transferase ◽

Induced Apoptosis ◽

Coa Reductase

AbstractHMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway leading to the formation of cholesterol and isoprenoids such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). The inhibition of HMG-CoA reductase by lovastatin induced apoptosis in plasma cell lines and tumor cells from patients with multiple myeloma. Here we show that cotreatment with mevalonate or geranylgeranyl moieties, but not farnesyl groups, rescued myeloma cells from lovastatin-induced apoptosis. In addition, the inhibition of geranylgeranylation by specific inhibition of geranylgeranyl transferase I (GGTase I) induced the apoptosis of myeloma cells. Apoptosis triggered by the inhibition of geranylgeranylation was associated with reduction of Mcl-1 protein expression, collapse of the mitochondrial transmembrane potential, expression of the mitochondrial membrane protein 7A6, cytochrome c release from mitochondria into the cytosol, and stimulation of caspase-3 activity. These results imply that protein geranylgeranylation is critical for regulating myeloma tumor cell survival, possibly through regulating Mcl-1 expression. Our results show that pharmacologic agents such as lovastatin or GGTase inhibitors may be useful in the treatment of multiple myeloma.

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A niche-dependent myeloid transcriptome signature defines dormant myeloma cells

Blood ◽

10.1182/blood.2018880930 ◽

2019 ◽

Vol 134 (1) ◽

pp. 30-43 ◽

Cited By ~ 22

Author(s):

Weng Hua Khoo ◽

Guy Ledergor ◽

Assaf Weiner ◽

Daniel L. Roden ◽

Rachael L. Terry ◽

...

Keyword(s):

Multiple Myeloma ◽

Overall Survival ◽

Disease Progression ◽

Drug Targets ◽

Myeloid Cell ◽

Progression Free Survival ◽

Healthy Human ◽

Myeloma Cells ◽

Depth Of Response ◽

Transcriptome Signature

Abstract The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.

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Kinome-Wide RNAi Studies in Human Multiple Myeloma Identify a Lymphoid Restricted Kinase GRK6 as a Selectively Vulnerable Target That Regulates STAT3/MCL1.

Blood ◽

10.1182/blood.v114.22.601.601 ◽

2009 ◽

Vol 114 (22) ◽

pp. 601-601

Author(s):

Rodger E. Tiedemann ◽

Yuan Xiao Zhu ◽

Jessica Schmidt ◽

Hongwei Yin ◽

Quick Que ◽

...

Keyword(s):

Multiple Myeloma ◽

Epithelial Cells ◽

Tumor Cells ◽

Research Funding ◽

Kinase Inhibitors ◽

Lymphoid Tissues ◽

Myeloma Cells ◽

Stat3 Phosphorylation ◽

Human Multiple Myeloma

Abstract Abstract 601 A paucity of validated kinase targets in human multiple myeloma (MM) has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in MM tumor lines bearing common t(4;14), t(14;16) and t(11;14) translocations to identify critically vulnerable kinases in MM tumor cells without regard to preconceived mechanistic notions. Primary screening was performed in duplicate using an 1800-oligo siRNA library in a single-siRNA-per-well format. siRNA were transfected at low concentration (13nM) to minimize off-target effects using conditions that resulted in transfection of >95% cells and <5% background cytotoxicity. After 96 hours, viability was measured by ATP-dependent luminescence. Fifteen kinases were consistently vulnerable in MM cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. While several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly the G-protein coupled receptor kinase, GRK6, appeared selectively vulnerable in MM. GRK6 inhibition is selectively lethal to KMS11, OPM1, H929, KMS18 and OCI-MY5 myeloma cells and has minimal effect on 293, MCF7, SF767, A375 or A549 epithelial cells. Persistent expression of FLAG-GRK6 via cDNA rescued KMS11 cells from the lethal effect of a 3'UTR-targeted GRK6 siRNA, but not from control siRNA, validating identification of GRK6 as an essential myeloma survival kinase. Furthermore, concordant results were obtained using four different exon-based GRK6 siRNA, all of which induced GRK6 silencing and similar inhibition of KMS11 proliferation and viability. Significantly, GRK6 is ubiquitously expressed in lymphoid tissues and myeloma, but by comparison appears absent or only weakly expressed in most primary human somatic tissues. From co-immunoprecipitation experiments we demonstrate that GRK6 is highly expressed in myeloma cells via direct association with the HSP90 chaperone. Inhibition of HSP90 with geldanamycin blocks GRK6 protein expression. Importantly, direct GRK6 silencing causes rapid and selective suppression of STAT3 phosphorylation that is associated with sustained reductions in total MCL1 protein levels and MCL1 phosphorylation (within 24 hours), providing a potent mechanism for the cytotoxicity of GRK6 inhibition in MM tumor cells. GF109203X is an inhibitor of both protein kinase C and of GRK6 that causes near total inhibition of these kinases in vitro at distinct concentrations of 0.1μM and 1-10μM respectively. Notably, GF109203X was substantially cytotoxic to 10/14 myeloma tumor lines at concentrations most consistent with GRK6 inhibition (5-20μM), and was selectively more cytotoxic to myeloma tumor cells than to non-myeloma cell lines (P=0.01), highlighting the potential of GRK6 as a pharmaceutical target for selective therapeutic intervention in myeloma. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. Disclosures: Perkins: MMRC: Employment. Reeder:Celgene: Research Funding; Millennium: Research Funding. Fonseca:Otsuka: Consultancy; BMS: Consultancy; Amgen: Consultancy; Medtronic: Consultancy; Genzyme: Consultancy.

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The Proteasome Inhibitor Bortezomib Stimulates Osteoblastic Differentiation of Human Osteoblast Precursor Cells Via Upregulation of Vitamin D Receptor Signaling

Blood ◽

10.1182/blood.v116.21.1899.1899 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1899-1899

Author(s):

Martin Kaiser ◽

Maren Mieth ◽

Orhan Sezer ◽

Ulrike Heider

Keyword(s):

Multiple Myeloma ◽

Vitamin D ◽

Proteasome Inhibitor ◽

Research Funding ◽

Fold Increase ◽

Osteoblastic Differentiation ◽

Luciferase Reporter ◽

Human Osteoblast ◽

Myeloma Cells ◽

Osteoblast Precursors

Abstract Abstract 1899 Introduction In multiple myeloma (MM), interactions of the malignant plasma cell clone with the bone marrow microenvironment lead to an enhanced osteoclast recruitment and impaired osteoblast activity. The proteasome inhibitor bortezomib has been shown to suppress osteoclast activity, and there is recent evidence that bortezomib enhances osteoblast differentiation. The aim of this study was to investigate the effects of bortezomib on human osteoblast precursors, focusing on vitamin D (VD) dependent osteoblastic differentiation. Since vitamin D receptor (VDR) is degraded by the proteasome, we hypothesized that bortezomib could influence its signaling and hence vitamin D induced osteoblastic differentiation. This might be of clinical importance, since an increased rate of vitamin D deficiency has recently been reported in patients with MM. Methods Primary human mesenchymal stem cells (hMSC) and primary human osteoblasts (hOB) were isolated from bone marrow aspirates or from bone fragments of healthy donors undergoing orthopedic surgery, respectively. Ascorbic acid and β-glycerolphosphate were used for osteoblastic stimulation (OS), either in combination with or without vitamin D. In order to analyze the effects of proteasome inhibition on osteoblastic differentiation and activity, hMSC and hOB were incubated with bortezomib at subapoptotic doses (1 - 5 nM). In addition, coculture experiments of hMSC, hOB and myeloma cells were performed. Expression of osteocalcin and osteopontin (OPN) were quantified by real-time PCR as markers of osteoblastic lineage differentiation. Expression of VDR was analyzed by western blot in subcellular fractions and VDR signaling was investigated using luciferase reporter assays. Results In coculture experiments, myeloma cells inhibited the vitamin D dependent differentiation and activity of osteoblast precursors, e.g. coculture of hMSC with the myeloma cell line LP-1 for 4 days decreased their osteocalcin expression by 58%. Treatment with bortezomib led to an increased osteoblastic differentiation of hMSC and hOB by OS, represented by an enhanced expression of osteoblast markers osteocalcin and OPN. Importantly, this effect could be further increased, when vitamin D was added. In hMSC stimulated with OS only, addition of 5 nM bortezomib led to an 18.3 fold increase in OPN mRNA expression. In comparison, hMSC stimulated with OS + vitamin D showed a 27.5 fold increase in OPN mRNA with the addition of bortezomib. Osteocalcin expression was increased 1.9 fold by bortezomib in the presence of OS and vitamin D, but not with OS alone. Similar results were obtained with osteoblasts: Incubation with bortezomib slightly increased osteocalcin and OPN mRNA expression in cells stimulated with OS only (1.3 fold and 2.4 fold, respectively). In comparison, in cells stimulated with OS and vitamin D, bortezomib elevated osteocalcin and OPN expression 2.9 fold and 5.5 fold, respectively. Bortezomib led to an increase in nuclear VDR levels in hMSC in western blot analyses. Primary hMSC transfected with a VDR luciferase reporter construct showed a 3.7 fold increase in VDR signaling with bortezomib. Conclusion Our data show that bortezomib stimulates osteoblastic differentiation of hMSCs and hOBs and acts, at least in part, through VDR signaling. Substitution of vitamin D in multiple myeloma patients treated with bortezomib may be beneficial for bone turnover and needs clinical evaluation. Disclosures: Kaiser: Johnson & Johnson: Research Funding. Mieth:Johnson & Johnson: Research Funding. Sezer:Johnson & Johnson: Research Funding. Heider:Johnson & Johnson: Research Funding.

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mRNA and Protein Expression Levels of Secretory Leukocyte Protease Inhibitor (SLPI) Are Severely Reduced in Patients with Severe Congenital Neutropenia (CN)

Blood ◽

10.1182/blood.v118.21.2165.2165 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2165-2165

Author(s):

Wienke Ellerbeck ◽

Olga Klimenkova ◽

Julia Skokowa ◽

Karl Welte

Keyword(s):

Protease Inhibitor ◽

Cell Line ◽

Myeloid Cells ◽

Myeloid Cell ◽

Myeloid Differentiation ◽

Severe Congenital Neutropenia ◽

Healthy Individuals ◽

Congenital Neutropenia ◽

Protein Levels ◽

Myeloid Cell Line

Abstract Abstract 2165 Secretory Leukocyte Protease Inhibitor (SLPI) is a cationic serine protease inhibitor with antiprotease, primarily anti-Neutrophil ELastase (NE), activities. Moreover, SLPI modulates intracellular signal transduction pathways such as NF-kB and Erk. The molecular interaction and the balance between NE and SLPI is tightly regulated. On the one side, NE upregulates the SLPI expression and at the other hand SLPI inhibits the NE-induced degradation of proteins. We identified severe diminished levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN) harbouring either ELANE or HAX1 mutations, as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. SLPI protein levels in plasma of CN patients were also significantly reduced. We further analysed whether diminished levels of SLPI are associated with the „maturation arrest“ of myeloid cells seen in CN patients. We inhibited SLPI using lentivirus-based transduction of the myeloid cell line NB4 with SLPI-specific shRNA and analysed ATRA-triggered myeloid differentiation. Indeed, myeloid differentiation was severely affected in NB4 cells transduced with SLPI-specific shRNA, as compared to control shRNA transduced cells. Further, we analysed the mechanisms leading to SLPI downregulation. Previously, we identified severely reduced mRNA and protein levels of NE in myeloid cells and in plasma of CN patients with either ELANE or HAX1 mutations, as compared to healthy individuals. Knowing that NE induces SLPI expression, we assumed that diminished NE levels may be responsible for the low SLPI expression in CN patients. Indeed, inhibition of NE in the myeloid cell line NB4 using NE-specific shRNAs led to diminished expression of SLPI mRNA, as compared to ctrl shRNA transduced cells. At the same time, we also found that transduction of the myeloid cell line NB4 with wild type (WT) NE resulted in the increased expression of SLPI mRNA but mutated (MUT) forms of NE as found in CN patients were not able to induce SLPI mRNA, as compared to ctrl transduced cells. Taken together, both diminished NE levels and mutations in ELANE gene may cause downregulation of SLPI. In summary, SLPI is severely downregulated in CN patients due to defective NE protein levels and ELANE mutations. As a consequence, the anti-microbial and antiinflammatory activities of SLPI are diminished in CN patients. Disclosures: No relevant conflicts of interest to declare.

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Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v118.21.136.136 ◽

2011 ◽

Vol 118 (21) ◽

pp. 136-136

Author(s):

Ze Tian ◽

Jian-Jun Zhao ◽

Jianhong Lin ◽

Dharminder Chauhan ◽

Kenneth C. Anderson

Keyword(s):

Multiple Myeloma ◽

Tumor Suppressor ◽

Board Of Directors ◽

Expression Profiling ◽

Research Funding ◽

Advisory Committees ◽

Myeloma Cells ◽

Investigational Agent ◽

Reporter Activity

Abstract Abstract 136 Investigational Agent MLN9708 Target Tumor Suppressor MicroRNA-33b in Multiple Myeloma Cells Ze Tian, Jianjun Zhao, Jianhong Lin, Dharminder Chauhan, Kenneth C. Anderson Medical Oncology, Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, 02115 MicroRNAs (miRNAs) are 19–25 nucleotide-long noncoding RNA molecules that regulate gene expression both at the level of messenger RNA degradation and translation. Emerging evidence shows that miRNAs play a critical role in tumor pathogenesis by functioning as either oncogene or tumor suppressor genes. The role of miRNA and their regulation in response to proteasome inhibitors treatment in Multiple Myeloma (MM) is unclear. Here, we utilized MLN9708, a selective orally bio-available proteasome inhibitor to examine its effects on miRNA alterations in MM.1S MM cells. Upon exposure to aqueous solutions or plasma, MLN9708 rapidly hydrolyzes to its biologically active form MLN2238. Our previous study using both in vitro and in vivo models showed that MLN2238 inhibits tumor growth and triggers apoptosis via activation of caspases. Moreover, MLN2238 triggered apoptosis in bortezomib-resistant MM cells, and induced synergistic anti-MM activity when combined with HDAC inhibitor SAHA, dexamethasone, and lenalidomide. In the current study, we treated MM.1S cells with MLN2238 (12 nM) for 3 hours and harvested; total RNA was subjected to miRNA profiling using TaqMan® Array Human miRNA A-Card Set v3.0 and the data was analyzed using dChip analysis. Results showed that MLN2238 modulates miRNA expression with a total of 36 miRNA changing their expression profiling (δδCT>1.5 or δδCT <-1.5; 19 were upregulated and 17 showed a downregulation). Among all miRNA, miR-33b was highly (δδCT>7) upregulated in response to MLN2238 treatment. We therefore hypothesized that miR-33b may play a role in MM pathogenesis as well as during MLN2238-induced proteasome inhibition in MM cells. We first utilized quantitative polymerase chain reaction (q-PCR) to validate the changes in miRNA expression profiling. Results confirmed that MLN2238 treatment triggers significant increase in the miR-33b expression in MM.1S cells (2.1 and 2.2 folds at 3h and 6h, respectively; P<0.001). Examination of normal PBMCs and plasma cells showed higher expression of miR-33b than patient MM cells (P<0.001). We further investigated the functional role of miR-33b in MM cells at baseline and during MLN2238 treatment. Drug sensitivity, cell viability, apoptosis, colony formation, and migration assays were performed using cell TilTer-Glo, Annexin V-FITC/PI staining, MTT staining, and Transwell assays, respectively. Signaling pathways modulated post miR-33b overexpression were evaluated by q-PCR, immunoblot, and reporter assays. Our findings show that overexpression of miR-33b significantly decreased cell viability, cell migration, colony formation, as well as increased apoptosis and sensitivity of MM cells to MLN2238 treatment. Targetscan analysis predicted pim-1 as a putative downstream target of miR-33b. Overexpression of miR-33b downregulated pim-1 mRNA and protein expression. To further corroborate these data, we co-tranfected miR-33b and Pim-1-wt or Pim-1-mt in 293T and MM.1S cell lines. In concert with our earlier findings, miR-33b decreases pim-1-wt, but not pim-1-mt reporter activity in both cell lines. Reflecting the overexpression study results, MLN2238 treatment also decreases pim-1-wt, but not pim1-mt reporter activity. Moreover, a biochemical inhibitor of pim1/2 triggered apoptosis in MM cells. Finally, overexpression of miR-33b inhibits tumor growth (P<0.001) and prolongs survival (P<0.001) in both subcutaneous and disseminated human MM xenograft models. In summary, our study suggests that miR-33b is a tumor suppressor, which plays a role during MLN2238-induced apoptotic signaling in MM cells, and provide the basis for novel therapeutic strategies targeting miR-33b in MM. Disclosures: Anderson: Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership.

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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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CD38-Targeted Immunochemotherapy Of Multiple Myeloma: Preclinical Evidence For Its Combinatorial Use In Lenalidomide and Bortezomib Refractory/Intolerant MM Patients

Blood ◽

10.1182/blood.v122.21.277.277 ◽

2013 ◽

Vol 122 (21) ◽

pp. 277-277 ◽

Cited By ~ 3

Author(s):

Inger S. Nijhof ◽

Willy A. Noort ◽

Jeroen Lammerts van Bueren ◽

Berris van Kessel ◽

Joost M. Bakker ◽

...

Keyword(s):

Multiple Myeloma ◽

Research Funding ◽

Plasma Cells ◽

Cell Lysis ◽

In Vitro Assays ◽

Human Antibody ◽

Myeloma Cells ◽

Clinical Phase

Abstract Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

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Targeting EZH2 in Multiple Myeloma Could be Promising for a Subgroup of MM Patients in Combination with IMiDs

Blood ◽

10.1182/blood.v128.22.311.311 ◽

2016 ◽

Vol 128 (22) ◽

pp. 311-311 ◽

Cited By ~ 4

Author(s):

Laurie Herviou ◽

Alboukadel Kassambara ◽

Stephanie Boireau ◽

Nicolas Robert ◽

Guilhem Requirand ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Board Of Directors ◽

Research Funding ◽

Plasma Cells ◽

Newly Diagnosed ◽

Advisory Committees ◽

Myeloma Cells ◽

Ezh2 Expression ◽

Bone Marrow Plasma

Abstract Multiple Myeloma is a B cell neoplasia characterized by the accumulation of clonal plasma cells within the bone marrow.Epigenetics is characterized by a wide range of changes that are reversible and orchestrate gene expression. Recent studies have shown that epigenetic modifications play a role in multiple myeloma (MM) by silencing various cancer-related genes. We investigated the epigenetic genes differentially expressed between normal bone marrow plasma cells (BMPC ; N=5) and MM plasma cells from patients (N=206). Using SAM (Significance Analysis of Microarrays) analysis, only 12 genes significantly differentially expressed between BMPC and MM cells (ratio > 2 and FDR (false discovery rate) < 5%) were identified, including the EZH2 histone methyltransferase. EZH2, the enzymatic subunit of Polycomb Repressive Complex 2, is a histone methyltransferases able to repress gene expression by catalyzing H3K27me3 histone mark. EZH2 overexpression has been associated with numerous hematological malignancies, including MM. We thus studied EZH2 role in MM physiopathology and drug resistance. EZH2 expression was analyzed in normal bone marrow plasma cells (BMPCs; N=5), primary myeloma cells from newly diagnosed patients (MMCs; N=206) and human myeloma cell lines (HMCLs; N=40) using Affymetrix microarrays. EZH2 gene is significantly overexpressed in MMCs of patients (median 574, range 105 - 4562) compared to normal BMPCs (median = 432; range: 314 - 563) (P < 0.01). The expression is even higher in HMCLs (median 4481, range 581 - 8455) compared to primary MMCs or BMPCs (P < 0.001). High EZH2 expression is associated with a poor prognosis in 3 independent cohorts of newly diagnosed patients (Heidelberg-Montpellier cohort - N=206, UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N =158). Furthermore, GSEA analysis of patients with high EZH2 expression highlighted a significant enrichment of genes involved in cell cycle, downregulated in mature plasma cells vs plasmablasts, and EZH2 targets. Specific EZH2 inhibition by EPZ-6438 EZH2 inhibitor induced a significant decrease of global H3K27me3 in all the HMCLs tested (P < 0.01) and inhibited MM cell growth in 5 out of the 6 HMCLs tested. The inhibitory effect of EZH2 inhibitor on MM cell growth appeared at day 6 suggesting that it is mediated by epigenetic reprogramming. To confirm that EZH2 is also required for the survival of primary MMCs from patients, primary MM cells (n = 17 patients) co-cultured with their bone marrow microenvironment and recombinant IL-6 were treated with EPZ-6438. As identified in HMCLs, EZH2 inhibition significantly reduced the median number of viable myeloma cells by 35% (P = 0.004) from a subset of patients (n=9) while the other group (n=8) was resistant. Of interest, EPZ-6438 induced a significant global H3K27me3 decrease in both groups of patient. RNA sequencing of 6 HMCLs treated with EPZ-6438 combined with H3K27me3 ChIP analyses allowed us to create an EZ GEP-based score able to predict HMCLs and primary MM cells sensitivity to EZH2 inhibitors. We also observed a synergy between EPZ-6438 and Lenalidomide, a conventional drug used for MM treatment. More interestingly, pretreatment of myeloma cells with EPZ-6438 significantly re-sensitize drug-resistant MM cells to Lenalidomide. Investigating the effect of EPZ-6438/Lenalidomide combination in MMC, we identified that IKZF1, IRF4 and MYC protein levels were significantly more inhibited by the combination treatment (65.5%, 63.9% and 14.8% respectively) compared with Lenalidomide (51.5%, 43% and 2.2%) or EPZ-6438 (45.2%, 38.7% and 6.2%) alone. Clinical trials are ongoing with EZH2 inhibitors in lymphoma and could be promising for a subgroup of MM patients in combination with IMiDs. Furthermore, the EZ score enables identification of MM patients with an adverse prognosis and who could benefit from treatment with EZH2 inhibitors. Disclosures Goldschmidt: Celgene: 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; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Hose:EngMab: Research Funding; Takeda: Other: Travel grant; Sanofi: Research Funding.

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Safety and Efficacy of Letetresgene Autoleucel (lete-cel; GSK3377794) Alone or in Combination with Pembrolizumab in Relapsed and Refractory Multiple Myeloma

Blood ◽

10.1182/blood-2021-153004 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3865-3865

Author(s):

Taiga Nishihori ◽

James E. Hoffman ◽

Jonathan L. Kaufman ◽

Anne Huff ◽

Charlotte Snape ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cell ◽

Cancer Therapy ◽

Research Funding ◽

Cell Kinetics ◽

Publicly Traded ◽

Myeloma Cells ◽

Safety And Efficacy ◽

Anti Cancer ◽

Grade 3

Abstract Background: Outcomes remain poor for patients with relapsed and refractory multiple myeloma (RRMM) progressing on conventional therapy (proteasome inhibitors, IMiDs, anti-CD38 antibodies, BCMA targeting agents, corticosteroids). Lete-cel, an autologous T-cell therapy, targets NY-ESO-1/LAGE-1a+ tumors using a genetically modified, high-affinity T-cell receptor. NY-ESO-1 and LAGE-1a are immunogenic cancer/testis antigens frequently overexpressed in MM and are linked to poor clinical outcomes. Additionally, PD-1 expression, which may limit adaptive immune response, has been observed previously in RRMM patients following treatment with lete-cel (Stadtmauer et al. Blood Adv, 2019; 3: 2022-2034). This open-label, pilot study evaluated the safety and efficacy of lete-cel +/- pembrolizumab (pembro) in patients with RRMM. Study design and methods: Key eligibility criteria include: age ≥18 yr; HLA-A*02:01; A*02:05, and/or A*02:06; NY-ESO-1+ and/or LAGE-1a+; protocol-required prior regimens; specified washouts from prior therapy; no active/chronic/intercurrent illness. Following lymphodepletion (LD), patients received lete-cel (Arm 1) or lete-cel + pembro (Arm 2). Planned pembro dosing was 200 mg/dose Q3 weeks (wks) starting at Wk 3. Primary endpoint was safety and tolerability. Key efficacy endpoint was investigator-assessed overall response rate (ORR) by International Myeloma Working Group uniform response criteria for MM (2016); response was assessed Q3 wks from Wk 3 to Wk 24, then Q6 wks to Wk 72, then every 3 months (mo) to disease progression/death/withdrawal. NY-ESO-1/LAGE-1a expression was assessed by qRT-PCR on myeloma cells. Transduced cell kinetics were assessed by qPCR of transgene vector copies in DNA from peripheral blood mononuclear cells. Results: Six patients (all male; median age 63 yr) were enrolled; 3 per arm. All had prior systemic anti-cancer therapy; 3 patients had received ≥5 prior regimens. Five of 6 patients received systemic anti-cancer therapy between leukapheresis and LD. All received reduced LD due to age and, in some patients, renal impairment. Patients in each arm received similar numbers of transduced T cells. Each of the 3 patients in Arm 2 received a median of 3 pembro doses (range: 3-4 doses). Start of pembro dosing was delayed to Wk 6 in 2 patients due to ongoing toxicities. There were no Grade 5 AEs. Grade 3/4 T-cell related AEs were reported in 3 patients, and all patients had Grade 3/4 LD-related AEs. Hematopoietic cytopenias were the most common treatment-emergent and treatment-related Grade 3/4 AE, occurring in all patients. All cytopenias were reported to have resolved for 4 patients or to have improved to Grade 1 at final patient follow-up for 2 patients. Three patients had cytokine release syndrome (Arm 1: 1 patient, Grade 2; Arm 2: 2 patients; 1 Grade 1 and 1 Grade 2); all patients recovered. There was 1 event of graft vs host disease (GvHD skin; Grade 1) and, in a separate patient, 1 event of Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) (Grade 1). Both events resolved. All patients had reduction in tumor burden. Arm 1 (lete-cel alone) had an ORR of 33.3% (1 CR, 2 SD) and median progression-free survival (PFS) of 2.79 mo, while Arm 2 (combination) had an ORR of 66.7% (1 VGPR, 1 PR, 1 SD) and median PFS of 2.78 mo. Time to response for all responders was 3 weeks. Pembro dosing for the 2 Arm 2 responders began at Wk 6. Duration of response in each responder was 2.1 mo. Overall survival data are not mature. Two of 3 responders exhibited clearance of antigen positive myeloma cells in the bone marrow for up to 6 weeks after lete-cel infusion. T cell kinetics trended toward higher peak expansion (Cmax) and area under the curve (AUC) over the first 28 days post-dose (AUC0-28d) in responders vs. non-responders. Serum cytokine profiles in relation to response and CRS will be discussed. Conclusions: A single lete-cel infusion was associated with antitumor activity in 6/6 heavily pretreated RRMM patients, including 1 CR, 1 VGPR, 1PR. Both responses in Arm 2 occurred prior to pembro initiation. The associated safety profile was manageable and consistent with that seen in other lete-cel studies. Responders showed a trend toward higher Cmax and AUC0-28d as compared to non-responders. The study was closed in November 2020 due to protracted enrollment. This study (208470; NCT03168438) was funded by GlaxoSmithKline. Submission support was provided by Fishawack Health. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Kaufman: Janssen: Honoraria; Heidelberg Pharma: Research Funding; Fortis Therapeutics: Research Funding; Tecnofarma SAS, AbbVie: Honoraria; Amgen: Research Funding; BMS: Consultancy, Research Funding; Incyte, celgene: Consultancy; Incyte, TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Genentech, AbbVie, Janssen: Consultancy, Research Funding; Novartis: Research Funding; Roche/Genetech, Tecnopharma: Consultancy, Honoraria; Sutro, Takeda: Research Funding. Huff: GSK: Current Employment, Current equity holder in publicly-traded company. Snape: Veramed: Current Employment. Jain: Butterfly Network: Current equity holder in publicly-traded company; Marker Therapeutics: Current equity holder in publicly-traded company; 23 and Me: Current equity holder in publicly-traded company; Sema4 Holdings: Current equity holder in publicly-traded company; GSK: Current Employment, Current equity holder in publicly-traded company. Kapoor: GSK: Current equity holder in publicly-traded company. Zajic: GSK: Current Employment, Current equity holder in publicly-traded company. Suchindran: GSK: Current Employment, Current equity holder in publicly-traded company. Chisamore: Merck & Co. Inc: Current Employment, Current equity holder in publicly-traded company. Rapoport: GSK: Other: Support received as site principal investigator for this study.

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