Kathrin M. Bernt

Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor.

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.

MLL-Rearranged Leukemia Is Dependent on Aberrant H3K79 Methylation by DOT1L

The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.

Chromatin Modifying Enzymes as Modulators of Reprogramming

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l

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.

AF10 regulates progressive H3K79 methylation and HOX gene expression in diverse AML subtypes.

Homeotic (HOX) genes are dysregulated in multiple malignancies, including several AML subtypes. We demonstrate that H3K79 dimethylation (H3K79me2) is converted to monomethylation (H3K79me1) at HOX loci as hematopoietic cells mature, thus coinciding with a decrease in HOX gene expression. We show that H3K79 methyltransferase activity as well as H3K79me1-to-H3K79me2 conversion is regulated by the DOT1L cofactor AF10. AF10 inactivation reverses leukemia-associated epigenetic profiles, precludes abnormal HOXA gene expression, and impairs the transforming ability of MLL-AF9, MLL-AF6, and NUP98-NSD1 fusions-mechanistically distinct HOX-activating oncogenes. Furthermore, NUP98-NSD1-transformed cells are sensitive to small-molecule inhibition of DOT1L. Our findings demonstrate that pharmacological inhibition of the DOT1L/AF10 complex may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression.

Leukemia Stem Cells and Human Acute Lymphoblastic Leukemia

Leukemias and other cancers have been proposed to contain a subpopulation of cells that display characteristics of stem cells and maintain tumor growth. The fact that most anticancer therapy is directed against the bulk of the tumor, and possibly spares the cancer stem cells, may lie at the heart of treatment failures with conventional modalities. Leukemia stem cells are fairly well described for acute myeloid leukemia (AML), but their existence and relevance for acute lymphoblastic leukemia (ALL) is less clear. Several reports describe subpopulations with primitive phenotypes in clinical ALL samples. However, it has also been suggested that the majority of leukemic subfractions can propagate leukemia in the appropriate experimental setting, and that their hierarchical organization is less strict than in AML. In addition, it is uncertain whether cancer stem cells arise from malignant transformation of a tissue-specific stem cell, or from committed progenitors or differentiated cells that re-acquire a stem cell-like program. In common childhood ALL, current evidence points towards the cell of origin being a committed lymphoid progenitor. In this review, we highlight recent findings relating to the question of leukemia stem cells in ALL.

A role for DOT1L in MLL-rearranged leukemias

Leukemias harboring rearrangements of the MLL gene carry a poor prognosis. Over the past 6 years, it has become increasingly clear that fusions of MLL induce widespread epigenetic dysregulation that may mediate much of their transforming activity. The histone methyltransferase DOT1L, which methylates histone 3 on lysine 79 (H3K79) has received particular attention. Several MLL fusions may physically interact with DOT1L. Genome-wide H3K79 methylation profiles in MLL-rearranged leukemias are abnormal, and can serve to distinguish MLL-rearranged from other types of leukemias. Loss of H3K79 methylation affects expression of MLL-target loci and is detrimental to the leukemogenic activity of MLL-rearranged cells, suggesting that transformation in these leukemias is driven by a DOT1L dependent, aberrant epigenetic program. The ‘histone code hypothesis’ proposes that histone modifications, along with proteins that recognize, place and remove these marks, form a sophisticated regulatory network that directly control gene expression. Histone modifications may prime genes for rapid induction after signaling receptor engagement, coordinate individual genes to genetic programs that are coregulated, or organize a sequence of transcriptional events during development. MLL-rearranged leukemias have recently been proposed to rely heavily on epigenetic dysregulation during malignant transformation. In contrast to most other cancers, MLL-rearranged leukemias display a remarkable paucity of DNA sequence alterations: frequently, the only genetic abnormality uncovered by genome-wide technologies in these leukemias is the MLL-translocation. The induction of widespread epigenetic changes could explain the apparent lack of a need for cooperating mutagenesis on a DNA-sequence level. This is significant, since most MLL-rearranged

Abstract IA11: Meningioma-1 cooperates with MLL and DOT1L to induce leukemia

Meningioma-1 (MN1) is frequently overexpressed in AML, and associated with a poor prognosis. In addition, MN1-TEL fusions are found in AML, underscoring the importance and possible driver function of MN1 in AML. Forced expression of MN1 in murine hematopoietic progenitors induces a highly aggressive leukemia as a single hit. The mechanism by which MN1 induces AML is unclear. MN1 is a transcriptional co-activator with almost no sequence or structural similarity to any other protein, and no targeted approaches to MN1-high AML are currently available. We sought to understand the mechanism by which MN1 induces AML with the goal to identify targetable downstream effectors. We found that the gene expression program induced by forced expression of MN1 in hematopoietic progenitors substantially overlaps with a set of genes that is downregulated in response of loss of the histone methyltransferase Dot1l in normal hematopoietic progenitors. This led us to hypothesize that the MN1-induced leukemogenic gene expression program might be dependent on Dot1l. We established MN1 leukemias on a Dot1l conditional background and found that loss of Dot1l indeed induced cell cycle arrest, differentiation and apoptosis, and prolonged the survival of transplanted mice in vivo. This was associated with the downregulation of the MN1-induced gene expression program. We next sought to investigate a possible mechanism for this observation. MN1 has been reported to be recruited to its target genes by ChIP-seq, but it does not itself possess sequence specific DNA binding capacity. The mediator of this recruitment is thus unclear. Since Dot1l has been shown to be required for the high level expression of MLL-fusion target genes in MLL -rearranged leukemias, we asked whether MN1 might cooperate with wild type MLL1, explaining the dependence on Dot1L. In order to test whether wild-type MLL1 is required for MN1 leukemias, we established AML on a Mll1 -conditional background. Results largely phenocopied the loss of Dot1l , suggesting that MN1 cooperates with both MLL1 and Dot1L to induce leukemia. We are currently investigating whether MN1 binds MLL1 and/or DOT1L directly to exert this function using protein pull downs and ChIP-Seq. Finally, we asked whether our findings had relevance for human AML. MN1 overexpression is found over a wide range of different molecular subgroups but is relatively under-represented in MLL -rearranged AML, suggesting redundant pathways. A subgroup that frequently displays very high levels of MN1 expression are AML with a complex karyotype with loss of 5q or 7 sequences, and high expression of HOXA cluster genes. We analyzed the response of a human cell line and two patient samples with high MN1/high HOXA9 expression to inhibition of DOT1L, and found induction of differentiation and apoptosis similar to our mouse model. In summary, our data suggest that MN1 cooperates with wild type MLL1 to induce a leukemogenic gene expression program that results in AML, and that this program may be targetable by inhibiting DOT1L. Citation Format: Simone Riedel, Jessica Haladyna, Brett Stevens, Daniel Pollyea, Qi Wei, Craig Jordan, Patricia Ernst, Tobias Neff, Kathrin Bernt. Meningioma-1 cooperates with MLL and DOT1L to induce leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA11.