RESUMO
The impact of exosome-mediated crosstalk between multiple myeloma (MM) cells and osteoclasts (OCs) on bone lesions remains to be investigated. Here, we identified NSUN2 and YBX1-mediated m5C modifications upregulated LncRNA MALAT1 expression in MM cells, which could be transported to OCs via exosomes and promote bone lesions. Methodologically, RNA-seq was carried out to detect the cargoes of exosomes. TRAP staining and WB were used to evaluate osteoclastogenesis in vitro. Micro-CT and bone histomorphometric analyses were performed to identify bone destruction in vivo. RNA pull-down, RIP, MeRIP, and luciferase reporter assays were used to test the interactions between molecules. The clinical features of MALAT1, NSUN2 and YBX1 were verified through public datasets and clinicopathological data analyses. Mechanistically, MALAT1 was the highest expressed lncRNA in U266 exosomes and could be transported to RAW264.7 cells. MALAT1 could enhance the differentiation of RAW264.7 cells into OCs by stimulating RANKL expression and its downstream AKT and MAPKs signaling pathways via a ceRNA mechanism. Additionally, MALAT1 could be modified by NSUN2, an m5C methyltransferase, which in turn stabilized MALAT1 through the "reader" YBX1. Clinical studies indicated a notable positive correlation between MALAT1, NSUN2, YBX1 levels and bone destruction features, as well as with RANKL expression.
Assuntos
Exossomos , Mieloma Múltiplo , Osteoclastos , Ligante RANK , RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Animais , Camundongos , Humanos , Exossomos/metabolismo , Exossomos/genética , Ligante RANK/metabolismo , Ligante RANK/genética , Osteoclastos/metabolismo , Osteoclastos/patologia , Células RAW 264.7 , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , MasculinoRESUMO
OBJECTIVE: The drug resistance of multiple myeloma (MM) cells is one of the main causes of relapse, refractory and progression of MM. METHODS: First, Western blot analysis was used to detect the expression levels of NLRP3, ASC, pro-IL-1ß and cleaved IL-1ß, and RT-qPCR was used to detect the mRNA expression levels of them. The expression levels of IL-1ß and IL-18 in the supernatant were detected by ELISA, and the expression levels of these factors in the activated group and the control group were compared to verify the activation of BMMCs and KM3. RESULT: 1. The protein expression of NLRP3 and cleavd-IL-1ß in the BMMCs cells was significantly higher than that of the control group (P < 0.05). The mRNA expression levels of caspase-1 and IL-1ß were higher than those of the control group (P = 0.03, P = 0.02). 2. The protein expression levels of NLRP3 and cleaved-IL-1ß in the KM3 cells were significantly higher than those of the control group (P < 0.05). The expressions of caspase-1 mRNA(P = 0.016) and IL-1ß mRNA(P = 0.037) were significantly increased compared with the control group. 3. The early apoptosis results of BMMCs showed that the apoptosis rate of the LPS+ATP+Dex group was lower than that of the Dex group (P = 0.017). The early apoptosis rate of the LPS+ATP+Dex+Vel group was decreased compared with the Dex+Vel group (P = 0.045). 4. The early apoptosis rate of KM3 in the LPS+ATP+Dex group was lower than that in the Dex group (P = 0.03). CONCLUSION: 1. LPS+ATP can activate NLRP3 inflammasome in multiple myeloma cells. 2. Activation of NLRP3 inflammasome inhibits the early apoptosis of myeloma cells induced by dexamethasone and bortezomib.
Assuntos
Inflamassomos , Mieloma Múltiplo , Proteína 3 que Contém Domínio de Pirina da Família NLR , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Linhagem Celular Tumoral , Masculino , Feminino , Pessoa de Meia-IdadeRESUMO
Multiple myeloma (MM) is a hematological malignancy whose curability is greatly challenged by recurrent patient relapses and therapy resistance. We have previously proposed the high expression of ADAM8, ADAM9 and ADAM15 (A Disintegrin And Metalloproteinase 8/9/15) as adverse prognostic markers in MM. This study focused on the so far scarcely researched role of ADAM8/9/15 in MM using two patient cohorts and seven human MM cell lines (HMCL). High ADAM8/9/15 expression was associated with high-risk cytogenetic abnormalities and extramedullary disease. Furthermore, ADAM8/15 expression increased with MM progression and in relapsed/refractory MM compared to untreated patient samples. RNA sequencing and gene set enrichment analysis comparing ADAM8/9/15high/low patient samples revealed an upregulation of proliferation markers and proliferation-associated gene sets in ADAM8/9/15high patient samples. High ADAM8/9/15 expression correlated with high Ki67 and high ADAM8/15 expression with high MYC protein expression in immunohistochemical stainings of patient tissue. Conversely, siRNA-mediated knockdown of ADAM8/9/15 in HMCL downregulated proliferation-related gene sets. Western blotting revealed that ADAM8 knockdown regulated IGF1R/AKT signaling and ADAM9 knockdown decreased mTOR activation. Lastly, high ADAM8/9/15 expression levels were verified as prognostic markers independent of Ki67/MYC expression and/or high-risk abnormalities. Overall, these findings suggest that ADAM8/9/15 play a role in MM progression and proliferation signaling.
Assuntos
Proteínas ADAM , Proliferação de Células , Progressão da Doença , Proteínas de Membrana , Mieloma Múltiplo , Transdução de Sinais , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Masculino , Feminino , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Pessoa de Meia-Idade , Biomarcadores Tumorais , IdosoRESUMO
Background: Immune checkpoint ligand-receptor interactions appear to be associated with multiple myeloma (MM) progression. Simultaneously, previous studies showed the possibility of PD-1 and TIM-3 expression on T cells upon stimulation with common γ-chain family cytokines in vitro and during homeostatic proliferation. The aim of the present work was to study the impact of homeostatic proliferation on the expansion of certain T cell subsets up-regulating PD-1 and TIM-3 checkpoint molecules. Methods: The expression of CD25, CD122, CD127 common γ-chain cytokine receptors, phosphorylated signal transducer and activator of transcription-5 (pSTAT5) and eomesodermin (EOMES) was comparatively assessed with flow cytometry in PD-1- and TIM-3-negative and positive T cells before the conditioning and during the first post-transplant month in peripheral blood samples of MM patients. Results: Substantial proportions of PD-1- and TIM-3-positive T lymphocytes expressed common γ-chain cytokine receptors and pSTAT5. Frequencies of cytokine receptor expressing cells were significantly higher within TIM-3+ T cells compared to PD-1+TIM-3- subsets. Considerable proportions of both PD-1-/TIM-3-negative and positive CD8+ T cells express EOMES, while only moderate frequencies of CD4+ PD-1+/TIM-3+ T cells up-regulate this transcription factor. Besides, the surface presence of CD25 and intranuclear expression of EOMES in CD4+ T cells were mutually exclusive regardless of PD-1 and TIM-3 expression. The stimulation with common γ-chain cytokines up-regulates PD-1 and TIM-3 during the proliferation of initially PD-1/TIM-3-negative T cells but fails to expand initially PD-1+ and TIM-3+ T cell subsets in vitro. Conclusions: Both PD-1 and TIM-3 expressing T cells appear to be able to respond to homeostatic cytokine stimulation. Differences in common γ-chain cytokine receptor expression between PD-1+ and TIM-3+ T cells may reflect functional dissimilarity of these cell subsets. Checkpoint blockade appears to alleviate lymphopenia-induced proliferation of PD-1+ T cells but may raise the possibility of immune-mediated adverse events.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Mieloma Múltiplo , Receptor de Morte Celular Programada 1 , Humanos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Pessoa de Meia-Idade , Masculino , Feminino , Idoso , Interleucina-7/metabolismo , Interleucina-15/farmacologia , Interleucina-15/metabolismo , Regulação para Cima , Adulto , Receptores de Citocinas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Objective Myeloma-related bone disease (MBD) is one of the most common complications of multiple myeloma (MM). This study aims to investigate the correlation between serum bone metabolism indexes (BMIs), the clinical characteristics and prognosis of newly diagnosed MM (NDMM) patients. METHODS: The serum BMIs of 148 patients with NDMM in a single hematological disease treatment center from April 2014 to December 2019 were analyzed retrospectively, including type I collagen amino terminal elongation peptide (PINP), ß-C-terminal telopeptide of type I collagen (ß-CTX) and N-terminal osteocalcin (N-MID). Other clinical indexes were simultaneously collected and the degree of bone damage in patients was evaluated. We explored the effect of serum BMIs on the prognosis and identified independent prognostic factors. Another 77 NDMM patients from April 2018 to February 2021 served as the validation cohort. RESULTS: The area under the curve (AUC) predicted by ß-C-terminal telopeptide of type I collagen (ß-CTX), type I collagen amino terminal elongation peptide (PINP), and N-terminal osteocalcin (N-MID) for overall survival (OS) were 0.708, 0.613, and 0.538, respectively. Patients with high serum levels had shorter OS (p < .001, p = .004, p = .027, respectively). Cox multivariate analysis indicated that serum ß- CTXãlactic dehydrogenaseãhemoglobin and the degree of bone injury were independent prognostic factors. A COX regression model was established with a C-index of 0.782 and validated with a C-index of 0.711. CONCLUSION: The serum BMIs are correlated with the patients' OS, and ß- CTX can be an independent prognostic factor.
Assuntos
Doenças Ósseas , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/sangue , Mieloma Múltiplo/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Prognóstico , Idoso , Doenças Ósseas/etiologia , Doenças Ósseas/mortalidade , Doenças Ósseas/sangue , Doenças Ósseas/metabolismo , Estudos Retrospectivos , Colágeno Tipo I/sangue , Colágeno Tipo I/metabolismo , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Biomarcadores/sangue , Osteocalcina/sangue , Osteocalcina/metabolismo , Adulto , Idoso de 80 Anos ou mais , PeptídeosRESUMO
BACKGROUND: Adrenomedullin (AM) is a multifunctional peptide which under basal conditions mainly regulates vasodilation and maintains vascular integrity but is also implicated in the pathogenesis of several malignancies, including multiple myeloma (MM). It has been shown that adrenomedullin is expressed by human myeloma cell lines and that it enhances MM-driven angiogenesis. However, the clinical impact of AM remains unknown. MATERIALS AND METHODS: On that basis, we enrolled 32 newly diagnosed multiple myeloma patients (NDMM) and studied the potential of AM as a prognostic biomarker. RESULTS: We report that elevated levels of AM trend with suboptimal treatment response and inferior survival of NDMM patients.
Assuntos
Adrenomedulina , Mieloma Múltiplo , Neovascularização Patológica , Humanos , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/mortalidade , Adrenomedulina/metabolismo , Feminino , Masculino , Prognóstico , Pessoa de Meia-Idade , Idoso , Neovascularização Patológica/metabolismo , Biomarcadores Tumorais/metabolismo , Idoso de 80 Anos ou mais , AngiogêneseRESUMO
BACKGROUND: Cellular immunotherapy using modified T cells offers new avenues for cancer treatment. T-cell receptor (TCR) engineering of CD8 T cells enables these cells to recognize tumor-associated antigens and tumor-specific neoantigens. Improving TCR T-cell therapy through increased potency and in vivo persistence will be critical for clinical success. METHODS: We evaluated a novel drug combination to enhance TCR therapy in mouse models for acute myeloid leukemia (AML) and multiple myeloma (MM). RESULTS: Combining TCR therapy with the SUMO E1 inhibitor TAK981 and the DNA methylation inhibitor 5-Aza-2' deoxycytidine resulted in strong antitumor activity in a persistent manner against two in vivo tumor models of established AML and MM. We uncovered that the drug combination caused strong T-cell proliferation, increased cytokine signaling in T cells, improved persistence of T cells, and reduced differentiation towards exhausted phenotype. Simultaneously the drug combination enhanced immunogenicity of the tumor by increasing HLA and co-stimulation and surprisingly reducing inhibitory ligand expression. CONCLUSION: Combining T-cell therapy with TAK981 and 5-Aza-2' deoxycytidine may be an important step towards improved clinical outcome.
Assuntos
Decitabina , Epigênese Genética , Receptores de Antígenos de Linfócitos T , Animais , Decitabina/farmacologia , Decitabina/uso terapêutico , Camundongos , Humanos , Epigênese Genética/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/metabolismo , Processamento de Proteína Pós-Traducional , Linhagem Celular Tumoral , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismoRESUMO
Objective: To study the expressions of C-C class chemokine 17 (CCL17), C-C class chemokine 22 (CCL22), and C-C chemokine receptor 4 (CCR4) in newly diagnosed multiple myeloma (NDMM) for analyzing their correlations with clinical features and to preliminarily explore their roles in the development of NDMM. Methods: The study included 40 patients with NDMM and 20 healthy volunteers from the Department of Hematology of the Affiliated Hospital of Zunyi Medical University from July 2020 to December 2022. Peripheral blood, bone marrow, and bone marrow biopsy tissue samples were collected from the two groups. The expression levels of CCL17, CCL22, and CCR4 in patients with NDMM were analyzed using real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry. The mRNA expression levels of CCL17, CCL22, and CCR4 in the bone marrow mononuclear cell (BMMNC) of patients with NDMM were analyzed to assess their correlations with clinical indicators. Results: The mRNA expression levels of CCL17, CCL22, and CCR4 in BMMNC were higher in patients with NDMM than in controls (all P<0.05). The protein expression levels of CCL17 and CCL22 in peripheral blood supernatants and bone marrow supernatants were higher in patients with NDMM than in controls (all P<0.05). The expression levels of CCL17, CCL22, and CCR4 in bone marrow biopsy tissues were higher in patients with NDMM than in controls (all P<0.05). The mRNA expression level of CCL17 was increased in NDMM patients with combined anemia, bone damage, renal damage, and M protein level ≥30 g/L (all P<0.05). The mRNA expression level of CCL22 was increased in NDMM patients with combined anemia, bone damage, and renal damage (all P<0.05). The mRNA expression level of CCR4 was increased in NDMM patients with combined anemia and renal damage (all P<0.05) . Conclusion: CCL17, CCL22, and CCR4 were highly expressed in clinical samples from patients with NDMM compared to those from controls, and they may be involved in the occurrence and development of NDMM.
Assuntos
Quimiocina CCL17 , Quimiocina CCL22 , Mieloma Múltiplo , Receptores CCR4 , Humanos , Receptores CCR4/metabolismo , Quimiocina CCL17/metabolismo , Quimiocina CCL17/genética , Quimiocina CCL22/metabolismo , Quimiocina CCL22/genética , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Medula Óssea/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Relevância ClínicaRESUMO
TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a Multiple Myeloma Research Foundation (MMRF) cohort of 858 patients, persisting across primary and recurrent MM cases. TRIM44 expression notably increases in advanced MM stages, indicating its potential role in disease progression. Single-cell RNA sequencing across MM stages showed significant TRIM44 upregulation in smoldering MM (SMM) and MM compared to normal bone marrow, especially in patients with t(4;14) cytogenetic abnormalities. This analysis further identified high TRIM44 expression as predictive of lower responsiveness to proteasome inhibitor (PI) treatments, underscoring its critical function in the unfolded protein response (UPR) in TRIM44-high MM cells. Our findings also demonstrate that TRIM44 facilitates SQSTM1 oligomerization under oxidative stress, essential for its phosphorylation and subsequent autophagic degradation. This process supports the survival of PI-resistant MM cells by activating the NRF2 pathway, which is crucial for oxidative stress response and, potentially, other chemotherapy-induced stressors. Additionally, TRIM44 counters the TRIM21-mediated suppression of the antioxidant response, enhancing MM cell survival under oxidative stress. Collectively, our discoveries highlight TRIM44's significant role in MM progression and resistance to therapy, suggesting its potential value as a therapeutic target.
Assuntos
Mieloma Múltiplo , Complexo de Endopeptidases do Proteassoma , Proteínas com Motivo Tripartido , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/genética , Humanos , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Prognóstico , Linhagem Celular Tumoral , Complexo de Endopeptidases do Proteassoma/metabolismo , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genética , Autofagia/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Inibidores de Proteassoma/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Targeted protein degradation of neosubstrates plays a crucial role in hematological cancer treatment involving immunomodulatory imide drugs (IMiDs) therapy. Nevertheless, the persistence of inevitable drug resistance and hematological toxicities represents a significant obstacle to their clinical effectiveness. METHODS: Phenotypic profiling of a small molecule compounds library in multiple hematological cancer cell lines was conducted to screen for hit degraders. Molecular dynamic-based rational design and cell-based functional assays were conducted to develop more potent degraders. Multiple myeloma (MM) tumor xenograft models were employed to investigate the antitumor efficacy of the degraders as single or combined agents with standard of care agents. Unbiased proteomics was employed to identify multiple therapeutically relevant neosubstrates targeted by the degraders. MM patient-derived cell lines (PDCs) and a panel of solid cancer cell lines were utilized to investigate the effects of candidate degrader on different stage of MM cells and solid malignancies. Unbiased proteomics of IMiDs-resistant MM cells, cell-based functional assays and RT-PCR analysis of clinical MM specimens were utilized to explore the role of BRD9 associated with IMiDs resistance and MM progression. RESULTS: We identified a novel cereblon (CRBN)-dependent lead degrader with phthalazinone scaffold, MGD-4, which induced the degradation of Ikaros proteins. We further developed a novel potent candidate, MGD-28, significantly inhibited the growth of hematological cancer cells and induced the degradation of IKZF1/2/3 and CK1α with nanomolar potency via a Cullin-CRBN dependent pathway. Oral administration of MGD-4 and MGD-28 effectively inhibited MM tumor growth and exhibited significant synergistic effects with standard of care agents. MGD-28 exhibited preferentially profound cytotoxicity towards MM PDCs at different disease stages and broad antiproliferative activity in multiple solid malignancies. BRD9 modulated IMiDs resistance, and the expression of BRD9 was significant positively correlated with IKZF1/2/3 and CK1α in MM specimens at different stages. We also observed pronounced synergetic efficacy between the BRD9 inhibitor and MGD-28 for MM treatment. CONCLUSIONS: Our findings present a strategy for the multi-targeted degradation of Ikaros proteins and CK1α against hematological cancers, which may be expanded to additional targets and indications. This strategy may enhance efficacy treatment against multiple hematological cancers and solid tumors.
Assuntos
Neoplasias Hematológicas , Humanos , Animais , Linhagem Celular Tumoral , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/metabolismo , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteólise/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Fator de Transcrição Ikaros/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de SinalRESUMO
OBJECTIVE: Multiple myeloma (MM) is caused by abnormal cloning of plasma cells. miR-184 is abnormally expressed in several types of tumors, but its expression and role in MM have not been reported. METHODS: The bone marrow samples of healthy controls and MM patients were collected, and plasma cells were sorted. The multiple myeloma cell line OPM-2 was cultured and assigned into miR-NC+siRNA-NC group, miR-184 inhibitor+siRNA-NC group, and miR-184 inhibitor+siRNA-Notch1 group. Cell proliferation was assessed by MTT assay. Clone formation was evaluated by colony formation assay. Cell apoptosis activity was tested with flow cytometry. Notch1 and cleaved caspase3 protein expressions were detected. RESULTS: MiR-184 expression was increased in myeloma plasma cells (P<0.05). Transfection of miR-184 inhibitor can downregulate miR-184 expression, increase the levels of Notch1 and cleaved caspase3, inhibit OPM-2 cell proliferation, restrain colony formation, enhance caspase3 activity, and suppress tumor cell invasion (P<0.05). However, administration of siRNA-Notch1 retarded the effect of miR-184 inhibitor by decreasing the expressions of Notch1 and cleaved caspase3, enhancing colony formation and tumor cell invasion, as well as inhibiting caspase3 activity and cell proliferation. CONCLUSION: Our data indicated that miR-184 expression is increased in myeloma plasma cells. Down-regulation of miR-184 promotes MM cell apoptosis and inhibits proliferation and colony formation by regulating Notch1 expression.
Assuntos
Apoptose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , MicroRNAs , Mieloma Múltiplo , Receptor Notch1 , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Receptor Notch1/metabolismo , Receptor Notch1/genética , Proliferação de Células/genética , Apoptose/genética , Linhagem Celular Tumoral , Masculino , Feminino , Caspase 3/metabolismo , Caspase 3/genética , Pessoa de Meia-Idade , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
In this study, we employed the dithiothreitol-based protein equalisation technique and analytical proteomics to better understand myeloma diseases by comparing the proteomes of pellets and supernatants formed upon application of DTT on serum samples. The number of unique proteins found in pellets was 252 for healthy individuals and 223 for multiple myeloma patients. The comparison of these proteomes showed 97 dysregulated proteins. The unique proteins found for supernatants were 264 for healthy individuals and 235 for multiple myeloma patients. The comparison of these proteomes showed 87 dysregulated proteins. The analytical proteomic comparison of both groups of dysregulated proteins is translated into parallel dysregulated pathways, including chaperone-mediated autophagy and protein folding, addressing potential therapeutic interventions. Future research endeavours in personalised medicine should prioritize refining analytical proteomic methodologies using serum dithiothreitol-based protein equalisation to explore innovative therapeutic strategies. We highlight the advanced insights gained from this analytical strategy in studying multiple myeloma, emphasising its complex nature and the critical role of personalised, targeted analytical techniques in enhancing therapeutic efficacy in personalised medicine.
Assuntos
Ditiotreitol , Mieloma Múltiplo , Proteômica , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Humanos , Ditiotreitol/química , Proteômica/métodos , Proteoma/análise , Proteoma/metabolismoRESUMO
The role of programmed cell death 4 (PDCD4) in multiple myeloma (MM) development remains unknown. Here, we investigated its role and action mechanism in MM. Bioinformatic analysis indicated that patients with MM and high PDCD4 expression had higher overall survival than those with low PDCD4 expression. PDCD4 expression promoted MM cell apoptosis and inhibited their viability in vitro and tumor growth in vivo. RNA-binding protein immunoprecipitation sequencing analysis showed that PDCD4 is bound to the 5' UTR of the apoptosis-related genes PIK3CB, Cathepsin Z (CTSZ), and X-chromosome-linked apoptosis inhibitor (XIAP). PDCD4 knockdown reduced the cell apoptosis rate, which was rescued by adding PIK3CB, CTSZ, or XIAP inhibitors. Dual luciferase reporter assays confirmed the internal ribosome entry site (IRES) activity of the 5' UTRs of PIK3CB and CTSZ. An RNA pull-down assay confirmed binding of the 5' UTR of PIK3CB and CTSZ to PDCD4, identifying the specific binding fragments. PDCD4 is expected to promote MM cell apoptosis by binding to the IRES domain in the 5' UTR of PIK3CB and CTSZ and inhibiting their translation. Our findings suggest that PDCD4 plays an important role in MM development by regulating the expression of PIK3CB, CTSZ, and XIAP, and highlight new potential molecular targets for MM treatment.
Assuntos
Proteínas Reguladoras de Apoptose , Apoptose , Mieloma Múltiplo , Proteínas de Ligação a RNA , Animais , Humanos , Masculino , Camundongos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Classe I de Fosfatidilinositol 3-Quinases/genética , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
Proteasome inhibitors (PIs), bortezomib, carfilzomib, and ixazomib, are the first-line treatment for multiple myeloma (MM). They inhibit cytosolic protein degradation in cells, which leads to the accumulation of misfolded and malfunctioned proteins in the cytosol and endoplasmic reticulum, resulting in cell death. Despite being a breakthrough in MM therapy, malignant cells develop resistance to PIs via different mechanisms. Understanding these mechanisms drives research toward new anticancer agents to overcome PI resistance. In this review, we summarize the mechanism of action of PIs and how MM cells adapt to these drugs to develop resistance. Finally, we explore these mechanisms to present strategies to interfere with PI resistance. The strategies include new inhibitors of the ubiquitin-proteasome system, drug efflux inhibitors, autophagy disruption, targeting stress response mechanisms, affecting survival and cell cycle regulators, bone marrow microenvironment modulation, and immunotherapy. We list potential pharmacological targets examined in in vitro, in vivo, and clinical studies. Some of these strategies have already provided clinicians with new anti-MM medications, such as panobinostat and selinexor. We hope that further exploration of the subject will broaden the range of therapeutic options and improve patient outcomes.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo , Inibidores de Proteassoma , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Complexo de Endopeptidases do Proteassoma/metabolismo , Autofagia/efeitos dos fármacosRESUMO
Multiple myeloma is the second most hematological cancer. RUVBL1 and RUVBL2 form a subcomplex of many chromatin remodeling complexes implicated in cancer progression. As an inhibitor specific to the RUVBL1/2 complex, CB-6644 exhibits remarkable anti-tumor activity in xenograft models of Burkitt's lymphoma and multiple myeloma (MM). In this work, we defined transcriptional signatures corresponding to CB-6644 treatment in MM cells and determined underlying epigenetic changes in terms of chromatin accessibility. CB-6644 upregulated biological processes related to interferon response and downregulated those linked to cell proliferation in MM cells. Transcriptional regulator inference identified E2Fs as regulators for downregulated genes and MED1 and MYC as regulators for upregulated genes. CB-6644-induced changes in chromatin accessibility occurred mostly in non-promoter regions. Footprinting analysis identified transcription factors implied in modulating chromatin accessibility in response to CB-6644 treatment, including ATF4/CEBP and IRF4. Lastly, integrative analysis of transcription responses to various chemical compounds of the molecular signature genes from public gene expression data identified CB-5083, a p97 inhibitor, as a synergistic candidate with CB-6644 in MM cells, but experimental validation refuted this hypothesis.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , DNA Helicases , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , DNA Helicases/genética , DNA Helicases/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proliferação de Células/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Deregulation of transcription factors (TFs) leading to uncontrolled proliferation of tumor cells within the microenvironment represents a hallmark of cancer. However, the biological and clinical impact of transcriptional interference, particularly in multiple myeloma (MM) cells, remains poorly understood. The present study shows for the first time that MYC and JUNB, two crucial TFs implicated in MM pathogenesis, orchestrate distinct transcriptional programs. Specifically, our data revealed that expression levels of MYC, JUNB, and their respective downstream targets do not correlate and that their global chromatin-binding patterns are not significantly overlapping. Mechanistically, MYC expression was not affected by JUNB knockdown, and conversely, JUNB expression and transcriptional activity were not affected by MYC knockdown. Moreover, suppression of MYC levels in MM cells via targeting the master regulator BRD4 by either siRNA-mediated knockdown or treatment with the novel proteolysis targeting chimera (PROTAC) MZ-1 overcame bone marrow (BM) stroma cell/IL-6-induced MYC- but not MEK-dependent JUNB-upregulation and transcriptional activity. Consequently, targeting of the two non-overlapping MYC- and JUNB-transcriptoms by MZ-1 in combination with genetic or pharmacological JUNB-targeting approaches synergistically enhanced MM cell death, both in 2D and our novel dynamic 3D models of the BM milieu as well as in murine xenografts. In summary, our data emphasize the opportunity to employ MYC and JUNB dual-targeting treatment strategies in MM as another exciting approach to further improve patient outcomes.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo , Proteínas Proto-Oncogênicas c-myc , Fatores de Transcrição , Mieloma Múltiplo/genética , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Mieloma Múltiplo/metabolismo , Humanos , Animais , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-jun/genéticaRESUMO
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
B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and multiple myeloma (MM), remain incurable, with MM particularly prone to relapse. Our study introduces a novel mouse model with active RANK signaling and the TCL1 oncogene, displaying both CLL and MM phenotypes. In younger mice, TCL1 and RANK expression expands CLL-like B1-lymphocytes, while MM originates from B2-cells, becoming predominant in later stages and leading to severe disease progression and mortality. The induced MM mimics human disease, exhibiting features like clonal plasma cell expansion, paraproteinemia, anemia, and kidney and bone failure, as well as critical immunosurveillance strategies that promote a tumor-supportive microenvironment. This research elucidates the differential impacts of RANK activation in B1- and B2-cells and underscores the distinct roles of single versus combined oncogenes in B-cell malignancies. We also demonstrate that human MM cells express RANK and that inhibiting RANK signaling can reduce MM progression in a xenotransplantation model. Our study provides a rationale for further investigating the effects of RANK signaling in B-cell transformation and the shaping of a tumor-promoting microenvironment.
Assuntos
Linfócitos B , Transformação Celular Neoplásica , Mieloma Múltiplo , Proteínas Proto-Oncogênicas , Transdução de Sinais , Animais , Humanos , Camundongos , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linhagem da Célula , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genética , Microambiente TumoralRESUMO
Proteasome inhibitors (PIs), such as bortezomib and calfizomib, were backbone agents in the treatment of multiple myeloma (MM). In this study, we investigated bortezomib interactors in MM cells and identified dihydrolipoamide dehydrogenase (DLD) as a molecular target of bortezomib. DLD catalyzes the oxidation of dihydrolipoamide to form lipoamide, a reaction that also generates NADH. Our data showed that bortezomib bound to DLD and inhibited DLD's enzymatic function in MM cells. DLD knocked down MM cells (DLD-KD) had decreased levels of NADH. Reduced NADH suppressed assembly of proteasome complex in cells. As a result, DLD-KD MM cells had decreased basal-level proteasome activity and were more sensitive to bortezomib. Since PIs were used in many anti-MM regimens in clinics, we found that high expression of DLD correlated with inferior prognosis of MM. Considering the regulatory role of DLD in proteasome assembly, we evaluated DLD targeting therapy in MM cells. DLD inhibitor CPI-613 showed a synergistic anti-MM effect with bortezomib in vitro and in vivo. Overall, our findings elucidated DLD as an alternative molecular target of bortezomib in MM. DLD-targeting might increase MM sensitivity to PIs.