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
Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor-associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8-LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Animais , Autofagia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mitofagia , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/fisiologiaRESUMO
FLT3-mutant acute myeloid leukemia (AML) is an aggressive form of leukemia with poor prognosis. Treatment with FLT3 inhibitors frequently produces a clinical response, but the disease nevertheless often recurs. Recent studies have revealed system-wide gene expression changes in FLT3-mutant AML cell lines in response to drug treatment. Here we sought a systems-level understanding of how these cells mediate these drug-induced changes. Using RNAseq data from AML cells with an internal tandem duplication FLT3 mutation (FLT3-ITD) under six drug treatment conditions including quizartinib and dexamethasone, we identified seven distinct gene programs representing diverse biological processes involved in AML drug-induced changes. Based on the literature knowledge about genes from these modules, along with public gene regulatory network databases, we constructed a network of FLT3-ITD AML. Applying the BooleaBayes algorithm to this network and the RNAseq data, we created a probabilistic, data-driven dynamical model of acquired resistance to these drugs. Analysis of this model reveals several interventions that may disrupt targeted parts of the system-wide drug response. We anticipate co-targeting these points may result in synergistic treatments that can overcome resistance and prevent eventual recurrence.
RESUMO
Acute myeloid leukemia (AML) is a heterogeneous disease caused by several gene mutations and cytogenetic abnormalities affecting differentiation and proliferation of myeloid lineage cells. FLT3 is a receptor tyrosine kinase commonly overexpressed or mutated, and its mutations are associated with poor prognosis in AML. Although aggressive chemotherapy often followed by hematopoietic stem cell transplant is the current standard of care, the recent approval of FLT3-targeted drugs is revolutionizing AML treatment that had remained unchanged since the 1970s. However, despite the dramatic clinical response to targeted agents, such as FLT3 inhibitors, remission is almost invariably short-lived and ensued by relapse and drug resistance. Hence, there is an urgent need to understand the molecular mechanisms driving drug resistance in order to prevent relapse. In this review, we discuss FLT3 as a target and highlight current understanding of FLT3 inhibitor resistance.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Terapia de Alvo Molecular , Mutação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/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
Autophagosomal membranes are emerging as platforms for various cell survival and death signaling networks beyond autophagy. While autophagy-dependent cell death has been reported in response to a variety of stimuli, the underlying molecular mechanisms remain far from clear. Here, we demonstrate that inhibition of autophagosome completion by Atg2A/B deletion accumulates immature autophagosomal membranes that promote non-canonical caspase-8 activation in response to nutrient starvation via an intracellular death-inducing signaling complex (iDISC). Importantly, iDISC-induced caspase-8 dimerization and activation occurs on accumulated autophagosomal membranes and requires the LC3 conjugation machinery but is independent from the extrinsic pathway of apoptosis. Moreover, we have identified NF-κB signaling and c-FLIP as negative regulators of iDISC-mediated caspase-8 activation and apoptosis. Collectively, these findings reveal autophagosomal membrane completion as a novel target to switch cytoprotective autophagy to apoptosis.