RESUMO
Venetoclax is a highly potent, selective BCL2 inhibitor capable of inducing apoptosis in cells dependent on BCL2 for survival. Most myeloma is MCL1-dependent; however, a subset of myeloma enriched for translocation t(11;14) is codependent on BCL2 and thus sensitive to venetoclax. The biology underlying this heterogeneity remains poorly understood. We show that knockdown of cyclin D1 does not induce resistance to venetoclax, arguing against a direct role for cyclin D1 in venetoclax sensitivity. To identify other factors contributing to venetoclax response, we studied a panel of 31 myeloma cell lines and 25 patient samples tested for venetoclax sensitivity. In cell lines, we corroborated our previous observation that BIM binding to BCL2 correlates with venetoclax response and further showed that knockout of BIM results in decreased venetoclax sensitivity. RNA-sequencing analysis identified expression of B-cell genes as enriched in venetoclax-sensitive myeloma, although no single gene consistently delineated sensitive and resistant cells. However, a panel of cell surface makers correlated well with ex vivo prediction of venetoclax response in 21 patient samples and may serve as a biomarker independent of t(11;14). Assay for transposase-accessible chromatin sequencing of myeloma cell lines also identified an epigenetic program in venetoclax-sensitive cells that was more similar to B cells than that of venetoclax-resistant cells, as well as enrichment for basic leucine zipper domain-binding motifs such as BATF. Together, these data indicate that remnants of B-cell biology are associated with BCL2 dependency and point to novel biomarkers of venetoclax-sensitive myeloma independent of t(11;14).
Assuntos
Linfócitos B/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Mieloma Múltiplo , Sulfonamidas/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem Celular Tumoral , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/metabolismo , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Translocação Genética/efeitos dos fármacosRESUMO
Multiple myeloma is highly dependent on the bone marrow microenvironment until progressing to very advanced extramedullary stages of the disease such as plasma cell leukemia. Stromal cells in the bone marrow secrete a variety of cytokines that promote plasma cell survival by regulating antiapoptotic members of the Bcl-2 family including Mcl-1, Bcl-xL, and Bcl-2. Although the antiapoptotic protein on which a cell depends is typically consistent among normal cells of a particular phenotype, Bcl-2 family dependence is highly heterogeneous in multiple myeloma. Although normal plasma cells and most multiple myeloma cells require Mcl-1 for survival, a subset of myeloma is codependent on Bcl-2 and/or Bcl-xL We investigated the role of the bone marrow microenvironment in determining Bcl-2 family dependence in multiple myeloma. We used the Bcl-2/Bcl-xL inhibitor ABT-737 to study the factors regulating whether myeloma is Mcl-1 dependent, and thus resistant to ABT-737-induced apoptosis, or Bcl-2/Bcl-xL codependent, and thus sensitive to ABT-737. We demonstrate that bone marrow stroma is capable of inducing Mcl-1 dependence through the production of the plasma cell survival cytokine interleukin-6 (IL-6). IL-6 upregulates Mcl-1 transcription in a STAT3-dependent manner, although this occurred in a minority of the cells tested. In all cells, IL-6 treatment results in posttranslational modification of the proapoptotic protein Bim. Phosphorylation of Bim shifts its binding from Bcl-2 and Bcl-xL to Mcl-1, an effect reversed by MEK inhibition. Blocking IL-6 or downstream signaling restored Bcl-2/Bcl-xL dependence and may therefore represent a clinically useful strategy to enhance the activity of Bcl-2 inhibitors.
Assuntos
Medula Óssea/metabolismo , Mieloma Múltiplo/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Transdução de Sinais , Microambiente Tumoral , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Compostos de Bifenilo/farmacologia , Medula Óssea/patologia , Linhagem Celular Tumoral , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Sulfonamidas/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Speciation of arsenic is usually carried out using chromatography-based methods coupled with spectroscopic determination; however, the inevitable procedures involving sample preparation and separation could potentially alter the integrity of the arsenic metabolites present in biological samples. Surface-enhanced Raman spectroscopy (SERS) could be a promising alternative for providing a reliable arsenic analysis under the influence of a cellular matrix. A method for arsenic speciation using SERS in cellular matrix was developed in this study and four arsenicals were selected, including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV). Silver nanoparticles in the form of colliodal suspension with different surface charges, i.e., coated with citrate (AgNPs-Citrate) and spermine (AgNPs-Spermine) were employed as SERS substrates. Adsorption of arsenicals on nanoparticles in colloidal suspensions and the cellular matrix and the pH, size, and zeta potential of the colloidal suspensions were investigated for a better understanding of the SERS signal response of arsenicals in the colloidal suspensions or under the influence of cellular matrix. Arsenicals showed substantially different SERS responses in the two colloidal suspensions, mainly because of the distinct difference in the interaction between the arsenicals and the nanoparticles. Arsenic speciation in cell lysate could be successfully carried out in AgNPs-Spermine suspension, while AgNPs-Citrate could not yield significant SERS signals under the experimental conditions. This study proved that AgNPs-Spermine colloidal suspension could be a promising SERS substrate for studying arsenic metabolism in a biological matrix, reducing the bias caused by traditional techniques that involve sample extraction and pretreatment.
Assuntos
Arsênio/química , Análise Espectral Raman/métodos , Arsênio/análise , Coloides , Espectrometria de Massas/métodos , Nanopartículas Metálicas/químicaRESUMO
Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Transportador de Glucose Tipo 4/antagonistas & inibidores , Transportador de Glucose Tipo 4/metabolismo , Animais , Simulação por Computador , Bases de Dados de Produtos Farmacêuticos , Glucose/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/química , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/química , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Conformação Proteica , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The function and survival of normal and malignant plasma cells depends on the elaborately regulated ubiquitin proteasome system. Proteasome inhibitors such as bortezomib have proved to be highly effective in the treatment of multiple myeloma (MM), and their effects are related to normal protein homeostasis which is critical for plasma cell survival. Many ubiquitin ligases are regulated by conjugation with NEDD8. Therefore, neddylation may also impact survival and proliferation of malignant plasma cells. Here, we show that MLN4924, a potent NEDD8 activating enzyme (NAE) inhibitor, induced cytotoxicity in MM cell lines, and its antitumor effect is associated with suppression of the AKT and mammalian target of rapamycin (mTOR) signaling pathways through increased expression of REDD1. Combining MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MM cell lines which can overcome the prosurvival effects of growth factors such as interleukin-6 and insulin-like growth factor-1. Altogether, our findings demonstrate an important function for REDD1 in MLN4924-induced cytotoxicity in MM and also provide a promising therapeutic combination strategy for myeloma.
Assuntos
Antineoplásicos/farmacologia , Ciclopentanos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Regulação para Cima/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Enzimas Ativadoras de Ubiquitina/metabolismoAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Terapia de Alvo Molecular , Mieloma Múltiplo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Terapia de Salvação , Sulfonamidas/uso terapêutico , Adulto , Idoso , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/efeitos adversos , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 14/genética , Cromossomos Humanos Par 14/ultraestrutura , Ensaios Clínicos Fase III como Assunto/estatística & dados numéricos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Seleção de Pacientes , Prognóstico , Intervalo Livre de Progressão , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversos , Translocação GenéticaRESUMO
Here, we report the identification of dimethylarsinothioyl glutathione (DMMTA(V)(GS)) as a metabolite in cellular extracts of dimethyarsinous glutathione (Darinaparsin, DMA(III)(GS)) treated human multiple myeloma (MM) cell lines. Co-elution of sulfur and arsenic on the inductively coupled plasma mass spectrometer (ICP-MS) indicated the presence of sulfur along with arsenic in the newly observed unidentified molecule on the speciation chromatograms of cell lines treated with DMA(III)(GS). Liquid chromatography-electrospray ionization-mass spectrometry of the unknown peak in the MS and tandem MS modes revealed molecular ion peaks at m/z = 443.9 and 466.0, corresponding to [DMMTA(V)(GS) + H](+) and [DMMTA(V)(GS) + Na](+), as well as peaks at 314.8 for the loss of glutamic acid and 231.1 for the loss of glycine. In addition, peaks were observed at 176.9 corresponding to cysteine and glycine adducts and at 137.1 for the [C2H6AsS](+) ion. An increase in the peak area of the unidentified peak was observed upon spiking the cell extracts with a standard of DMMTA(V)(GS). Heat deactivation of MM cells prevented the formation of DMMTA(V)(GS) raising the possibility of its formation via an enzymatic reaction. Formation studies in DMA(III)(GS) treated MM cells revealed the dependence of DMMTA(V)(GS) formation on the depletion of DMA(III)(GS). The presence of 5 mM glutathione prevented its formation, indicating that DMA(III), a dissociation product of DMA(III)(GS), is likely a precursor for the formation of DMMTA(V)(GS). DMMTA(V)(GS) was observed to form under acidic and neutral pH conditions (pH 3.0-7.4). In addition, DMMTA(V)(GS) was found to be stable in cell extracts at both acidic and neutral pH conditions. When assessing the toxicity by exposing multiple myeloma cells to arsenicals externally, DMMTA(V)(GS) was found to be much less toxic than DMA(III)(GS) and DMMTA(V), potentially due to its limited uptake in the cells (10 and 16% of the uptakes of DMA(III)(GS) and DMMTA(V), respectively).
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Arsenicais/metabolismo , Arsenicais/uso terapêutico , Glutationa/análogos & derivados , Mieloma Múltiplo/tratamento farmacológico , Antineoplásicos/análise , Apoptose/efeitos dos fármacos , Arsenicais/análise , Linhagem Celular Tumoral , Glutationa/análise , Glutationa/metabolismo , Glutationa/uso terapêutico , Humanos , Mieloma Múltiplo/metabolismoRESUMO
Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-x(L)/Bcl-2 dependence. Surprisingly, analysis of Mcl-1-dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-x(L) in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Membrana/fisiologia , Mieloma Múltiplo/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteína bcl-X/fisiologia , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Compostos de Bifenilo/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/farmacologia , Sulfonamidas/farmacologia , Distribuição Tecidual , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
The BCL2 inhibitor venetoclax promotes apoptosis in blood cancer cells and is approved for treatment of chronic lymphocytic leukemia and acute myeloid leukemia. However, multiple myeloma cells are frequently more dependent on MCL-1 for survival, conferring resistance to venetoclax. Here we report that mevalonate pathway inhibition with statins can overcome resistance to venetoclax in multiple myeloma cell lines and primary cells. In addition, statins sensitize to apoptosis induced by MCL-1 inhibitor, S63845. In retrospective analysis of venetoclax clinical studies in multiple myeloma, background statin use was associated with a significantly enhanced rate of stringent complete response and absence of progressive disease. Statins sensitize multiple myeloma cells to venetoclax by upregulating two proapoptotic proteins: PUMA via a p53-independent mechanism and NOXA via the integrated stress response. These findings provide rationale for prospective testing of statins with venetoclax regimens in multiple myeloma. SIGNIFICANCE: BH3 mimetics including venetoclax hold promise for treatment of multiple myeloma but rational combinations are needed to broaden efficacy. This study presents mechanistic and clinical data to support addition of pitavastatin to venetoclax regimens in myeloma. The results open a new avenue for repurposing statins in blood cancer.
Assuntos
Antineoplásicos , Neoplasias Hematológicas , Inibidores de Hidroximetilglutaril-CoA Redutases , Mieloma Múltiplo , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Mieloma Múltiplo/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Estudos Retrospectivos , Estudos Prospectivos , Antineoplásicos/farmacologia , Neoplasias Hematológicas/tratamento farmacológicoRESUMO
The connections between metabolic state and therapy resistance in multiple myeloma (MM) are poorly understood. We previously reported that electron transport chain (ETC) suppression promotes sensitivity to the BCL-2 antagonist venetoclax. Here, we show that ETC suppression promotes resistance to proteasome inhibitors (PIs). Interrogation of ETC-suppressed MM reveals integrated stress response-dependent suppression of protein translation and ubiquitination, leading to PI resistance. ETC and protein translation gene expression signatures from the CoMMpass trial are down-regulated in patients with poor outcome and relapse, corroborating our in vitro findings. ETC-suppressed MM exhibits up-regulation of the cystine-glutamate antiporter SLC7A11, and analysis of patient single-cell RNA-seq shows that clusters with low ETC gene expression correlate with higher SLC7A11 expression. Furthermore, erastin or venetoclax treatment diminishes mitochondrial stress-induced PI resistance. In sum, our work demonstrates that mitochondrial stress promotes PI resistance and underscores the need for implementing combinatorial regimens in MM cognizant of mitochondrial metabolic state.
Assuntos
Mieloma Múltiplo , Inibidores de Proteassoma , Antiporters , Compostos Bicíclicos Heterocíclicos com Pontes , Linhagem Celular Tumoral , Cistina/metabolismo , Cistina/uso terapêutico , Glutamatos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , SulfonamidasRESUMO
BACKGROUND: The CCL2 chemokine is involved in promoting cancer angiogenesis, proliferation and metastasis by malignancies that express CCR2 receptor. Thus the CCL2/CCR2 axis is an attractive molecular target for anticancer drug development. METHODS: We have generated a novel fusion protein using GMCSF and an N-terminal truncated version of MCP1/CCL2 (6-76) [hereafter GMME1] and investigated its utility as a CCR2-specific tumoricidal agent. RESULTS: We found that distinct to full length CCL2 or its N-truncated derivative (CCL2 5-76), GMME1 bound to CCR2 on mouse lymphoma EG7, human multiple myeloma cell line U266, or murine and human medulloblastoma cell lines, and led to their death by apoptosis. We demonstrated that GMME1 specifically blocked CCR2-associated STAT3 phosphorylation and up-regulated pro-apoptotic BAX. Furthermore, GMME1 significantly inhibited EG7 tumor growth in C57BL/6 mice, and induced apoptosis of primary myeloma cells from patients. CONCLUSION: Our data demonstrate that GMME1 is a fusokine with a potent, CCR2 receptor-mediated pro-apoptotic effect on tumor cells and could be exploited as a novel biological therapy for CCR2+ malignancies including lymphoid and central nervous system malignancies.
Assuntos
Antineoplásicos/farmacologia , Quimiocina CCL2/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Receptores CCR2/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antígenos CD/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Linfoma , Meduloblastoma , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Multiple myeloma is a malignancy of plasma cells. Extensive genetic and transcriptional characterization of myeloma has identified subtypes with prognostic and therapeutic implications. In contrast, relatively little is known about the myeloma epigenome. EXPERIMENTAL DESIGN: CD138+CD38+ myeloma cells were isolated from fresh bone marrow aspirate or the same aspirate after freezing for 1-6 months. Gene expression and chromatin accessibility were compared between fresh and frozen samples by RNA sequencing (RNA-seq) and assay for transpose accessible chromatin sequencing (ATAC-seq). Chromatin accessible regions were used to identify regulatory RNA expression in more than 700 samples from newly diagnosed patients in the Multiple Myeloma Research Foundation CoMMpass trial (NCT01454297). RESULTS: Gene expression and chromatin accessibility of cryopreserved myeloma recapitulated that of freshly isolated samples. ATAC-seq performed on a series of biobanked specimens identified thousands of chromatin accessible regions with hundreds being highly coordinated with gene expression. More than 4,700 of these chromatin accessible regions were transcribed in newly diagnosed myelomas from the CoMMpass trial. Regulatory element activity alone recapitulated myeloma gene expression subtypes, and in particular myeloma subtypes with immunoglobulin heavy chain translocations were defined by transcription of distal regulatory elements. Moreover, enhancer activity predicted oncogene expression implicating gene regulatory mechanisms in aggressive myeloma. CONCLUSIONS: These data demonstrate the feasibility of using biobanked specimens for retrospective studies of the myeloma epigenome and illustrate the unique enhancer landscapes of myeloma subtypes that are coupled to gene expression and disease progression.
Assuntos
Cromatina/genética , Regulação Neoplásica da Expressão Gênica/genética , Expressão Gênica , Mieloma Múltiplo/genética , RNA/genética , Progressão da Doença , Epigenoma , Estudos de Viabilidade , Humanos , Prognóstico , Análise de Sequência de RNARESUMO
The BCL-2 antagonist venetoclax is highly effective in multiple myeloma (MM) patients exhibiting the 11;14 translocation, the mechanistic basis of which is unknown. In evaluating cellular energetics and metabolism of t(11;14) and non-t(11;14) MM, we determine that venetoclax-sensitive myeloma has reduced mitochondrial respiration. Consistent with this, low electron transport chain (ETC) Complex I and Complex II activities correlate with venetoclax sensitivity. Inhibition of Complex I, using IACS-010759, an orally bioavailable Complex I inhibitor in clinical trials, as well as succinate ubiquinone reductase (SQR) activity of Complex II, using thenoyltrifluoroacetone (TTFA) or introduction of SDHC R72C mutant, independently sensitize resistant MM to venetoclax. We demonstrate that ETC inhibition increases BCL-2 dependence and the 'primed' state via the ATF4-BIM/NOXA axis. Further, SQR activity correlates with venetoclax sensitivity in patient samples irrespective of t(11;14) status. Use of SQR activity in a functional-biomarker informed manner may better select for MM patients responsive to venetoclax therapy.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Complexo II de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Sulfonamidas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Linhagem Celular Tumoral , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 14/genética , Resistencia a Medicamentos Antineoplásicos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Mutação , Oxirredução/efeitos dos fármacos , Seleção de Pacientes , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/uso terapêutico , Tenoiltrifluoracetona/farmacologia , Translocação GenéticaRESUMO
Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).
Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Animais , Bortezomib/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ratos , Ratos Nus , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer cells consume more glucose to fuel metabolic programs fundamental to sustaining their survival, growth and proliferation. Among the fourteen SLC2A family members, GLUTs 1 and 4 are high-affinity glucose transporters. GLUT4 (SLC2A4) is highly expressed in muscle and adipose tissue. Basally retained within the cell, GLUT4 traffics to the plasma membrane (PM) in response to insulin and exercise-stimulation. The plasma cell malignancy multiple myeloma (MM) exhibits increased constitutive expression of GLUT4 on the PM, co-opting use of GLUT4 for survival and proliferation. GLUT4 inhibition by knockdown or treatment with the FDA-approved HIV protease inhibitor ritonavir leads to cytostatic and/or cytotoxic and chemosensitizing effects in tumor cells both in vitro and in vivo. We recently reported our generation of GLUT4 homology models and virtual high-throughput screening (vHTS) to identify multiple series of novel GLUT4 antagonists. In this report, we describe our initial hit-to-lead optimization to synthesize new analogs with improved potency and selectivity for GLUT4, and the biological characterization of these compounds in a variety of assays. We show that our lead compound (compound 20) decreases glucose uptake and cell proliferation as well as inhibits the expression of pro-survival MCL-1 in MM similar to the effect observed via knockdown of GLUT4 expression. Compound 20 is also effective at chemosensitizing multiple myeloma cell lines and patient samples to venetoclax, dexamethasone and melphalan. In sum, we report development of selective GLUT4 inhibitors lacking inhibitory activity against GLUT1 and GLUT8. We show that selective pharmacological inhibition of GLUT4 is feasible and this may represent a novel strategy for the treatment and chemosensitization of multiple myeloma to standard therapeutics.
Assuntos
Antineoplásicos/farmacologia , Transportador de Glucose Tipo 4/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Transportador de Glucose Tipo 4/metabolismo , Células HEK293 , Humanos , Camundongos , Estrutura Molecular , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Relação Estrutura-Atividade , Células Tumorais CultivadasAssuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Sulfonamidas , Protocolos de Quimioterapia Combinada Antineoplásica , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Humanos , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas c-bcl-2 , Sulfonamidas/uso terapêuticoRESUMO
Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl-1 is amplified in many human cancers, and knockdown of Mcl-1 using RNAi can lead to apoptosis. Thus, Mcl-1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl-1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl-1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl-1 inhibitors. PDB ID CODES: Comp. 2: 5IEZ; Comp. 5: 5IF4.
Assuntos
Antineoplásicos/farmacologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Animais , Antineoplásicos/química , Proteína 11 Semelhante a Bcl-2/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Descoberta de Drogas , Humanos , Imunoprecipitação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína bcl-X/metabolismoRESUMO
During the metabolism of different arsenic-containing compounds in human, a variety of metabolites are produced with significantly varying toxicities. Currently available analytical methods can only detect a limited number of human metabolites in biological samples during one run due to their diverse characteristics. In addition, co-elution of species is often unnoticeable with most detection techniques leading to inaccurate metabolic profiles and assessment of toxicity. A high performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS) method was developed that can identify thirteen common arsenic metabolites possibly present in human with special attention dedicated to thiolated or thiol conjugated arsenicals. The thirteen species included in this study are arsenite (As(III)), arsino-glutathione (As(GS)3), arsenate (As(V)), monomethylarsonous acid (MMA(III)), monomethylarsino-glutathione (MMA(III)(GS) 2), monomethylarsonic acid (MMA(V)), dimethylarsinous acid (DMA(III) (from DMA(III)I)), S-(dimethylarsinic)cysteine (DMA(III) (Cys)), dimethylarsino-glutathione (DMA(III)(GS)), dimethylarsinic acid (DMA(V)), dimethylmonothioarsinic acid (DMMTA(V)), dimethyldithioarsinic acid (DMDTA(V)), dimethylarsinothioyl glutathione (DMMTA(V)(GS)). The developed method was applied for the analysis of cancer cells that were incubated with darinaparsin (DMA(III)(GS)), a novel chemotherapeutic agent for refractory malignancies, and the arsenic metabolic profile obtained was compared to results using a previously developed method. This method provides a useful analytical tool which is much needed in unequivocally identifying the arsenicals formed during the metabolism of environmental arsenic exposure or therapeutic arsenic administration.