The ability of MLL to bind RUNX1 and methylate H3K4 at PU.1 regulatory regions is impaired by MDS/AML-associated RUNX1/AML1 mutations

Abstract

The mixed-lineage leukemia (MLL) H3K4 methyltransferase protein, and the heterodimeric RUNX1/CBF␤ transcription factor complex, are critical for definitive and adult hematopoiesis, and both are frequently targeted in human acute leukemia. We identified a physical and functional interaction between RUNX1 (AML1) and MLL and show that both are required to maintain the histone lysine 4 trimethyl mark (H3K4me3) at 2 critical regulatory regions of the AML1 target gene PU. IntroductionThe transcriptional regulation of hematopoiesis requires coordinate changes in gene expression to control the processes of stem cell self-renewal, differentiation, and maturation. The primary importance of transcriptional regulation in hematopoiesis is exemplified by human acute myelogenous leukemia, where recurrent chromosomal translocations are found that affect transcriptional regulators including transcription factors and histone modifying enzymes, such as AML1, CBF␤, RAR␣, TEL, MLL, MOZ, CBP, and p300. 1,2 Acute leukemias accompanied by MLL translocations have traditionally been thought of being distinct from the CBF leukemias (those associated with AML1 or CBF␤ translocations) at least in part because of their different gene expression profile, different prognosis, and different frequency of occurrence in de novo versus secondary AML. Yet mutations and translocations involving the mixed-lineage leukemia and AML1 genes are found in both AML and ALL, and it has been postulated that MLL and AML1/CBF␤ may both participate in regulating some common target genes. 3 The MLL gene was isolated as a common target of chromosomal translocations involving 11q23. [4][5][6] These translocations, which are observed in adult, childhood, and infant acute leukemias, fuse MLL with more than 60 different partner proteins. However, the most common translocations generate the MLL/AF6, MLL/ AF9, MLL/ENL(s), MLL/AF10, and the MLL/AF17 fusion proteins in AML, and the MLL/AF4, MLL/LAF4, MLL/5q31, and MLL/ENL(l) fusion proteins in B-ALL. 7 MLL is a functional ortholog of the Drosophila trithorax (trx) protein, 8 which is involved in maintaining epigenetic transcriptional memory at homeobox (Hox) gene loci. 9 The SET domain of MLL has histone methyltransferase activity 10 and MLL forms a multicomponent complex that specifically methylates lysine 4 on histone H3 (H3-K4), a modification typically associated with transcriptionally active regions of chromatin. The best-studied downstream targets of MLL (and trx) are the Hox genes, which control segment pecificity and cell fate in the developing embryo. 11 Targeted homozygous disruption of MLL in mice was embryonic lethal at day 10.5-14, 12 and similar to loss of trx function in flies, Hox gene expression was initiated but not maintained in these mice. 13 Another group, who targeted exons 12-14 of Mll, obtained a slight delay in lethality, 14 but both groups reported that Mll-deficient mice have defective yolk sac and fetal liver hematopoiesis. 15 Animals carrying a single normal Mll allele are phenotypically abnor...

The Runx-PU.1 pathway preserves normal and AML/ETO9a leukemic stem cells

“…42 Reducing PU.1 levels can induce AML in mice, 34 and low PU.1 levels have also been implicated mechanistically in specific forms of leukemia such as acute promyelocytic leukemia 43,44 and core-binding factor leukemia ( Figure 5A-C). [8][9][10][11]14 However, the observation that the Runx-PU.1 pathway is essential in HSC biology led us to investigate whether normal and leukemic stem cells might share the requirement of Runx-induced PU.1 for their stemness. As proof of principle, we disrupted the Runx-PU.1 axis in AML/ETO9a-induced leukemia and observed a dramatic decrease in leukemic stem cell function.…”

“…[3][4][5][6][7] In leukemia with chromosomal translocations, a dominant negative effect of the fusion protein and inactivation of the Runx downstream target PU.1 has been considered as a critical mechanism of leukemia development. [8][9][10][11][12][13][14][15] Runx1 knockout mice lack definitive hematopoiesis, 16 mainly because of the essential role of Runx1 in the endothelial-tohematopoietic cell transition during embryonic development. [17][18][19][20] However, studies of its function in adult hematopoietic stem cells (HSCs) have been inconsistent, with some demonstrating that Runx1 deficiency results in HSC defects 21,22 and others suggesting minimal impact on HSCs.…”

Disordered epigenetic regulation in MLL-related leukemia

“…Some transcription factors, such as Menin, LEDGF, HCF1/2, E2F, NFE2, p53, and c-Myb, have been shown to interact with MLL family members, which could lead to methylation of H3K4 in a locus-or DNA sequence-specific manner [16,17,[25][26][27][28][29]. A recent study by our group identified a physical and functional interaction between RUNX1 and MLL, and showed that both are required for maintenance of the H3K4me3 mark at two critical regulatory regions of the RUNX1 target gene PU.1 locus [30].…”

RUNX1 deficiency (familial platelet disorder with predisposition to myeloid leukemia, FPDMM)

“…Moreover, RUNX1 binds MLL and methylated H3K4 at PU.1-regulatory regions. RUNX1 mutations impair this interaction resulting in loss of the H3K4me3 mark within PU.1-regulatory regions, and decreased PU.1expression [31].…”