ABSTRACT
IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we found that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress in vitro and to radiation in vitro and in vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.
Subject(s)
Glioma/metabolism , Glutamic Acid/biosynthesis , Transaminases/physiology , Cell Line, Tumor , Glioma/physiopathology , Glutamic Acid/drug effects , Glutarates/metabolism , Glutarates/pharmacology , Homeostasis/drug effects , Humans , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/physiology , Minor Histocompatibility Antigens/genetics , Minor Histocompatibility Antigens/physiology , Mutation , Oxidation-Reduction/drug effects , Pregnancy Proteins/genetics , Pregnancy Proteins/physiology , Transaminases/antagonists & inhibitors , Transaminases/geneticsABSTRACT
Multiple myeloma (MM), a hematologic malignancy that preferentially colonizes the bone marrow, remains incurable with a survival rate of 3 to 6 mo for those with advanced disease despite great efforts to develop effective therapies. Thus, there is an urgent clinical need for innovative and more effective MM therapeutics. Insights suggest that endothelial cells within the bone marrow microenvironment play a critical role. Specifically, cyclophilin A (CyPA), a homing factor secreted by bone marrow endothelial cells (BMECs), is critical to MM homing, progression, survival, and chemotherapeutic resistance. Thus, inhibition of CyPA provides a potential strategy to simultaneously inhibit MM progression and sensitize MM to chemotherapeutics, improving therapeutic response. However, inhibiting factors from the bone marrow endothelium remains challenging due to delivery barriers. Here, we utilize both RNA interference (RNAi) and lipid-polymer nanoparticles to engineer a potential MM therapy, which targets CyPA within blood vessels of the bone marrow. We used combinatorial chemistry and high-throughput in vivo screening methods to engineer a nanoparticle platform for small interfering RNA (siRNA) delivery to bone marrow endothelium. We demonstrate that our strategy inhibits CyPA in BMECs, preventing MM cell extravasation in vitro. Finally, we show that siRNA-based silencing of CyPA in a murine xenograft model of MM, either alone or in combination with the Food and Drug Administration (FDA)-approved MM therapeutic bortezomib, reduces tumor burden and extends survival. This nanoparticle platform may provide a broadly enabling technology to deliver nucleic acid therapeutics to other malignancies that home to bone marrow.
Subject(s)
Multiple Myeloma , United States , Humans , Animals , Mice , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Bone Marrow , RNA, Small Interfering/genetics , Endothelial Cells , Cyclophilin A , Lipids , Tumor MicroenvironmentABSTRACT
PSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.
Subject(s)
Multiple Myeloma , Proteasome Endopeptidase Complex , Humans , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Proteomics , Leukocytes, Mononuclear/metabolism , Carrier Proteins/genetics , Proteins/metabolism , RNA-Binding ProteinsABSTRACT
Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies.
Subject(s)
Lymphoma, Large B-Cell, Diffuse/genetics , MicroRNAs/genetics , Neoplasms, Plasma Cell/genetics , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Chromosome Deletion , Chromosome Disorders/genetics , Chromosome Disorders/pathology , Chromosomes, Human, Pair 13/genetics , Gene Deletion , Gene Expression Regulation, Neoplastic , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Mice, Inbred C57BL , Multigene Family , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasms, Plasma Cell/pathology , Plasma Cells/metabolism , Plasma Cells/pathology , Plasmacytoma/genetics , Plasmacytoma/pathologyABSTRACT
ABSTRACT: Mycosis fungoides (MF) is primarily characterized by epidermotropic CD3+/CD4+/CD45RO+ memory T cells. CD4/CD8 double-negative MF is an uncommon variant with no presumed prognostic significance. Despite the variability in the clinical course and presentation of MF, most cases behave indolently. About 5% of patients, however, advance to stage IV with visceral organ involvement. Central nervous system metastasis in MF is rare with no known cases of direct central nervous system invasion by MF to date. We report an exceedingly rare locally aggressive case of CD4/CD8 double-negative MF with direct dural invasion and underline pertinent diagnostic challenges encountered in our case.
Subject(s)
Dura Mater/pathology , Head and Neck Neoplasms/pathology , Lymphoma, T-Cell, Cutaneous/pathology , Mycosis Fungoides/pathology , Scalp/pathology , Skin Neoplasms/pathology , Adult , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Dura Mater/immunology , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/immunology , Head and Neck Neoplasms/therapy , Humans , Lymphoma, T-Cell, Cutaneous/genetics , Lymphoma, T-Cell, Cutaneous/immunology , Lymphoma, T-Cell, Cutaneous/therapy , Mycosis Fungoides/genetics , Mycosis Fungoides/immunology , Mycosis Fungoides/therapy , Neoplasm Invasiveness , Scalp/immunology , Skin Neoplasms/genetics , Skin Neoplasms/immunology , Skin Neoplasms/therapy , Treatment OutcomeABSTRACT
Reed-Sternberg (RS) cells of classical Hodgkin lymphoma (cHL) express multiple immunoregulatory proteins that shape the cHL microenvironment and allow tumor cells to evade immune surveillance. Expression of certain immunoregulatory proteins is modulated by prosurvival transcription factors, such as NFκB and STATs. Because these factors also induce expression of the oncogenic PIM1/2/3 serine/threonine kinases, and as PIMs modulate transcriptional activity of NFκB and STATs, we hypothesized that these kinases support RS cell survival and foster their immune privilege. Here, we investigated PIM1/2/3 expression in cHL and assessed their role in developing RS cell immune privilege and survival. PIM1/2/3 were ubiquitously expressed in primary and cultured RS cells, and their expression was driven by JAK-STAT and NFκB activity. Genetic or chemical PIM inhibition with a newly developed pan-PIM inhibitor, SEL24-B489, induced RS cell apoptosis. PIM inhibition decreased cap-dependent protein translation, blocked JAK-STAT signaling, and markedly attenuated NFκB-dependent gene expression. In a cHL xenograft model, SEL24-B489 delayed tumor growth by 95.8% (P = .0002). Furthermore, SEL24-B489 decreased the expression of multiple molecules engaged in developing the immunosuppressive microenvironment, including galectin-1 and PD-L1/2. In coculture experiments, T cells incubated with SEL24-B489-treated RS cells exhibited higher expression of activation markers than T cells coincubated with control RS cells. Taken together, our data indicate that PIM kinases in cHL exhibit pleiotropic effects, orchestrating tumor immune escape and supporting RS cell survival. Inhibition of PIM kinases decreases RS cell viability and disrupts signaling circuits that link these cells with their niches. Thus, PIM kinases are promising therapeutic targets in cHL.
Subject(s)
Hodgkin Disease/enzymology , Hodgkin Disease/immunology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-pim-1/metabolism , Proto-Oncogene Proteins/metabolism , Reed-Sternberg Cells/enzymology , Reed-Sternberg Cells/pathology , Cell Line, Tumor , Cell Survival , Chemokines/metabolism , Down-Regulation , Hodgkin Disease/pathology , Humans , Immunomodulation , Janus Kinases/metabolism , Lymphocyte Activation/immunology , NF-kappa B/metabolism , Protein Biosynthesis , RNA Caps/metabolism , STAT Transcription Factors/metabolism , Signal Transduction , T-Lymphocytes/immunologyABSTRACT
Lymph node microenvironment provides chronic lymphocytic leukaemia (CLL) cells with signals promoting their survival and granting resistance to chemotherapeutics. CLL cells overexpress PIM kinases, which regulate apoptosis, cell cycle and migration. We demonstrate that BCR crosslinking, CD40 stimulation, and coculture with stromal cells increases PIMs expression in CLL cells, indicating microenvironment-dependent PIMs regulation. PIM1 and PIM2 expression at diagnosis was higher in patients with advanced disease (Binet C vs. Binet A/B) and in those, who progressed after first-line treatment. In primary CLL cells, inhibition of PIM kinases with a pan-PIM inhibitor, SEL24-B489, decreased PIM-specific substrate phosphorylation and induced dose-dependent apoptosis in leukaemic, but not in normal B cells. Cytotoxicity of SEL24-B489 was similar in TP53-mutant and TP53 wild-type cells. Finally, inhibition of PIM kinases decreased CXCR4-mediated cell chemotaxis in two related mechanisms-by decreasing CXCR4 phosphorylation and surface expression, and by limiting CXCR4-triggered mTOR pathway activity. Importantly, PIM and mTOR inhibitors similarly impaired migration, indicating that CXCL12-triggered mTOR is required for CLL cell chemotaxis. Given the microenvironment-modulated PIM expression, their pro-survival function and a role of PIMs in CXCR4-induced migration, inhibition of these kinases might override microenvironmental protection and be an attractive therapeutic strategy in this disease.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Proto-Oncogene Proteins c-pim-1/metabolism , Receptors, CXCR4/metabolism , TOR Serine-Threonine Kinases/metabolism , Adult , Aged , Aged, 80 and over , Cell Movement/drug effects , Female , Gene Expression Regulation, Leukemic , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Male , Middle Aged , Prognosis , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/genetics , Tumor Cells, Cultured , Tumor MicroenvironmentABSTRACT
Inhibition of spleen tyrosine kinase (SYK) in tonic B-cell receptor (BCR) signal-dependent diffuse large B-cell lymphomas (DLBCLs) inhibits cellular proliferation, decreases cholesterol biosynthesis, and triggers apoptosis, at least in part via a mechanism involving decreased activity of phosphatidylinositol 3-kinase/AKT axis. Because forkhead box O1 (FOXO1) is a major effector of this pathway, we investigated the role of FOXO1 in toxicity of BCR pathway inhibition. Inhibition of SYK in DLBCL cells with tonic BCR signaling decreased phospho-AKT and phospho-FOXO1 levels and triggered FOXO1-driven gene expression. Introduction of constitutively active FOXO1 mutant triggered cell cycle arrest and apoptosis, indicating that increased FOXO1 activity is toxic to these DLBCL cells. Depletion of FOXO1 with short hairpin RNA led to almost complete resistance to chemical SYK inhibitor R406, demonstrating that FOXO1 is also required for R406-induced cell death. FOXO1 in these cells is also involved in regulation of expression of the critical master regulator of cholesterol biosynthesis, SREBP1. Because HRK is the key effector of SYK inhibition, we characterized a mechanism linking FOXO1 activation and HRK induction that involves caspase-dependent cleavage of HRK's transcriptional repressor DREAM. Because AKT in lymphoma cells can be regulated by other signals than BCR, we assessed the combined effects of the AKT inhibitor MK-2206 with R406 and found markedly synergistic FOXO1-dependent toxicity. In primary DLBCLs, FOXO1 expression was present in 80% of tumors, correlated with SYK activity, and was associated with longer overall survival. These results demonstrate that FOXO1 is required for SYK and AKT inhibitor-induced toxicity.
Subject(s)
Forkhead Transcription Factors/physiology , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptors, Antigen, B-Cell/genetics , Apoptosis/genetics , Cell Cycle/genetics , Forkhead Box Protein O1 , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoma, Large B-Cell, Diffuse/pathology , Microarray Analysis , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptors, Antigen, B-Cell/metabolism , Signal Transduction/genetics , Syk Kinase , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
MYC is one of the most frequently deregulated oncogenes in human malignancies. It encodes a leucine zipper transcription factor that modulates a broad spectrum of cellular genes responsible for enhancing cell proliferation, cellular metabolism, growth, angiogenesis, metastasis, genomic instability, stem cell self-renewal and reduced differentiation. MYC functions predominantly as an amplifier of expression of already active genes, potentiating the pre-existing transcriptional program, although it can also repress certain transcriptional targets. In mouse models, MYC induces lymphomas, but requires cooperation with other lesions, including inactivation of the p53 pathway, structural alterations of BCL2 family members, or increased PI3K activity. In human B-cell tumors, MYC rearrangements involving the 8q24 region and immunoglobulin heavy or light genes are a hallmark of Burkitt lymphoma (BL), but can also occur in other lymphoid malignancies, that include diffuse large B-cell lymphoma (DLBCL), B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma (BCLU), plasma cell myeloma (PCM), mantle cell lymphoma (MCL) and plasmablastic lymphoma. For non-BL lymphoid malignancies, MYC fusions represent secondary genetic events and exist in the context of complex karyotypes. Regardless of the mechanism deregulating MYC, lymphomas over-expressing MYC are addicted to this oncogene, highlighting the potential clinical utility of MYC targeting strategies. Several promising approaches for pharmaceutical intervention have been suggested which are now in preclinical or clinical development. Herein, we therefore review the molecular pathogenetic mechanisms associated with MYC deregulation in human B-cell lymphomas and their implications for therapies targeting MYC.
Subject(s)
Lymphoma, B-Cell/genetics , Proto-Oncogene Proteins c-myc/physiology , Animals , Gene Expression Regulation, Neoplastic , Genes, myc , Humans , Lymphoma, B-Cell/etiology , Lymphoma, B-Cell/therapy , MiceABSTRACT
Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma in adults, exhibiting highly heterogenous clinical behavior and complex molecular background. In addition to the genetic complexity, different DLBCL subsets exhibit phenotypic features independent of the genetic background. For example, a subset of DLBCLs is distinguished by increased oxidative phosphorylation and unique transcriptional features, including overexpression of certain mitochondrial genes and a molecular chaperone, heat shock protein HSP90α (termed "OxPhos" DLBCLs). In this study, we identified a feed-forward pathogenetic circuit linking HSP90α and SIRT1 in OxPhos DLBCLs. The expression of the inducible HSP90α isoform remains under SIRT1-mediated regulation. SIRT1 knockdown or chemical inhibition reduced HSP90α expression in a mechanism involving HSF1 transcription factor, whereas HSP90 inhibition reduced SIRT1 protein stability, indicating that HSP90 chaperones SIRT1. SIRT1-HSP90α interaction in DLBCL cells was confirmed by co-immunoprecipitation and proximity ligation assay (PLA). The number of SIRT1-HSP90α complexes in PLA was significantly higher in OxPhos- dependent than -independent cells. Importantly, SIRT1-HSP90α interactions in OxPhos DLBCLs markedly increased in mitosis, suggesting a specific role of the complex during this cell cycle phase. RNAi-mediated and chemical inhibition of SIRT1 and/or HSP90 significantly increased the number of cells with chromosome segregation errors (multipolar spindle formation, anaphase bridges and lagging chromosomes). Finally, chemical SIRT1 inhibitors induced dose-dependent cytotoxicity in OxPhos-dependent DLBCL cell lines and synergized with the HSP90 inhibitor. Taken together, our findings define a new OxPhos-DLBCL-specific pathogenetic loop involving SIRT1 and HSP90α that regulates chromosome dynamics during mitosis and may be exploited therapeutically.
Subject(s)
Chromosome Segregation , HSP90 Heat-Shock Proteins , Lymphoma, Large B-Cell, Diffuse , Sirtuin 1 , Humans , HSP90 Heat-Shock Proteins/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Molecular Chaperones/metabolism , Sirtuin 1/metabolismABSTRACT
Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy. SIGNIFICANCE: Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease. This article is highlighted in the In This Issue feature, p. 101.
Subject(s)
Leukemia, Lymphocytic, Chronic, B-Cell , Lymphoma, Large B-Cell, Diffuse , Lymphoma, Non-Hodgkin , Humans , Animals , Mice , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Phosphatidylinositol 3-Kinases/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , B-LymphocytesABSTRACT
Dysregulated Wnt/ß-catenin signaling is implicated in the pathogenesis of many human cancers, including colorectal cancer (CRC), making it an attractive clinical target. With the aim of inhibiting oncogenic Wnt activity, we developed a high-throughput screening AlphaScreen assay to identify selective small-molecule inhibitors of the interaction between ß-catenin and its coactivator BCL9. We identified a compound that consistently bound to ß-catenin and specifically inhibited in vivo native ß-catenin/BCL9 complex formation in CRC cell lines. This compound inhibited Wnt activity, down-regulated expression of the Wnt/ß-catenin signature in gene expression studies, disrupted cholesterol homeostasis, and significantly reduced the proliferation of CRC cell lines and tumor growth in a xenograft mouse model of CRC. This study has therefore identified a specific small-molecule inhibitor of oncogenic Wnt signaling, which may have value as a probe for functional studies and has important implications for the development of novel therapies in patients with CRC.
Subject(s)
Colorectal Neoplasms , beta Catenin , Animals , Cholesterol , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Homeostasis , Humans , Mice , Neoplasm Proteins/metabolism , Transcription Factors/genetics , Wnt Signaling Pathway/genetics , beta Catenin/geneticsABSTRACT
Immunomodulatory drugs (IMiDs) have markedly improved patient outcome in multiple myeloma (MM); however, resistance to IMiDs commonly underlies relapse of disease. Here, we identify that tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) knockdown (KD)/knockout (KO) in MM cells mediates IMiD resistance via activation of noncanonical nuclear factor κB (NF-κB) and extracellular signal-regulated kinase (ERK) signaling. Within MM bone marrow (BM) stromal cell supernatants, TNF-α induces proteasomal degradation of TRAF2, noncanonical NF-κB, and downstream ERK signaling in MM cells, whereas interleukin-6 directly triggers ERK activation. RNA sequencing of MM patient samples shows nearly universal ERK pathway activation at relapse on lenalidomide maintenance therapy, confirming its clinical relevance. Combination MEK inhibitor treatment restores IMiD sensitivity of TRAF2 KO cells both in vitro and in vivo. Our studies provide the framework for clinical trials of MEK inhibitors to overcome IMiD resistance in the BM microenvironment and improve patient outcome in MM.
Subject(s)
Immunomodulating Agents , Multiple Myeloma , Bone Marrow/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinase Kinases/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , NF-kappa B/metabolism , Neoplasm Recurrence, Local , TNF Receptor-Associated Factor 2/metabolism , Tumor MicroenvironmentABSTRACT
Proteasome inhibitor bortezomib induces apoptosis in multiple myeloma (MM) cells, and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine MM models, we here show that bortezomib also triggers immunogenic cell death (ICD) characterized by exposure of calreticulin on dying MM cells, phagocytosis of tumor cells by dendritic cells, and induction of MM specific immunity. We identify a bortezomib-triggered specific ICD-gene signature associated with better outcome in two independent MM patient cohorts. Importantly, bortezomib stimulates MM cells immunogenicity via activation of cGAS/STING pathway and production of type-I interferons; and STING agonists significantly potentiate bortezomib-induced ICD. Our studies therefore delineate mechanisms whereby bortezomib exerts immunotherapeutic activity, and provide the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in MM.
Subject(s)
Multiple Myeloma , Animals , Bortezomib/pharmacology , Humans , Immunity , Membrane Proteins/genetics , Mice , Multiple Myeloma/drug therapy , Nucleotidyltransferases/genetics , Signal TransductionABSTRACT
The bone marrow (BM) microenvironment actively promotes multiple myeloma (MM) pathogenesis and therapies targeting both cancer cells and the niche are highly effective. We were interested in identifying novel signaling pathways supporting MM-BM crosstalk. Mutations in the transmembrane receptor Roundabout 1 (ROBO1) were recently identified in MM patients, however their functional consequences are uncertain. Through protein structure-function studies, we discovered that ROBO1 is necessary for MM adhesion to BM stromal and endothelial cells and ROBO1 knock out (KO) compromises BM homing and engraftment in a disseminated mouse model. ROBO1 KO significantly decreases MM proliferation in vitro and intra- and extramedullary tumor growth, in vivo. Mechanistically, ROBO1 C-terminus is cleaved in a ligand-independent fashion and is sufficient to promote MM proliferation. Viceversa, mutants lacking the cytoplasmic domain, including the human-derived G674* truncation, act dominantly negative. Interactomic and RNA sequencing studies suggest ROBO1 may be involved in RNA processing, supporting further studies.
Subject(s)
Bone Marrow , Multiple Myeloma , Nerve Tissue Proteins , Receptors, Immunologic , Animals , Bone Marrow/metabolism , Bone Marrow Cells , Endothelial Cells/metabolism , Humans , Mice , Multiple Myeloma/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Tumor Microenvironment/genetics , Roundabout ProteinsABSTRACT
Hotspot mutation of IKZF3 (IKZF3-L162R) has been identified as a putative driver of chronic lymphocytic leukemia (CLL), but its function remains unknown. Here, we demonstrate its driving role in CLL through a B cell-restricted conditional knockin mouse model. Mutant Ikzf3 alters DNA binding specificity and target selection, leading to hyperactivation of B cell receptor (BCR) signaling, overexpression of nuclear factor κB (NF-κB) target genes, and development of CLL-like disease in elderly mice with a penetrance of ~40%. Human CLL carrying either IKZF3 mutation or high IKZF3 expression was associated with overexpression of BCR/NF-κB pathway members and reduced sensitivity to BCR signaling inhibition by ibrutinib. Our results thus highlight IKZF3 oncogenic function in CLL via transcriptional dysregulation and demonstrate that this pro-survival function can be achieved by either somatic mutation or overexpression of this CLL driver. This emphasizes the need for combinatorial approaches to overcome IKZF3-mediated BCR inhibitor resistance.
Subject(s)
B-Lymphocytes/pathology , Ikaros Transcription Factor/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Mutation/genetics , Transcription, Genetic/genetics , Animals , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , NF-kappa B/genetics , Receptors, Antigen, B-Cell/genetics , Signal Transduction/geneticsABSTRACT
Colorectal cancer (CRC) is the third most commonly diagnosed cancer, which despite recent advances in treatment, remains incurable due to molecular heterogeneity of tumor cells. The B-cell lymphoma 9 (BCL9) oncogene functions as a transcriptional co-activator of the Wnt/ß-catenin pathway, which plays critical roles in CRC pathogenesis. Here we have identified a ß-catenin-independent function of BCL9 in a poor-prognosis subtype of CRC tumors characterized by expression of stromal and neural associated genes. In response to spontaneous calcium transients or cellular stress, BCL9 is recruited adjacent to the interchromosomal regions, where it stabilizes the mRNA of calcium signaling and neural associated genes by interacting with paraspeckle proteins. BCL9 subsequently promotes tumor progression and remodeling of the tumor microenvironment (TME) by sustaining the calcium transients and neurotransmitter-dependent communication among CRC cells. These data provide additional insights into the role of BCL9 in tumor pathogenesis and point towards additional avenues for therapeutic intervention.
Subject(s)
Colorectal Neoplasms/metabolism , Transcription Factors/metabolism , Animals , Calcium/metabolism , Cell Communication , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/physiopathology , Gene Expression Regulation, Neoplastic , Humans , Mice , Protein Binding , Transcription Factors/genetics , Wnt Proteins/metabolism , Wnt Signaling Pathway/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
MYD88 L265P is the most common mutation in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) and one of the most frequent in poor-prognosis subtypes of diffuse large B-cell lymphoma (DLBCL). Although inhibition of the mutated MYD88 pathway has an adverse impact on LPL/WM and DLBCL cell survival, its role in lymphoma initiation remains to be clarified. We show that in mice, human MYD88L265P promotes development of a non-clonal, low-grade B-cell lymphoproliferative disorder with several clinicopathologic features that resemble human LPL/WM, including expansion of lymphoplasmacytoid cells, increased serum immunoglobulin M (IgM) concentration, rouleaux formation, increased number of mast cells in the bone marrow, and proinflammatory signaling that progresses sporadically to clonal, high-grade DLBCL. Murine findings regarding differences in the pattern of MYD88 staining and immune infiltrates in the bone marrows of MYD88 wild-type (MYD88WT) and MYD88L265P mice are recapitulated in the human setting, which provides insight into LPL/WM pathogenesis. Furthermore, histologic transformation to DLBCL is associated with acquisition of secondary genetic lesions frequently seen in de novo human DLBCL as well as LPL/WM-transformed cases. These findings indicate that, although the MYD88L265P mutation might be indispensable for the LPL/WM phenotype, it is insufficient by itself to drive malignant transformation in B cells and relies on other, potentially targetable cooperating genetic events for full development of lymphoma.
Subject(s)
Amino Acid Substitution , B-Lymphocytes/metabolism , Biomarkers, Tumor , Cell Transformation, Neoplastic/genetics , Mutation , Myeloid Differentiation Factor 88/genetics , Alleles , Animals , B-Lymphocytes/pathology , Biopsy , Disease Models, Animal , Gene Expression , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Immunohistochemistry , Immunophenotyping , Mice , Mice, Transgenic , Myeloid Differentiation Factor 88/metabolism , Neoplasm Grading , Transcriptome , Waldenstrom Macroglobulinemia/etiology , Waldenstrom Macroglobulinemia/metabolism , Waldenstrom Macroglobulinemia/pathologyABSTRACT
The immune cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received significant attention as a cancer therapeutic due to its ability to selectively trigger cancer cell apoptosis without causing toxicity in vivo. While TRAIL has demonstrated significant promise in preclinical studies in mice as a cancer therapeutic, challenges including poor circulation half-life, inefficient delivery to target sites, and TRAIL resistance have hindered clinical translation. Recent advances in drug delivery, materials science, and nanotechnology are now being exploited to develop next-generation nanoparticle platforms to overcome barriers to TRAIL therapeutic delivery. Here, we review the design and implementation of nanoparticles to enhance TRAIL-based cancer therapy. The platforms we discuss are diverse in their approaches to the delivery problem and provide valuable insight into guiding the design of future nanoparticle-based TRAIL cancer therapeutics to potentially enable future translation into the clinic.
Subject(s)
Nanoparticles/chemistry , Neoplasms/drug therapy , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Drug Delivery Systems , Humans , Neoplasms/pathology , Particle Size , Surface Properties , TNF-Related Apoptosis-Inducing Ligand/chemistryABSTRACT
Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in acute myeloid leukemia patients (AML). Although FLT3 tyrosine kinase inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. For such reasons, we have developed SEL24-B489 - a potent, dual PIM and FLT3-ITD inhibitor. SEL24-B489 exhibited significantly broader on-target activity in AML cell lines and primary AML blasts than selective FLT3-ITD or PIM inhibitors. SEL24-B489 also demonstrated marked activity in cells bearing FLT3 tyrosine kinase domain (TKD) mutations that lead to FLT3 inhibitor resistance. Moreover, SEL24-B489 inhibited the growth of a broad panel of AML cell lines in xenograft models with a clear pharmacodynamic-pharmacokinetic relationship. Taken together, our data highlight the unique dual activity of the SEL24-B489 that abrogates the activity of signaling circuits involved in proliferation, inhibition of apoptosis and protein translation/metabolism. These results underscore the therapeutic potential of the dual PIM/FLT3-ITD inhibitor for the treatment of AML.