RESUMO
The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fatores Imunológicos/farmacologia , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Chaperonas Moleculares/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Células Tumorais CultivadasRESUMO
ABSTRACT: Patients with multiple myeloma (MM) treated with B-cell maturation antigen (BCMA)-specific chimeric antigen receptor (CAR) T cells usually relapse with BCMA+ disease, indicative of CAR T-cell suppression. CD200 is an immune checkpoint that is overexpressed on aberrant plasma cells (aPCs) in MM and is an independent negative prognostic factor for survival. However, CD200 is not present on MM cell lines, a potential limitation of current preclinical models. We engineered MM cell lines to express CD200 at levels equivalent to those found on aPCs in MM and show that these are sufficient to suppress clinical-stage CAR T-cells targeting BCMA or the Tn glycoform of mucin 1 (TnMUC1), costimulated by 4-1BB and CD2, respectively. To prevent CD200-mediated suppression of CAR T cells, we compared CRISPR-Cas9-mediated knockout of the CD200 receptor (CD200RKO), to coexpression of versions of the CD200 receptor that were nonsignaling, that is, dominant negative (CD200RDN), or that leveraged the CD200 signal to provide CD28 costimulation (CD200R-CD28 switch). We found that the CD200R-CD28 switch potently enhanced the polyfunctionality of CAR T cells, and improved cytotoxicity, proliferative capacity, CAR T-cell metabolism, and performance in a chronic antigen exposure assay. CD200RDN provided modest benefits, but surprisingly, the CD200RKO was detrimental to CAR T-cell activity, adversely affecting CAR T-cell metabolism. These patterns held up in murine xenograft models of plasmacytoma, and disseminated bone marrow predominant disease. Our findings underscore the importance of CD200-mediated immune suppression in CAR T-cell therapy of MM, and highlight a promising approach to enhance such therapies by leveraging CD200 expression on aPCs to provide costimulation via a CD200R-CD28 switch.
Assuntos
Imunoterapia Adotiva , Mieloma Múltiplo , Humanos , Camundongos , Animais , Mieloma Múltiplo/metabolismo , Antígenos CD28/metabolismo , Linfócitos T , Antígeno de Maturação de Linfócitos B/metabolismo , Recidiva Local de Neoplasia/metabolismoRESUMO
ABSTRACT: Genomic instability contributes to cancer progression and is at least partly due to dysregulated homologous recombination (HR). Here, we show that an elevated level of ABL1 kinase overactivates the HR pathway and causes genomic instability in multiple myeloma (MM) cells. Inhibiting ABL1 with either short hairpin RNA or a pharmacological inhibitor (nilotinib) inhibits HR activity, reduces genomic instability, and slows MM cell growth. Moreover, inhibiting ABL1 reduces the HR activity and genomic instability caused by melphalan, a chemotherapeutic agent used in MM treatment, and increases melphalan's efficacy and cytotoxicity in vivo in a subcutaneous tumor model. In these tumors, nilotinib inhibits endogenous as well as melphalan-induced HR activity. These data demonstrate that inhibiting ABL1 using the clinically approved drug nilotinib reduces MM cell growth, reduces genomic instability in live cell fraction, increases the cytotoxicity of melphalan (and similar chemotherapeutic agents), and can potentially prevent or delay progression in patients with MM.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Melfalan/farmacologia , Instabilidade Genômica , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
ABSTRACT: Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2). A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high-energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased nicotinamide adenine dinucleotide phosphate (NADP[H]) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage, and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine, compromising the synthesis of creatine from its precursor, guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage, and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high-energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum, consistent with impaired use of mitochondrial adenosine triphosphate (ATP). Accordingly, AK2 suppression increased the sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.
Assuntos
Adenilato Quinase , Histona-Lisina N-Metiltransferase , Mieloma Múltiplo , Translocação Genética , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Adenilato Quinase/metabolismo , Adenilato Quinase/genética , Cromossomos Humanos Par 14/genética , Epigenoma , Cromossomos Humanos Par 4/genética , Carbono/metabolismo , Linhagem Celular Tumoral , Proteínas RepressorasRESUMO
ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Pirimidinas , Tiofenos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linhagem Celular Tumoral , Apoptose , Antineoplásicos/uso terapêuticoRESUMO
Macrophages are a dominant leukocyte population in the tumor microenvironment and actively promote cancer progression. However, the molecular mechanism underlying the role of macrophages remains poorly understood. Here we show that polarized M2 macrophages enhance 3-phosphoinositide-dependent protein kinase 1 (PDPK1)-mediated phosphoglycerate kinase 1 (PGK1) threonine (T) 243 phosphorylation in tumor cells by secreting interleukin-6 (IL-6). This phosphorylation facilitates a PGK1-catalyzed reaction toward glycolysis by altering substrate affinity. Inhibition of PGK1 T243 phosphorylation or PDPK1 in tumor cells or neutralization of macrophage-derived IL-6 abrogates macrophage-promoted glycolysis, proliferation, and tumorigenesis. In addition, PGK1 T243 phosphorylation correlates with PDPK1 activation, IL-6 expression, and macrophage infiltration in human glioblastoma multiforme (GBM). Moreover, PGK1 T243 phosphorylation also correlates with malignance and prognosis of human GBM. Our findings demonstrate a novel mechanism of macrophage-promoted tumor growth by regulating tumor cell metabolism, implicating the therapeutic potential to disrupt the connection between macrophages and tumor cells by inhibiting PGK1 phosphorylation.
Assuntos
Macrófagos/metabolismo , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glicólise , Humanos , Macrófagos/patologia , Camundongos , Camundongos Nus , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Fosforilação , Prognóstico , Microambiente TumoralRESUMO
Although translocation events between chromosome 4 (NSD2 gene) and chromosome 14 (immunoglobulin heavy chain [IgH] locus) (t(4;14)) is considered high risk in newly diagnosed multiple myeloma (NDMM), only â¼30% to 40% of t(4;14) patients are clinically high risk. We generated and compared a large whole genome sequencing (WGS) and transcriptome (RNA sequencing) from 258 t(4;14) (n = 153 discovery, n = 105 replication) and 183 non-t(4;14) NDMM patients with associated clinical data. A landmark survival analysis indicated only â¼25% of t(4;14) patients had an overall survival (OS) <24 months, and a comparative analysis of the patient subgroups identified biomarkers associated with this poor outcome, including translocation breakpoints located in the NSD2 gene and expression of IgH-NSD2 fusion transcripts. Three breakpoints were identified and are designated as: "no-disruption" (upstream of NSD2), "early-disruption" (in the 5' UTR), and "late-disruption" (within the NSD2 gene). Our results show a significant difference in OS based on the location of DNA breakpoints (median OS 28.6 "late-disruption" vs 59.2 "early disruption" vs 75.1 months "no disruption"). These findings have been replicated in an independent replication dataset. Also, univariate and multivariate analysis suggest high-risk markers such as del17p, 1p independently contribute to poor outcome in t(4;14) MM patients.
Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/metabolismo , Sequência de Bases , Translocação Genética , TranscriptomaRESUMO
In a short time, single-cell platforms have become the norm in many fields of research, including multiple myeloma (MM). In fact, the large amount of cellular heterogeneity in MM makes single-cell platforms particularly attractive because bulk assessments can miss valuable information about cellular subpopulations and cell-to-cell interactions. The decreasing cost and increasing accessibility of single-cell platform, combined with breakthroughs in obtaining multiomics data for the same cell and innovative computational programs for analyzing data, have allowed single-cell studies to make important insights into MM pathogenesis; yet, there is still much to be done. In this review, we will first focus on the types of single-cell profiling and the considerations for designing a single-cell profiling experiment. Then, we will discuss what have learned from single-cell profiling about myeloma clonal evolution, transcriptional reprogramming, and drug resistance, and about the MM microenvironment during precursor and advanced disease.
Assuntos
Mieloma Múltiplo , Humanos , Mieloma Múltiplo/metabolismo , Comunicação Celular , Evolução Clonal , Análise de Célula Única , Microambiente TumoralRESUMO
Immunomodulatory agents (IMiDs) are a cornerstone of treatment for patients with multiple myeloma. IMiDs are used in therapeutic combinations at all stages of disease and are approved as a single-agent maintenance treatment after autologous stem cell transplantation. However, patients become resistant to ongoing therapy over time and inevitably relapse. It is only in the last decade that the mechanism of IMiD action has been elucidated; through binding to the cereblon component of the CRL4CRBN E3 ubiquitin ligase, a set of neosubstrates is designated for degradation by the proteasome. In myeloma cells, this includes the zinc-finger B-cell transcription factors Ikaros and Aiolos, which, in turn, lead to decreased levels of IRF4 and c-MYC and cell death. As our knowledge of IMiD mechanism of action has advanced, the ability to study resistance mechanisms has also developed. This review explores the existing work on IMiD resistance and proposes areas of future research that may advance our understanding and management of this common clinical condition.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Agentes de Imunomodulação , Transplante Autólogo , Recidiva Local de Neoplasia , Biologia , Ubiquitina-Proteína LigasesRESUMO
Mitochondrial damage-associated molecular patterns (mtDAMPs) include proteins, lipids, metabolites, and DNA and have various context-specific immunoregulatory functions. Cell-free mitochondrial DNA (mtDNA) is recognized via pattern recognition receptors and is a potent activator of the innate immune system. Cell-free mtDNA is elevated in the circulation of trauma patients and patients with cancer; however, the functional consequences of elevated mtDNA are largely undefined. Multiple myeloma (MM) relies upon cellular interactions within the bone marrow (BM) microenvironment for survival and progression. Here, using in vivo models, we describe the role of MM cell-derived mtDAMPs in the protumoral BM microenvironment and the mechanism and functional consequence of mtDAMPs in myeloma disease progression. Initially, we identified elevated levels of mtDNA in the peripheral blood serum of patients with MM compared with those of healthy controls. Using the MM1S cells engrafted into nonobese diabetic severe combined immunodeficient gamma mice, we established that elevated mtDNA was derived from MM cells. We further show that BM macrophages sense and respond to mtDAMPs through the stimulator of interferon genes (STING) pathway, and inhibition of this pathway reduces MM tumor burden in the KaLwRij-5TGM1 mouse model. Moreover, we found that MM-derived mtDAMPs induced upregulation of chemokine signatures in BM macrophages, and inhibition of this signature resulted in egress of MM cells from the BM. Here, we demonstrate that malignant plasma cells release mtDNA, a form of mtDAMPs, into the myeloma BM microenvironment, which in turn activates macrophages via STING signaling. We establish the functional role of these mtDAMP-activated macrophages in promoting disease progression and retaining MM cells in the protumoral BM microenvironment.
Assuntos
Mieloma Múltiplo , Animais , Camundongos , Mieloma Múltiplo/metabolismo , Plasmócitos/patologia , Macrófagos/metabolismo , DNA Mitocondrial/genética , Progressão da Doença , Microambiente TumoralRESUMO
Multiple myeloma (MM) is the second most common hematological tumor in adults. Immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide (Len), are effective drugs for the treatment of multiple myeloma. Len can recruit IKZF1 and IKZF3 to cereblon (CRBN), a substrate receptor of the cullin 4-RING E3 ligase (CRL4), promote their ubiquitination and degradation, and finally inhibit the proliferation of myeloma cells. However, MM patients develop resistance to IMiDs over time, leading to disease recurrence and deterioration. To explore the possible approaches that may enhance the sensitivity of IMiDs to MM, in this study, we used the proximity labeling technique TurboID and quantitative proteomics to identify Lys-63-specific deubiquitinase BRCC36 as a CRBN-interacting protein. Biochemical experiments demonstrated that BRCC36 in the BRISC complex protects CRBN from lysosomal degradation by specifically cleaving the K63-linked polyubiquitin chain on CRBN. Further studies found that a small-molecule compound SHIN1, which binds to BRISC complex subunit SHMT2, can upregulate CRBN by elevating BRCC36. The combination of SHIN1 and Len can further increase the sensitivity of MM cells to IMiDs. Therefore, this study provides the basis for the exploration of a possible strategy for the SHIN1 and Len combination treatment for MM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Lenalidomida , Lisossomos , Mieloma Múltiplo , Ubiquitina-Proteína Ligases , Humanos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Lenalidomida/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Linhagem Celular Tumoral , Ubiquitinação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidoresRESUMO
Period circadian regulator 3 (PER3) functions as a tumor suppressor in various cancers. However, the role of PER3 in multiple myeloma (MM) has not been reported yet. Through this study, we aimed to investigate the potential role of PER3 in MM and the underlying mechanisms. RT-qPCR and western blotting were used to determine the mRNA and protein expression levels of PER3. Glyoxylate reductase 1 homolog (GLYR1) was predicted to be a transcription factor of PER3. The binding sites of GLYR1 on the promoter region of PER3 were analyzed using UCSC and confirmed using luciferase and chromatin immunoprecipitation assays. Viability, apoptosis, and metathesis were determined using CCK-8, colony formation, TUNEL, and transwell assays. We found that PER3 expression decreased in MM. Low PER3 levels may predict poor survival rates; PER3 overexpression suppresses the viability and migration of MM cells and promotes apoptosis. Moreover, GLYR1 transcriptionally activates PER3, and the knockdown of PER3 alleviates the effects of GLYR1 and induces its malignant behavior in MM cells. To conclude, GLYR1 upregulates PER3 and suppresses the aggressive behavior of MM cells, suggesting that GLYR1/PER3 signaling may be a potential therapeutic target for MM.
Assuntos
Movimento Celular , Proliferação de Células , Mieloma Múltiplo , Proteínas Circadianas Period , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Linhagem Celular Tumoral , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/genética , Apoptose , Regulação Neoplásica da Expressão GênicaRESUMO
Multiple myeloma (MM) is a neoplasia of B plasma cells that often induces bone pain. However, the mechanisms underlying myeloma-induced bone pain (MIBP) are mostly unknown. Using a syngeneic MM mouse model, we show that periosteal nerve sprouting of calcitonin gene-related peptide (CGRP+) and growth associated protein 43 (GAP43+) fibers occurs concurrent to the onset of nociception and its blockade provides transient pain relief. MM patient samples also showed increased periosteal innervation. Mechanistically, we investigated MM induced gene expression changes in the dorsal root ganglia (DRG) innervating the MM-bearing bone of male mice and found alterations in pathways associated with cell cycle, immune response and neuronal signaling. The MM transcriptional signature was consistent with metastatic MM infiltration to the DRG, a never-before described feature of the disease that we further demonstrated histologically. In the DRG, MM cells caused loss of vascularization and neuronal injury, which may contribute to late-stage MIBP. Interestingly, the transcriptional signature of a MM patient was consistent with MM cell infiltration to the DRG. Overall, our results suggest that MM induces a plethora of peripheral nervous system alterations that may contribute to the failure of current analgesics and suggest neuroprotective drugs as appropriate strategies to treat early onset MIBP.SIGNIFICANCE STATEMENT Multiple myeloma (MM) is a painful bone marrow cancer that significantly impairs the quality of life of the patients. Analgesic therapies for myeloma-induced bone pain (MIBP) are limited and often ineffective, and the mechanisms of MIBP remain unknown. In this manuscript, we describe cancer-induced periosteal nerve sprouting in a mouse model of MIBP, where we also encounter metastasis to the dorsal root ganglia (DRG), a never-before described feature of the disease. Concomitant to myeloma infiltration, the lumbar DRGs presented blood vessel damage and transcriptional alterations, which may mediate MIBP. Explorative studies on human tissue support our preclinical findings. Understanding the mechanisms of MIBP is crucial to develop targeted analgesic with better efficacy and fewer side effects for this patient population.
Assuntos
Doenças Ósseas , Mieloma Múltiplo , Tecido Nervoso , Humanos , Camundongos , Masculino , Animais , Mieloma Múltiplo/complicações , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Qualidade de Vida , Dor/metabolismo , Tecido Nervoso/metabolismo , Tecido Nervoso/patologia , Gânglios Espinais/metabolismoRESUMO
MafA and c-Maf are close members of the Maf transcription factor family and indicators of poor prognosis of multiple myeloma (MM). Our previous study finds that the ubiquitin ligase HERC4 induces c-Maf degradation but stabilizes MafA, and the mechanism is elusive. In the present study, we find that HERC4 interacts with MafA and mediates its K63-linked polyubiquitination at K33. Moreover, HERC4 inhibits MafA phosphorylation and its transcriptional activity triggered by glycogen synthase kinase 3ß (GSK3ß). The K33R MafA variant prevents HERC4 from inhibiting MafA phosphorylation and increases MafA transcriptional activity. Further analyses reveal that MafA can also activate the STAT3 signaling, but it is suppressed by HERC4. Lastly, we demonstrate that lithium chloride, a GSK3ß inhibitor, can upregulate HERC4 and synergizes dexamethasone, a typical anti-MM drug, in inhibiting MM cell proliferation and xenograft growth in nude mice. These findings thus highlight a novel regulation of MafA oncogenic activity in MM and provide the rationale by targeting HERC4/GSK3ß/MafA for the treatment of MM.
Assuntos
Glicogênio Sintase Quinase 3 beta , Fatores de Transcrição Maf Maior , Mieloma Múltiplo , Poliubiquitina , Ubiquitina-Proteína Ligases , Ubiquitinação , Animais , Humanos , Camundongos , Proliferação de Células , Dexametasona/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Cloreto de Lítio/farmacologia , Fatores de Transcrição Maf Maior/antagonistas & inibidores , Fatores de Transcrição Maf Maior/metabolismo , Camundongos Nus , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Fosforilação , Poliubiquitina/metabolismo , Fator de Transcrição STAT3/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Targeting DNA damage repair factors, such as DNA-dependent protein kinase catalytic subunit (DNA-PKcs), may offer an opportunity for effective treatment of multiple myeloma (MM). In combination with DNA damage-inducing agents, this strategy has been shown to improve chemotherapies partially via activation of cGAS-STING pathway by an elevated level of cytosolic DNA. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated into the cytoplasm upon DNA damage, leading to cGAS-STING activation. METHODS: We examined the role of DNA-PKcs inhibition on cGAS-STING-mediated MM chemosensitivity by performing mass spectrometry and mechanism study. RESULTS: Here, we found DNA-PKcs inhibition potentiated DNA damage-inducing agent doxorubicin-induced anti-MM effect by activating cGAS-STING signaling. The cGAS-STING activation in MM cells caused cell death partly via IRF3-NOXA-BAK axis and induced M1 polarization of macrophages. Moreover, this activation was not caused by defective classical non-homologous end joining (c-NHEJ). Instead, upon DNA damage induced by doxorubicin, inhibition of DNA-PKcs promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of cytosolic cGAS and DNA, activating cGAS-STING. CONCLUSIONS: Inhibition of DNA-PKcs could improve the efficacy of doxorubicin in treatment of MM by de-sequestrating cGAS in damaged chromatin.
Assuntos
Cromatina , Dano ao DNA , Proteína Quinase Ativada por DNA , Doxorrubicina , Proteínas de Membrana , Mieloma Múltiplo , Nucleotidiltransferases , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteína Quinase Ativada por DNA/metabolismo , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Cromatina/metabolismo , Cromatina/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Doxorrubicina/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Linhagem Celular Tumoral , Camundongos , Animais , Transdução de Sinais/efeitos dos fármacosRESUMO
Sarcopenia manifests as muscle atrophy and loss that is complicated with malignancy. This study explored the mechanism of extracellular vesicles (EVs) in multiple myeloma (MM) with sarcopenia. SP2/0 conditioned medium (CM) was collected to isolate SP2/0-EVs. C2C12 cells were incubated with SP2/0 CM or SP2/0-EVs. ROS, TNF-α, IL-6, MuRF1 and MyHC levels were detected by DCF-DA fluorescent probe, ELISA, and Western blot. GW4869 was used to inhibit EV secretion in SP2/0 to confirm its effect on muscle atrophy. Serum was collected from MM patients with or without sarcopenia to detect RAGE mRNA expression. SP2/0 cells were transfected with RAGE siRNA and C2C12 cells were treated with the isolated si-RAGE-EVs or/and TLR4 agonist. SP2/0 tumor-bearing mouse model was established. Healthy mice and SP2/0-tumor bearing mice were treated with SP2/0-EVs or si-RAGE-EVs. SP2/0 CM or SP2/0-EVs stimulated ROS, inflammatory responses, and myotube atrophy in C2C12 cells. GW4869 blocked EV secretion and the effects of SP2/0 CM. RAGE mRNA expression in serum EVs was increased in MM&Sarcopenia patients and RAGE knockdown in SP2/0-EVs partially nullified SP2/0-EVs' effects. SP2/0-EVs activated the TLR4/NF-κB p65 pathway by translocating RAGE. SP2/0-EVs-derived RAGE elevated ROS production, inflammation, and myotube atrophy in C2C12 cells and caused muscle loss in SP2/0 tumor-bearing mice by activating the TLR4/NF-κB p65 pathway. SP2/0-EVs partially recapitulated muscle loss in healthy mice. SP2/0-EVs-derived RAGE increased ROS production, inflammation, and myotube atrophy in MM through TLR4/NF-κB p65 pathway activation.
Assuntos
Vesículas Extracelulares , Inflamação , Mieloma Múltiplo , Atrofia Muscular , Receptor para Produtos Finais de Glicação Avançada , Transdução de Sinais , Receptor 4 Toll-Like , Fator de Transcrição RelA , Animais , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Humanos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/genética , Camundongos , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor para Produtos Finais de Glicação Avançada/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Inflamação/metabolismo , Inflamação/patologia , Inflamação/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Atrofia Muscular/genética , Linhagem Celular Tumoral , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Masculino , FemininoRESUMO
BACKGROUND: Despite the advances of therapies, multiple myeloma (MM) remains an incurable hematological cancer that most patients experience relapse. Tumor angiogenesis is strongly correlated with cancer relapse. Human leukocyte antigen G (HLA-G) has been known as a molecule to suppress angiogenesis. We aimed to investigate whether soluble HLA-G (sHLA-G) was involved in the relapse of MM. METHODS: We first investigated the dynamics of serum sHLA-G, vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6) in 57 successfully treated MM patients undergoing remission and relapse. The interactions among these angiogenesis-related targets (sHLA-G, VEGF and IL-6) were examined in vitro. Their expression at different oxygen concentrations was investigated using a xenograft animal model by intra-bone marrow and skin grafts with myeloma cells. RESULTS: We found that HLA-G protein degradation augmented angiogenesis. Soluble HLA-G directly inhibited vasculature formation in vitro. Mechanistically, HLA-G expression was regulated by hypoxia-inducible factor-1α (HIF-1α) in MM cells under hypoxia. We thus developed two mouse models of myeloma xenografts in intra-bone marrow (BM) and underneath the skin, and found a strong correlation between HLA-G and HIF-1α expressions in hypoxic BM, but not in oxygenated tissues. Yet when stimulated with IL-6, both HLA-G and HIF-1α could be targeted to ubiquitin-mediated degradation via PARKIN. CONCLUSION: These results highlight the importance of sHLA-G in angiogenesis at different phases of multiple myeloma. The experimental evidence that sHLA-G as an angiogenesis suppressor in MM may be useful for future development of novel therapies to prevent relapse.
Assuntos
Antígenos HLA-G , Interleucina-6 , Mieloma Múltiplo , Neovascularização Patológica , Mieloma Múltiplo/sangue , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Humanos , Animais , Neovascularização Patológica/metabolismo , Antígenos HLA-G/sangue , Antígenos HLA-G/metabolismo , Camundongos , Interleucina-6/sangue , Interleucina-6/metabolismo , Masculino , Feminino , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/sangue , Pessoa de Meia-Idade , Linhagem Celular Tumoral , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Idoso , Modelos Animais de Doenças , AngiogêneseRESUMO
Multiple myeloma (MM) is an incurable plasma cell cancer in the bone marrow. Immunomodulatory drugs, such as lenalidomide (LEN) and pomalidomide, are backbone agents in MM treatment, and LEN resistance is commonly seen in the MM clinic. In this study, we presented that heterogeneous nuclear ribonucleoprotein U (hnRNPU) affected MM resistance to LEN via the regulation of target mRNA translation. hnRNPULow MM cells exhibited upregulated CRBN and IKZF1 proteins, stringent IKZF1/3 protein degradation upon LEN addition and increased sensitivity to LEN. RNA pulldown assays and RNA electrophoretic mobility shift assays revealed that hnRNPU bound to the 3'-untranslated region of CRBN and IKZF1 mRNA. A sucrose gradient assay suggested that hnRNPU specifically regulated CRBN and IKZF1 mRNA translation. The competition of hnRNPU binding to its target mRNAs by small RNAs with hnRNPU-binding sites restored MM sensitivity to LEN. hnRNPU function in vivo was confirmed in an immunocompetent MM mouse model constructed by the inoculation of Crbn-humanized murine 5TGM1 cells into CrbnI391V/+ mice. Overall, this study suggests a novel mechanism of LEN sensitivity in which hnRNPU represses CRBN and IKZF1 mRNA translation.
Assuntos
Lenalidomida , Mieloma Múltiplo , Lenalidomida/farmacologia , Lenalidomida/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Humanos , Camundongos , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismoRESUMO
Peptide separations that combine high sensitivity, robustness, peak capacity, and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with offline gradient generation. One binary pump generates gradients in an accelerated fashion to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshooting. A high degree of parallelization is possible, as one sample undergoes separation while the next sample plus its corresponding mobile phase gradient are transferred into the storage loop and a third sample is loaded into a sample loop. Selective offline elution from the trap column into the sample loop prevents salts and hydrophobic species from entering the analytical column, thus greatly enhancing column lifetime and system robustness. With this design, samples can be analyzed as fast as every 20 min at a flow rate of just 40 nL/min with close to 100% MS utilization time and continuously for as long as several months without column replacement. We utilized the system to analyze the proteomes of single cells from a multiple myeloma cell line upon treatment with the immunomodulatory imide drug lenalidomide.
Assuntos
Proteoma , Análise de Célula Única , Humanos , Proteoma/análise , Nanotecnologia , Proteômica/métodos , Cromatografia Líquida/métodos , Linhagem Celular Tumoral , Lenalidomida/farmacologia , Talidomida/farmacologia , Talidomida/análogos & derivados , Mieloma Múltiplo/metabolismoRESUMO
Drug resistance in multiple myeloma (MM) poses a significant challenge to treatment efficacy, primarily attributed to P-glycoprotein (P-gp) dysfunction. This study delves into the elusive spatial organization of P-gp, aiming to enhance our understanding of its role in MM drug resistance by exploring the intricate relationship between molecular function and spatial arrangement. Employing super-resolution imaging of P-gp with the inhibitor probe Tariquidar-TAMR labeling on MM cell membranes, the research uncovered a more pronounced clustering distribution of P-gp in drug-resistant cells (MM1R) compared to drug-sensitive counterparts (MM1S). Further exploration revealed the clustering distribution of P-gp was heightened as cellular drug resistance increased in hypoxic condition, directly emphasizing the strong correlation between P-gp cluster morphology and drug resistance. Additionally, stable P-gp cluster formation was influenced by cross-linking of membrane carbohydrates, and disrupting these glycoprotein clusters could reduce cellular drug resistance, suggesting that altering distribution patterns of P-gp can modulate drug responsiveness. Finally, dexamethasone (Dex) treatment was revealed to enhance P-gp clustering distribution, particularly in MM1S cells, indicating that change degree in P-gp distribution correlate with the modifiable space of cellular drug responsiveness. This study provides insights into the correlation between P-gp assembly and cellular drug responsiveness, deepening our understanding of functional changes in MM drug resistance and offering valuable perspectives for overcoming this challenge.