RESUMO
FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.
Assuntos
Antineoplásicos/uso terapêutico , Glucocorticoides/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2/biossíntese , Proteína 11 Semelhante a Bcl-2/genética , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Simulação por Computador , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Inflamação/genética , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Seleção Genética , Transcriptoma , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
BACKGROUND: Anti-GD2 monoclonal antibody immunotherapy has significantly improved the overall survival rate for high-risk neuroblastoma patients. However, 40% of patients fail to respond or develop resistance to treatment, and the molecular mechanisms by which this occurs remain poorly understood. Tumor-derived small extracellular vesicles (sEVs) have emerged as critical regulators in modulating the response to immunotherapy. In this study, we investigated the role of neuroblastoma-derived sEVs in promoting resistance to the anti-GD2 monoclonal antibody dinutuximab. Moreover, to determine whether pharmacologic inhibition of sEV secretion sensitizes tumors to dinutuximab treatment, we combined dinutuximab with tipifarnib, a farnesyltransferase inhibitor that inhibits sEV secretion. METHODS: We investigated the role of neuroblastoma-derived sEVs in modulating the response to dinutuximab by utilizing the syngeneic 9464D-GD2 mouse model. The effect of neuroblastoma-derived sEVs in modulating the tumor microenvironment (TME) and host immune system were evaluated by RNA-sequencing and flow cytometry. Importantly, we used this mouse model to investigate the efficacy of tipifarnib in sensitizing neuroblastoma tumors to dinutuximab. The effect of tipifarnib on both the TME and host immune system were assessed by flow cytometry. RESULTS: We demonstrated that neuroblastoma-derived sEVs significantly attenuated the efficacy of dinutuximab in vivo and modulated tumor immune cell infiltration upon dinutuximab treatment to create an immunosuppressive TME that contains more tumor-associated macrophages and fewer tumor-infiltrating NK cells. In addition, we demonstrated that neuroblastoma-derived sEVs suppress splenic NK cell maturation in vivo and dinutuximab-induced NK cell-mediated antibody-dependent cellular cytotoxicity in vitro. Importantly, tipifarnib drastically enhanced the efficacy of dinutuximab-mediated inhibition of tumor growth and prevented the immunosuppressive effects of neuroblastoma-derived sEVs in vivo. CONCLUSIONS: These preclinical findings uncover a novel mechanism by which neuroblastoma-derived sEVs modulate the immune system to promote resistance to dinutuximab and suggest that tipifarnib-mediated inhibition of sEV secretion may serve as a viable treatment strategy to enhance the antitumor efficacy of anti-GD2 immunotherapy in high-risk neuroblastoma patients.
Assuntos
Antineoplásicos , Vesículas Extracelulares , Neuroblastoma , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Fatores Imunológicos/uso terapêutico , Imunoterapia , Camundongos , Neuroblastoma/patologia , Quinolonas , Microambiente TumoralRESUMO
Autophagosomal membranes can serve as activation platforms for intracellular death-inducing signaling complexes (iDISCs) to initiate Caspase-8-dependent apoptosis. In this study, we explore the impact of ESCRT-III-dependent phagophore closure on iDISC assemblies and cell death in osteosarcoma and neuroblastoma cells. Inhibition of phagophore closure by conditional depletion of CHMP2A, an ESCRT-III component, stabilizes iDISCs on immature autophagosomal membranes and induces Caspase-8-dependent cell death. Importantly, suppression of the iDISC formation via deletion of ATG7, an E1 enzyme for ubiquitin-like autophagy-related proteins, blocks Caspase-8 activation and cell death following CHMP2A depletion. Although DR5 expression and TRAIL-induced apoptosis are enhanced in CHMP2A-depleted cells, the canonical extrinsic pathway of apoptosis is not responsible for the initiation of cell death by CHMP2A depletion. Furthermore, the loss of CHMP2A impairs neuroblastoma tumor growth associated with decreased autophagy and increased apoptosis in vivo. Together, these findings indicate that inhibition of the ESCRT-III-dependent autophagosome sealing process triggers noncanonical Caspase-8 activation and apoptosis, which may open new avenues for therapeutic targeting of autophagy in cancer.
Assuntos
Autofagia , Caspase 8/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Osteossarcoma/metabolismo , Transdução de Sinais , Animais , Apoptose , Autofagossomos/metabolismo , Linhagem Celular Tumoral , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Feminino , Humanos , Masculino , Camundongos , Neuroblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although neoadjuvant chemotherapy is a standard component of breast cancer treatment, recent evidence suggests that chemotherapeutic drugs can promote metastasis through poorly defined mechanisms. Here we utilize xenograft mouse models of triple-negative breast cancer to explore the importance of chemotherapy-induced tumor-derived small extracellular vesicles (sEV) in metastasis. Doxorubicin (DXR) enhanced tumor cell sEV secretion to accelerate pulmonary metastasis by priming the premetastatic niche. Proteomic analysis and CRISPR/Cas9 gene editing identified the inflammatory glycoprotein PTX3 enriched in DXR-elicited sEV as a critical regulator of chemotherapy-induced metastasis. Both genetic inhibition of sEV secretion from primary tumors and pharmacologic inhibition of sEV uptake in secondary organs suppressed metastasis following chemotherapy. Taken together, this research uncovers a mechanism of chemotherapy-mediated metastasis by which drug-induced upregulation of sEV secretion and PTX3 protein cargo primes the premetastatic niche and suggests that inhibition of either sEV uptake in secondary organs or secretion from primary tumor cells may be promising therapeutic strategies to suppress metastasis. SIGNIFICANCE: These findings show that chemotherapy-induced small extracellular vesicles accelerate breast cancer metastasis, and targeted inhibition of tumor-derived vesicles may be a promising therapeutic strategy to improve the efficacy of chemotherapy treatment.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteína C-Reativa/metabolismo , Doxorrubicina/efeitos adversos , Vesículas Extracelulares/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Componente Amiloide P Sérico/metabolismo , Animais , Antibióticos Antineoplásicos/efeitos adversos , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Proteína C-Reativa/genética , Movimento Celular , Proliferação de Células , Vesículas Extracelulares/efeitos dos fármacos , Feminino , Humanos , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Componente Amiloide P Sérico/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transient receptor potential melastatin 2 (TRPM2) ion channel has an essential function in maintaining cell survival following oxidant injury. Here, we show that TRPM2 is highly expressed in acute myeloid leukemia (AML). The role of TRPM2 in AML was studied following depletion with CRISPR/Cas9 technology in U937 cells. In in vitro experiments and in xenografts, depletion of TRPM2 in AML inhibited leukemia proliferation, and doxorubicin sensitivity was increased. Mitochondrial function including oxygen consumption rate and ATP production was reduced, impairing cellular bioenergetics. Mitochondrial membrane potential and mitochondrial calcium uptake were significantly decreased in depleted cells. Mitochondrial reactive oxygen species (ROS) were significantly increased, and Nrf2 was decreased, reducing the antioxidant response. In TRPM2-depleted cells, ULK1, Atg7, and Atg5 protein levels were decreased, leading to autophagy inhibition. Consistently, ATF4 and CREB, two master transcription factors for autophagosome biogenesis, were reduced in TRPM2-depleted cells. In addition, Atg13 and FIP200, which are known to stabilize ULK1 protein, were decreased. Reconstitution with TRPM2 fully restored proliferation, viability, and autophagy; ATF4 and CREB fully restored proliferation and viability but only partially restored autophagy. TRPM2 expression reduced the elevated ROS found in depleted cells. These data show that TRPM2 has an important role in AML proliferation and survival through regulation of key transcription factors and target genes involved in mitochondrial function, bioenergetics, the antioxidant response, and autophagy. Targeting TRPM2 may represent a novel therapeutic approach to inhibit myeloid leukemia growth and enhance susceptibility to chemotherapeutic agents through multiple pathways.
Assuntos
Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Canais de Cátion TRPM/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Neuroblastoma is the most common extracranial solid malignancy in the pediatric population, accounting for over 9% of all cancer-related deaths in children. Autophagy is a cell self-protective mechanism that promotes tumor cell growth and survival, making it an attractive target for treating cancer. However, the role of autophagy in neuroblastoma tumor growth and metastasis is largely undefined. Here we demonstrate that targeted inhibition of an essential autophagy kinase, unc-51 like autophagy kinase 1 (ULK1), with a recently developed small-molecule inhibitor of ULK1, SBI-0206965, significantly reduces cell growth and promotes apoptosis in SK-N-AS, SH-SY5Y, and SK-N-DZ neuroblastoma cell lines. Furthermore, inhibition of ULK1 by a dominant-negative mutant of ULK1 (dnULK1K46N) significantly reduces growth and metastatic disease and prolongs survival of mice bearing SK-N-AS xenograft tumors. We also show that SBI-0206965 sensitizes SK-N-AS cells to TRAIL treatment, but not to mTOR inhibitors (INK128, Torin1) or topoisomerase inhibitors (doxorubicin, topotecan). Collectively, these findings demonstrate that ULK1 is a viable drug target and suggest that inhibitors of ULK1 may provide a novel therapeutic option for the treatment of neuroblastoma. Mol Cancer Ther; 17(11); 2365-76. ©2018 AACR.
Assuntos
Apoptose , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neuroblastoma/enzimologia , Neuroblastoma/patologia , Animais , Apoptose/efeitos dos fármacos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Benzamidas/química , Benzamidas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Pirimidinas/química , Pirimidinas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Inibidores da Topoisomerase/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. Here, we report the HaloTag-LC3 autophagosome completion assay that specifically detects phagophores, nascent autophagosomes, and mature autophagic structures. Using this assay, we identify the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A as a critical regulator of phagophore closure. During autophagy, CHMP2A translocates to the phagophore and regulates the separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes. Consistently, inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion. The ESCRT-mediated membrane abscission appears to be a critical step in forming functional autolysosomes by preventing mislocalization of lysosome-associated membrane glycoprotein 1 to the inner autophagosomal membrane. Collectively, our work reveals a function for the ESCRT machinery in the final step of autophagosome formation and provides a useful tool for quantitative analysis of autophagosome biogenesis and maturation.
Assuntos
Autofagia , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica , Lisossomos/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , RNA Interferente Pequeno/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Acute myeloid leukemia (AML) is a hierarchical hematopoietic malignancy originating from leukemic stem cells (LSCs). Autophagy is a lysosomal degradation pathway that is hypothesized to be important for the maintenance of AML as well as contribute to chemotherapy response. Here we employ a mouse model of AML expressing the fusion oncogene MLL-AF9 and explore the effects of Atg5 deletion, a key autophagy protein, on the malignant transformation and progression of AML. Consistent with a transient decrease in colony-forming potential in vitro, the in vivo deletion of Atg5 in MLL-AF9-transduced bone marrow cells during primary transplantation prolonged the survival of recipient mice, suggesting that autophagy has a role in MLL-AF9-driven leukemia initiation. In contrast, deletion of Atg5 in malignant AML cells during secondary transplantation did not influence the survival or chemotherapeutic response of leukemic mice. Interestingly, autophagy was found to be involved in the survival of differentiated myeloid cells originating from MLL-AF9-driven LSCs. Taken together, our data suggest that Atg5-dependent autophagy may contribute to the development but not chemotherapy sensitivity of murine AML induced by MLL-AF9.