RESUMO
CRISPR-Cas9 is a useful tool for inserting precise genetic alterations through homology-directed repair (HDR), although current methods rely on provision of an exogenous repair template. Here, we tested the possibility of repairing heterozygous single nucleotide variants (SNVs) using the cell's own wild-type allele rather than an exogenous template. Using high-fidelity Cas9 to perform allele-specific CRISPR across multiple human leukemia cell lines as well as in primary hematopoietic cells from patients with leukemia, we find high levels of reversion to wild-type in the absence of exogenous template. Moreover, we demonstrate that bulk treatment to revert a truncating mutation in ASXL1 using CRISPR-mediated interallelic gene conversion (IGC) is sufficient to prolong survival in a human cell line-derived xenograft model (median survival 33 days vs 27.5 days; p = 0.0040). These results indicate that IGC can be applied to numerous types of leukemia and can meaningfully alter cellular phenotypes at scale. Because our method targets single-base mutations, rather than larger variants targeted by IGC in prior studies, it greatly expands the pool of risk-increasing genetic lesions which could potentially be targeted by IGC. This technique may reduce cost and complexity for experiments modeling phenotypic consequences of SNVs. The principles of SNV-specific IGC demonstrated in this proof-of-concept study could be applied to investigate the phenotypic effects of targeted clonal reduction of leukemogenic SNV driver mutations.
RESUMO
Impairing the BET family coactivator BRD4 with small-molecule inhibitors (BETi) showed encouraging preclinical activity in treating acute myeloid leukemia (AML). However, dose-limiting toxicities and limited clinical activity dampened the enthusiasm for BETi as a single agent. BETi resistance in AML myeloblasts was found to correlate with maintaining mitochondrial respiration, suggesting that identifying the metabolic pathway sustaining mitochondrial integrity could help develop approaches to improve BETi efficacy. Herein, we demonstrated that mitochondria-associated lactate dehydrogenase allows AML myeloblasts to utilize lactate as a metabolic bypass to fuel mitochondrial respiration and maintain cellular viability. Pharmacologically and genetically impairing lactate utilization rendered resistant myeloblasts susceptible to BET inhibition. Low-dose combinations of BETi and oxamate, a lactate dehydrogenase inhibitor, reduced in vivo expansion of BETi-resistant AML in cell line and patient-derived murine models. These results elucidate how AML myeloblasts metabolically adapt to BETi by consuming lactate and demonstrate that combining BETi with inhibitors of lactate utilization may be useful in AML treatment. SIGNIFICANCE: Lactate utilization allows AML myeloblasts to maintain metabolic integrity and circumvent antileukemic therapy, which supports testing of lactate utilization inhibitors in clinical settings to overcome BET inhibitor resistance in AML. See related commentary by Boët and Sarry, p. 950.
Assuntos
Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Animais , Camundongos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Ácido Láctico , Linhagem Celular Tumoral , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Lactato Desidrogenases , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo CelularRESUMO
CD47-SIRPa is a myeloid check point pathway that promotes phagocytosis of cells lacking markers for self-recognition. Tumor cells can overexpress CD47 and bind to SIRPa on macrophages, preventing phagocytosis. CD47 expression is enhanced and correlated with a negative prognosis in acute myeloid leukemia (AML), with its blockade leading to cell clearance. ALX90 is an engineered fusion protein with high affinity for CD47. Composed of the N-terminal D1 domain of SIRPα genetically linked to an inactive Fc domain from human immunoglobulin (Ig) G, ALX90 is designed to avoid potential toxicity of CD47-expressing red blood cells. Venetoclax (VEN) is a specific B-cell lymphoma-2 (BCL-2) inhibitor that can restore apoptosis in malignant cells. In AML, VEN is combined with azanucleosides to induce superior remission rates, however treatment for refractory/relapse is an unmet need. We questioned whether the anti-tumor activity of a VENbased regimen can be augmented through CD47 inhibition (CD47i) in AML and how this triplet may be enhanced. Human AML cell lines were sensitive to ALX90 and its addition increased efficacy of a VEN plus azacitidin (VEN+AZA) regimen in vivo. However, CD47i failed to clear bone marrow tumor burden in PDX models. We hypothesized that the loss of resident macrophages in the bone marrow in AML reduced efficiency of CD47i. Therefore, we attempted to enhance this medullary macrophage population with agonism of TLR3 via polyinosinic:polycytidylic acid (poly(I:C)), which led to expansion and activation of medullary macrophages in in vivo AML PDX models and potentiated CD47i. In summary, the addition of poly(I:C) can enhance medullary macrophage populations to potentiate the phagocytosis merited by therapeutic inhibition of CD47.
Assuntos
Antígeno CD47 , Leucemia Mieloide Aguda , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Humanos , Animais , Camundongos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Sulfonamidas/farmacologia , Receptores Imunológicos/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Antígenos de Diferenciação/metabolismo , Fagocitose/efeitos dos fármacos , Poli I-C/farmacologiaRESUMO
Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.
Assuntos
Leucemia Mieloide Aguda , Animais , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Modelos Animais de Doenças , Células-Tronco Neoplásicas/metabolismoRESUMO
Hematopoiesis integrates cytokine signaling, metabolism, and epigenetic modifications to regulate blood cell generation. These processes are linked, as metabolites provide essential substrates for epigenetic marks. In this study, we demonstrate that ATP citrate lyase (Acly), which metabolizes citrate to generate cytosolic acetyl-CoA and is of clinical interest, can regulate chromatin accessibility to limit myeloid differentiation. Acly was tested for a role in murine hematopoiesis by small-molecule inhibition or genetic deletion in lineage-depleted, c-Kit-enriched hematopoietic stem and progenitor cells from Mus musculus. Treatments increased the abundance of cell populations that expressed the myeloid integrin CD11b and other markers of myeloid differentiation. When single-cell RNA sequencing was performed, we found that Acly inhibitor-treated hematopoietic stem and progenitor cells exhibited greater gene expression signatures for macrophages and enrichment of these populations. Similarly, the single-cell assay for transposase-accessible chromatin sequencing showed increased chromatin accessibility at genes associated with myeloid differentiation, including CD11b, CD11c, and IRF8. Mechanistically, Acly deficiency altered chromatin accessibility and expression of multiple C/EBP family transcription factors known to regulate myeloid differentiation and cell metabolism, with increased Cebpe and decreased Cebpa and Cebpb. This effect of Acly deficiency was accompanied by altered mitochondrial metabolism with decreased mitochondrial polarization but increased mitochondrial content and production of reactive oxygen species. The bias to myeloid differentiation appeared due to insufficient generation of acetyl-CoA, as exogenous acetate to support alternate compensatory pathways to produce acetyl-CoA reversed this phenotype. Acly inhibition thus can promote myelopoiesis through deprivation of acetyl-CoA and altered histone acetylome to regulate C/EBP transcription factor family activity for myeloid differentiation.
Assuntos
ATP Citrato (pro-S)-Liase , Montagem e Desmontagem da Cromatina , Epigênese Genética , Mielopoese , Animais , Camundongos , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , ATP Citrato (pro-S)-Liase/deficiência , ATP Citrato (pro-S)-Liase/genética , Cromatina/metabolismo , Mielopoese/genéticaRESUMO
Dnm2fl/fl Pf4-Cre (Dnm2Plt-/- ) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt-/- platelets. Additionally, Dnm2Plt-/- platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt-/- mice was due to JAK2 constitutive activation or to elevated circulating TPO levels. In unstimulated Dnm2Plt-/- platelets, STAT3 and, to a lower extent, STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt-/- mice in the Mpl-/- background to generate Mpl-/-Dnm2Plt-/- mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl-/- Dnm2Plt-/- platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl-/- Dnm2Plt-/- mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl-/- Dnm2Plt-/- mice had severe erythroblast (EB) maturation defects, decreased expression of hemoglobin and heme homeostasis genes and increased expression of ribosome biogenesis and protein translation genes in spleen EBs, and developed anemia with grossly elevated plasma erythropoietin (EPO) levels, leading to early fatality by postnatal day 25. Mpl-/- Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.
RESUMO
Cancer cells have long been recognized to exhibit unique bioenergetic requirements. The apoptolidin family of glycomacrolides are distinguished by their selective cytotoxicity towards oncogene-transformed cells, yet their molecular mechanism remains uncertain. We used photoaffinity analogs of the apoptolidins to identify the F1 subcomplex of mitochondrial ATP synthase as the target of apoptolidin A. Cryogenic electron microscopy (cryo-EM) of apoptolidin and ammocidin-ATP synthase complexes revealed a novel shared mode of inhibition that was confirmed by deep mutational scanning of the binding interface to reveal resistance mutations which were confirmed using CRISPR-Cas9. Ammocidin A was found to suppress leukemia progression in vivo at doses that were tolerated with minimal toxicity. The combination of cellular, structural, mutagenesis, and in vivo evidence defines the mechanism of action of apoptolidin family glycomacrolides and establishes a path to address oxidative phosphorylation-dependent cancers.
Assuntos
Leucemia , Neoplasias , Trifosfato de Adenosina , Humanos , Leucemia/tratamento farmacológico , Macrolídeos , ATPases Mitocondriais Próton-Translocadoras/química , Neoplasias/tratamento farmacológicoRESUMO
Eltrombopag (ELT) is a thrombopoietic agent approved for immune thrombocytopenia and also a potent iron chelator. Here we found that ELT exhibited dose-dependent opposing effects on in vitro megakaryopoiesis: low concentrations (≤6 µM, ELT6) stimulated megakaryopoiesis, but high concentrations (30 µM, ELT30) suppressed megakaryocyte (MK) differentiation and proliferation. The suppressive effects of ELT30 were reproduced by other iron chelators, supporting iron chelation as a likely mechanism. During MK differentiation, committed MK progenitors (CD34+/CD41+ and CD34-/CD41+ cells) were significantly more sensitive than undifferentiated progenitors (CD34+/CD41- cells) to the suppressive effects of ELT30, which resulted from both decreased proliferation and increased apoptosis. The antiproliferative effects of ELT30 were reversed by increased iron in the culture, as were the proapoptotic effects when exposure to ELT30 was short. Because committed MK progenitors exhibited the highest proliferative rate and the highest sensitivity to iron chelation, we tested whether their iron status influenced their response to ELT during rapid cell expansion. In these studies, iron deficiency reduced the proliferation of CD41+ cells in response to all ELT concentrations. Severe iron deficiency also reduced the number of MKs generated in response to high thrombopoietin concentrations by â¼50%, compared with iron-replete cultures. Our findings support the hypothesis that although iron deficiency can stimulate certain cells and steps in megakaryopoiesis, it can also limit the proliferation of committed MK progenitors, with severity of iron deficiency and degree of thrombopoietic stimulation influencing the ultimate output. Further studies are needed to clarify how megakaryopoiesis, iron deficiency, and ELT stimulation are clinically interrelated.
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Sangue Fetal , Células Progenitoras de Megacariócitos , Benzoatos , Diferenciação Celular , Hidrazinas , Ferro/farmacologia , PirazóisRESUMO
Dysregulation of apoptotic machinery is one mechanism by which acute myeloid leukemia (AML) acquires a clonal survival advantage. B-cell lymphoma protein-2 (BCL2) overexpression is a common feature in hematologic malignancies. The selective BCL2 inhibitor, venetoclax (VEN) is used in combination with azacitidine (AZA), a DNAmethyltransferase inhibitor (DNMTi), to treat patients with AML. Despite promising response rates to VEN/AZA, resistance to the agent is common. One identified mechanism of resistance is the upregulation of myeloid cell leukemia-1 protein (MCL1). Pevonedistat (PEV), a novel agent that inhibits NEDD8-activating enzyme, and AZA both upregulate NOXA (PMAIP1), a BCL2 family protein that competes with effector molecules at the BH3 binding site of MCL1. We demonstrate that PEV/AZA combination induces NOXA to a greater degree than either PEV or AZA alone, which enhances VEN-mediated apoptosis. Herein, using AML cell lines and primary AML patient samples ex vivo, including in cells with genetic alterations linked to treatment resistance, we demonstrate robust activity of the PEV/VEN/AZA triplet. These findings were corroborated in preclinical systemic engrafted models of AML. Collectively, these results provide rational for combining PEV/VEN/AZA as a novel therapeutic approach in overcoming AML resistance in current therapies.
Assuntos
Azacitidina , Leucemia Mieloide Aguda , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Ciclopentanos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Pirimidinas , SulfonamidasRESUMO
BACKGROUND: All-trans retinoic acid (ATRA), a derivate of vitamin A, has been successfully used as a therapy to induce differentiation in M3 acute promyelocytic leukemia (APML), and has led to marked improvement in outcomes. Previously, attempts to use ATRA in non-APML in the clinic, however, have been underwhelming, likely due to persistent signaling through other oncogenic drivers. Dysregulated JAK/STAT signaling is known to drive several hematologic malignancies, and targeting JAK1 and JAK2 with the JAK1/JAK2 inhibitor ruxolitinib has led to improvement in survival in primary myelofibrosis and alleviation of vasomotor symptoms and splenomegaly in polycythemia vera and myelofibrosis. OBJECTIVE: While dose-dependent anemia and thrombocytopenia limit the use of JAK2 inhibition, selectively targeting JAK1 has been explored as a means to suppress inflammation and STAT-associated pathologies related to neoplastogenesis. The objective of this study is to employ JAK1 inhibition (JAK1i) in the presence of ATRA as a potential therapy in non-M3 acute myeloid leukemia (AML). METHODS: Efficacy of JAK1i using INCB52793 was assessed by changes in cell cycle and apoptosis in treated AML cell lines. Transcriptomic and proteomic analysis evaluated effects of JAK1i. Synergy between JAK1i+ ATRA was assessed in cell lines in vitro while efficacy in vivo was assessed by tumor reduction in MV-4-11 cell line-derived xenografts. RESULTS: Here we describe novel synergistic activity between JAK1i inhibition and ATRA in non-M3 leukemia. Transcriptomic and proteomic analysis confirmed structural and functional changes related to maturation while in vivo combinatory studies revealed significant decreases in leukemic expansion. CONCLUSIONS: JAK1i+ ATRA lead to decreases in cell cycle followed by myeloid differentiation and cell death in human leukemias. These findings highlight potential uses of ATRA-based differentiation therapy of non-M3 human leukemia.
Assuntos
Leucemia Mieloide Aguda , Leucemia , Diferenciação Celular , Humanos , Janus Quinase 1 , Proteômica , Fator de Transcrição STAT5 , Tretinoína/farmacologiaRESUMO
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic syndrome/myeloproliferative neoplasm overlap syndrome characterized by monocytic proliferation in the presence of dysplastic bone marrow changes, inflammatory symptoms, and propensity for transformation to acute myeloid leukemia (AML), with a poor prognosis and limited treatment options. Unlike the α and ß isoforms, the phosphatidylinositol-3-kinase (PI3K)-δ signaling protein is predominantly expressed by hematopoietic cells and therefore has garnered interest as a potential target for the treatment of lymphomas and leukemias. We revealed a pattern of increased PIK3CD:PIK3CA ratio in monocytic M5 AML patients and cell lines, and this ratio correlated with responsiveness to pharmacological PI3K-δ inhibition in vitro. Because CMML is a disease defined by monocytic clonal proliferation, we tested the PI3K-δ inhibitor umbralisib as a single agent and in combination with the JAK1/2 inhibitor ruxolitinib, in CMML. Our ex vivo experiments with primary CMML patient samples revealed synergistic inhibition of viability and clonogenicity with this combination. Phospho-specific flow cytometry revealed that dual inhibition had the unique ability to decrease STAT5, ERK, AKT, and S6 phosphorylation simultaneously, which offers a mechanistic hypothesis for the enhanced efficacy of the combination treatment. These preclinical data indicate promising activity by co-inhibition of PI3K-δ and JAK1/2 and support the use of ruxolitinibâ¯+â¯umbralisib combination therapy in CMML under active clinical investigation.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pirazóis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Sinergismo Farmacológico , Humanos , Leucemia Mielomonocítica Crônica/enzimologia , Terapia de Alvo Molecular , Nitrilas , PirimidinasRESUMO
PURPOSE: The BCL2 inhibitor, venetoclax, has transformed clinical care in acute myeloid leukemia (AML). However, subsets of patients do not respond or eventually acquire resistance. Venetoclax-based regimens can lead to considerable marrow suppression in some patients. Bromodomain and extraterminal inhibitors (BETi) are potential treatments for AML, as regulators of critical AML oncogenes. We tested the efficacy of novel BET inhibitor INCB054329, and its synergy with venetoclax to reduce AML without induction of hematopoietic toxicity. EXPERIMENTAL DESIGN: INCB054329 efficacy was assessed by changes in cell cycle and apoptosis in treated AML cell lines. In vivo efficacy was assessed by tumor reduction in MV-4-11 cell line-derived xenografts. Precision run-on and sequencing (PRO-seq) evaluated effects of INCB054329. Synergy between low-dose BETi and venetoclax was assessed in cell lines and patient samples in vitro and in vivo while efficacy and toxicity was assessed in patient-derived xenograft (PDX) models. RESULTS: INCB054329 induced dose-dependent apoptosis and quiescence in AML cell lines. PRO-seq analysis evaluated the effects of INCB054329 on transcription and confirmed reduced transcriptional elongation of key oncogenes, MYC and BCL2, and genes involved in the cell cycle and metabolism. Combinations of BETi and venetoclax led to reduced cell viability in cell lines and patient samples. Low-dose combinations of INCB054329 and venetoclax in cell line and PDX models reduced AML burden, regardless of the sensitivity to monotherapy without development of toxicity. CONCLUSIONS: Our findings suggest low dose combinations of venetoclax and BETi may be more efficacious for patients with AML than either monotherapy, potentially providing a longer, more tolerable dosing regimen.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leucemia Mieloide/tratamento farmacológico , Compostos Orgânicos/farmacologia , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas/farmacologia , Doença Aguda , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Drugs targeting chromatin-modifying enzymes have entered clinical trials for myeloid malignancies, including INCB059872, a selective irreversible inhibitor of Lysine-Specific Demethylase 1 (LSD1). While initial studies of LSD1 inhibitors suggested these compounds may be used to induce differentiation of acute myeloid leukemia (AML), the mechanisms underlying this effect and dose-limiting toxicities are not well understood. Here, we used precision nuclear run-on sequencing (PRO-seq) and ChIP-seq in AML cell lines to probe for the earliest regulatory events associated with INCB059872 treatment. The changes in nascent transcription could be traced back to a loss of CoREST activity and activation of GFI1-regulated genes. INCB059872 is in phase I clinical trials, and we evaluated a pre-treatment bone marrow sample of a patient who showed a clinical response to INCB059872 while being treated with azacitidine. We used single-cell RNA-sequencing (scRNA-seq) to show that INCB059872 caused a shift in gene expression that was again associated with GFI1/GFI1B regulation. Finally, we treated mice with INCB059872 and performed scRNA-seq of lineage-negative bone marrow cells, which showed that INCB059872 triggered accumulation of megakaryocyte early progenitor cells with gene expression hallmarks of stem cells. Accumulation of these stem/progenitor cells may contribute to the thrombocytopenia observed in patients treated with LSD1 inhibitors.
Assuntos
Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos C57BL , RNA-Seq , Análise de Célula Única/métodos , Células-Tronco/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequenciamento do Exoma/métodosRESUMO
BACKGROUND: DNA methyltransferase inhibitors (DNMTis) improve survival for patients with myelodysplastic syndromes (MDS) and those with acute myeloid leukemia (AML) unable to receive standard cytotoxic chemotherapy and are, accordingly, the backbone of standard-of-care treatment for these conditions. Standard regimens with DNMTIs, decitabine (DEC) or azacitidine (AZA) include daily subcutaneous (s.c.) or intravenous (i.v.) administration for 5-7 consecutive days. Attempts to provide the therapy orally have been limited given rapid clearance of the agents by the enzyme cytidine deaminase (CDA), which is ubiquitous in the gut and liver as part of first-pass metabolism. Recently, cedazuridine (CDZ), an oral inhibitor of CDA, was successfully combined with DEC to approximate the pharmacokinetics of i.v. DEC in patients. OBJECTIVE: To determine if an oral dosing strategy might be feasible in the clinic with AZA, we attempted to increase the bioavailability of oral AZA through the use of CDZ, in a murine model. METHODS: Following pharmacokinetic and pharmacodynamic assessment of oral AZA dosed with CDZ in murine and monkey models, we tested this regimen in vivo with a human cell line-derived xenograft transplantation experiment (CDX). Following this we combined the regimen with venetoclax (VEN) to test the efficacy of an all-oral regimen in a patient-derived xenograft (PDX) model. RESULTS: Parenteral AZA and oral AZA + CDZ exhibited similar pharmacokinetic profiles, and efficacy against human AML cells. Tumor regression was seen with AZA + CDZ in MOLM-13 CDX and PDX models. CONCLUSIONS: We conclude that oral AZA when combined with CDZ achieves successful tumor regression in both CDX and PDX models. Furthermore, the combination of AZA + CDZ with VEN in a PDX model emulated responses seen with VEN + AZA in the clinic, implying a potential all-oral VEN-based therapy opportunity in myeloid diseases.
Assuntos
Azacitidina/uso terapêutico , Uridina/análogos & derivados , Administração Oral , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Modelos Animais de Doenças , Feminino , Haplorrinos , Humanos , Infusões Parenterais , Camundongos , Resultado do Tratamento , Uridina/uso terapêuticoRESUMO
The selective inhibitor of nuclear export (SINE) compounds selinexor (KPT-330) and eltanexor (KPT-8602) are from a novel class of small molecules that target exportin-1 (XPO1 [CRM1]), an essential nucleo-cytoplasmic transport protein responsible for the nuclear export of major tumor suppressor proteins and growth regulators such as p53, p21, and p27. XPO1 also affects the translation of messenger RNAs for critical oncogenes, including MYC, BCL2, MCL1, and BCL6, by blocking the export of the translation initiation factor eIF4E. Early trials with venetoclax (ABT-199), a potent, selective inhibitor of BCL2, have revealed responses across a variety of hematologic malignancies. However, many tumors are not responsive to venetoclax. We used models of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) to determine in vitro and in vivo responses to treatment with venetoclax and SINE compounds combined. Cotreatment with venetoclax and SINE compounds demonstrated loss of viability in multiple cell lines. Further in vitro analyses showed that this enhanced cell death was the result of an increase in apoptosis that led to a loss of clonogenicity in methylcellulose assays, coinciding with activation of p53 and loss of MCL1. Treatment with SINE compounds and venetoclax combined led to a reduction in tumor growth in both AML and DLBCL xenografts. Immunohistochemical analysis of tissue sections revealed that the reduction in tumor cells was partly the result of an induction of apoptosis. The enhanced effects of this combination were validated in primary AML and DLBCL patient cells. Our studies reveal synergy with SINE compounds and venetoclax in aggressive hematologic malignancies and provide a rationale for pursuing this approach in a clinical trial.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Neoplasias Hematológicas , Transporte Ativo do Núcleo Celular , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Neoplasias Hematológicas/tratamento farmacológico , Humanos , SulfonamidasRESUMO
Venetoclax-based therapy can induce responses in approximately 70% of older previously untreated patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. In the present study, we report that responses to venetoclax +azacitidine in patients with AML correlate closely with developmental stage, where phenotypically primitive AML is sensitive, but monocytic AML is more resistant. Mechanistically, resistant monocytic AML has a distinct transcriptomic profile, loses expression of venetoclax target BCL2, and relies on MCL1 to mediate oxidative phosphorylation and survival. This differential sensitivity drives a selective process in patients which favors the outgrowth of monocytic subpopulations at relapse. Based on these findings, we conclude that resistance to venetoclax + azacitidine can arise due to biological properties intrinsic to monocytic differentiation. We propose that optimal AML therapies should be designed so as to independently target AML subclones that may arise at differing stages of pathogenesis. SIGNIFICANCE: Identifying characteristics of patients who respond poorly to venetoclax-based therapy and devising alternative therapeutic strategies for such patients are important topics in AML. We show that venetoclax resistance can arise due to intrinsic molecular/metabolic properties of monocytic AML cells and that such properties can potentially be targeted with alternative strategies.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Sulfonamidas/uso terapêutico , Idoso , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Humanos , Sulfonamidas/farmacologiaRESUMO
Overexpression of myeloid cell leukemia-1 (Mcl-1) in cancers correlates with high tumor grade and poor survival. Additionally, Mcl-1 drives intrinsic and acquired resistance to many cancer therapeutics, including B cell lymphoma 2 family inhibitors, proteasome inhibitors, and antitubulins. Therefore, Mcl-1 inhibition could serve as a strategy to target cancers that require Mcl-1 to evade apoptosis. Herein, we describe the use of structure-based design to discover a novel compound (42) that robustly and specifically inhibits Mcl-1 in cell culture and animal xenograft models. Compound 42 binds to Mcl-1 with picomolar affinity and inhibited growth of Mcl-1-dependent tumor cell lines in the nanomolar range. Compound 42 also inhibited the growth of hematological and triple negative breast cancer xenografts at well-tolerated doses. These findings highlight the use of structure-based design to identify small molecule Mcl-1 inhibitors and support the use of 42 as a potential treatment strategy to block Mcl-1 activity and induce apoptosis in Mcl-1-dependent cancers.
Assuntos
Antineoplásicos/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Azepinas/química , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Simulação de Dinâmica Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Suppression of apoptosis by expression of antiapoptotic BCL2 family members is a hallmark of acute myeloblastic leukemia (AML). Induced myeloid leukemia cell differentiation protein (MCL1), an antiapoptotic BCL2 family member, is commonly upregulated in AML cells and is often a primary mode of resistance to treatment with the BCL2 inhibitor venetoclax. Here, we describe VU661013, a novel, potent, selective MCL1 inhibitor that destabilizes BIM/MCL1 association, leads to apoptosis in AML, and is active in venetoclax-resistant cells and patient-derived xenografts. In addition, VU661013 was safely combined with venetoclax for synergy in murine models of AML. Importantly, BH3 profiling of patient samples and drug-sensitivity testing ex vivo accurately predicted cellular responses to selective inhibitors of MCL1 or BCL2 and showed benefit of the combination. Taken together, these data suggest a strategy of rationally using BCL2 and MCL1 inhibitors in sequence or in combination in AML clinical trials. SIGNIFICANCE: Targeting antiapoptotic proteins in AML is a key therapeutic strategy, and MCL1 is a critical antiapoptotic oncoprotein. Armed with novel MCL1 inhibitors and the potent BCL2 inhibitor venetoclax, it may be possible to selectively induce apoptosis by combining or thoughtfully sequencing these inhibitors based on a rational evaluation of AML.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacologia , Leucemia Mieloide/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Pirazinas/farmacologia , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Doença Aguda , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Células HL-60 , Humanos , Indóis/química , Células K562 , Leucemia Mieloide/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirazinas/química , Pirazóis/química , Células THP-1 , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Chronic inflammation plays a crucial role in the pathogenesis of obesity and insulin resistance. However, the primary mediators that affect energy homeostasis remain ill defined. Here, we report an unexpected role for immediate early response gene X-1 (IEX-1), a downstream target of NF-κB, in energy metabolism. We found that IEX-1 expression was highly induced in white adipose tissue (WAT) in both epidydmal and subcutaneous depots but not in interscapular brown adipose tissue (BAT) in mice fed a high fat diet (HFD). Null mutation of IEX-1 protected mice against HFD-induced adipose and hepatic inflammation, hepatic steatosis, and insulin resistance. Unexpectedly, IEX-1 knockout (IEX-1(-/-)) mice gained markedly less weight on HFD for 20 weeks as compared to wild-type (WT) littermates (37 ± 3 versus 48 ± 2 gm) due to increased energy expenditure. Mechanistically, we showed that IEX-1 deficiency induced browning and activated thermogenic genes program in WAT but not in BAT by promoting alternative activation of adipose macrophages. Consequently, IEX-1(-/-) mice exhibited enhanced thermogenesis (24 ± 0.1 versus 22 ± 0.1 kcal/hour/kg in WT mice) explaining increased energy expenditure and lean phenotype in these mice. In conclusion, the present study suggests that IEX-1 is a novel physiological regulator of energy homeostasis via its action in WAT.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Dieta Hiperlipídica , Proteínas Imediatamente Precoces/genética , Obesidade/etiologia , Animais , Calorimetria , Metabolismo Energético , Fígado Gorduroso/etiologia , Expressão Gênica , Teste de Tolerância a Glucose , Proteínas Imediatamente Precoces/deficiência , Imuno-Histoquímica , Imunofenotipagem , Insulina/sangue , Resistência à Insulina , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/metabolismo , TermogêneseRESUMO
BACKGROUND: Liver regeneration is clinically of major importance in the setting of liver injury, resection or transplantation. We have demonstrated that the NF-κB inhibitory protein A20 significantly improves recovery of liver function and mass following extended liver resection (LR) in mice. In this study, we explored the Systems Biology modulated by A20 following extended LR in mice. METHODOLOGY AND PRINCIPAL FINDINGS: We performed transcriptional profiling using Affymetrix-Mouse 430.2 arrays on liver mRNA retrieved from recombinant adenovirus A20 (rAd.A20) and rAd.ßgalactosidase treated livers, before and 24 hours after 78% LR. A20 overexpression impacted 1595 genes that were enriched for biological processes related to inflammatory and immune responses, cellular proliferation, energy production, oxidoreductase activity, and lipid and fatty acid metabolism. These pathways were modulated by A20 in a manner that favored decreased inflammation, heightened proliferation, and optimized metabolic control and energy production. Promoter analysis identified several transcriptional factors that implemented the effects of A20, including NF-κB, CEBPA, OCT-1, OCT-4 and EGR1. Interactive scale-free network analysis captured the key genes that delivered the specific functions of A20. Most of these genes were affected at basal level and after resection. We validated a number of A20's target genes by real-time PCR, including p21, the mitochondrial solute carriers SLC25a10 and SLC25a13, and the fatty acid metabolism regulator, peroxisome proliferator activated receptor alpha. This resulted in greater energy production in A20-expressing livers following LR, as demonstrated by increased enzymatic activity of cytochrome c oxidase, or mitochondrial complex IV. CONCLUSION: This Systems Biology-based analysis unravels novel mechanisms supporting the pro-regenerative function of A20 in the liver, by optimizing energy production through improved lipid/fatty acid metabolism, and down-regulated inflammation. These findings support pursuit of A20-based therapies to improve patients' outcomes in the context of extreme liver injury and extensive LR for tumor treatment or donation.