Author(s): Lukasz P. Gondek
All cells in a single organism share the identical genetic information encoded in DNA, yet they differ tremendously in function. These variations in cellular fate are mainly dictated by the distinct transcriptional programs. Gene expression is orchestrated by epigenetic information that interconnects genome sequence and environmental/extracellular factors. This fine-tuning of cellular fate and function can happen at multiple levels: DNA modifications, post-translational modifications of histones, and higher-order chromatin structures.
DNA methylation is a process of covalent modification of cytosine at the DNA level that is usually associated with gene silencing. This process is catalyzed by one of the three enzymes: DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B). These repressive marks can be erased either by cell division or by enzymes ten-eleven translocation (TET) dioxygenases. Thus, DNMT and TET confer opposing effect on DNA methylation status. Somatic mutations in DNMT3A and TET2 are among the most frequently mutated genes in hematologic malignancies and premalignant clonal hematopoiesis.
Post-translational histone modifications include methylation, acetylation, phosphorylation, ubiquitination, and sumoylation of histones at different residues. The functional consequences of such modifications rely not only on the type of modification but also on the modified residues. Thus, the same chemical modification may have distinct effects on gene expression. The most common histone marks and their transcriptional effects are depicted in Figure 7.1.
Both DNA methylation and histone modifications participate in the third layer of epigenetic modification, that is, chromatin remodeling. Certain histone marks, particularly acetylation, result in chromatin relaxation (euchromatin) and active transcription. Conversely, deacetylation of histones is associated with chromatin compaction (heterochromatin) that blocks transcriptional machinery from binding to DNA and results in repression of gene expression (Figure 7.2).
Lysine-specific histone demethylase (LSD1) is a monoamine oxidase involved in demethylation of lysine 4 and 9 on histone H3 (H3K4/9). LSD1 is a component of several multiprotein complexes and can act both as a transcriptional repressor or activator. High expression of LSD1 has been found in AML stem cells. Preclinical studies suggested that AML with MLL and RUNX1 rearrangements as well as erythroleukemia may be particularly sensitive to LSD1 inhibition. MLL rearrangements are present in 5% to 10% of acute leukemia patients and indicate a very poor prognosis. MLL rearrangements result in an in-frame fusion of the N-terminal region of MLL to 1 of over 60 fusion partners that interact directly with DOT1L. DOT1L is a histone methyltransferase enzyme that catalyzes mono-, di-, or tri-methylation of H3K79 at MLL-target genes that results in activation of leukemogenic genes including HOXA9 and MEIS1. DOT1L inhibitor EPZ5676 has been tested in phase 1 clinical trials and showed modest activity in relapsed/refractory AML. Menin is a ubiquitously expressed nuclear protein that facilitates binding of MLL/MLL-FP to target genes such as HOXA genes. The disruption of this binding was shown to exert a strong antileukemic effect in preclinical models, especially in combination with DOT1L inhibition.
Bromodomain and extraterminal (BET) proteins, including BRD4, are “reader” proteins that bind to acetylated histones and recruit a number of chromatin modifiers and mediators of transcription. BRD4 after binding to acetylated histone recruits P-TEFb and CDK9, enabling transcript elongation by RNA polymerase II. Thus, BRD4 links histone acetylation with active transcription especially at the enhancers and promoters of such oncogenes as c-MYC and BCL2. ASXL1 frameshift mutations are relatively frequent in myeloid neoplasm. The aberrant interaction of truncated ASXL1 and BRD4 enhances the expression of genes involved in stem cell maintenance and differentiation. In preclinical models, ASXL1 mutated cells were explicitly sensitive to BET bromodomain inhibitors.
Somatic mutations, amplifications, and translocations involving genes that encode proteins responsible for epigenetic modifications of DNA have been recently found in myeloid malignancies. This includes genes involved in DNA methylation (TET2, DNMT3A, IDH1/2) and post-translational histone modifications (EZH2, ASXL1, KMT2A, DOT1L). In this chapter, we focused on the function of these proteins as well as available targeted therapies. The recent efforts to develop more selective and less toxic therapies focused on direct inhibition of mutated proteins or disruption of downstream pathways may result in restoration of normal epigenetic patterns and improvement in hematopoietic differentiation rather than direct cytotoxic effect on leukemic cells.