RESUMEN
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
Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Regulación Leucémica de la Expresión Génica , Genes ras , Complejo Represivo Polycomb 2/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Factor de Transcripción STAT3/genética , Animales , Modelos Animales de Enfermedad , Proteína Potenciadora del Homólogo Zeste 2/deficiencia , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Ratones , Ratones Transgénicos , Fosforilación , Complejo Represivo Polycomb 2/deficiencia , Complejo Represivo Polycomb 2/metabolismo , Células Precursoras de Linfocitos T/metabolismo , Células Precursoras de Linfocitos T/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
Genome scale sequencing in patients with cancer has revealed a lower frequency of genetic aberrations in hematologic disorders compared with most other malignancies, suggesting a prominent role for epigenetic mechanisms. In parallel, epigenetic modifiers that are altered in cancer play critical roles in normal hematopoietic development, influencing both self-renewal of hematopoietic stem cells and differentiation into the different lineages. In this review, we aim to compare the role of several key DNA or histone modifying enzymes and complexes in normal development and hematopoietic malignancies, including DNMT3A, TET2, IDH1, IDH2, MLL1, MLL4, DOT1L, PRC1/2 and WSHC1/NSD2/MMSET. Insights into their biological mechanisms led to the development of therapies designed to target mutant IDH1 and IDH2, DOT1L in MLL-rearranged leukemias and EZH2 in several cancer types including lymphomas. Inhibitors for these enzymes are currently in clinical trials.
Asunto(s)
Epigénesis Genética , Neoplasias Hematológicas/genética , Hematopoyesis/genética , Animales , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/fisiología , ADN Metiltransferasa 3A , Proteínas de Unión al ADN/genética , Dioxigenasas , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Isocitrato Deshidrogenasa/genética , Metiltransferasas/genética , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas del Grupo Polycomb/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genéticaRESUMEN
Polycomb repressive complex 2 (PRC2) is a chromatin regulator with central roles in development and cancer. The canonical function of PRC2 is the trimethylation of histone 3 on lysine residue 27. This epigenetic modification is associated with gene silencing. Both tumor suppressor and oncogenic functions have been reported for PRC2, depending on cellular context. In leukemia mediated by the leukemogenic fusion MLL-AF9, complete ablation of canonical PRC2 function by genetic inactivation of the core component embryonic ectoderm development (Eed) or by combined pharmacologic inhibition of the PRC2 methyltransferases EZH2 and EZH1 has a strong anti-leukemic effect, and this effect has been linked to de-repression of the PRC2 target locus Cdkn2a. We asked whether inactivation of Cdkn2a is sufficient to restore leukemic activity of Eed-inactivated MLL-AF9 leukemia cells, using combined genetic inactivation of Cdkn2a and Eed. We found that Cdkn2a inactivation partially rescues in vitro and in vivo growth of Eed-inactivated MLL-AF9 cells. However, the growth of Eed-null Cdkn2a-null MLL-AF9 cells in the absence of Cdkn2a remained severely compromised in vitro and in vivo, compared with that of their Eed-floxed Cdkn2a-null counterparts. RNA sequencing analysis revealed that several genes previously implicated in inefficient growth of MLL-AF9-transformed cells, including Gata2, Egr1, and Cdkn2b were de-repressed as a consequence of Eed inactivation. Furthermore, we found that direct binding targets of MLL fusion proteins are negatively enriched in Eed-inactivated Cdkn2a-null MLL-AF9-transformed cells. Our data indicate that interference with PRC2 function affects MLL-AF9-mediated leukemogenesis by both Cdkn2a-dependent and Cdkn2a-independent mechanisms.