RESUMO
Despite recent advances in acute myeloid leukemia (AML) molecular characterization and targeted therapies, a majority of AML cases still lack therapeutically actionable targets. In 127 AML cases with unmet therapeutic needs, as defined by the exclusion of ELN favorable cases and of FLT3-ITD mutations, we identified 51 (40%) cases with alterations in RAS pathway genes (RAS+, mostly NF1, NRAS, KRAS, and PTPN11 genes). In 79 homogeneously treated AML patients from this cohort, RAS+ status were associated with higher white blood cell count, higher LDH, and reduced survival. In AML models of oncogenic addiction to RAS-MEK signaling, the MEK inhibitor trametinib demonstrated antileukemic activity in vitro and in vivo. However, the efficacy of trametinib was heterogeneous in ex vivo cultures of primary RAS+ AML patient specimens. From repurposing drug screens in RAS-activated AML cells, we identified pyrvinium pamoate, an anti-helminthic agent efficiently inhibiting the growth of RAS+ primary AML cells ex vivo, preferentially in trametinib-resistant PTPN11- or KRAS-mutated samples. Metabolic and genetic complementarity between trametinib and pyrvinium pamoate translated into anti-AML synergy in vitro. Moreover, this combination inhibited the propagation of RA+ AML cells in vivo in mice, indicating a potential for future clinical development of this strategy in AML.
Assuntos
Leucemia Mieloide Aguda , Mutações Sintéticas Letais , Animais , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Mutação , Estresse Oxidativo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Imidazóis/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Pirimidinonas/farmacologia , Sulfonamidas/farmacologia , Resposta a Proteínas não Dobradas/fisiologia , eIF-2 Quinase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Apoptose/fisiologia , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Células THP-1 , Células U937 , Adulto JovemRESUMO
Given the poor prognosis of relapsed/refractory myeloid malignancies, the concept of sequential conditioning before allogeneic hematopoietic stem cell transplantation (allo-HSCT) has proven to be an effective approach. We sought to evaluate a sequential scheme combining fludarabine, amsacrine, and cytarabine (FLAMSA) for cytoreduction, followed by reduced-intensity conditioning with busulfan and melphalan (FLAMSA-BuMel), which was designed to be suitable for both HLA-matched and haploidentical HSCT. This single-center retrospective study included 36 adult patients with high-risk myeloid malignancies who underwent allo-HSCT from HLA-matched (n = 19) or haploidentical (n = 17) donors. Along with the standard prophylaxis for graft-versus-host disease (GVHD), patients with a haploidentical donor received post-transplantation high-dose cyclophosphamide. A post-transplantation consolidation treatment with low-dose 5-azacytidine and prophylactic donor lymphocyte infusions was provided whenever possible. Thirty patients (83%) achieved complete remission on day +30. With a median follow-up of 30.0 months, the 2-year overall survival was 89% in the HLA-matched group versus 34% in the haploidentical group (P = .0018). The 2-year disease-free survival in these 2 groups was 68% and 34%, respectively (P = .013). At 2 years, the probability of relapse was 32% and 20%, respectively, and nonrelapse mortality was 0% and 58%, respectively (P = .0003). The leading cause of death was relapse in the HLA-matched group (3 of 19) and hemorrhagic events (5 of 17) in the haploidentical group, favored by significantly delayed platelet reconstitution and a severe GVHD context. These data confirm the feasibility of FLAMSA-BuMel as a sequential conditioning in allo-HSCT for high-risk myeloid malignancies. The use of bone marrow as the preferred graft source might reduce the incidence of acute GVHD and nonrelapse mortality in the haploidentical transplantation setting.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Bussulfano , Humanos , Leucemia Mieloide Aguda/terapia , Melfalan , Recidiva Local de Neoplasia , Estudos RetrospectivosRESUMO
The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Junções Íntimas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Células CACO-2 , Cálcio/metabolismo , Permeabilidade da Membrana Celular , Análise Mutacional de DNA , Humanos , Sistema de Sinalização das MAP Quinases , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
AMP-activated protein kinase (AMPK) is a critical energy sensor, regulating signaling networks involved in pathology including metabolic diseases and cancer. This increasingly recognized role of AMPK has prompted tremendous research efforts to develop new pharmacological AMPK activators. To precisely study the role of AMPK, and the specificity and activity of AMPK activators in cellular models, genetic AMPK inactivating tools are required. We report here methods for genetic inactivation of AMPK α1/α2 catalytic subunits in human cell lines by the CRISPR/Cas9 technology, a recent breakthrough technique for genome editing.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Silenciamento de Genes/métodos , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Subunidades Proteicas/genética , RNA Guia de Cinetoplastídeos/genética , Transdução de Sinais/genética , Transfecção/métodosRESUMO
Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. (clinicaltrials identifiers: 00209209 and 00209222).
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/genética , Linfoma de Célula do Manto/diagnóstico , Adulto , Idoso , Antígenos CD/sangue , Antígenos CD/genética , Biomarcadores Tumorais/sangue , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/mortalidade , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Projetos Piloto , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Recidiva , Indução de Remissão , Análise de SobrevidaRESUMO
AMPK is a master regulator of cellular metabolism that exerts either oncogenic or tumor suppressor activity depending on context. Here, we report that the specific AMPK agonist GSK621 selectively kills acute myeloid leukemia (AML) cells but spares normal hematopoietic progenitors. This differential sensitivity results from a unique synthetic lethal interaction involving concurrent activation of AMPK and mTORC1. Strikingly, the lethality of GSK621 in primary AML cells and AML cell lines is abrogated by chemical or genetic ablation of mTORC1 signaling. The same synthetic lethality between AMPK and mTORC1 activation is established in CD34-positive hematopoietic progenitors by constitutive activation of AKT or enhanced in AML cells by deletion of TSC2. Finally, cytotoxicity in AML cells from GSK621 involves the eIF2α/ATF4 signaling pathway that specifically results from mTORC1 activation. AMPK activation may represent a therapeutic opportunity in mTORC1-overactivated cancers.