RESUMO
ABSTRACT: SETBP1 mutations are found in various clonal myeloid disorders. However, it is unclear whether they can initiate leukemia, because SETBP1 mutations typically appear as later events during oncogenesis. To answer this question, we generated a mouse model expressing mutated SETBP1 in hematopoietic tissue: this model showed profound alterations in the differentiation program of hematopoietic progenitors and developed a myeloid neoplasm with megakaryocytic dysplasia, splenomegaly, and bone marrow fibrosis, prompting us to investigate SETBP1 mutations in a cohort of 36 triple-negative primary myelofibrosis (TN-PMF) cases. We identified 2 distinct subgroups, one carrying SETBP1 mutations and the other completely devoid of somatic variants. Clinically, a striking difference in disease aggressiveness was noted, with patients with SETBP1 mutation showing a much worse clinical course. In contrast to myelodysplastic/myeloproliferative neoplasms, in which SETBP1 mutations are mostly found as a late clonal event, single-cell clonal hierarchy reconstruction in 3 patients with TN-PMF from our cohort revealed SETBP1 to be a very early event, suggesting that the phenotype of the different SETBP1+ disorders may be shaped by the opposite hierarchy of the same clonal SETBP1 variants.
Assuntos
Sistema Hematopoético , Doenças Mieloproliferativas-Mielodisplásicas , Transtornos Mieloproliferativos , Mielofibrose Primária , Animais , Camundongos , Humanos , Mielofibrose Primária/genética , Transtornos Mieloproliferativos/genética , Mutação , Proteínas de Transporte/genética , Proteínas Nucleares/genéticaRESUMO
Recent investigations have improved our understanding of the molecular aberrations supporting Waldenström macroglobulinemia (WM) biology; however, whether the immune microenvironment contributes to WM pathogenesis remains unanswered. First, we showed how a transgenic murine model of human-like lymphoplasmacytic lymphoma/WM exhibits an increased number of regulatory T cells (Tregs) relative to control mice. These findings were translated into the WM clinical setting, in which the transcriptomic profiling of Tregs derived from patients with WM unveiled a peculiar WM-devoted messenger RNA signature, with significant enrichment for genes related to nuclear factor κB-mediated tumor necrosis factor α signaling, MAPK, and PI3K/AKT, which was paralleled by a different Treg functional phenotype. We demonstrated significantly higher Treg induction, expansion, and proliferation triggered by WM cells, compared with their normal cellular counterpart; with a more profound effect within the context of CXCR4C1013G-mutated WM cells. By investigating the B-cell-to-T-cell cross talk at single-cell level, we identified the CD40/CD40-ligand as a potentially relevant axis that supports WM cell-Tregs interaction. Our findings demonstrate the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.
Assuntos
Linfoma de Células B , Macroglobulinemia de Waldenstrom , Humanos , Animais , Camundongos , Macroglobulinemia de Waldenstrom/patologia , Ligante de CD40/genética , Fosfatidilinositol 3-Quinases , Ligantes , Transdução de Sinais , Linfoma de Células B/complicações , Microambiente TumoralRESUMO
Fibroblast growth factors (FGFs) act as proangiogenic and mitogenic cytokines in several cancers, including multiple myeloma (MM). Indeed, corrupted FGF autocrine and paracrine secretion induces an aberrant activation of the FGF receptor (FGFR) signaling sustaining cancer cell spreading and resistance to pharmacological treatments. Thus, FGF traps may represent a promising anti-cancer strategy to hamper the ligand-dependent activation of the FGF/FGFR system. We previously identified NSC12 as the first orally available small molecule FGF trap able to inhibit the growth and progression of several FGF-dependent tumor models. NSC12 is a pregnenolone derivative carrying a 1,1-bis-trifluoromethyl-1,3-propanediol chain in position 17 of the steroid nucleus. Investigation of structure-activity relationships (SARs) provided more potent and specific NSC12 steroid derivatives and highlighted that the C17-side chain is pivotal for the FGF trap activity. Here, a scaffold hopping approach allowed to obtain two FGF trap compounds (22 and 57) devoid of the steroid nucleus and able to efficiently bind FGF2 and to inhibit FGFR activation in MM cells. Accordingly, these compounds exert a potent anti-tumor activity on MM cell lines both in vitro and in vivo and on MM patient-derived primary cells, strongly affecting the survival of both proteasome-inhibitor sensitive and resistant MM cells. These results propose a new therapeutic option for relapsed/refractory MM patients and set the bases for the development of novel FGF traps prone to chemical diversification to be used in the clinic for the treatment of those tumors in which the FGF/FGFR system plays a pivotal role, including MM.
Assuntos
Antineoplásicos , Fatores de Crescimento de Fibroblastos , Mieloma Múltiplo , Receptores de Fatores de Crescimento de Fibroblastos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Humanos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Linhagem Celular Tumoral , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Descoberta de Drogas , Camundongos , Fator 2 de Crescimento de Fibroblastos/metabolismoRESUMO
Clonal evolution drives tumor progression, dissemination, and relapse in multiple myeloma (MM), with most patients dying of relapsed disease. This multistage process requires tumor cells to enter the circulation, extravasate, and colonize distant bone marrow (BM) sites. Here, we developed a fluorescent or DNA-barcode clone-tracking system on MM PrEDiCT (progression through evolution and dissemination of clonal tumor cells) xenograft mouse model to study clonal behavior within the BM microenvironment. We showed that only the few clones that successfully adapt to the BM microenvironment can enter the circulation and colonize distant BM sites. RNA sequencing of primary and distant-site MM tumor cells revealed a progression signature sequentially activated along human MM progression and significantly associated with overall survival when evaluated against patient data sets. A total of 28 genes were then computationally predicted to be master regulators (MRs) of MM progression. HMGA1 and PA2G4 were validated in vivo using CRISPR-Cas9 in the PrEDiCT model and were shown to be significantly depleted in distant BM sites, indicating their role in MM progression and dissemination. Loss of HMGA1 and PA2G4 also compromised the proliferation, migration, and adhesion abilities of MM cells in vitro. Overall, our model successfully recapitulates key characteristics of human MM disease progression and identified potential new therapeutic targets for MM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores Tumorais/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Proteína HMGA1a/metabolismo , Mieloma Múltiplo/patologia , Recidiva Local de Neoplasia/patologia , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Biomarcadores Tumorais/genética , Medula Óssea/metabolismo , Medula Óssea/patologia , Sistemas CRISPR-Cas , Adesão Celular , Movimento Celular , Proliferação de Células , Evolução Clonal , Progressão da Doença , Feminino , Proteína HMGA1a/antagonistas & inibidores , Proteína HMGA1a/genética , Humanos , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
The human fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb because of disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule (NSC12). Wide-transcriptome profiling confirmed inhibition of FGFR signaling in NSC12-treated WM cells; unveiling a significant inhibition of MYD88 was also confirmed at the protein level. Importantly, the NSC12-dependent silencing of MYD88 was functionally active, as it led to inhibition of MYD88-driven pathways, such as BTK and SYK, as well as the MYD88-downstream target HCK. Of note, both canonical and noncanonical NF-κB cascades were downregulated in WM cells upon NSC12 treatment. Functional sequelae exerted by NSC12 in WM cells were studied, demonstrating significant inhibition of WM cell growth, induction of WM cell apoptosis, halting MAPK, JAK/STAT3, and PI3K-Akt pathways. Importantly, NSC12 exerted an anti-WM effect even in the presence of bone marrow microenvironment, both in vitro and in vivo. Our studies provide the evidence for using NSC12 as a specific FGF/FGFR system inhibitor, thus representing a novel therapeutic strategy in WM.
Assuntos
Biomarcadores Tumorais/metabolismo , Colesterol/análogos & derivados , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Macroglobulinemia de Waldenstrom/prevenção & controle , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Colesterol/farmacologia , Perfilação da Expressão Gênica , Humanos , Camundongos , Transdução de Sinais , Células Tumorais Cultivadas , Microambiente Tumoral , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/metabolismo , Macroglobulinemia de Waldenstrom/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MYD88 and CXCR4 mutations are common in Waldenström macroglobulinemia (WM). Mutated CXCR4 (CXCR4Mut) impacts BTK-inhibitor response. We conducted a phase 1 trial of the CXCR4-antagonist ulocuplumab with ibrutinib in this first-ever study to target CXCR4Mut in WM. Ibrutinib was initiated at 420 mg/d with cycle 1 and continued until intolerance or progression; ulocuplumab was given cycles 1 to 6, with a 3 + 3 dose-escalation design. Each cycle was 4 weeks. Thirteen symptomatic patients, of whom 9 were treatment-naive patients were enrolled. Twelve were evaluable for response. At best response, their median serum immunoglobulin M declined from 5574 to 1114 mg/dL; bone marrow disease decreased from 65% to 10%, and hemoglobin increased from 10.1 to 14.2 g/dL (P < .001). The major and VGPR response rates were 100% and 33%, respectively, with VGPRs observed at lower ulocuplumab dose cohorts. Median times to minor and major responses were 0.9 and 1.2 months, respectively. With a median follow-up of 22.4 months, the estimated 2-year progression-free survival was 90%. The most frequent recurring grade ≥2 adverse events included reversible thrombocytopenia, rash, and skin infections. Ulocuplumab dose-escalation did not impact adverse events. The study demonstrates the feasibility of combining a CXCR4-antagonist with ibrutinib and provides support for the development of CXCR4-antagonists for CXCR4Mut WM. This trial was registered at www.clinicaltrials.gov as #NCT03225716.
Assuntos
Adenina/análogos & derivados , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos/uso terapêutico , Piperidinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Receptores CXCR4/genética , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Adenina/efeitos adversos , Adenina/uso terapêutico , Adulto , Idoso , Anticorpos Monoclonais Humanizados/efeitos adversos , Antineoplásicos/efeitos adversos , Humanos , Pessoa de Meia-Idade , Mutação/efeitos dos fármacos , Piperidinas/efeitos adversos , Inibidores de Proteínas Quinases/efeitos adversos , Receptores CXCR4/antagonistas & inibidores , Macroglobulinemia de Waldenstrom/genéticaRESUMO
Alterations in KRAS have been identified as the most recurring somatic variants in the multiple myeloma (MM) mutational landscape. Combining DNA and RNA sequencing, we studied 756 patients and observed KRAS as the most frequently mutated gene in patients at diagnosis; in addition, we demonstrated the persistence or de novo occurrence of the KRAS aberration at disease relapse. Small-molecule inhibitors targeting KRAS have been developed; however, they are selective for tumors carrying the KRASG12C mutation. Therefore, there is still a need to develop novel therapeutic approaches to target the KRAS mutational events found in other tumor types, including MM. We used AZD4785, a potent and selective antisense oligonucleotide that selectively targets and downregulates all KRAS isoforms, as a tool to dissect the functional sequelae secondary to KRAS silencing in MM within the context of the bone marrow niche and demonstrated its ability to significantly silence KRAS, leading to inhibition of MM tumor growth, both in vitro and in vivo, and confirming KRAS as a driver and therapeutic target in MM.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mutação/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Camundongos SCID , Terapia de Alvo Molecular , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Oligonucleotídeos Antissenso/uso terapêutico , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
Exosomes, secreted by several cell types, including cancer cells, can be isolated from the peripheral blood and have been shown to be powerful markers of disease progression in cancer. In this study, we examined the prognostic significance of circulating exosomal microRNAs (miRNAs) in multiple myeloma (MM). A cohort of 156 patients with newly diagnosed MM, uniformly treated and followed, was studied. Circulating exosomal miRNAs were isolated and used to perform a small RNA sequencing analysis on 10 samples and a quantitative reverse transcription polymerase chain reaction (qRT-PCR) array on 156 samples. We studied the relationship between miRNA levels and patient outcomes, including progression-free survival (PFS) and overall survival (OS). We identified miRNAs as the most predominant small RNAs present in exosomes isolated from the serum of patients with MM and healthy controls by small RNA sequencing of circulating exosomes. We then analyzed exosomes isolated from serum samples of 156 patients using a qRT-PCR array for 22 miRNAs. Two of these miRNAs, let-7b and miR-18a, were significantly associated with both PFS and OS in the univariate analysis and were still statistically significant after adjusting for the International Staging System and adverse cytogenetics in the multivariate analysis. Our findings support the use of circulating exosomal miRNAs to improve the identification of patients with newly diagnosed MM with poor outcomes. The results require further validation in other independent prospective MM cohorts.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Mieloma Múltiplo/diagnóstico , Adulto , Idoso , Biomarcadores Tumorais/sangue , Bortezomib/uso terapêutico , Estudos de Casos e Controles , Linhagem Celular Tumoral , Dexametasona/uso terapêutico , Exossomos/química , Exossomos/metabolismo , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Cariotipagem , Masculino , Melfalan/uso terapêutico , MicroRNAs/sangue , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Prognóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Análise de Sobrevida , Transplante Autólogo , Resultado do TratamentoRESUMO
Familial aggregation of Waldenström macroglobulinemia (WM) cases, and the clustering of B-cell lymphoproliferative disorders among first-degree relatives of WM patients, has been reported. Nevertheless, the possible contribution of inherited susceptibility to familial WM remains unrevealed. We performed whole exome sequencing on germ line DNA obtained from 4 family members in which coinheritance for WM was documented in 3 of them, and screened additional independent 246 cases by using gene-specific mutation sequencing. Among the shared germ line variants, LAPTM5(c403t) and HCLS1(g496a) were the most recurrent, being present in 3/3 affected members of the index family, detected in 8% of the unrelated familial cases, and present in 0.5% of the nonfamilial cases and in <0.05 of a control population. LAPTM5 and HCLS1 appeared as relevant WM candidate genes that characterized familial WM individuals and were also functionally relevant to the tumor clone. These findings highlight potentially novel contributors for the genetic predisposition to familial WM and indicate that LAPTM5(c403t) and HCLS1(g496a) may represent predisposition alleles in patients with familial WM.
Assuntos
Proteínas Sanguíneas/genética , Exoma , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Proteínas de Membrana/genética , Macroglobulinemia de Waldenstrom/genética , Proteínas Adaptadoras de Transdução de Sinal , Família , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MasculinoRESUMO
The tumor suppressors B-lymphocyte-induced maturation protein-1 (BLIMP-1) and p53 play a crucial role in B-cell lymphomas, and their inactivation contributes to the pathogenesis of a wide spectrum of lymphoid malignancies, including diffuse large B-cell lymphomas (DLBCLs). Patients with activated B-cell-like (ABC) DLBCL may present with loss of BLIMP-1, c-Myc over-expression, decreased p53, and poor prognosis. Nevertheless, there is a lack of in vivo models recapitulating the biology of high-grade ABC DLBCL. We therefore aimed to develop an in vivo model aiming to recapitulate the phenotype observed in this cohort of patients. A Cre-Lox approach was used to achieve inactivation of both p53 and BLIMP-1 in murine B-cells. Contextual ablation of BLIMP-1 and p53 led to development of IgM-positive B-cell lymphoma with an aggressive phenotype, supported by c-Myc up-regulation, and accumulation of somatic mutations, as demonstrated by whole exome sequencing. Sensitivity of B-tumor cells to BTK inhibition was demonstrated. This model mirrors what reported in patients with ABC DLBLC, and therefore represents a novel model for studying the biology of ABC-DLBCL harboring the dual loss of BLIMP-1/p53 and c-Myc over-expression.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Proteínas Repressoras/deficiência , Proteína Supressora de Tumor p53/deficiência , Animais , Linfócitos B/efeitos dos fármacos , Biomarcadores , Evolução Clonal/genética , Modelos Animais de Doenças , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes myc , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Camundongos Transgênicos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Inibidores de Proteínas Quinases/farmacologiaRESUMO
PURPOSE OF THE REVIEW: Herein we dissect mechanisms behind the dissemination of cancer cells from primary tumor site to the bone marrow, which are necessary for metastasis development, with a specific focus on multiple myeloma. RECENT FINDINGS: The ability of tumor cells to invade vessels and reach the systemic circulation is a fundamental process for metastasis development; however, the interaction between clonal cells and the surrounding microenvironment is equally important for supporting colonization, survival, and growth in the secondary sites of dissemination. The intrinsic propensity of tumor cells to recognize a favorable milieu where to establish secondary growth is the basis of the "seed and soil" theory. This theory assumes that certain tumor cells (the "seeds") have a specific affinity for the milieu of certain organs (the "soil"). Recent literature has highlighted the important contributions of the vascular niche to the hospitable "soil" within the bone marrow. In this review, we discuss the crucial role of stromal cells and endothelial cells in supporting primary growth, homing, and metastasis to the bone marrow, in the context of multiple myeloma, a plasma cell malignancy with the unique propensity to primarily grow and metastasize to the bone marrow.
Assuntos
Medula Óssea/irrigação sanguínea , Neoplasias Ósseas/secundário , Tecido Conjuntivo/irrigação sanguínea , Células Endoteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/patologia , Medula Óssea/metabolismo , Tecido Conjuntivo/metabolismo , Células Endoteliais/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Mieloma Múltiplo/metabolismo , Metástase Neoplásica , Microambiente TumoralRESUMO
Bone is a favorable microenvironment for tumor growth and a frequent destination for metastatic cancer cells. Targeting cancers within the bone marrow remains a crucial oncologic challenge due to issues of drug availability and microenvironment-induced resistance. Herein, we engineered bone-homing polymeric nanoparticles (NPs) for spatiotemporally controlled delivery of therapeutics to bone, which diminish off-target effects and increase local drug concentrations. The NPs consist of poly(D,L-lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and bisphosphonate (or alendronate, a targeting ligand). The engineered NPs were formulated by blending varying ratios of the synthesized polymers: PLGA-b-PEG and alendronate-conjugated polymer PLGA-b-PEG-Ald, which ensured long circulation and targeting capabilities, respectively. The bone-binding ability of Ald-PEG-PLGA NPs was investigated by hydroxyapatite binding assays and ex vivo imaging of adherence to bone fragments. In vivo biodistribution of fluorescently labeled NPs showed higher retention, accumulation, and bone homing of targeted Ald-PEG-PLGA NPs, compared with nontargeted PEG-PLGA NPs. A library of bortezomib-loaded NPs (bone-targeted Ald-Bort-NPs and nontargeted Bort-NPs) were developed and screened for optimal physiochemical properties, drug loading, and release profiles. Ald-Bort-NPs were tested for efficacy in mouse models of multiple myeloma (MM). Results demonstrated significantly enhanced survival and decreased tumor burden in mice pretreated with Ald-Bort-NPs versus Ald-Empty-NPs (no drug) or the free drug. We also observed that bortezomib, as a pretreatment regimen, modified the bone microenvironment and enhanced bone strength and volume. Our findings suggest that NP-based anticancer therapies with bone-targeting specificity comprise a clinically relevant method of drug delivery that can inhibit tumor progression in MM.
Assuntos
Antineoplásicos , Neoplasias Ósseas/tratamento farmacológico , Ácidos Borônicos , Sistemas de Liberação de Medicamentos , Ácido Láctico , Mieloma Múltiplo/tratamento farmacológico , Nanopartículas , Polietilenoglicóis , Ácido Poliglicólico , Pirazinas , Microambiente Tumoral/efeitos dos fármacos , Alendronato/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Ácidos Borônicos/química , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Ácido Láctico/síntese química , Ácido Láctico/química , Ácido Láctico/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Nanopartículas/química , Nanopartículas/ultraestrutura , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Polietilenoglicóis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Ácido Poliglicólico/síntese química , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Pirazinas/química , Pirazinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Clonal proliferation of plasma cells within the bone marrow (BM) affects local cells, such as mesenchymal stromal cells (MSCs), leading to osteolysis and fatality in multiple myeloma (MM). Consequently, there is an urgent need to find better mechanisms of inhibiting myeloma growth and osteolytic lesion development. To meet this need and accelerate clinical translation, better models of myeloma within the BM are required. Herein we have developed a clinically relevant, three-dimensional (3D) myeloma BM coculture model that mimics bone cell/cancer cell interactions within the bone microenvironment. The coculture model and clinical samples were used to investigate myeloma growth, osteogenesis inhibition, and myeloma-induced abnormalities in MM-MSCs. This platform demonstrated myeloma support of capillary-like assembly of endothelial cells and cell adhesion-mediated drug resistance (CAM-DR). Also, distinct normal donor (ND)- and MM-MSC miRNA (miR) signatures were identified and used to uncover osteogenic miRs of interest for osteoblast differentiation. More broadly, our 3D platform provides a simple, clinically relevant tool to model cancer growth within the bone-useful for investigating skeletal cancer biology, screening compounds, and exploring osteogenesis. Our identification and efficacy validation of novel bone anabolic miRs in MM opens more opportunities for novel approaches to cancer therapy via stromal miR modulation.
Assuntos
Células da Medula Óssea/patologia , Mieloma Múltiplo/patologia , Osteogênese/fisiologia , Cultura Primária de Células/métodos , Nicho de Células-Tronco , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Modelos Biológicos , Osteoblastos/citologia , Osteoblastos/fisiologia , Nicho de Células-Tronco/fisiologia , Alicerces TeciduaisRESUMO
The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating cell trafficking in hematopoietic stem cells and clonal B cells. We screened 418 patients with B-cell lymphoproliferative disorders and described the presence of the C1013G/CXCR4 warts, hypogammaglobulinemia, infections, and myelokathexis-associated mutation in 28.2% (37/131) of patients with lymphoplasmacytic lymphoma (Waldenström macroglobulinemia [WM]), being either absent or present in only 7% of other B-cell lymphomas. In vivo functional characterization demonstrates its activating role in WM cells, as demonstrated by significant tumor proliferation and dissemination to extramedullary organs, leading to disease progression and decreased survival. The use of a monoclonal antibody anti-CXCR4 led to significant tumor reduction in a C1013G/CXCR4 WM model, whereas drug resistance was observed in mutated WM cells exposed to Bruton's tyrosine kinase, mammalian target of rapamycin, and phosphatidylinositol 3-kinase inhibitors, but not proteasome inhibitors. These findings demonstrate that C1013G/CXCR4 is an activating mutation in WM and support its role as a critical regulator of WM molecular pathogenesis and as an important therapeutic target.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Mutação de Sentido Incorreto , Receptores CXCR4/metabolismo , Macroglobulinemia de Waldenstrom/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Intervalo Livre de Doença , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Receptores CXCR4/genética , Taxa de Sobrevida , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Macroglobulinemia de Waldenstrom/genética , Macroglobulinemia de Waldenstrom/mortalidade , Macroglobulinemia de Waldenstrom/patologiaRESUMO
Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family that has been recently linked to tumor development. However, its role in modulating multiple myeloma (MM) biology and disease progression remains unexplored. We first demonstrated that patients with MM present with higher expression of Pyk2 compared with healthy individuals. By using loss-of-function approaches, we found that Pyk2 inhibition led to reduction of MM tumor growth in vivo as well as decreased cell proliferation, cell-cycle progression, and adhesion ability in vitro. In turn, overexpression of Pyk2 promoted the malignant phenotype, substantiated by enhanced tumor growth and reduced survival. Mechanistically, inhibition of Pyk2 reduced activation of Wnt/ß-catenin signaling by destabilizing ß-catenin, leading to downregulation of c-Myc and Cyclin D1. Furthermore, treatment of MM cells with the FAK/Pyk2 inhibitor VS-4718 effectively inhibited MM cell growth both in vitro and in vivo. Collectively, our findings describe the tumor-promoting role of Pyk2 in MM, thus providing molecular evidence for a novel tyrosine kinase inhibitor as a new therapeutic option in MM.
Assuntos
Aminopiridinas/farmacologia , Quinase 2 de Adesão Focal/antagonistas & inibidores , Mieloma Múltiplo/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Progressão da Doença , Feminino , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Immunoblotting , Medições Luminescentes , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The role of altered sialylation in multiple myeloma (MM) cell trafficking has not been previously investigated. In the present study we identified high expression of ß-galactoside α-2,3-sialyltransferase, ST3GAL6, in MM cell lines and patients. This gene plays a key role in selectin ligand synthesis in humans through the generation of functional sialyl Lewis X. In MRC IX patients, high expression of this gene is associated with inferior overall survival. In this study we demonstrate that knockdown of ST3GAL6 results in a significant reduction in levels of α-2,3-linked sialic acid on the surface of MM cells with an associated significant reduction in adhesion to MM bone marrow stromal cells and fibronectin along with reduced transendothelial migration in vitro. In support of our in vitro findings, we demonstrate significantly reduced homing and engraftment of ST3GAL6 knockdown MM cells to the bone marrow niche in vivo, along with decreased tumor burden and prolonged survival. This study points to the importance of altered glycosylation, particularly sialylation, in MM cell adhesion and migration.
Assuntos
Mieloma Múltiplo/enzimologia , Proteínas de Neoplasias/metabolismo , Sialiltransferases/metabolismo , Migração Transendotelial e Transepitelial , Animais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Ácido N-Acetilneuramínico/biossíntese , Ácido N-Acetilneuramínico/genética , Proteínas de Neoplasias/genética , Sialiltransferases/genética , Células Estromais/enzimologia , Células Estromais/patologia , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Multiple myeloma is characterized by clonal proliferation of plasma cells within the bone marrow resulting in anemia, lytic bone lesions, hypercalcemia, and renal impairment. Despite advanced in our understanding of this complex disease in recent years, it is still considered an incurable malignancy. This is, in part, due to the highly heterogenous genomic and phenotypic nature of the disease, which is to date incompletely understood. It is clear that a deeper level of knowledge of the biological events underlying the development of these diseases is needed to identify new targets and generate effective novel therapies. MicroRNAs (miRNAs), which are single strand, 20-nucleotide, noncoding RNA's, are key regulators of gene expression and have been reported to exert transcriptional control in multiple myeloma. miRNAs are now recognized to play a role in many key areas such as cellular proliferation, differentiation, apoptosis and stress response. Substantial advances have been made in recent years in terms of our understanding of the biological role of miRNAs in a diverse range of hematological and solid malignancues, In multiple myeloma these advances have yielded new information of prognostic and diagnostic relevance which have helped to shed light on epigenetic regulation in this disease.
Assuntos
Epigênese Genética , Mieloma Múltiplo/genética , HumanosRESUMO
Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells, making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis, Nampt, in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells, but not normal donor as well as MM patients PBMCs. Importantly, FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6, IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly, cytotoxicity of FK866 triggered autophagy, but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally, FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model, associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology, providing the basis for a novel targeted therapeutic approach.