ABSTRACT
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.
Subject(s)
Antineoplastic Agents/administration & dosage , Benzimidazoles/administration & dosage , DNA Methylation/drug effects , Drug Carriers , Enzyme Inhibitors/administration & dosage , Epigenesis, Genetic/drug effects , Leukemia, Biphenotypic, Acute/drug therapy , Methyltransferases/antagonists & inhibitors , Administration, Oral , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Benzimidazoles/blood , Benzimidazoles/chemistry , Benzimidazoles/pharmacokinetics , Biological Availability , Chemistry, Pharmaceutical , Delayed-Action Preparations , Dogs , Drug Administration Schedule , Enzyme Inhibitors/blood , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Gene Expression Regulation, Leukemic/drug effects , Histone-Lysine N-Methyltransferase , Humans , Infusions, Intravenous , Injections, Subcutaneous , Leukemia, Biphenotypic, Acute/enzymology , Leukemia, Biphenotypic, Acute/genetics , Leukemia, Biphenotypic, Acute/pathology , Male , Methyltransferases/metabolism , Mice , Rats, Sprague-Dawley , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Enhancer of zeste homolog 2 (EZH2) and related EZH1 control gene expression and promote tumorigenesis via methylating histone H3 at lysine 27 (H3K27). These methyltransferases are ideal therapeutic targets due to their frequent hyperactive mutations and overexpression found in cancer, including hematopoietic malignancies. Here, we characterized a set of small molecules that allow pharmacologic manipulation of EZH2 and EZH1, which include UNC1999, a selective inhibitor of both enzymes, and UNC2400, an inactive analog compound useful for assessment of off-target effect. UNC1999 suppresses global H3K27 trimethylation/dimethylation (H3K27me3/2) and inhibits growth of mixed lineage leukemia (MLL)-rearranged leukemia cells. UNC1999-induced transcriptome alterations overlap those following knockdown of embryonic ectoderm development, a common cofactor of EZH2 and EZH1, demonstrating UNC1999's on-target inhibition. Mechanistically, UNC1999 preferentially affects distal regulatory elements such as enhancers, leading to derepression of polycomb targets including Cdkn2a. Gene derepression correlates with a decrease in H3K27me3 and concurrent gain in H3K27 acetylation. UNC2400 does not induce such effects. Oral administration of UNC1999 prolongs survival of a well-defined murine leukemia model bearing MLL-AF9. Collectively, our study provides the detailed profiling for a set of chemicals to manipulate EZH2 and EZH1 and establishes specific enzymatic inhibition of polycomb repressive complex 2 (PRC2)-EZH2 and PRC2-EZH1 by small-molecule compounds as a novel therapeutics for MLL-rearranged leukemia.
Subject(s)
Antineoplastic Agents/pharmacology , Leukemia, Biphenotypic, Acute/enzymology , Polycomb Repressive Complex 2/antagonists & inhibitors , Animals , Chromatin Immunoprecipitation , Disease Models, Animal , Enhancer of Zeste Homolog 2 Protein , Enzyme Inhibitors/pharmacology , Immunoblotting , Mass Spectrometry , Mice , Mice, Inbred BALB C , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Real-Time Polymerase Chain ReactionABSTRACT
Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.
Subject(s)
Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Leukemia, Biphenotypic, Acute/enzymology , Myeloid-Lymphoid Leukemia Protein/metabolism , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Humans , Intracellular Signaling Peptides and Proteins , Mice , Myeloid-Lymphoid Leukemia Protein/chemistry , Myeloid-Lymphoid Leukemia Protein/genetics , Oligopeptides/chemistry , Oligopeptides/physiology , Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Transcriptome/drug effectsSubject(s)
Base Sequence/genetics , Fusion Proteins, bcr-abl/genetics , Leukemia, Biphenotypic, Acute/genetics , Piperazines/administration & dosage , Protein Kinase Inhibitors/administration & dosage , Protein-Tyrosine Kinases/genetics , Pyrimidines/administration & dosage , Sequence Deletion , Thiazoles/administration & dosage , Benzamides , Dasatinib , Drug Resistance, Neoplasm , Female , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Leukemia, Biphenotypic, Acute/drug therapy , Leukemia, Biphenotypic, Acute/enzymology , Middle Aged , Protein Structure, Tertiary/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolismABSTRACT
Biphenotypic acute leukemia co-expressing T-lymphoid and myeloid markers is rare, accounting for less than 1% of acute leukemias. However, several clinical characteristics including male predominance, frequent lymphadenopathy and unfavorable outcome have been identified. Recurrence of monosomies 7p and/or 12p in T/myeloid biphenotypic acute leukemia has been reported. We treated a patient with T/myeloid biphenotypic acute leukemia showing clonal chromosomal and genetic abnormalities including dic(7;12)(p11;p11) and Fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication. Cytogenetic analysis of both bone marrow and lymph node cells disclosed that the patient's lymph node leukemia cells had chromosomal abnormalities in addition to dic(7;12). Our findings suggest that the leukemia cells of systemic lymphadenopathy had evolved as secondary cells from marrow leukemia cells. The patient was successfully treated with induction chemotherapy for acute myeloid leukemia followed by allogeneic bone marrow transplantation.
Subject(s)
Biomarkers, Tumor/genetics , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 7/genetics , Gene Duplication , Leukemia, Biphenotypic, Acute/diagnosis , Leukemia, Biphenotypic, Acute/enzymology , fms-Like Tyrosine Kinase 3/metabolism , Base Sequence , Biomarkers, Tumor/metabolism , Biopsy , Humans , Leukemia, Biphenotypic, Acute/genetics , Leukemia, Biphenotypic, Acute/surgery , Male , Middle Aged , Molecular Sequence Data , fms-Like Tyrosine Kinase 3/geneticsABSTRACT
The objective of this study was to investigate the expression and function of indoleamine 2, 3-dioxygenase (IDO) in leukemia. The IDO expressions in human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia (ALL) were detected by immunofluorescence staining. Constructed leukemia mouse model was used to observe whether the IDO inhibitor, 1-methyl tryptophan (1-MT), has any effect in treating leukemia. The experimental group were fed with 1-MT solution every day while the mice in control group had no further treatment. The results showed that the average ratios of IDO expression were 29.4 +/- 11.2% in M(5) patients and 24.7 +/- 7.96% in ALL patients respectively. After statistical test, IDO expression level in leukemia cells was significantly higher than that of normal mononuclear cells. The tumor decreased gradually in mice treated with 1-MT. At the terminal point of the experiment (88 days after vaccination), the average survival time in the experimental group was 42.3 days while the mice in control group only lived 15.1 days in average, which difference was statistically significant (P < 0.05). Some of the leukemia mice in the experimental group long-term survived without tumor (more than three months after vaccination). It is concluded that human acute monocyte leukemia (M(5)) and acute lymphocyte leukemia (ALL) express IDO, and both can be treated by 1-MT in mice.
Subject(s)
Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Leukemia, Biphenotypic, Acute/enzymology , Leukemia, Monocytic, Acute/enzymology , Tryptophan/analogs & derivatives , Adolescent , Adult , Animals , Child , Female , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/antagonists & inhibitors , Leukemia L1210/drug therapy , Leukemia, Biphenotypic, Acute/drug therapy , Leukemia, Monocytic, Acute/drug therapy , Male , Mice , Mice, Inbred DBA , Middle Aged , Tryptophan/therapeutic use , Young AdultABSTRACT
Accurate identification and classification of leukemic blast cells is a very important prerequisite of the precise diagnosis of acute leukemia and has a great impact on therapy and prognosis. The purpose of this review is to consider, in the broad sense of the word, the present possibilities and limitations of enzyme cytochemistry and to emphasize how cytochemistry may contribute, on integration with the other methods of study, to the final classification and differential diagnosis of acute leukemia, a highly variable hematological disorder. In this review, the role of conventional enzyme cytochemistry, either dominant or subsidiary, in the discrimination of acute leukemia subtypes is discussed. The survey confirms the absolute necessity of immunologic marker analysis in the accurate diagnosis of acute lymphoblastic leukemia, undifferentiated or minimally differentiated leukemia and mixed-lineage leukemia because in these cases, the cytochemical evaluation provides insufficiently relevant information regarding blast cell origin, specificity of leukemia subtypes and the discrete stages of leukemic cell maturation. On the other hand, cytochemical investigation is appreciated to be dominant over immunophenotyping in characterizing acute myeloid leukemia, because of the lack of specificity of the majority of immunological markers against myeloid antigens and, because of the availability of standardized and sufficiently specific cytochemical reactions. The cytogenetic, molecular biological and electron microscopic studies mentioned in this review supplement the important information for correct differential diagnosis of acute leukemia. The prognostic impact of enzyme cytochemistry in correlation to other methods is evaluated.
Subject(s)
Histocytochemistry/methods , Leukemia/diagnosis , Leukemia/pathology , Acute Disease/classification , Humans , Leukemia/classification , Leukemia/enzymology , Leukemia, Biphenotypic, Acute/classification , Leukemia, Biphenotypic, Acute/diagnosis , Leukemia, Biphenotypic, Acute/enzymology , Leukemia, Biphenotypic, Acute/pathology , Leukemia, Lymphoid/classification , Leukemia, Lymphoid/diagnosis , Leukemia, Lymphoid/enzymology , Leukemia, Lymphoid/pathology , Leukemia, Myeloid/classification , Leukemia, Myeloid/diagnosis , Leukemia, Myeloid/enzymology , Leukemia, Myeloid/pathologyABSTRACT
Seven adult patients with untreated acute lymphocytic leukemia (ALL) who manifested 5% to 40% myeloperoxidase (MPO)-positive blasts by electron microscopy (EM) are reported. Six patients had an L2 morphology, and one had an L1 morphology by the French-American-British (FAB) classification. The immunophenotype was T cell in four patients. Molecular analysis showed rearrangement of the immunoglobulin JH in four patients, three of them also having rearrangement of the T-cell receptor beta or gamma. Induction chemotherapy with vincristine-doxorubicin-dexamethasone (VAD) produced a complete remission in five of six patients (83%). Our findings suggest the existence of a previously undescribed subtype of mixed-lineage leukemia, which by morphology and immunophenotype often appears as T-cell ALL but exhibits MPO-positive blasts by EM.