ABSTRACT
ABSTRACT: Immunogenic cell death (ICD) is a form of cell death by which cancer treatments can induce a clinically relevant antitumor immune response in a broad range of cancers. In multiple myeloma (MM), the proteasome inhibitor bortezomib is an ICD inducer and creates durable therapeutic responses in patients. However, eventual relapse and resistance to bortezomib appear inevitable. Here, by integrating patient transcriptomic data with an analysis of calreticulin (CRT) protein interactors, we found that GABA type A receptor-associated protein (GABARAP) is a key player whose loss prevented tumor cell death from being perceived as immunogenic after bortezomib treatment. GABARAP is located on chromosome 17p, which is commonly deleted in patients with high risk MM. GABARAP deletion impaired the exposure of the eat-me signal CRT on the surface of dying MM cells in vitro and in vivo, thus reducing tumor cell phagocytosis by dendritic cells and the subsequent antitumor T-cell response. Low GABARAP was independently associated with shorter survival in patients with MM and reduced tumor immune infiltration. Mechanistically, we found that GABARAP deletion blocked ICD signaling by decreasing autophagy and altering Golgi apparatus morphology, with consequent defects in the downstream vesicular transport of CRT. Conversely, upregulating autophagy using rapamycin restored Golgi morphology, CRT exposure, and ICD signaling in GABARAPKO cells undergoing bortezomib treatment. Therefore, coupling an ICD inducer, such as bortezomib, with an autophagy inducer, such as rapamycin, may improve patient outcomes in MM, in which low GABARAP in the form of del(17p) is common and leads to worse outcomes.
Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis Regulatory Proteins , Drug Resistance, Neoplasm , Microtubule-Associated Proteins , Multiple Myeloma , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Multiple Myeloma/immunology , Multiple Myeloma/genetics , Humans , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Mice , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Bortezomib/pharmacology , Bortezomib/therapeutic use , Calreticulin/metabolism , Calreticulin/genetics , Immunogenic Cell Death/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Autophagy/drug effectsABSTRACT
Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.
Subject(s)
MicroRNAs , Multiple Myeloma , RNA, Long Noncoding , Humans , Animals , Mice , RNA, Long Noncoding/genetics , Multiple Myeloma/genetics , Chromatin , MicroRNAs/metabolism , Cell Proliferation , Gene Expression Regulation, NeoplasticABSTRACT
Given the profound multiple myeloma (MM) heterogeneity in clonal proliferation of malignant plasma cells (PCs) and anti-MM therapeutic potential of nanotherapies, it is inevitable to develop treatment plan for patients with MM. Two composite nanoparticles (NPs), As4S4/Fe3O4 (4:1) and As4S4/Fe3O4 (1:1) demonstrated effective anti-MM activity in in vitro, ex vivo, and in vivo in xenograft mouse model. Composite NPs triggered activation of p-ERK1/2/p-JNK, and downregulation of c-Myc, p-PI3K, p-4E-BP1; G2/M cell cycle arrest with increase in cyclin B1, histones H2AX/H3, activation of p-ATR, p-Chk1/p-Chk2, p-H2AX/p-H3; and caspase- and mitochondria-dependent apoptosis induction. NPs attenuated the stem cell-like side population in MM cells, both alone and in the presence of stroma. For a higher clinical response rate, As4S4/Fe3O4 (4:1) observed synergism with dexamethasone and melphalan, while As4S4/Fe3O4 (1:1) showed synergistic effects in combination with bortezomib, lenalidomide and pomalidomide anti-MM agents, providing the framework for further clinical evaluation of composite NPs in MM.
ABSTRACT
Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells exhibited profound expansion of clonal cells in both unswitched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper, effector CTL and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1&PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. Our study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies.
Subject(s)
Lymphoma, B-Cell , Waldenstrom Macroglobulinemia , Humans , Waldenstrom Macroglobulinemia/drug therapy , Waldenstrom Macroglobulinemia/metabolism , Tumor Microenvironment , Plasma Cells/pathology , B-Lymphocytes/pathologyABSTRACT
Anti-CD38 monoclonal antibody (MoAb) treatments including daratumumab (DARA) are effective therapies for both newly diagnosed and relapsed multiple myeloma (MM). In this study, we examined the soluble factors that modulate CD38 expression and are associated with sensitivity to DARA-mediated antibody-dependent cellular cytotoxicity (ADCC) in the bone marrow (BM) microenvironment. Importantly, primary BM stromal cell (BMSC) culture supernatant (BMSC-sup) and interleukin-6 (IL-6) downregulated CD38 expression and reduced DARA-mediated ADCC. Both cytokine profiling of the BMSC-sup and genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout screening in MM cell lines identified and validated the JAK-STAT3 signaling pathway mediating CD38 downregulation, whereas the JAK-STAT1 pathway mediated CD38 upregulation. STAT3 knockdown abrogated BMSC-sup- and IL-6-induced CD38 downregulation on MM cell lines. We also confirmed that STAT3 and CD38 is negatively correlated in primary MM cells. To assess potential clinical relevance, pharmacological inhibition of the JAK-STAT pathway on BMSC-sup-induced CD38 downregulation was further examined. JAK inhibitor ruxolitinib inhibited STAT3 phosphorylation in MM cell lines, upregulated CD38 expression in MM cell lines and primary patient MM cells, and augmented DARA-mediated ADCC against MM cell lines. Taken together, our results suggest that CD38 expression on MM cells in the BM microenvironment is regulated by both STAT1 (positively) and STAT3 (negatively), and that inhibition of the JAK-STAT3 pathway represents a novel therapeutic option to enhance CD38 expression and anti-CD38 MoAb-mediated MM cytotoxicity.
Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Janus Kinases/metabolism , Membrane Glycoproteins/metabolism , Multiple Myeloma/metabolism , STAT Transcription Factors/metabolism , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Bone Marrow/metabolism , Bone Marrow/pathology , Humans , Janus Kinases/drug effects , Multiple Myeloma/pathology , Nitriles , Pyrazoles/pharmacology , Pyrimidines , STAT Transcription Factors/drug effects , Signal Transduction/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcriptase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-type-p53 MM.1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Multiple Myeloma/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/immunology , Apoptosis/physiology , Blotting, Western , Gene Knockdown Techniques , Humans , Immunologic Factors/pharmacology , Multiple Myeloma/mortality , Multiple Myeloma/pathology , Prognosis , Signal Transduction/drug effectsABSTRACT
Multiple myeloma (MM) has proven clinically susceptible to modulation of pathways of protein homeostasis. Blockade of proteasomal degradation of polyubiquitinated misfolded proteins by the proteasome inhibitor bortezomib (BTZ) achieves responses and prolongs survival in MM, but long-term treatment with BTZ leads to drug-resistant relapse in most patients. In a proof-of-concept study, we previously demonstrated that blocking aggresomal breakdown of polyubiquitinated misfolded proteins with the histone deacetylase 6 (HDAC6) inhibitor tubacin enhances BTZ-induced cytotoxicity in MM cells in vitro. However, these foundational studies were limited by the pharmacologic liabilities of tubacin as a chemical probe with only in vitro utility. Emerging from a focused library synthesis, a potent, selective, and bioavailable HDAC6 inhibitor, WT161, was created to study the mechanism of action of HDAC6 inhibition in MM alone and in combination with BTZ. WT161 in combination with BTZ triggers significant accumulation of polyubiquitinated proteins and cell stress, followed by caspase activation and apoptosis. More importantly, this combination treatment was effective in BTZ-resistant cells and in the presence of bone marrow stromal cells, which have been shown to mediate MM cell drug resistance. The activity of WT161 was confirmed in our human MM cell xenograft mouse model and established the framework for clinical trials of the combination treatment to improve patient outcomes in MM.
Subject(s)
Antineoplastic Agents/therapeutic use , Bortezomib/therapeutic use , Histone Deacetylase Inhibitors/therapeutic use , Hydroxamic Acids/therapeutic use , Multiple Myeloma/drug therapy , Proteasome Inhibitors/therapeutic use , Terphenyl Compounds/therapeutic use , Anilides/pharmacology , Anilides/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Drug Resistance, Neoplasm , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase 6/metabolism , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydroxamic Acids/pharmacology , Male , Mice , Multiple Myeloma/metabolism , Proteasome Inhibitors/pharmacology , Terphenyl Compounds/pharmacology , Tubulin/metabolism , Tumor Cells, CulturedABSTRACT
Proteasome inhibitors are one of the most important classes of agents to have emerged for the treatment of multiple myeloma in the past two decades, and now form one of the backbones of treatment. Three agents in this class have been approved by the United States Food and Drug Administration-the first-in-class compound bortezomib, the second-generation agent carfilzomib, and the first oral proteasome inhibitor, ixazomib. The success of this class of agents is due to the exquisite sensitivity of myeloma cells to the inhibition of the 26S proteasome, which plays a critical role in the pathogenesis and proliferation of the disease. Proteasome inhibition results in multiple downstream effects, including the inhibition of NF-κB signaling, the accumulation of misfolded and unfolded proteins, resulting in endoplasmic reticulum stress and leading to the unfolded protein response, the downregulation of growth factor receptors, suppression of adhesion molecule expression, and inhibition of angiogenesis; resistance to proteasome inhibition may arise through cellular responses mediating these downstream effects. These multiple biologic consequences of proteasome inhibition result in synergistic or additive activity with other chemotherapeutic and targeted agents for myeloma, and proteasome inhibitor-based combination regimens have become established as a cornerstone of therapy throughout the myeloma treatment algorithm, incorporating agents from the other key classes of antimyeloma agents, including the immunomodulatory drugs, monoclonal antibodies, and histone deacetylase inhibitors. This review gives an overview of the critical role of the proteasome in myeloma and the characteristics of the different proteasome inhibitors and provides a comprehensive summary of key clinical efficacy and safety data with the currently approved proteasome inhibitors.
Subject(s)
Multiple Myeloma/drug therapy , Multiple Myeloma/enzymology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Drug Resistance, Neoplasm , Humans , Proteasome Inhibitors/administration & dosage , Randomized Controlled Trials as TopicABSTRACT
INTRODUCTION: Proteasome inhibitors (PIs) are therapeutic backbones of multiple myeloma treatment, with PI-based therapies being standards of care throughout the treatment algorithm. Proteasome inhibition affects multiple critical signaling pathways in myeloma cells and interacts synergistically with mechanisms of action of other conventional and novel agents, resulting in substantial anti-myeloma activity and at least additive effects. Areas covered: This review summarizes the biologic effects of proteasome inhibition in myeloma and provides an overview of the importance of proteasome inhibition to the current treatment algorithm. It reviews key clinical data on three PIs, specifically bortezomib, carfilzomib, and ixazomib; assesses ongoing phase 3 trials with these agents; and looks ahead to the increasingly broad role of both approved PIs and PIs under investigation in the frontline and relapsed settings. Expert commentary: Progress to date with PIs in multiple myeloma has been impressive, but there remain unmet needs and challenges, as well as increasing opportunities to optimize the use of these agents. Understanding discrepancies between PIs in terms of efficacy and safety profile is a key goal of ongoing research, along with proteomics-based efforts to identify potential biomarkers of sensitivity and resistance, thereby enabling increasingly personalized treatment approaches in the future.
Subject(s)
Antineoplastic Agents/therapeutic use , Multiple Myeloma/drug therapy , Proteasome Inhibitors/therapeutic use , Antineoplastic Agents/adverse effects , Clinical Trials, Phase III as Topic , Humans , Proteasome Inhibitors/adverse effectsABSTRACT
Multiple myeloma (MM) cell lines and primary tumor cells are addicted to the MYC oncoprotein for survival. Little is known, however, about how MYC expression is upregulated in MM cells. The mucin 1 C-terminal subunit (MUC1-C) is an oncogenic transmembrane protein that is aberrantly expressed in MM cell lines and primary tumor samples. The present studies demonstrate that targeting MUC1-C with silencing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 editing or with the GO-203 inhibitor is associated with downregulation of MYC messenger RNA and protein. The results show that MUC1-C occupies the MYC promoter and thereby activates the MYC gene by a ß-catenin/transcription factor 4 (TCF4)-mediated mechanism. In this way, MUC1-C (1) increases ß-catenin occupancy on the MYC promoter, (2) forms a complex with ß-catenin and TCF4, and, in turn, (3) drives MYC transcription. Analysis of MM cells using quantitative real-time reverse transcription polymerase chain reaction arrays further demonstrated that silencing MUC1-C is associated with downregulation of MYC target genes, including CCND2, hTERT, and GCLC Analysis of microarray data sets further demonstrated that MUC1 levels positively correlate with MYC expression in MM progression and in primary cells from over 800 MM patients. These findings collectively provide convincing evidence that MUC1-C drives MYC expression in MM.
Subject(s)
Down-Regulation , Gene Expression Regulation, Neoplastic , Mucin-1/biosynthesis , Multiple Myeloma/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Response Elements , Transcription, Genetic , CRISPR-Cas Systems , Cell Line, Tumor , Cyclin D2/biosynthesis , Cyclin D2/genetics , Glutamate-Cysteine Ligase/biosynthesis , Glutamate-Cysteine Ligase/genetics , Humans , Mucin-1/genetics , Multiple Myeloma/genetics , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-myc/genetics , Telomerase/biosynthesis , Telomerase/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Multiple myeloma (MM) is characterized by a highly unstable genome, with aneuploidy observed in nearly all patients. The mechanism causing this karyotypic instability is largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage response. Here, we show that the NAD(+)-dependent deacetylase SIRT6 is highly expressed in MM cells, as an adaptive response to genomic stability, and that high SIRT6 levels are associated with adverse prognosis. Mechanistically, SIRT6 interacts with the transcription factor ELK1 and with the ERK signaling-related gene. By binding to their promoters and deacetylating H3K9 at these sites, SIRT6 downregulates the expression of mitogen-activated protein kinase (MAPK) pathway genes, MAPK signaling, and proliferation. In addition, inactivation of ERK2/p90RSK signaling triggered by high SIRT6 levels increases DNA repair via Chk1 and confers resistance to DNA damage. Using genetic and biochemical studies in vitro and in human MM xenograft models, we show that SIRT6 depletion both enhances proliferation and confers sensitization to DNA-damaging agents. Our findings therefore provide insights into the functional interplay between SIRT6 and DNA repair mechanisms, with implications for both tumorigenesis and the treatment of MM.
Subject(s)
DNA Damage , Multiple Myeloma/enzymology , Multiple Myeloma/pathology , Sirtuins/metabolism , Acetylation , Cell Line, Tumor , Cell Proliferation , DNA Repair , Doxorubicin/pharmacology , Histones/metabolism , Humans , Lysine/metabolism , MAP Kinase Signaling System , Models, Biological , Mutagens/toxicity , Prognosis , ets-Domain Protein Elk-1/metabolismABSTRACT
Lenalidomide (LEN) acts directly on multiple myeloma (MM) cells by inducing cereblon-mediated degradation of interferon regulatory factor 4, Ikaros (IKZF)1 and IKZF3, transcription factors that are essential for MM cell survival. The mucin 1 (MUC1) C-terminal transmembrane subunit (MUC1-C) oncoprotein is aberrantly expressed by MM cells and protects against reactive oxygen species (ROS)-mediated MM cell death. The present studies demonstrate that targeting MUC1-C with GO-203, a cell-penetrating peptide inhibitor of MUC1-C homodimerization, is more than additive with LEN in downregulating the WNT/ß-catenin pathway, suppressing MYC, and inducing late apoptosis/necrosis. We show that the GO-203/LEN combination acts by synergistically increasing ROS and, in turn, suppressing ß-catenin. LEN resistance has been linked to activation of the WNT/ß-cateninâCD44 pathway. In this regard, our results further demonstrate that targeting MUC1-C is effective against LEN-resistant MM cells. Moreover, GO-203 resensitized LEN-resistant MM cells to LEN treatment in association with suppression of ß-catenin and CD44. Targeting MUC1-C also resulted in downregulation of CD44 on the surface of primary MM cells. These findings, and the demonstration that expression of MUC1 and CD44 significantly correlate in microarrays from primary MM cells, provide support for combining GO-203 with LEN in the treatment of MM and in LEN-resistance.
Subject(s)
Mucin-1/drug effects , Multiple Myeloma/pathology , Thalidomide/analogs & derivatives , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Computational Biology/methods , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hyaluronan Receptors/metabolism , Lenalidomide , Molecular Targeted Therapy/methods , Mucin-1/metabolism , Multiple Myeloma/metabolism , Oxidation-Reduction/drug effects , Peptides/administration & dosage , Peptides/pharmacology , Reactive Oxygen Species/metabolism , Thalidomide/administration & dosage , Thalidomide/pharmacology , Tumor Cells, Cultured/drug effectsABSTRACT
Multiple myeloma (MM), a B cell malignancy characterized by clonal proliferation of plasma cells in the bone marrow, remains incurable despite the use of novel and conventional therapies. In this study, we demonstrated MM cell cytotoxicity triggered by realgar (REA; As4 S4 ) nanoparticles (NREA) versus Arsenic trioxide (ATO) against MM cell lines and patient cells. Both NREA and ATO showed in vivo anti-MM activity, resulting in significantly decreased tumour burden. The anti-MM activity of NREA and ATO is associated with apoptosis, evidenced by DNA fragmentation, depletion of mitochondrial membrane potential, cleavage of caspases and anti-apoptotic proteins. NREA induced G2 /M cell cycle arrest and modulation of cyclin B1, p53 (TP53), p21 (CDKN1A), Puma (BBC3) and Wee-1 (WEE1). Moreover, NREA induced modulation of key regulatory molecules in MM pathogenesis including JNK activation, c-Myc (MYC), BRD4, and histones. Importantly, NREA, but not ATO, significantly depleted the proportion and clonogenicity of the MM stem-like side population, even in the context of the bone marrow stromal cells. Finally, our study showed that both NREA and ATO triggered synergistic anti-MM activity when combined with lenalidomide or melphalan. Taken together, the anti-MM activity of NREA was more potent compared to ATO, providing the preclinical framework for clinical trials to improve patient outcome in MM.
Subject(s)
Antineoplastic Agents/administration & dosage , Arsenicals/administration & dosage , Multiple Myeloma/drug therapy , Oxides/administration & dosage , Sulfides/administration & dosage , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Arsenic Trioxide , Arsenicals/pharmacology , Arsenicals/therapeutic use , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Delivery Systems/methods , Humans , Mice, SCID , Molecular Targeted Therapy/methods , Multiple Myeloma/pathology , Nanoparticles , Neoplastic Stem Cells/drug effects , Oxides/pharmacology , Oxides/therapeutic use , Prohibitins , Sulfides/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
In this prospective, multicenter, phase 2 study, 64 patients with relapsed or relapsed and refractory multiple myeloma (MM) received up to 8 21-day cycles of bortezomib 1.0 mg/m(2) (days 1, 4, 8, and 11), lenalidomide 15 mg/day (days 1-14), and dexamethasone 40/20 mg/day (cycles 1-4) and 20/10 mg/day (cycles 5-8) (days of/after bortezomib dosing). Responding patients could receive maintenance therapy. Median age was 65 years; 66% were male, 58% had relapsed and 42% had relapsed and refractory MM, and 53%, 75%, and 6% had received prior bortezomib, thalidomide, and lenalidomide, respectively. Forty-eight of 64 patients (75%; 90% confidence interval, 65-84) were alive without progressive disease at 6 months (primary end point). The rate of partial response or better was 64%; median duration of response was 8.7 months. Median progression-free and overall survivals were 9.5 and 30 months, respectively (median follow-up: 44 months). Common treatment-related toxicities included sensory neuropathy (53%), fatigue (50%), and neutropenia (42%); common grade 3/4 treatment-related toxicities included neutropenia (30%), thrombocytopenia (22%), and lymphopenia (11%). Grade 3 motor neuropathy was reported in 2 patients. Lenalidomide-bortezomib-dexamethasone appears effective and tolerable in patients with relapsed or relapsed and refractory MM, demonstrating substantial activity among patients with diverse prior therapies and adverse prognostic characteristics. This trial is registered with www.clinicaltrials.gov as #NCT00378209.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Boronic Acids/administration & dosage , Bortezomib , Dexamethasone/administration & dosage , Female , Humans , Lenalidomide , Male , Middle Aged , Multiple Myeloma/mortality , Neoplasm Staging , Pyrazines/administration & dosage , Recurrence , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Treatment OutcomeABSTRACT
Multiple myeloma is a plasma cell malignancy characterized by complex heterogeneous cytogenetic abnormalities. The bone marrow microenvironment promotes multiple myeloma cell growth and resistance to conventional therapies. Although multiple myeloma remains incurable, novel targeted agents, used alone or in combination, have shown great promise to overcome conventional drug resistance and improve patient outcome. Recent oncogenomic studies have further advanced our understanding of the molecular pathogenesis of multiple myeloma, providing the framework for new prognostic classification and identifying new therapeutic targets.
Subject(s)
Antineoplastic Agents/therapeutic use , Bone Marrow/pathology , Multiple Myeloma/pathology , Drug Resistance, Neoplasm , Genomics , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/geneticsABSTRACT
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous, immature myeloid cell population with the ability to suppress immune responses. MDSCs have been characterized in infections, inflammatory diseases, and solid tumors; however, their presence and role in the tumor-promoting, immune-suppressive microenvironment in hematologic malignancies remains unclear. We assessed the presence, frequency, and functional characteristics of MDSCs in patients with newly diagnosed, relapsed, and relapsed/refractory multiple myeloma (MM) compared with healthy donors. Additionally, we evaluated the immunomodulatory effects of lenalidomide and bortezomib on MDSCs in MM. CD11b(+)CD14(-)HLA-DR(-/low)CD33(+)CD15(+) MDSCs were significantly increased in both the peripheral blood and the bone marrow of patients with active MM compared with healthy donors. Furthermore, MDSCs induced MM growth while suppressing T-cell-mediated immune responses. Conversely, MM cells induced the development of MDSCs from healthy donor peripheral blood mononuclear cells, confirming a bidirectional interaction between MDSCs and MM cells and immune effector cells. Our results further suggest that MDSCs may be associated with the activity of disease in MM. Importantly, our studies suggest that inhibition of the tumor-promoting and immune-suppressive functions of MDSCs in MM may represent a promising novel immune-based therapeutic strategy.
Subject(s)
Multiple Myeloma/immunology , Myeloid Cells/immunology , T-Lymphocytes/immunology , Tumor Microenvironment/immunology , Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Bortezomib , CD11b Antigen/immunology , CD11b Antigen/metabolism , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Coculture Techniques , Cytokines/immunology , Cytokines/metabolism , Flow Cytometry , HLA-DR Antigens/immunology , HLA-DR Antigens/metabolism , Humans , Immunologic Factors/pharmacology , Lenalidomide , Lewis X Antigen/immunology , Lewis X Antigen/metabolism , Lipopolysaccharides/immunology , Lipopolysaccharides/pharmacology , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Pyrazines/pharmacology , Reactive Oxygen Species/immunology , Reactive Oxygen Species/metabolism , Sialic Acid Binding Ig-like Lectin 3/immunology , Sialic Acid Binding Ig-like Lectin 3/metabolism , T-Lymphocytes/metabolism , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Tumor Burden/immunology , Tumor Microenvironment/drug effectsABSTRACT
We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD⺠content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD⺠depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD⺠depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD⺠level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib.
Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Multiple Myeloma/drug therapy , NAD/metabolism , Neoplasm Recurrence, Local/drug therapy , Nicotinamide Phosphoribosyltransferase/metabolism , Pyrazines/pharmacology , Acrylamides/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Autophagy/drug effects , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Bortezomib , Case-Control Studies , Caspases/genetics , Caspases/metabolism , Cell Proliferation/drug effects , Drug Synergism , Female , Fluorescent Antibody Technique , Gene Expression Profiling , Humans , Male , Mice , Mice, SCID , Multiple Myeloma/metabolism , Multiple Myeloma/mortality , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/mortality , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Nicotinamide Phosphoribosyltransferase/genetics , Oligonucleotide Array Sequence Analysis , Piperidines/pharmacology , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Condensation of Igs has been observed in pharmaceutical formulations and in vivo in cases of cryoglobulinemia. We report a study of monoclonal IgG cryoglobulins overexpressed by two patients with multiple myeloma. These cryoglobulins form crystals, and we measured their solubility lines. Depending on the supersaturation, we observed a variety of condensate morphologies consistent with those reported in clinical investigations. Remarkably, the crystallization can occur at quite low concentrations. This suggests that, even within the regular immune response to infections, cryoprecipitation of Ig can be possible.
Subject(s)
Cryoglobulinemia/immunology , Cryoglobulinemia/pathology , Cryoglobulins/chemistry , Buffers , Crystallization , Crystallography, X-Ray , Humans , Hydrogen-Ion Concentration , SolubilityABSTRACT
Everolimus, an oral mammalian target of rapamycin (mTOR) inhibitor, has been studied in multiple myeloma (MM) but lacks significant single agent activity. Based on preclinical studies showing synergistic activity of mTOR inhibitors with lenalidomide, we studied the combination of lenalidomide and everolimus in relapsed or refractory MM in a phase I clinical trial. We assessed patient samples using gene expression, Western blotting and immunohistochemistry to probe the mTOR pathway. Twenty-six patients were evaluable for toxicity. Dose-limiting toxicities included grade 4 neutropenia and thrombocytopenia. The maximum tolerated dose was lenalidomide 15 mg and everolimus 5 mg for 21 d with a 7 d rest period. Grade 3/4 adverse events included thrombocytopenia (35%) and neutropenia (42%). The overall response rate was 65% (1 complete response + 4 partial response + 10 minimal response). The median progression-free survival was 5·5 months and median overall survival was 29·5 months. Biomarker data demonstrated downregulation of phosphorylated p70S6K. Gene expression profiling suggested activation of mTOR in responders versus non-responders. The combination of lenalidomide and everolimus was well tolerated with predictable toxicities and showed responses in a heavily pretreated population. When confirmed with larger patient numbers, this analysis may guide patient selection for future clinical trials of mTOR inhibition in MM.