RESUMEN
BACKGROUND: The combination of ivosidenib - an inhibitor of mutant isocitrate dehydrogenase 1 (IDH1) - and azacitidine showed encouraging clinical activity in a phase 1b trial involving patients with newly diagnosed IDH1-mutated acute myeloid leukemia. METHODS: In this phase 3 trial, we randomly assigned patients with newly diagnosed IDH1-mutated acute myeloid leukemia who were ineligible for intensive induction chemotherapy to receive oral ivosidenib (500 mg once daily) and subcutaneous or intravenous azacitidine (75 mg per square meter of body-surface area for 7 days in 28-day cycles) or to receive matched placebo and azacitidine. The primary end point was event-free survival, defined as the time from randomization until treatment failure (i.e., the patient did not have complete remission by week 24), relapse from remission, or death from any cause, whichever occurred first. RESULTS: The intention-to-treat population included 146 patients: 72 in the ivosidenib-and-azacitidine group and 74 in the placebo-and-azacitidine group. At a median follow-up of 12.4 months, event-free survival was significantly longer in the ivosidenib-and-azacitidine group than in the placebo-and-azacitidine group (hazard ratio for treatment failure, relapse from remission, or death, 0.33; 95% confidence interval [CI], 0.16 to 0.69; P = 0.002). The estimated probability that a patient would remain event-free at 12 months was 37% in the ivosidenib-and-azacitidine group and 12% in the placebo-and-azacitidine group. The median overall survival was 24.0 months with ivosidenib and azacitidine and 7.9 months with placebo and azacitidine (hazard ratio for death, 0.44; 95% CI, 0.27 to 0.73; P = 0.001). Common adverse events of grade 3 or higher included febrile neutropenia (28% with ivosidenib and azacitidine and 34% with placebo and azacitidine) and neutropenia (27% and 16%, respectively); the incidence of bleeding events of any grade was 41% and 29%, respectively. The incidence of infection of any grade was 28% with ivosidenib and azacitidine and 49% with placebo and azacitidine. Differentiation syndrome of any grade occurred in 14% of the patients receiving ivosidenib and azacitidine and 8% of those receiving placebo and azacitidine. CONCLUSIONS: Ivosidenib and azacitidine showed significant clinical benefit as compared with placebo and azacitidine in this difficult-to-treat population. Febrile neutropenia and infections were less frequent in the ivosidenib-and-azacitidine group than in the placebo-and-azacitidine group, whereas neutropenia and bleeding were more frequent in the ivosidenib-and-azacitidine group. (Funded by Agios Pharmaceuticals and Servier Pharmaceuticals; AGILE ClinicalTrials.gov number, NCT03173248.).
Asunto(s)
Antineoplásicos , Azacitidina , Leucemia Mieloide Aguda , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Azacitidina/administración & dosificación , Azacitidina/efectos adversos , Azacitidina/uso terapéutico , Neutropenia Febril/inducido químicamente , Glicina/análogos & derivados , Humanos , Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucopenia/inducido químicamente , Piridinas/administración & dosificación , Piridinas/efectos adversos , Piridinas/uso terapéutico , RecurrenciaRESUMEN
Pinometostat (EPZ-5676) is a first-in-class small-molecule inhibitor of the histone methyltransferase disrupter of telomeric silencing 1-like (DOT1L). In this phase 1 study, pinometostat was evaluated for safety and efficacy in adult patients with advanced acute leukemias, particularly those involving mixed lineage leukemia (MLL) gene rearrangements (MLL-r) resulting from 11q23 translocations. Fifty-one patients were enrolled into 6 dose-escalation cohorts (n = 26) and 2 expansion cohorts (n = 25) at pinometostat doses of 54 and 90 mg/m2 per day by continuous intravenous infusion in 28-day cycles. Because a maximum tolerated dose was not established in the dose-escalation phase, the expansion doses were selected based on safety and clinical response data combined with pharmacodynamic evidence of reduction in H3K79 methylation during dose escalation. Across all dose levels, plasma pinometostat concentrations increased in an approximately dose-proportional fashion, reaching an apparent steady-state by 4-8 hours after infusion, and rapidly decreased following treatment cessation. The most common adverse events, of any cause, were fatigue (39%), nausea (39%), constipation (35%), and febrile neutropenia (35%). Overall, 2 patients, both with t(11;19), experienced complete remission at 54 mg/m2 per day by continuous intravenous infusion, demonstrating proof of concept for delivering clinically meaningful responses through targeting DOT1L using the single agent pinometostat in MLL-r leukemia patients. Administration of pinometostat was generally safe, with the maximum tolerated dose not being reached, although efficacy as a single agent was modest. This study demonstrates the therapeutic potential for targeting DOT1L in MLL-r leukemia and lays the groundwork for future combination approaches in this patient population. This clinical trial is registered at www.clinicaltrials.gov as NCT01684150.
Asunto(s)
Antineoplásicos/uso terapéutico , Bencimidazoles/uso terapéutico , Histonas/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Metiltransferasas/antagonistas & inhibidores , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Bencimidazoles/efectos adversos , Femenino , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Metilación/efectos de los fármacos , Metiltransferasas/metabolismo , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE: ARID1A is commonly mutated in pancreatic ductal adenocarcinoma (PDAC), but the functional effects of ARID1A mutations in the pancreas are unclear. Understanding the molecular mechanisms that drive PDAC formation may lead to novel therapies. DESIGN: Concurrent conditional Arid1a deletion and Kras activation mutations were modelled in mice. Small-interfering RNA (siRNA) and CRISPR/Cas9 were used to abrogate ARID1A in human pancreatic ductal epithelial cells. RESULTS: We found that pancreas-specific Arid1a loss in mice was sufficient to induce inflammation, pancreatic intraepithelial neoplasia (PanIN) and mucinous cysts. Concurrent Kras activation accelerated the development of cysts that resembled intraductal papillary mucinous neoplasm. Lineage-specific Arid1a deletion confirmed compartment-specific tumour-suppressive effects. Duct-specific Arid1a loss promoted dilated ducts with occasional cyst and PDAC formation. Heterozygous acinar-specific Arid1a loss resulted in accelerated PanIN and PDAC formation with worse survival. RNA-seq showed that Arid1a loss induced gene networks associated with Myc activity and protein translation. ARID1A knockdown in human pancreatic ductal epithelial cells induced increased MYC expression and protein synthesis that was abrogated with MYC knockdown. ChIP-seq against H3K27ac demonstrated an increase in activated enhancers/promoters. CONCLUSIONS: Arid1a suppresses pancreatic neoplasia in a compartment-specific manner. In duct cells, this process appears to be associated with MYC-facilitated protein synthesis.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Animales , Carcinoma Ductal Pancreático/metabolismo , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Humanos , Ratones , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de TranscripciónRESUMEN
Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute myeloid leukemia and portend a poor prognosis; thus, new therapeutic strategies are needed. The likely mechanism by which DNMT3A loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes; however, our current understanding is incomplete. We observed that murine hematopoietic stem cells (HSCs) in which Dnmt3a had been conditionally deleted markedly overexpress the histone 3 lysine 79 (H3K79) methyltransferase, Dot1l. We demonstrate that Dnmt3a(-/-) HSCs have increased H3K79 methylation relative to wild-type (WT) HSCs, with the greatest increases noted at DNA methylation canyons, which are regions highly enriched for genes dysregulated in leukemia and prone to DNA methylation loss with Dnmt3a deletion. These findings led us to explore DOT1L as a therapeutic target for the treatment of DNMT3A-mutant AML. We show that pharmacologic inhibition of DOT1L resulted in decreased expression of oncogenic canyon-associated genes and led to dose- and time-dependent inhibition of proliferation, induction of apoptosis, cell-cycle arrest, and terminal differentiation in DNMT3A-mutant cell lines in vitro. We show in vivo efficacy of the DOT1L inhibitor EPZ5676 in a nude rat xenograft model of DNMT3A-mutant AML. DOT1L inhibition was also effective against primary patient DNMT3A-mutant AML samples, reducing colony-forming capacity (CFC) and inducing terminal differentiation in vitro. These studies suggest that DOT1L may play a critical role in DNMT3A-mutant leukemia. With pharmacologic inhibitors of DOT1L already in clinical trials, DOT1L could be an immediately actionable therapeutic target for the treatment of this poor prognosis disease.
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ADN (Citosina-5-)-Metiltransferasas/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Terapia Molecular Dirigida , Mutación/genética , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina/uso terapéutico , Animales , Apoptosis , Puntos de Control del Ciclo Celular , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN Metiltransferasa 3A , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Lisina/metabolismo , Metilación , Ratones Endogámicos C57BL , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Ratas , Factores de Tiempo , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6-transformed cells as well as the human MLL-AF6-positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.
Asunto(s)
Transformación Celular Neoplásica/genética , N-Metiltransferasa de Histona-Lisina/genética , Cinesinas/genética , Metiltransferasas/fisiología , Proteína de la Leucemia Mieloide-Linfoide/genética , Miosinas/genética , Proteínas de Fusión Oncogénica/genética , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/fisiología , Lisina/metabolismo , Metiltransferasas/antagonistas & inhibidores , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Compuestos de Fenilurea/farmacologíaRESUMEN
Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.
Asunto(s)
Antineoplásicos/farmacología , Bencimidazoles/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Leucemia/genética , Leucemia/terapia , Metiltransferasas/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Metilación de ADN , Relación Dosis-Respuesta a Droga , Femenino , Histona Metiltransferasas , Histonas/metabolismo , Humanos , Trasplante de Neoplasias , Conformación Proteica , Ratas , Ratas DesnudasRESUMEN
EPZ-5676 [(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol], a small-molecule inhibitor of the protein methyltransferase DOT1L, is currently under clinical investigation for acute leukemias bearing MLL-rearrangements (MLL-r). In this study, we evaluated EPZ-5676 in combination with standard of care (SOC) agents for acute leukemias as well as other chromatin-modifying drugs in cellular assays with three human acute leukemia cell lines: MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), and SKM-1 (non-MLL-r). Studies were performed to evaluate the antiproliferative effects of EPZ-5676 combinations in a cotreatment model in which the second agent was added simultaneously with EPZ-5676 at the beginning of the assay, or in a pretreatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. EPZ-5676 was found to act synergistically with the acute myeloid leukemia (AML) SOC agents cytarabine or daunorubicin in MOLM-13 and MV4-11 MLL-r cell lines. EPZ-5676 is selective for MLL-r cell lines as demonstrated by its lack of effect either alone or in combination in the nonrearranged SKM-1 cell line. In MLL-r cells, the combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the chemotherapeutic agents, suggesting that EPZ-5676 sets up a durable, altered chromatin state that enhances the chemotherapeutic effects. Our evaluation of EPZ-5676 in conjunction with other chromatin-modifying drugs also revealed a consistent combination benefit, including synergy with DNA hypomethylating agents. These results indicate that EPZ-5676 is highly efficacious as a single agent and synergistically acts with other chemotherapeutics, including AML SOC drugs and DNA hypomethylating agents in MLL-r cells.
Asunto(s)
Antineoplásicos/administración & dosificación , Bencimidazoles/administración & dosificación , Proliferación Celular/efectos de los fármacos , Inhibidores de Crecimiento/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Metiltransferasas/antagonistas & inhibidores , Línea Celular Tumoral , Sinergismo Farmacológico , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia Mieloide Aguda/patología , Metilación/efectos de los fármacos , Metiltransferasas/metabolismoRESUMEN
(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of distribution 2-3 fold higher than total body water. EPZ-5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half-life (t1/2 ) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro-in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu -corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ-5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated product (M4) as well as an N-oxide (M6).
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Antineoplásicos/farmacocinética , Bencimidazoles/farmacocinética , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Metiltransferasas/antagonistas & inhibidores , Animales , Antineoplásicos/sangre , Bencimidazoles/sangre , Proteínas Sanguíneas/metabolismo , Perros , Hepatocitos/metabolismo , Células de Riñón Canino Madin Darby , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Permeabilidad , Ratas Sprague-DawleyRESUMEN
Approximately 20% of acute myeloid leukemia (AML) patients carry mutations in IDH1 or IDH2 that result in over-production of the oncometabolite D-2-hydroxyglutarate (2-HG). Small molecule inhibitors that block 2-HG synthesis can induce complete morphological remission; however, almost all patients eventually acquire drug resistance and relapse. Using a multi-allelic mouse model of IDH1-mutant AML, we demonstrate that the clinical IDH1 inhibitor AG-120 (ivosidenib) exerts cell-type-dependent effects on leukemic cells, promoting delayed disease regression. Although single-agent AG-120 treatment does not fully eradicate the disease, it increases cycling of rare leukemia stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitizes IDH1-mutant AML to azacitidine, with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non-genetic heterogeneity on treatment response and provide a mechanistic rationale for the observed combinatorial effect of AG-120 and azacitidine in patients.
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Isocitrato Deshidrogenasa , Leucemia Mieloide Aguda , Animales , Azacitidina/farmacología , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Ratones , Mutación/genética , Células Madre/metabolismoRESUMEN
PURPOSE: Ivosidenib is an oral inhibitor of the mutant isocitrate dehydrogenase 1 (IDH1) enzyme, approved for treatment of IDH1-mutant (mIDH1) acute myeloid leukemia (AML). Preclinical work suggested that addition of azacitidine to ivosidenib enhances mIDH1 inhibition-related differentiation and apoptosis. PATIENTS AND METHODS: This was an open-label, multicenter, phase Ib trial comprising dose-finding and expansion stages to evaluate safety and efficacy of combining oral ivosidenib 500 mg once daily continuously with subcutaneous azacitidine 75 mg/m2 on days 1-7 in 28-day cycles in patients with newly diagnosed mIDH1 AML ineligible for intensive induction chemotherapy (ClinicalTrials.gov identifier: NCT02677922). RESULTS: Twenty-three patients received ivosidenib plus azacitidine (median age, 76 years; range, 61-88 years). Treatment-related grade ≥ 3 adverse events occurring in > 10% of patients were neutropenia (22%), anemia (13%), thrombocytopenia (13%), and electrocardiogram QT prolongation (13%). Adverse events of special interest included all-grade IDH differentiation syndrome (17%), all-grade electrocardiogram QT prolongation (26%), and grade ≥ 3 leukocytosis (9%). Median treatment duration was 15.1 months (range, 0.3-32.2 months); 10 patients remained on treatment as of February 19, 2019. The overall response rate was 78.3% (18/23 patients; 95% CI, 56.3% to 92.5%), and the complete remission rate was 60.9% (14/23 patients; 95% CI, 38.5% to 80.3%). With median follow-up of 16 months, median duration of response in responders had not been reached. The 12-month survival estimate was 82.0% (95% CI, 58.8% to 92.8%). mIDH1 clearance in bone marrow mononuclear cells by BEAMing (beads, emulsion, amplification, magnetics) digital polymerase chain reaction was seen in 10/14 patients (71.4%) achieving complete remission. CONCLUSION: Ivosidenib plus azacitidine was well tolerated, with an expected safety profile consistent with monotherapy with each agent. Responses were deep and durable, with most complete responders achieving mIDH1 mutation clearance.
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Azacitidina/administración & dosificación , Inhibidores Enzimáticos/administración & dosificación , Glicina/análogos & derivados , Leucemia Mieloide Aguda/tratamiento farmacológico , Piridinas/administración & dosificación , Anciano , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Quimioterapia Combinada , Femenino , Glicina/administración & dosificación , Humanos , Isocitrato Deshidrogenasa/genética , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana EdadAsunto(s)
Antineoplásicos/farmacología , Duplicación de Gen , Leucemia/genética , Metiltransferasas/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Secuencias Repetidas en Tándem , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patologíaRESUMEN
Diffuse large B-cell lymphoma (DLBCL) patients are typically treated with immunochemotherapy containing rituximab (rituximab, cyclophosphamide, hydroxydaunorubicin-vincristine (Oncovin), and prednisone [R-CHOP]); however, prognosis is extremely poor if R-CHOP fails. To identify genetic mechanisms contributing to primary or acquired R-CHOP resistance, we performed target-panel sequencing of 135 relapsed/refractory DLBCLs (rrDLBCLs), primarily comprising circulating tumor DNA from patients on clinical trials. Comparison with a metacohort of 1670 diagnostic DLBCLs identified 6 genes significantly enriched for mutations upon relapse. TP53 and KMT2D were mutated in the majority of rrDLBCLs, and these mutations remained clonally persistent throughout treatment in paired diagnostic-relapse samples, suggesting a role in primary treatment resistance. Nonsense and missense mutations affecting MS4A1, which encodes CD20, are exceedingly rare in diagnostic samples but show recurrent patterns of clonal expansion following rituximab-based therapy. MS4A1 missense mutations within the transmembrane domains lead to loss of CD20 in vitro, and patient tumors harboring these mutations lacked CD20 protein expression. In a time series from a patient treated with multiple rounds of therapy, tumor heterogeneity and minor MS4A1-harboring subclones contributed to rapid disease recurrence, with MS4A1 mutations as founding events for these subclones. TP53 and KMT2D mutation status, in combination with other prognostic factors, may be used to identify high-risk patients prior to R-CHOP for posttreatment monitoring. Using liquid biopsies, we show the potential to identify tumors with loss of CD20 surface expression stemming from MS4A1 mutations. Implementation of noninvasive assays to detect such features of acquired treatment resistance may allow timely transition to more effective treatment regimens.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Linfoma de Células B Grandes Difuso , Anticuerpos Monoclonales de Origen Murino , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Rituximab/uso terapéuticoRESUMEN
The Editor-in-Chief would like to alert readers that the ownership of some of the data presented in this article [1].
RESUMEN
PURPOSE: We sought to determine the mechanism of an exceptional response in a patient diagnosed with a SMARCB1/INI1-negative chordoma treated with tazemetostat, an EZH2 inhibitor, and followed by radiotherapy.Patient and Methods: In an attempt to investigate the mechanism behind this apparent abscopal effect, we interrogated tumor tissues obtained over the clinical course. We utilized next-generation sequencing, standard IHC, and employed a novel methodology of multiplex immunofluorescence analysis. RESULTS: We report an exceptional and durable response (2+ years) in a patient with SMARCB1-deleted, metastatic, poorly differentiated chordoma, a lethal disease with an overall survival of 6 months. The patient was treated for 4 weeks with tazemetostat, an EZH2 inhibitor, in a phase II clinical trial. At the time of progression she underwent radiation to the primary site and unexpectedly had a complete response at distant metastatic sites. We evaluated baseline and on-treatment tumor biopsies and demonstrate that tazemetostat resulted in pharmacodynamic inhibition of EZH2 as seen by decrease in histone trimethylation at H3K27. Tazemetostat resulted in a significant increase in intratumoral and stromal infiltration by proliferative (high Ki-67), CD8+ T cells, FoxP3+ regulatory T cells, and immune cells expressing checkpoint regulators PD-1 and LAG-3. These changes were pronounced in the stroma. CONCLUSIONS: These observations are the first demonstration in patient samples confirming that EZH2 inhibition can promote a sustained antitumor response that ultimately leads to T-cell exhaustion and checkpoint activation. This suggests that targeted alteration of the epigenetic landscape may sensitize some tumors to checkpoint inhibitors.
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Cordoma/etiología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Inmunomodulación , Proteína SMARCB1/deficiencia , Adulto , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biopsia , Línea Celular Tumoral , Cordoma/metabolismo , Cordoma/patología , Cordoma/terapia , Terapia Combinada , Modelos Animales de Enfermedad , Exones , Femenino , Humanos , Inmunohistoquímica , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Ratones , Mutación , Clasificación del Tumor , Estadificación de Neoplasias , Radioterapia , Proteína SMARCB1/metabolismo , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Leiomyosarcoma (LMS) is one of the most frequent soft tissue sarcoma subtypes and is characterized by a consistent deregulation of the PI3K/mTOR pathway. Cancer stem cells (CSCs) have been poorly studied in soft tissue sarcomas. In this study, we aimed to evaluate the association between CSCs, the outcome of LMS patients, and the resistance to PI3K/mTOR pathway inhibition. METHODS: We investigated the relationships between aldehyde dehydrogenase 1 (ALDH1) expression, a cancer stem cell marker, and the outcome of LMS patients in two independent cohorts. We assessed the impact of CSCs in resistance to PI3K/mTOR pathway inhibition using LMS cell lines, a xenograft mouse model, and human tumor samples. RESULTS: We found that enhanced ALDH1 activity is a hallmark of LMS stem cells and is an independent prognostic factor. We also identified that secondary resistance to PI3K/mTOR pathway inhibition was associated with the expansion of LMS CSCs. Interestingly, we found that EZH2 inhibition, a catalytic component of polycomb repressive complex which plays a critical role in stem cell maintenance, restored sensitivity to PI3K/mTOR pathway inhibition. Importantly, we confirmed the clinical relevance of our findings by analyzing tumor samples from patients who showed secondary resistance after treatment with a PI3Kα inhibitor. CONCLUSIONS: Altogether, our findings suggest that CSCs have a strong impact on the outcome of patients with LMS and that combining PI3K/mTOR and EZH2 inhibitors may represent a promising strategy in this setting.
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Resistencia a Antineoplásicos , Leiomiosarcoma/metabolismo , Leiomiosarcoma/patología , Células Madre Neoplásicas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Adulto , Anciano , Familia de Aldehído Deshidrogenasa 1/metabolismo , Animales , Línea Celular Tumoral , Estudios de Cohortes , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Femenino , Humanos , Imidazoles/farmacología , Imidazoles/uso terapéutico , Leiomiosarcoma/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Pronóstico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinolinas/farmacología , Quinolinas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DOT1L is a protein methyltransferase involved in the development and maintenance of MLL-rearranged (MLL-r) leukemia through its ectopic methylation of histones associated with well-characterized leukemic genes. Pinometostat (EPZ-5676), a selective inhibitor of DOT1L, is in clinical development in relapsed/refractory acute leukemia patients harboring rearrangements of the MLL gene. The observation of responses and subsequent relapses in the adult trial treating MLL-r patients motivated preclinical investigations into potential mechanisms of pinometostat treatment-emergent resistance (TER) in cell lines confirmed to have MLL-r. TER was achieved in five MLL-r cell lines, KOPN-8, MOLM-13, MV4-11, NOMO-1, and SEM. Two of the cell lines, KOPN-8 and NOMO-1, were thoroughly characterized to understand the mechanisms involved in pinometostat resistance. Unlike many other targeted therapies, resistance does not appear to be achieved through drug-induced selection of mutations of the target itself. Instead, we identified both drug efflux transporter dependent and independent mechanisms of resistance to pinometostat. In KOPN-8 TER cells, increased expression of the drug efflux transporter ABCB1 (P-glycoprotein, MDR1) was the primary mechanism of drug resistance. In contrast, resistance in NOMO-1 cells occurs through a mechanism other than upregulation of a specific efflux pump. RNA-seq analysis performed on both parental and resistant KOPN-8 and NOMO-1 cell lines supported two unique candidate pathway mechanisms that may explain the pinometostat resistance observed in these cell lines. These results are the first demonstration of TER models of the DOT1L inhibitor pinometostat and may provide useful tools for investigating clinical resistance. Mol Cancer Ther; 16(8); 1669-79. ©2017 AACR.
Asunto(s)
Bencimidazoles/uso terapéutico , Resistencia a Antineoplásicos , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/genética , Leucemia/tratamiento farmacológico , Leucemia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Bencimidazoles/farmacología , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.
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N-Metiltransferasa de Histona-Lisina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Metiltransferasas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Femenino , N-Metiltransferasa de Histona-Lisina/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Metiltransferasas/genética , Ratones , Ratones Noqueados , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Oncogénicas/genética , Transactivadores , Proteínas Supresoras de Tumor/genéticaRESUMEN
Protein methyltransferases are emerging as promising drug targets for therapeutic intervention in human cancers. Pinometostat (EPZ-5676) is a small molecule inhibitor of the DOT1L enzyme, a histone methyltransferase that methylates lysine 79 of histone H3. DOT1L activity is dysregulated in the pathophysiology of rearranged mixed lineage leukemia (MLL-r). Pinometostat is currently in Phase 1 clinical trials in relapsed refractory acute leukemia patients and is administered as a continuous IV infusion (CIV). The studies herein investigated alternatives to CIV administration of pinometostat to improve patient convenience. Various sustained release technologies were considered, and based on the required dose size as well as practical considerations, subcutaneous (SC) bolus administration of a solution formulation was selected for further evaluation in preclinical studies. SC administration offered improved exposure and complete bioavailability of pinometostat relative to CIV and oral administration. These findings warranted further evaluation in rat xenograft models of MLL-r leukemia. SC dosing in xenograft models demonstrated inhibition of MLL-r tumor growth and inhibition of pharmacodynamic markers of DOT1L activity. However, a dosing frequency of thrice daily (t.i.d) was required in these studies to elicit optimal inhibition of DOT1L target genes and tumor growth inhibition. Development of an extended release formulation may prove useful in the further optimization of the SC delivery of pinometostat, moving towards a more convenient dosing paradigm for patients.
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Antineoplásicos/administración & dosificación , Bencimidazoles/administración & dosificación , Metilación de ADN/efectos de los fármacos , Portadores de Fármacos , Inhibidores Enzimáticos/administración & dosificación , Epigénesis Genética/efectos de los fármacos , Leucemia Bifenotípica Aguda/tratamiento farmacológico , Metiltransferasas/antagonistas & inhibidores , Administración Oral , Animales , Antineoplásicos/sangre , Antineoplásicos/química , Antineoplásicos/farmacocinética , Bencimidazoles/sangre , Bencimidazoles/química , Bencimidazoles/farmacocinética , Disponibilidad Biológica , Química Farmacéutica , Preparaciones de Acción Retardada , Perros , Esquema de Medicación , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina , Humanos , Infusiones Intravenosas , Inyecciones Subcutáneas , Leucemia Bifenotípica Aguda/enzimología , Leucemia Bifenotípica Aguda/genética , Leucemia Bifenotípica Aguda/patología , Masculino , Metiltransferasas/metabolismo , Ratones , Ratas Sprague-Dawley , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DOT1L is the human protein methyltransferase responsible for catalyzing the methylation of histone H3 on lysine 79 (H3K79). The ectopic activity of DOT1L, associated with the chromosomal translocation that is a universal hallmark of MLL-rearranged leukemia, is a required driver of leukemogenesis in this malignancy. Here, we present studies on the structure-activity relationship of aminonucleoside-based DOT1L inhibitors. Within this series, we find that improvements in target enzyme affinity and selectivity are driven entirely by diminution of the dissociation rate constant for the enzyme-inhibitor complex, leading to long residence times for the binary complex. The biochemical K(i) and residence times measured for these inhibitors correlate well with their effects on intracellular H3K79 methylation and MLL-rearranged leukemic cell killing. Crystallographic studies reveal a conformational adaptation mechanism associated with high-affinity inhibitor binding and prolonged residence time; these studies also suggest that conformational adaptation likewise plays a critical role in natural ligand interactions with the enzyme, hence, facilitating enzyme turnover. These results provide critical insights into the role of conformational adaptation in the enzymatic mechanism of catalysis and in pharmacologic intervention for DOT1L and other members of this enzyme class.
Asunto(s)
Inhibidores Enzimáticos/química , Metiltransferasas/antagonistas & inhibidores , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Cinética , Metilación , Metiltransferasas/metabolismo , Simulación del Acoplamiento Molecular , Nucleósidos/química , Nucleósidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.