Project description:PURPOSE:Patients with mixed lineage leukemia (MLL)-rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL. EXPERIMENTAL DESIGN:Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation. RESULTS:Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLL-rearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells. CONCLUSION:In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification.
Project description:BACKGROUND:Mixed lineage leukemia (MLL) gene translocations are found in ~75% infant and 10% adult acute leukemia, showing a poor prognosis. Lysine-specific demethylase 1 (LSD1) has recently been implicated to be a drug target for this subtype of leukemia. More studies using potent LSD1 inhibitors against MLL-rearranged leukemia are needed. METHODS:LSD1 inhibitors were examined for their biochemical and biological activities against LSD1 and MLL-rearranged leukemia as well as other cancer cells. RESULTS:Potent LSD1 inhibitors with biochemical IC50 values of 9.8-77 nM were found to strongly inhibit proliferation of MLL-rearranged leukemia cells with EC50 of 10-320 nM, while these compounds are generally non-cytotoxic to several other tumor cells. LSD1 inhibition increased histone H3 lysine 4 (H3K4) methylation, downregulated expression of several leukemia-relevant genes, induced apoptosis and differentiation, and inhibited self-renewal of stem-like leukemia cells. Moreover, LSD1 inhibitors worked synergistically with inhibition of DOT1L, a histone H3 lysine 79 (H3K79) methyltransferase, against MLL-rearranged leukemia. The most potent LSD1 inhibitor showed significant in vivo activity in a systemic mouse model of MLL-rearranged leukemia without overt toxicities. Mechanistically, LSD1 inhibitors caused significant upregulation of several pathways that promote hematopoietic differentiation and apoptosis. CONCLUSIONS:LSD1 is a drug target for MLL-rearranged leukemia, and LSD1 inhibitors are potential therapeutics for the malignancy.
Project description:DOT1L, the only known histone H3-lysine 79 (H3K79) methyltransferase, has been shown to be essential for the survival and proliferation of mixed-linkage leukemia (MLL) gene rearranged leukemia cells, which are often resistant to conventional chemotherapeutic agents. To study the functions of DOT1L in MLL-rearranged leukemia, SYC-522, a potent inhibitor of DOT1L developed in our laboratory, was used to treat MLL-rearranged leukemia cell lines and patient samples. SYC-522 significantly inhibited methylation at H3K79, but not H3K4 or H3K27, and decreased the expression of two important leukemia-relevant genes, HOXA9 and MEIS1, by more than 50%. It also significantly reduced the expression of CCND1 and BCL2L1, which are important regulators of cell cycle and anti-apoptotic signaling pathways. Exposure of MLL-rearranged leukemia cells to this compound caused cell cycle arrest and promoted differentiation of those cells, both morphologically and by increased CD14 expression. SYC-522 did not induce apoptosis, even at 10 µM for as long as 6 days. However, treatment with this DOT1L inhibitor decreased the colony formation ability of primary MLL-rearranged AML cells by up to 50%, and promoted monocytic differentiation. Notably, SYC-522 treatment significantly increased the sensitivity of MLL-rearranged leukemia cells to chemotherapeutics, such as mitoxantrone, etoposide and cytarabine. A similar sensitization was seen with primary MLL-rearranged AML cells. SYC-522 did not affect chemotherapy-induced apoptosis in leukemia cells without MLL-rearrangement. Suppression of DOT1L activity inhibited the mitoxantrone-induced increase in the DNA damage response marker, ?H2AX, and increased the level of cPARP, an intracellular marker of apoptosis. These results demonstrated that SYC-522 selectively inhibited DOT1L, and thereby altered gene expression, promoted differentiation, and increased chemosensitivity by preventing DNA damage response. Therefore, inhibition of DOT1L, in combination with DNA damaging chemotherapy, represents a promising approach to improving outcomes for MLL-rearranged leukemia.
Project description: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.
Project description:Rearrangement of the mixed lineage leukemia (MLL; also known as lysine methyltransferase 2A) gene is a recurrent genomic aberration in acute myeloid leukemia (AML). MLLT3, super elongation complex subunit (AF9) is one of the most common MLL fusion partners in AML. The present study aimed to explore the aberrant expression of genes associated with the MLL?AF9 translocation and identified potential new targets for the therapy of AML with MLL?AF9 translocation. The transcriptomic and epigenetic datasets were downloaded from National Center of Biotechnology Information Gene Expression Omnibus (GEO) database. Differentially expressed genes were obtained from two independent datasets (GSE68643 and GSE73457). Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery. MLL?AF9?associated chromatin immunoprecipitation sequencing (ChIP?Seq) data was analyzed and identified binding sites for MLL?AF9 and wild type MLL (MLL WT). The ChIP?Seq of histone modification data was downloaded from the GEO database, including histone 3 lysine 4 trimethylation (H3K4me3), histone 3 lysine 79 dimethylation (H3K79me2) and histone 3 lysine 27 acetylation (H3K27ac), was used for comparing histone modification marks between the MLL?AF9 leukemia cells and normal hematopoietic cells at MLL?AF9 and MLL WT binding sites. The differentially expressed genes with the same trend in H3K79me2, H3K27ac and H3K4me3 alteration were identified as potential MLL?AF9 direct target genes. Upon validation using RNA?Seq data from the Therapeutically Applicable Research to Generate Effective Treatments AML project, eight potential direct target genes of MLL?AF9 were identified and further confirmed in MLL?AF9 mouse model using reverse transcription?quantitative polymerase chain reaction. These genes may have a critical role in AML with MLL?AF9 translocation.
Project description:Rearrangements of MLL (encoding lysine-specific methyltransferase 2A and officially known as KMT2A; herein referred to as MLL to denote the gene associated with mixed-lineage leukemia) generate MLL fusion proteins that bind DNA and drive leukemogenic gene expression. This gene expression program is dependent on the disruptor of telomeric silencing 1-like histone 3 lysine 79 (H3K79) methyltransferase DOT1L, and small-molecule DOT1L inhibitors show promise as therapeutics for these leukemias. However, the mechanisms underlying this dependency are unclear. We conducted a genome-scale RNAi screen and found that the histone deacetylase SIRT1 is required for the establishment of a heterochromatin-like state around MLL fusion target genes after DOT1L inhibition. DOT1L inhibits chromatin localization of a repressive complex composed of SIRT1 and the H3K9 methyltransferase SUV39H1, thereby maintaining an open chromatin state with elevated H3K9 acetylation and minimal H3K9 methylation at MLL fusion target genes. Furthermore, the combination of SIRT1 activators and DOT1L inhibitors shows enhanced antiproliferative activity against MLL-rearranged leukemia cells. These results indicate that the dynamic interplay between chromatin regulators controlling the activation and repression of gene expression could provide novel opportunities for combination therapy.
Project description:Leukemias driven by chromosomal translocation of the mixed-lineage leukemia gene (MLL or KMT2A) are highly prevalent in pediatric oncology. The poor survival rate and lack of an effective targeted therapy for patients with MLL-rearranged (MLL-r) leukemias emphasize an urgent need for improved knowledge and novel therapeutic approaches for these malignancies. The resulting chimeric products of MLL gene rearrangements, i.e., MLL-fusion proteins (MLL-FPs), are capable of transforming hematopoietic stem/progenitor cells (HSPCs) into leukemic blasts. The ability of MLL-FPs to reprogram HSPCs toward leukemia requires the involvement of multiple chromatin effectors, including the histone 3 lysine 79 methyltransferase DOT1L, the chromatin epigenetic reader BRD4, and the super elongation complex. These epigenetic regulators constitute a complicated network that dictates maintenance of the leukemia program, and therefore represent an important cluster of therapeutic opportunities. In this review, we will discuss the role of MLL and its fusion partners in normal HSPCs and hematopoiesis, including the links between chromatin effectors, epigenetic landscapes, and leukemia development, and summarize current approaches to therapeutic targeting of MLL-r leukemias.
Project description:Mixed-lineage leukemia (MLL) fusions are potent oncogenes that initiate aggressive forms of acute leukemia. As aberrant transcriptional regulators, MLL-fusion proteins alter gene expression in hematopoietic cells through interactions with the histone H3 lysine 79 (H3K79) methyltransferase DOT1L. Notably, interference with MLL-fusion cofactors like DOT1L is an emerging therapeutic strategy in this disease. Here, we identify the histone H2B E3 ubiquitin ligase ring finger protein 20 (RNF20) as an additional chromatin regulator that is necessary for MLL-fusion-mediated leukemogenesis. Suppressing the expression of Rnf20 in diverse models of MLL-rearranged leukemia leads to inhibition of cell proliferation, under tissue culture conditions as well as in vivo. Rnf20 knockdown leads to reduced expression of MLL-fusion target genes, effects resembling Dot1l inhibition. Using ChIP-seq, we found that H2B ubiquitination is enriched in the body of MLL-fusion target genes, correlating with sites of H3K79 methylation and transcription elongation. Furthermore, Rnf20 is required to maintain local levels of H3K79 methylation by Dot1l at Hoxa9 and Meis1. These findings support a model whereby cotranscriptional recruitment of Rnf20 at MLL-fusion target genes leads to amplification of Dot1l-mediated H3K79 methylation, thereby rendering leukemia cells dependent on Rnf20 to maintain their oncogenic transcriptional program.
Project description:The DOT1L histone H3 lysine 79 (H3K79) methyltransferase plays an oncogenic role in MLL-rearranged leukemogenesis. Here, we demonstrate that, in contrast to MLL-rearranged leukemia, DOT1L plays a protective role in ultraviolet radiation (UVR)-induced melanoma development. Specifically, the DOT1L gene is located in a frequently deleted region and undergoes somatic mutation in human melanoma. Specific mutations functionally compromise DOT1L methyltransferase enzyme activity leading to reduced H3K79 methylation. Importantly, in the absence of DOT1L, UVR-induced DNA damage is inefficiently repaired, so that DOT1L loss promotes melanoma development in mice after exposure to UVR. Mechanistically, DOT1L facilitates DNA damage repair, with DOT1L-methylated H3K79 involvement in binding and recruiting XPC to the DNA damage site for nucleotide excision repair (NER). This study indicates that DOT1L plays a protective role in UVR-induced melanomagenesis.
Project description: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.