RESUMO
The von Hippel-Lindau tumor suppressor pVHL is an E3 ligase that targets hypoxia-inducible factors (HIFs). Mutation of VHL results in HIF up-regulation and contributes to processes related to tumor progression such as invasion, metastasis, and angiogenesis. However, very little is known with regard to post-transcriptional regulation of pVHL. Here we show that WD repeat and SOCS box-containing protein 1 (WSB1) is a negative regulator of pVHL through WSB1's E3 ligase activity. Mechanistically, WSB1 promotes pVHL ubiquitination and proteasomal degradation, thereby stabilizing HIF under both normoxic and hypoxic conditions. As a consequence, WSB1 up-regulates the expression of HIF-1α's target genes and promotes cancer invasion and metastasis through its effect on pVHL. Consistent with this, WSB1 protein level negatively correlates with pVHL level and metastasis-free survival in clinical samples. This work reveals a new mechanism of pVHL's regulation by which cancer acquires invasiveness and metastatic tendency.
Assuntos
Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica , Proteínas/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Células HEK293 , Células HT29 , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Invasividade Neoplásica/genética , Neoplasias/genética , Neoplasias/fisiopatologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas/genética , Ubiquitinação , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.
Assuntos
DNA Mitocondrial , Inflamassomos , Síndromes Mielodisplásicas , Receptor Toll-Like 9 , Humanos , DNA Mitocondrial/metabolismo , Inflamassomos/metabolismo , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais/fisiologia , Receptor Toll-Like 9/metabolismoRESUMO
The mammalian target of rapamycin (mTOR), a kinase that regulates proliferation and apoptosis, has been extensively evaluated as a therapeutic target in multiple malignancies. Rapamycin analogs, which partially inhibit mTOR complex 1 (mTORC1), exhibit immunosuppressive and limited antitumor activity, but sometimes activate survival pathways through feedback mechanisms involving mTORC2. Thus, attention has turned to agents targeting both mTOR complexes by binding the mTOR active site. Here we show that disruption of either mTOR-containing complex is toxic to acute lymphocytic leukemia (ALL) cells and identify 2 previously unrecognized pathways leading to this cell death. Inhibition of mTORC1-mediated 4EBP1 phosphorylation leads to decreased expression of c-MYC and subsequent upregulation of the proapoptotic BCL2 family member PUMA, whereas inhibition of mTORC2 results in nuclear factor-κB-mediated expression of the Early Growth Response 1 (EGR1) gene, which encodes a transcription factor that binds and transactivates the proapoptotic BCL2L11 locus encoding BIM. Importantly, 1 or both pathways contribute to death of malignant lymphoid cells after treatment with dual mTORC1/mTORC2 inhibitors. Collectively, these observations not only provide new insight into the survival roles of mTOR in lymphoid malignancies, but also identify alterations that potentially modulate the action of mTOR dual inhibitors in ALL.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Inibidores Enzimáticos/farmacologia , NF-kappa B/metabolismo , Fosfoproteínas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
Bcl-2, the founding member of a family of apoptotic regulators, was initially identified as the protein product of a gene that is translocated and overexpressed in greater than 85% of follicular lymphomas (FLs). Thirty years later we now understand that anti-apoptotic Bcl-2 family members modulate the intrinsic apoptotic pathway by binding and neutralizing the mitochondrial permeabilizers Bax and Bak as well as a variety of pro-apoptotic proteins, including the cellular stress sensors Bim, Bid, Puma, Bad, Bmf and Noxa. Despite extensive investigation of all of these proteins, important questions remain. For example, how Bax and Bak breach the outer mitochondrial membrane remains poorly understood. Likewise, how the functions of anti-apoptotic Bcl-2 family members such as eponymous Bcl-2 are affected by phosphorylation or cancer-associated mutations has been incompletely defined. Finally, whether Bcl-2 family members can be successfully targeted for therapeutic advantage is only now being investigated in the clinic. Here we review recent advances in understanding Bcl-2 family biology and biochemistry that begin to address these questions.
Assuntos
Progressão da Doença , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
BACKGROUND: Some patients with low-grade lymphoid malignancies develop transformed disease, requiring stem cell transplantation (SCT). SCT outcomes in transformed low-grade lymphoid malignancies may differ from those of nontransformed disease or other aggressive non-Hodgkin lymphomas. We conducted a pooled analysis of the clinical outcomes of allogeneic versus high-dose therapy (HDT) with autologous SCT in adult patients with transformed low-grade lymphoid malignancies. METHODS: A PubMed, EMBASE, and Cochrane search yielded 4 comparative studies reporting allogeneic versus HDT with autologous SCT outcomes in adults (age ≥18) with transformed low-grade lymphoid malignancies, including follicular, chronic/small lymphocytic, and marginal zone lymphoma. Risk ratio (RR) and 95% CI were calculated using random-effects models. RESULTS: Rates for overall survival (OS) were 51.0 versus 69.5% (RR = 1.55, 95% CI 1.19-2.02, p = 0.001), rates of relapse were 37.3 versus 35.3% (RR = 1.04, 95% CI 0.70-1.55, p = 0.84), and rates of transplant-related mortality (TRM) were 33.3 versus 7.2% (RR = 4.52, 95% CI 2.75-7.43, p < 0.00001) for allogeneic versus autologous SCT. Previous rituximab treatment, reduced intensity conditioning regimen prior to SCT, or original pathology had no prognostic impact. CONCLUSION: HDT followed by autologous SCT was associated with lower TRM and a better OS, but there was no difference in relapse versus allogeneic SCT. Autologous SCT may be the better therapeutic option, considering the second chance of allogeneic SCT in the case of relapse.
Assuntos
Transplante Autólogo , Transplante Homólogo , Transplante de Células-Tronco Hematopoéticas , Humanos , Recidiva Local de Neoplasia , Transplante de Células-Tronco , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
BACKGROUND: Anthracyclines are commonly used chemotherapeutic agents with proven efficacy in such malignancies as breast cancer, Hodgkin and non-Hodgkin lymphomas. These agents are associated with irreversible accumulative dose-related cardiomyopathy. Many agents have been examined to reduce cardiotoxicity risk. AIMS: We performed a systematic review and meta-analysis to determine the efficacy of ß-blockers and angiotensin antagonists to prevent early-onset anthracyclines-induced left ventricular dysfunction and cardiac events. METHODS: Relevant articles were searched in PubMed, EMBASE and Cochrane database of systematic reviews up to July 2015. Eligible studies were limited to randomised controlled trials comparing the efficacy of cardioprotective agents (ß-blocker and angiotensin antagonist) with control (either no treatment or placebo) in adult patients (age >18 years) treated with anthracyclines-based regimens. RESULTS: Pooled analysis showed an association of ß-blockers and/or angiotensin antagonists treatment with higher post-chemotherapy left ventricular ejection fraction (LVEF) of 64.03% compared with 57.48% for control treatment. The mean difference estimate (95% CI) was 6.06% (0.54 to 11.58), p=0.03, with significant heterogeneity, I(2) (95% CI)=96% (82.7 to 109.3). Though the point estimate for the relative rate of cardiac events was lower in the experimental arm, the difference was not statistically significant. In an exploratory subgroup analysis, the benefit of experimental agents on LVEF preservation was prominent in patients treated with higher accumulative dose of anthracyclines, but not in the lower dose group. CONCLUSIONS: ß-Blockers and angiotensin antagonists treatments were associated with better LVEF preservation, and the benefit was prominent in patients treated with higher anthracyclines accumulative dose. Unexplained heterogeneity remains, indicating the need for more controlled studies. This analysis provides some support for the routine use of ß-blockers or angiotensin antagonists in patients undergoing anthracyclines treatment, especially when higher accumulative dose is expected.
Assuntos
Antagonistas Adrenérgicos beta/uso terapêutico , Angiotensinas/antagonistas & inibidores , Antraciclinas/efeitos adversos , Antibióticos Antineoplásicos/efeitos adversos , Cardiotônicos/uso terapêutico , Volume Sistólico/efeitos dos fármacos , Disfunção Ventricular Esquerda/induzido quimicamente , Disfunção Ventricular Esquerda/prevenção & controle , Antraciclinas/administração & dosagem , Antibióticos Antineoplásicos/administração & dosagem , Cardiotoxicidade , Humanos , Neoplasias/tratamento farmacológico , Ensaios Clínicos Controlados Aleatórios como Assunto , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
Novel combinations targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.
Assuntos
Proteínas de Ciclo Celular/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/genética , Proteínas Tirosina Quinases/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Pirimidinonas , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais , Ensaio Tumoral de Célula-TroncoRESUMO
Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPN) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for patients with triple-negative (TN) myelofibrosis (MF) who lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in TN-MF and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves numerous hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. Significance: This study establishes that MYC expression is increased in TN-MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in TN-MPNs.
Assuntos
Calgranulina B , Cromossomos Humanos Par 8 , Transtornos Mieloproliferativos , Proteínas Proto-Oncogênicas c-myc , Trissomia , Cromossomos Humanos Par 8/genética , Humanos , Trissomia/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Transtornos Mieloproliferativos/patologia , Animais , Camundongos , Mielofibrose Primária/genética , Mielofibrose Primária/patologia , Mielofibrose Primária/metabolismo , Transdução de Sinais/genéticaRESUMO
Multiple myeloma remains an incurable malignancy due to acquisition of intrinsic programs that drive therapy resistance. Here we report that casein kinase-1δ (CK1δ) and CK1ε are therapeutic targets in multiple myeloma that are necessary to sustain mitochondrial metabolism. Specifically, the dual CK1δ/CK1ε inhibitor SR-3029 had potent in vivo and ex vivo anti-multiple myeloma activity, including against primary multiple myeloma patient specimens. RNA sequencing (RNA-seq) and metabolic analyses revealed inhibiting CK1δ/CK1ε disables multiple myeloma metabolism by suppressing genes involved in oxidative phosphorylation (OxPhos), reducing citric acid cycle intermediates, and suppressing complexes I and IV of the electron transport chain. Finally, sensitivity of multiple myeloma patient specimens to SR-3029 correlated with elevated expression of mitochondrial genes, and RNA-seq from 687 multiple myeloma patient samples revealed that increased CSNK1D, CSNK1E, and OxPhos genes correlate with disease progression and inferior outcomes. Thus, increases in mitochondrial metabolism are a hallmark of multiple myeloma progression that can be disabled by targeting CK1δ/CK1ε. SIGNIFICANCE: CK1δ and CK1ε are attractive therapeutic targets in multiple myeloma whose expression increases with disease progression and connote poor outcomes, and that are necessary to sustain expression of genes directing OxPhos.
Assuntos
Caseína Quinase Idelta , Mieloma Múltiplo , Humanos , Caseína Quinase Idelta/genética , Caseína Quinase Idelta/metabolismo , Mieloma Múltiplo/genética , Sobrevivência Celular , Fosforilação , Progressão da DoençaRESUMO
NLRP3 inflammasome and IFN-stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in myelodysplastic syndromes (MDSs). Gene mutations involving mRNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDSs through undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS BM specimens, we showed accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor cyclic GMP-AMP synthase. Cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling caused ISG induction, NLRP3 inflammasome activation, and maturation of the effector protease caspase-1. Deregulation of RNA polymerase III drove cytosolic R-loop generation, which upon inhibition, extinguished ISG and inflammasome response. Mechanistically, caspase-1 degraded the master erythroid transcription factor, GATA binding protein 1, provoking anemia and myeloid lineage bias that was reversed by cGAS inhibition in vitro and in Tet2-/- hematopoietic stem and progenitor cell-transplanted mice. Together, these data identified a mechanism by which functionally distinct mutations converged upon the cGAS/STING/NLRP3 axis in MDS, directing ISG induction, pyroptosis, and myeloid lineage skewing.
Assuntos
Inflamassomos , Síndromes Mielodisplásicas , Animais , Caspases , DNA/metabolismo , Inflamassomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismoRESUMO
Recent studies demonstrated that MYC epigenetically regulates AML cell survival and differentiation by suppressing IDH1/2-TET2-5hmC signaling and that MYC overexpression is associated with poor survival outcomes in multiple AML patient cohorts. However, the oncogenic roles of MYC in MDS remain to be explored. A total of 41 patients with de novo MDS were retrospectively identified using the Total Cancer Care database at the Moffitt Cancer Center. A total of 61 % of patients had low MYC expression and 39 % of patients had high MYC expression defined as MYC reactivity by immunohistochemical staining in ≥5% of bone marrow (BM) cells at the time of MDS diagnosis. The median MDS-to-AML progression free survival (PFS) was significantly shorter in the high MYC group (median PFS 9.3 vs. 17.7 months, HR = 2.328, p = 0.013). Further, overall survival (OS) was also shorter in the high MYC patients (median OS 19.7 vs. 51.7 months, HR = 2.299, p = 0.053). Multivariate analyses demonstrated that high MYC expression is an independent poor prognostic factor for the MDS-to-AML progression (HR = 2.275, p = 0.046). Our observations indicate that MYC may play a crucial role in MDS transformation to AML and the underlying mechanisms of MYC-driven MDS clonal expansion and leukemic transformation require further investigation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mieloide Aguda/patologia , Mutação , Síndromes Mielodisplásicas/patologia , Segunda Neoplasia Primária/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Progressão da Doença , Feminino , Seguimentos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Segunda Neoplasia Primária/tratamento farmacológico , Segunda Neoplasia Primária/genética , Segunda Neoplasia Primária/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-myc/genética , Estudos Retrospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
MYC oncoproteins regulate transcription of genes directing cell proliferation, metabolism and tumorigenesis. A variety of alterations drive MYC expression in acute myeloid leukemia (AML) and enforced MYC expression in hematopoietic progenitors is sufficient to induce AML. Here we report that AML and myeloid progenitor cell growth and survival rely on MYC-directed suppression of Transcription Factor EB (TFEB), a master regulator of the autophagy-lysosome pathway. Notably, although originally identified as an oncogene, TFEB functions as a tumor suppressor in AML, where it provokes AML cell differentiation and death. These responses reflect TFEB control of myeloid epigenetic programs, by inducing expression of isocitrate dehydrogenase-1 (IDH1) and IDH2, resulting in global hydroxylation of 5-methycytosine. Finally, activating the TFEB-IDH1/IDH2-TET2 axis is revealed as a targetable vulnerability in AML. Thus, epigenetic control by a MYC-TFEB circuit dictates myeloid cell fate and is essential for maintenance of AML.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Isocitrato Desidrogenase , Leucemia Mieloide Aguda , Proteínas Proto-Oncogênicas c-myc , Transdução de Sinais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Diferenciação Celular/genética , Epigênese Genética , Humanos , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell malignancies that can phenotypically resemble other hematologic disorders. Thus, tools that may add to current diagnostic practices could aid in disease discrimination. Constitutive innate immune activation is a pathogenetic driver of ineffective hematopoiesis in MDS through Nod-like receptor protein 3 (NLRP3)-inflammasome-induced pyroptotic cell death. Oxidized mitochondrial DNA (ox-mtDNA) is released upon cytolysis, acts as a danger signal, and triggers inflammasome oligomerization via DNA sensors. By using immortalized bone marrow cells from murine models of common MDS somatic gene mutations and MDS primary samples, we demonstrate that ox-mtDNA is released upon pyroptosis. ox-mtDNA was significantly increased in MDS peripheral blood (PB) plasma compared with the plasma of healthy donors, and it was significantly higher in lower-risk MDS vs higher-risk MDS, consistent with the greater pyroptotic cell fraction in lower-risk patients. Furthermore, ox-mtDNA was significantly higher in MDS PB plasma compared with all other hematologic malignancies studied, with the exception of chronic lymphocytic leukemia (CLL). Receiver operating characteristic/area under the curve (ROC/AUC) analysis demonstrated that ox-mtDNA is a sensitive and specific biomarker for patients with MDS compared with healthy donors (AUC, 0.964), other hematologic malignancies excluding CLL (AUC, 0.893), and reactive conditions (AUC, 0.940). ox-mtDNA positively and significantly correlated with levels of known alarmins S100A9, S100A8, and apoptosis-associated speck-like protein containing caspase recruitment domain (CARD) specks, which provide an index of medullary pyroptosis. Collectively, these data indicate that quantifiable ox-mtDNA released into the extracellular space upon inflammasome activation serves as a biomarker for MDS and the magnitude of pyroptotic cell death.
Assuntos
Inflamassomos , Síndromes Mielodisplásicas , Animais , Biomarcadores , DNA Mitocondrial/genética , Humanos , Camundongos , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/genética , PiroptoseRESUMO
Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL-positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2-AP1-TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand-induced killing that can potentially be exploited for acute leukemia treatment. SIGNIFICANCE: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.
Assuntos
Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Pirazinas/farmacologia , Pirazóis/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with dismal clinical outcomes. Conventional chemotherapies such cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) and hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone alternating with high-dose cytarabine and methotrexate (CVAD) have been commonly used for the BPDCN treatment until a recent study showed promising outcomes in patients treated with SL-401 (Tagraxofusp). In this single-institution retrospective study, we identified a total of 49 consecutive BPDCN patients. Among 42 patients who received treatment, hyper-CVAD regimen was associated with higher complete response rate compared with CHOP-based regimens or SL-401 (91% vs 50% vs 50%), although the difference did not achieve statistical significance. Furthermore, there was no significant overall survival (OS) difference between patients treated with SL-401 vs other chemotherapies as their first-line treatment (hazard ratio = 1.597; 95% CI, 0.460-5.548; P = .431). Of note, patients who received allogeneic stem cell transplant (allo-SCT) had significantly longer OS (hazard ratio = 0.160; 95% CI, 0.0453-0.56; P = .041). Extent of disease (skin vs bone marrow vs both) or younger age (<60 years old) did not have significant prognostic impact on OS. Collectively, our study confirmed the survival benefit of allo-SCT and suggests that conventional and intensive chemotherapies such as CHOP and hyper-CVAD as well as SL-401 would be comparable first-line choice for the BPDCN patients.
Assuntos
Neoplasias Hematológicas , Transplante de Células-Tronco Hematopoéticas , Citarabina , Células Dendríticas , Neoplasias Hematológicas/terapia , Humanos , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
CPX-351 is a liposomally encapsulated 5:1 molar ratio of cytarabine and daunorubicin that recently received regulatory approval for the treatment of therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes based on improved overall survival compared to standard cytarabine/daunorubicin therapy. Checkpoint kinase 1 (CHK1), which is activated by DNA damage and replication stress, diminishes sensitivity to cytarabine and anthracyclines as single agents, suggesting that CHK1 inhibitors might increase the effectiveness of CPX-351. The present studies show that CPX-351 activates CHK1 as well as the S and G2/M cell cycle checkpoints. Conversely, CHK1 inhibition diminishes the cell cycle effects of CPX-351. Moreover, CHK1 knockdown or addition of a CHK1 inhibitor such as MK-8776, rabusertib or prexasertib enhances CPX-351-induced apoptosis in multiple TP53-null and TP53-wildtype AML cell lines. Likewise, CHK1 inhibition increases the antiproliferative effect of CPX-351 on primary AML specimens ex vivo, offering the possibility that CPX-351 may be well suited to combine with CHK1-targeted agents.
Assuntos
Apoptose , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Citarabina/farmacologia , Daunorrubicina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Leucócitos Mononucleares/patologia , Inibidores de Proteínas Quinases/farmacologia , Proliferação de Células , Humanos , Técnicas In Vitro , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Células Tumorais CultivadasRESUMO
MYC is an oncoprotein that coordinates the expression of genes involved in metabolism, cell differentiation and survival in various types of malignancies. However, the underlying oncogenic mechanisms and the clinical significance of MYC expression in the acute myeloid leukemia with myelodysplasia related changes (AML-MRC) remain to be answered. A total of 135 patients were retrospectively identified using Total Cancer Care (TCC) Moffitt Cancer Center (MCC) databases. Diagnosis of AML-MRC was based on the 2016 WHO classification utilizing bone marrow (BM) evaluation. MYC protein expression level was assessed by immunohistochemistry (IHC) staining using paraffin-embedded BM trephine biopsy samples obtained at the time of diagnosis or relapse. Concurrent somatic mutations were assessed using targeted next generation sequencing that include 54 genes. A total of 38% (nâ¯=â¯51) and 62% (nâ¯=â¯84) patients had high and low MYC expression, respectively. The most common somatic mutation in our cohort was TP53 followed by DNMT3A, and ASXL1. The median OS was significantly longer in low MYC patients (median OS 42.3 vs. 17.05 months, pâ¯=â¯0.0109). Multivariate analysis including MYC expression level, transplantation status, gender and age demonstrated high MYC expression (HR 1.77, 95% CI 1.004-3.104, pâ¯=â¯0.045) to be an independent poor prognostic factor. Further studies are needed to identify the underlying mechanism of MYC driven oncogenesis in AML-MRC. Additionally, the prognostic impact of MYC on the AML survival in a larger cohort that include diverse somatic mutations and chromosomal abnormalities requires further investigation.
Assuntos
Regulação da Expressão Gênica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/mortalidade , Proteínas Proto-Oncogênicas c-myc/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Biópsia , Aberrações Cromossômicas , Feminino , Humanos , Imuno-Histoquímica , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Mutação , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/terapia , Prognóstico , Modelos de Riscos ProporcionaisRESUMO
BACKGROUND: NLRP3 inflammasome-directed pyroptotic cell death drives ineffective haemopoiesis in myelodysplastic syndromes. During inflammasome assembly, the apoptosis-associated speck-like protein containing a CARD (PYCARD, commonly known as ASC) adaptor protein polymerises into large, filamentous clusters termed ASC specks that are released upon cytolysis. Specks are resistant to proteolytic degradation because of their prion-like structure, and therefore might serve as a biomarker for pyroptotic cell death in myelodysplastic syndromes. METHODS: This observational cohort study was done at the H Lee Moffitt Cancer Center (Tampa, FL, USA). Patients with myelodysplastic syndromes, healthy controls, and patients with non-myelodysplastic syndrome haematological cancers or type 2 diabetes were recruited. We used confocal and electron microscopy to visualise, and flow cytometry to quantify, ASC specks in peripheral blood and bone marrow plasma samples. Speck percentages were compared by t test or ANOVA, correlations were assessed by Spearman's rank correlation coefficient, and biomarker efficiency was assessed by receiver operating characteristics and area under the curve (AUC) analysis. FINDINGS: Between Jan 1, 2005, and Jan 12, 2017, we obtained samples from 177 patients with myelodysplastic syndromes and 29 healthy controls for the discovery cohort, and 113 patients with myelodysplastic syndromes and 31 healthy controls for the validation cohort. We also obtained samples from 22 patients with del(5q) myelodysplastic syndromes, 230 patients with non-myelodysplastic syndrome haematological cancers and 23 patients with type 2 diabetes. After adjustment for glucose concentration, the log10-transformed mean percentage of peripheral blood plasma-derived ASC specks was significantly higher in the 177 patients with myelodysplastic syndromes versus the 29 age-matched, healthy donors (-0·41 [SD 0·49] vs -0·67 [0·59], p=0·034). The percentages of ASC specks in samples from patients with myelodysplastic syndromes were significantly greater than those in samples from individuals with every other haematological cancer studied (all p<0·05) except myelofibrosis (p=0·19). The findings were confirmed in the independent validation cohort (p<0·0001). Peripheral blood plasma danger-associated molecular pattern protein S100-A8 and protein S100-A9 concentrations from 144 patients with myelodysplastic syndromes from the discovery cohort directly correlated with ASC speck percentage (r=0·4, p<0·0001 for S100-A8 and r=0·2, p=0·017 for S100-A9). Patients with at least two somatic gene mutations had a significantly greater mean percentage of peripheral blood plasma ASC specks than patients with one or no mutation (-0·22 [SD 0·63] vs -0·53 [0·44], p=0·008). The percentage of plasma ASC specks was a robust marker for pyroptosis in myelodysplastic syndromes (AUC=0·888), in which a cutoff of 0·80 maximised sensitivity at 0·84 (95% CI 0·65-0·91) and specificity at 0·87 (0·58-0·97). INTERPRETATION: Our results underscore the pathobiological relevance of ASC specks and suggest that ASC specks are a sensitive and specific candidate plasma biomarker that provides an index of medullary pyroptotic cell death and ineffective haemopoiesis in patients with myelodysplastic syndromes. FUNDING: T32 Training Grant (NIH/NCI 5T32 CA115308-08), Edward P Evans Foundation, The Taub Foundation Grants Program, the Flow Cytometry, Analytic Microscopy, and Tissue Core Facilities at the H Lee Moffitt Cancer Center and Research Institute, a National Cancer Institute-designated Comprehensive Cancer Center (P30-CA076292).