DOT1L as a Therapeutic Target: Insights into Epigenetic Regulation and Cancer Treatment

Mixed lineage leukemia (MLL) is an aggressive form of leukemia where the MLL gene is translocated and fused to more than 80 different nuclear, cytoplasmic and membrane proteins. MLL-AF9 is one of the most common MLL-fusions. This fusion partner is known to recruit several multiprotein complexes including the AEP, SEC and DOT1L complexes leading to transcriptional activation. In particular, the recruitment of Disruptor of telomeric silencing 1-like (DOT1L), a H3K79 histone methyltransferase, is essential for leukemogenesis by multiple MLL fusion proteins. We mapped the binding site to a 10 amino acid segment (865 - 874) on DOT1L where AF9 binds. Peptides derived from this region that show dose-dependent disruption of this interaction. We also demonstrated that DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. Encouraged by these results, we generated both genetic and chemical tools to elucidate the role of DOT1L recruitment to the MLL fusion partners and the mechanism of leukemogenic inhibition by disrupting the protein-protein interaction (PPI) between MLL-AF9 and DOT1L. Fl/Fl DOT1L MLL-AF9 Cre+ cell lines were generated with different constructs of DOT1L. These constructs consisted of DOT1L mutants lacking the 10 amino acid binding site, a I867A point mutant known to block DOT1L binding, and an enzymatic mutant known to yield a catalytically inactive protein. As control cell lines, MLL-AF6, a MLL-fusion containing a cytoplasmic protein, and E2A-HLF, a non-DOT1L dependent fusion, were generated to demonstrate the specific effects of generated DOT1L mutant constructs. Both DOT1L PPI mutants impaired the transformation by MLL-AF9 and induced cell death by inducing apoptosis and cell cycle arrest similarly to enzymatic inhibition. These results established a foundation for discovering small-molecule inhibitors that disrupting the AF9-DOT1L as potential disease-specific therapies that target chromatin modifications in this highly aggressive leukemia. A high throughput screening was conducted identifying several different chemical classes of small molecules that bind to the AF9 C-terminal hydrophobic binding site and disrupt the PPI between DOT1L and MLL-AF9 fusion protein. Identified small molecule inhibitors were validated with series of biochemical, functional and cell-based assays. Validated compounds selectively inhibit the growth of the DOT1L dependent murine cells and induce cell death in a similar manner to the genetic approach. The small molecules also showed specificity in killing human MLL-fusion cell lines in comparison to non-MLL fusion leukemia. These results show that blocking the recruitment of DOT1L by AF9 using both genetic and chemical tools eliminate MLL-AF9 mediated immortalization emphasizing an essential function for this interaction in leukemogenesis and warrant further development of the identified small-molecule inhibitors. Citation Format: Sierrah M. Grigsby, James Ropa, Justin Serio, Chenxi Shen, Jennifer Chase, Ivan Maillard, Andrew Muntean, Zaneta Nikolovska-Coleska. Using genetic and chemical approaches to probe the mechanism of DOT1L recruitment in MLL fusion leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2017-5489

An Epigenetic Regulator Screen Identifies Novel Targets That Sensitize MLL-Rearranged Leukemia to DOT1L Inhibition

MLL fusion proteins activate target genes in part via recruitment of the histone-modifying enzyme, DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase with selective activity for histone H3 lysine 79 (H3K79). The resulting hypermethylation of H3K79 at the HOX and MEIS1 loci appears to be pivotal for leukemogenesis in both AML and ALL. These findings emphasize the central role of DOT1L in leukemogenesis and strongly suggest that pharmacologic inhibition of DOT1L could be of therapeutic benefit for a number of ALL and AML subtypes. Applying a biochemical screening approach, two classes of small molecule DOT1L substrate competitive inhibitors were discovered. They selectively inhibit the cell growth in leukemic cells with MLL-translocation, in comparison with leukemic cells without MLL translocation, resulting in a dose and time dependent decrease of cellular H3K79 methylation, decrease in expression of MLL-target genes HOXA9 and MEIS1, inducing G0/G1 cell cycle arrest, an increase in expression of differentiation markers and death by apoptosis. Although the DOT1L inhibitors showed no toxicity on normal bone marrow, knockout studies show that DOT1L deficiency is associated with chromosomal instability and fatal progressive anemia. This suggested that developing therapeutic strategies other than global inhibition of DOT1L activity may be necessary. Therefore we characterized the protein-protein interaction (PPI) between DOT1L and ENL/AF9 and assessed its importance on MLL fusion protein-mediated transformation. We found that ENL and AF9 C-terminal domains bind to DOT1L with Kd of 238 nM and 111 nM respectively. We have mapped the binding site in DOT1L and found that only 10 amino acid of DOT1L are involved in interaction with ENL and AF9. Synthetic DOT1L 10mer peptide binds ENL/AF9 and disrupts the interaction between DOT1L/ENL and DOT1L/AF9. Alanine scanning mutagenesis studies showed that four hydrophobic residues are detrimental for the binding of DOT1L to both proteins ENL/AF9. To examine the critical role of the DOT1L-AF9 interaction in transformation by MLL fusion protein, colony forming unit (CFU) assay was performed using MLL-AF9 transformed cells lacking endogenous DOT1L. The colony forming potential of the MLL-immortalized cells was completely abolished by introducing DOT1L construct lacking 10 amino acid AF9 interacting residues (DOT1L (Δ10aa)), while the wild type DOT1L construct was able to restore CFU formation. Importantly global levels of H3K79 methylation were equivalent, but only the DOT1L (Δ10aa) construct failed to restore CFU formation. These results demonstrate that DOT1L interaction with MLL-AF9 in addition to H3K79 methylation is required for transformation of MLL-AF9. This suggests that selective disruption of this PPI is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL-fusion protein associated leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A158.

BackgroundEpigenetic regulations play pivotal roles in tumorigenesis and cancer development. Disruptor of telomeric silencing-1-like (DOT1L), also known as KMT4, is the only identified histone methyltransferase that catalyzes the mono-, di-, and tri-methylation of lysine 79 histone 3 (H3K79). However, little is known about the effect of H3K79 methylation on the modulation of colorectal cancer (CRC) development.MethodsDOT1L expression profiles in different subgroups of CRC tissues and its clinical significances were analyzed from some online datasheets. DOT1L in CRC cell lines was silenced by either lentivirus-mediated knockdown or inhibited by its specific inhibitor, EPZ004777. Then cell proliferation was detected by MTT assay, BrdU assay, and soft agar assay; cell cycle was detected by cytometry; and tumorigenicity was detected by using nude mice xenograft models. Clinical co-expression was analyzed between DOT1L and c-Myc. Chromatin immunoprecipitation (ChIP) assay was used to determine whether the translation of c-Myc was epigenetically regulated by H3K79me2 induced by DOT1L. c-Myc overexpression was used to rescue the cell cycle arrest and tumor growth induced by DOT1L silencing or inhibition in CRC.ResultsWe found that DOT1L was highly expressed in colorectal cancer and was negatively related to the prognosis of patients with CRC. Silencing or inhibition of DOT1L blocked cell proliferation, BrdU incorporation, self-renewal capability in vitro, and tumorigenicity in vivo. Besides, inhibition or silencing of DOT1L also induced cell cycle arrest at S phase, as well as decreased the expression of CDK2 and Cyclin A2. Furthermore, in the clinical databases of CRC, we found that the expression of DOT1L was positively correlated with that of c-Myc, a major regulator in the upstream of cell cycle–related factors. Besides, c-Myc expression was downregulated after DOT1L knockdown and c-Myc restoration rescued decrease of cell proliferation, BrdU corporation, self-renewal capability, cell cycle progression in vitro and tumorigenicity in vivo induced by DOT1L silencing. Then we found that H3K79 methylation was decreased after DOT1L knockdown. ChIP assay showed that H3K79me2 was enriched on the – 682~+ 284 region of c-Myc promoter, and the enrichment was decreased after DOT1L inhibition.ConclusionsOur results show that DOT1L epigenetically promotes the transcription of c-Myc via H3K79me2. DOT1L silencing or inhibition induces cell cycle arrest at S phase. DOT1L is a potential marker for colorectal cancer and EPZ004777 may be a potential drug for the treatment of colorectal cancer.

The AF9-Binding Domain of DOT1L Is Critical for Its Recruitment By MLL Fusion Proteins in Leukemia and Contributes to Its Functions in Normal Hematopoiesis

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangements of the MLL1 gene generate fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. To gain insights into the unique functions of DOT1L in MLL-driven leukemia, we elucidated the mechanisms of DOT1L recruitment to the MLL fusion partners. The binding site was mapped to a short segment of 10 amino acids in DOT1L and peptides derived from this region disrupted the interaction between DOT1L and MLL-AF9. DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. This discovery has established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias. Applying high throughput screening approach several different chemical classes of small molecules that disrupt the protein-protein interactions between DOT1L and oncogenic MLL-fusion proteins were identified and validated. To evaluate if the AF9-binding domain of DOT1L is critical for its functions in normal hematopoietic stem cells as opposed to leukemias driven by MLL fusion proteins, genetic tools were developed to functionally investigate the importance of the DOT1L AF9-binding domain in MLL-AF9-driven leukemia and its role in the physiological functions of DOT1L in normal hematopoiesis. Our findings demonstrate that pharmacological inhibition of the DOT1L complex through disrupting the AF9-DOT1L interactions may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression. Citation Format: Sierrah Grigsby, Jennifer Chase, James Ropa, Justin Serio, Chenxi Shen, Martha Larsen, Preston Donover, Melvin Reichman, Andrew Muntean, Ivan Maillard, Zaneta Nikolovska-Coleska. Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3803.

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangement of the MLL1 gene generates fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome-wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. We have mapped the binding site to a short segment of 10 amino acids in DOT1L and shown that DOT1L mutants lacking these residues did not support transformation by MLL-AF9. We hypothesized that by targeting the AF9-DOT1L protein-protein interactions (PPIs), we would selectively kill MLL-AF9 cells without effecting DOT1L role in normal hematopoiesis. Using established DOT1Lf/f MLL-AF9 with reintroduced WT-DOT1L, DOT1L missing 10aa AF9-binding domain (D10), DOT1L with a point mutation in the AF9-binding domain (I867A) and enzymatically inactive DOT1L (RCR), we were able to demonstrate that by disrupting the AF9-DOT1L PPIs, although we can inhibit leukemogenesis similarly to enzymatic inhibition, this interaction is not essential for normal hematopoiesis. Based on our initial studies to map the DOT1L interaction site and in conjunction with utilizing reported NMR structures of the AF9-DOT1L complex, we investigated the nature of the interactions and the minimum length of the peptide. Using different natural and unnatural amino acids, we successfully designed a 7mer peptide with KD of 10 nM and 25 nM against AF9 and ENL, respectively, showing similar potency as the originally identified and validated 10mer peptide. These results lay the groundwork for further optimization of the 7mer peptide towards developing DOT1L peptidomimetics with improved potency and cellular activity, to further validate the PPIs between DOT1L and MLL-fusion proteins as a potential therapeutic target for MLL rearranged leukemia. Citation Format: Sierrah Marie Grigsby, Jennifer Chase, Bridget Waas, Ann Friedman, Lei Du, Aihong Yao, James Ropa, Justin Serio, Andrew Muntean, Ivan Maillard, Haying Sun, Zaneta Nikolovska-Coleska. Towards peptidomimetics to target DOT1L recruitment in MLL-AF9 leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1380.

New effective combinational therapeutic strategies are an alternative choice for a successful translational study. Disruptor of telomeric silencing 1-like (DOT1L) is a histone 3 lysine 79 (H3K79) methyltransferase enzyme and its inhibition is being tested in phase 1 clinical trials. DOT1L has been implicated in many biological functions ranging from cell cycle regulation, transcriptional regulation, and heterochromatin formation, however, the functions of DOT1L in DNA-damage response remains to be unraveled. DNA double-strand breaks (DSB) are one of the most lethal forms of DNA damage and can lead to several disease phenotypes, including cancer. In this study, we investigated the role of DOT1L in the DNA double-strand break repair-pathway. Our results indicate that DOT1L is required for proper DNA-damage response and repair of DNA DSBs via a homologous recombination (HR) pathway. DOT1L activity prevents the proliferation of cancer cells; therefore this is a potential future cancer therapeutic target. And more importantly, our results show the combination of small molecule inhibitor PARP with other available chemotherapeutics agents shows synergism in the colorectal cancer cells. The data further suggest DOT1L plays a role HR-mediated DNA double strand break and loss of DOT1L functions leads to increased sensitivity to PARP inhibition. Therefore, we hypothesize that DOT1L activity (i.e. H3K79me3) may serve as a marker for molecular stratification of colorectal cancer.

Disruptor of telomeric silencing 1-like (DOT1L) is an epigenetic regulator that promotes gene expression by methylating lysine 79 on histone H3 and recruits transcription factors to gene targets. DOT1L is also an oncogenic driver in cancers that affect developing lymphocytes, yet how DOT1L activity regulates B-cell maturation remains poorly defined. Here, we use deep-sequencing and conditional knockout models to elucidate gene targets of H3K79me2, and the mechanistic contribution of DOT1L, during key stages of murine B-cell development. In the bone marrow, Dot1l was upregulated in pro B cells. Deep sequencing revealed that H3K79me2 accumulated during maturation. The genomic distribution of H3K79me2 peaks indicated that DOT1L regulates transcription and the cell cycle across different stages of B-cell development. As such, DOT1L was found to be essential for pre B-cell expansion, leading to a significant decrease in pre B cells in the absence of DOT1L and a skewing of the B-cell receptor repertoire to favor proximal VH usage. In addition to the effective generation of a diverse B-cell pool, DOT1L was also required to establish the marginal zone (MZ) B-cell gene expression program. Attenuation of MZ B cells and a bottlenecking at the pre-MZ B-cell stage in Dot1L-deficient mice correlated to H3K79me2 peaks at key MZ-regulatory genes such as Lfng and Dock10 in developing B cells. In contrast, Dot1l was reexpressed in germinal center (GC) B cells postimmunization to deposit H3K79me2 at key GC B-cell gene loci during GC differentiation. Together, these data demonstrate a vital role for DOT1L during B-cell lymphopoiesis, MZ B-cell generation, and GC B-cell biology.

BackgroundEpigenetics has been known to play a critical role in regulating the malignant phenotype. This study was designed to examine the expression of DOT1L (histone 3 lysine 79 methyltransferase) and H3K79 methylation in normal ovarian tissues and ovarian tumors and to explore the function of DOT1L and its underline mechanisms in ovarian cancer.MethodsThe expression of DOT1L and H3K79 methylation in 250 ovarian tumor samples and 24 normal ovarian samples was assessed by immunohistochemistry. The effects of DOT1L on cell proliferation in vitro were evaluated using CCK8, colony formation and flow cytometry. The DOT1L-targeted genes were determined using chromatin immune-precipitation coupled with high-throughput sequencing (ChIP-seq) and ChIP-PCR. Gene expression levels were measured by real-time PCR and immunoblotting. The effects of DOT1L on tumor growth in vivo were evaluated using an orthotopic ovarian tumor model.ResultsDOT1L expression and H3K79 methylation was significantly increased in malignant ovarian tumors. High DOT1L expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage, histologic grade, and lymphatic metastasis. DOT1L was an independent prognostic factor for the overall survival (OS) and progression-free survival (PFS) of ovarian cancer, and higher DOT1L expression was associated with poorer OS and PFS. Furthermore, DOT1L regulates the transcription of G1 phase genes CDK6 and CCND3 through H3K79 dimethylation; therefore, blocking DOT1L could result in G1 arrest and thereby impede the cell proliferation in vitro and tumor growth in vivo.ConclusionsOur findings first demonstrate that DOT1L over-expression has important clinical significance in ovarian cancer and also clarify that it drives cell cycle progression through transcriptional regulation of CDK6 and CCND3 through H3K79 methylation, suggesting that DOT1L might be potential target for prognostic assessment and therapeutic intervention in ovarian cancer.

Herein, the underlying role of disruptor of telomeric silencing 1-like (DOT1L) as a therapeutic target for mixed-lineage leukemia (MLL)-rearranged is comprehensively clarified. DOT1L can be aberrantly recruited by an MLL fusion partner, thereby causing the over-expression, of several leukemia relevant genes and eventually leading to leukemia. As the unique histone methyltransferase (HMT), DOT1L possesses the function to specifically methylate H3K79, which was identified as a hallmark of active transcription. Accordingly, blockading of DOT1L has been recognized as an effective approach for cancer treatment. Currently, nucleoside DOT1L inhibitors have been developed successfully with the only EPZ5676 entering phase I clinical trial in 2013, which was validated as 'orphan drug' toward MLL-rearranged leukemia by FDA. In order to find compounds with better pharmacokinetic properties as DOT1L inhibitors, other types of non-nucleoside skeletons have also been reported successively.

BackgroundPodocyte injury causes proteinuria and accelerates glomerular sclerosis during diabetic kidney disease (DKD). Disruptor of telomeric silencing 1-like (DOT1L), an evolutionarily conserved histone methyltransferase, has been reported in preventing kidney fibrosis in chronic kidney disease models. However, whether DOT1L exerts beneficial effects in diabetes induced podocyte injury and the underlying molecular mechanisms need further exploration.MethodsThe expression of DOT1L was confirmed by Western blotting in MPC-5 cells and cortex of kidney from db/db mice, as well as immunofluorescence staining in human renal biopsy samples. The effect of DOT1L on podocyte injury was obtained using MPC-5 cells and db/db mice. The potential target genes regulated by DOT1L was measured by RNA-sequencing. Then, a series of molecular biological experiments was performed to investigate the regulation of PLCL1 by DOT1L in MCP-5 cells and db/db mice. Lipid accumulation was assessed by UPLC-MS/MS analysis and Oil Red O staining.ResultsDOT1L expression was significantly declined in high glucose (HG)-treated MPC-5 cells, podocyte regions of kidney tissues from db/db mice and human renal biopsy samples. Subsequent investigations revealed that upregulation of DOT1L ameliorated HG-induced cell apoptosis in MPC-5 cells as well as primary podocytes. Furthermore, podocyte-specific DOT1L overexpression inhibited diabetic podocyte injury in db/db mice. Mechanistically, we revealed that DOT1L upregulated phospholipase C-like 1 (PLCL1) expression by mediating H3K79me2 at its promoter and PLCL1 silencing suppressed the protective role of DOT1L on podocyte injury. Moreover, DOT1L improved diabetes induced abnormal fatty acid metabolism in podocytes and PLCL1 knockdown reversed its protective effects.ConclusionsTaken together, our results indicate that DOT1L protects podocyte injury via PLCL1-mediated fatty acid metabolism and provides new insights into the therapeutic target of DKD.

Identification of DOT1L inhibitors by structure-based virtual screening adapted from a nucleoside-focused library

Discovery of potent DOT1L inhibitors by AlphaLISA based High Throughput Screening assay

Genome-Wide RNAi Screen Identifies The Mechanistic Role For DOT1L In MLL-Rearranged Leukemia