Disruptor Of Telomeric Silencing 1-like Research Articles

Disruptor of telomeric silencing 1-like promotes ovarian cancer tumor growth by stimulating pro-tumorigenic metabolic pathways and blocking apoptosis

ABSTRACTOvarian cancer is the leading cause of gynecological malignancy-related deaths. Current therapies for ovarian cancer do not provide meaningful and sustainable clinical benefits, highlighting the need for new therapies. We show that the histone H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is overexpressed in ovarian cancer and that a higher level of DOT1L expression correlates with shorter progression-free and overall survival (OS). Pharmacological inhibition of DOT1L (EPZ-5676, EPZ004777, and SGC0946) or genetic inhibition of DOT1L attenuates the growth of ovarian cancer cells in cell culture and in a mouse xenograft model of ovarian cancer. Transcriptome-wide mRNA expression profiling shows that DOT1L inhibition results in the downregulation of genes involved in cellular biosynthesis pathways and the upregulation of proapoptotic genes. Consistent with the results of transcriptome analysis, the unbiased large-scale metabolomic analysis showed reduced levels of several metabolites of the amino acid and nucleotide biosynthesis pathways after DOT1L inhibition. DOT1L inhibition also resulted in the upregulation of the NKG2D ligand ULBP1 and subsequent increase in natural killer (NK) cell-mediated ovarian cancer eradication. Collectively, our results demonstrate that DOT1L promotes ovarian cancer tumor growth by regulating apoptotic and metabolic pathways as well as NK cell-mediated eradication of ovarian cancer and identifies DOT1L as a new pharmacological target for ovarian cancer therapy.

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

Mixed lineage leukemia (MLL) is a very aggressive therapy resistant leukemia. MLL-AF9 is one of the most common MLL-fusion proteins that recruits disruptor of telomeric silencing 1-like (DOT1L), to MLL-target genes HOXA9/MEIS1. DOT1L is a histone methyltransferase that catalyzes methylation of H3K79, a gene activating mark, that is essential for leukemogenesis and normal hematopoiesis. Our lab and others mapped the 10 amino acid (865 - 874) binding site on DOT1L, which binds to the ANC1 homology domain (AHD) domain of AF9. We also identified a point mutation, I867A, sufficient to disrupt the AF9-DOT1L interaction in vitro. We demonstrated that DOT1L lacking the 10 amino acids (Δ10) were unable to support transformation by ML-AF9. We hypothesize that by disrupting the AF9-DOT1L protein-protein interaction (PPI), we will be able to inhibit leukemogenesis and potentially circumvent the negative consequences of DOT1L loss in normal bone marrow. We used a genetic approach to explore the role of DOT1L recruitment in leukemogenesis and normal hematopoiesis.We generated cell lines derived from MLL-AF9-driven DOT1Lf/f CreER(T2)+ bone marrow by transducing DOT1L constructs, including wild type (WT-DOT1L), deletion of 10 amino acids (Δ10-DOT1L), point mutant (I867A-DOT1L), and an enzymatically inactive mutant (RCR-DOT1L). Both Δ10 and I867A mutants inhibited leukemogenesis similarly to the RCR mutant. This inhibited proliferation was coupled with a decrease in H3K79me2, a decrease in HOXA9/Meis1 gene expression, cellular differentiation, and induction of apoptosis demonstrating that disruption of the AF9-DOT1L PPI mimics the phenotype of as the enzymatic inhibition, deeming it as a potential therapeutic target for this cancer.To explore the impact of disrupting the AF9-DOT1L interaction in normal bone marrow, we transduced Mx-Cre+ DOT1Lf/f bone marrow with either WT-DOT1L, Δ10-DOT1L, or RCR-DOT1L constructs and injected these cells into lethally irradiated B6-CD45.1 recipients. After hematopoietic reconstitution, recipients were treated with poly(I:C) to inactivate endogenous DOT1L. In the absence of DOT1L's methyltransferase activity (RCR-DOT1L), transduced hematopoietic progenitors underwent rapid failure. Interestingly, expression of the Δ10-DOT1L mutant was sufficient to support short-term hematopoiesis but insufficient to sustain long-term hematopoietic stem cells (LT-HSCs). Together these results show that blocking DOT1L's recruitment eliminates the transformative potential of the MLL-AF9 oncogene and that while DOT1L's function is critical for all hematopoietic progenitor cell survival, removing the DOT1L AF9 binding domain allows for short term hematopoiesis.To further explore the role of AF9 role in recruiting DOT1L in MLL-AF9 leukemia and normal hematopoiesis, using high throughput screening approach, we identified small molecules that bind to the AF9 AHD domain. These compounds inhibit proliferation of cells harboring the MLL-AF9 fusion, decrease H3K79me2, and induce apoptosis similar to Δ10-DOT1L and I867A-DOT1L mutants. We are currently working to further characterize their on-target activity in cells, and test their effects on normal bone marrow, with the ultimate goal to validate additional avenues for therapeutic intervention for MLL-rearranged leukemia. DisclosuresNo relevant conflicts of interest to declare.

DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A.

Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.

Inhibition of histone demethylases as an approach to restore deficient DOT1L activity in osteoarthritis

Purpose: Osteoarthritis (OA), the most common chronic joint disease worldwide, is characterized by progressive damage to the articular cartilage, increased joint-associated bone remodelling, and synovial inflammation. Current OA treatments are limited to pain relief, physiotherapy, or replacement surgeries in severe cases, yet disease-modifying drugs are lacking. A genome wide association study (GWAS) revealed a genetic association between polymorphisms in the DOT1L gene and OA. The Disruptor of telomeric silencing 1-like (DOT1L) gene encodes a histone methyltransferase that methylates Lysine 79 of Histone H3 (H3K79). We identified DOT1L as a key protector of cartilage homeostasis, using both human articular chondrocytes and different Dot1l genetic mouse models. Furthermore, we reported that DOT1L activity, indicated by the levels of H3K79 methylation (H3K79me), is reduced in OA as compared to non-OA cartilage. Therefore, maintaining H3K79me seems to be critical to preserve joint health and prevent the development or progression of OA. Here, we hypothesized that H3K79me could be restored or maintained by inhibiting demethylation at the H3K79 site, via targeting specific histone demethylases. There are two main families of histone demethylases: the lysine-specific demethylases (LSD) and Jumonji C (JmjC) demethylases. The LSD family contains two members: LSD1 and LSD2. The JmjC family is further classified into 6 subfamilies: from KDM2 to KDM7. In this new study, we aimed to investigate which histone demethylases are responsible for H3K79 demethylation and whether their specific targeting can lead to protective effects in OA. Methods: We determined the baseline mRNA expression of 28 reported histone demethylases in primary human articular chondrocytes (hACs) from non-OA patients, using real-time qPCR. We also determined the changes in histone demethylase expression in vitro upon OA-mimicking stimuli, namely proinflammatory cytokine IL-1β and the Wnt signalling activator CHIR99021. To interrogate the role of JmjC demethylase family in H3K79me, human articular chondrocyte C28/I2 cells were treated with the JmjC pan inhibitor JIB-04. In parallel, the LSD family members LSD1 and LSD2 were pharmacologically inhibited using LSD1 inhibitor II or silenced using siRNA against LSD2, respectively. The role of the different JmjC subfamilies on H3K79me was studied using selective pharmacological inhibitors. We assessed the levels of H3K79me by Western blot and immunofluorescence analysis. The translational impact of individual JmjC demethylase targeting was evaluated using a siRNA knockdown strategy in hACs from OA patients. In this setup, gene expression of healthy cartilage markers Collagen2a1 (COL2A1) and Aggrecan, as well as of catabolic markers MMP13 and ADAMTS5, was assessed using real-time qPCR. Results: The baseline expression of histone demethylases was mapped in hACs, showing differences in absolute expression among the different enzymes. Treatment with IL1-β resulted in an increase in KDM6B and KDM7A mRNA expression. Conversely, Wnt signalling activation by CHIR99021 led to a downregulation in most of histone demethylases’ mRNA expression. Interestingly, pharmacological inhibition of the JmjC family using JIB-04 resulted in increased H3K79me levels. However, the blockade of LSD family members did not lead to H3K79me changes in articular chondrocytes. Moreover, inhibition of all JmjC demethylase subfamilies increased H3K79me levels. Individual silencing of specific JmjC demethylases increased the expression of chondrocyte healthy markers while reducing the expression of catabolic markers. Conclusions: The discovery approach performed in this study unravels that histone demethylases from the JmjC family control H3K79me in the articular cartilage. Silencing of individual demethylases from this family exerted protective effects in the articular chondrocyte. Therefore, targeting specific histone demethylases could restore the loss of DOT1L activity in OA and lead to therapeutic benefits in this chronic disease.

DOT1L Regulates Thermogenic Adipocyte Differentiation and Function via Modulating H3K79 Methylation.

Brown and beige adipocytes are characterized as thermogenic adipocytes and have great potential for treating obesity and associated metabolic diseases. In this article, we identify a conserved mammalian lysine 79 of histone H3 (H3K79) methyltransferase, disruptor of telomeric silencing-1 like (DOT1L), as a new epigenetic regulator that controls thermogenic adipocyte differentiation and function. We show that deletion of DOT1L in thermogenic adipocytes potently protects mice from diet-induced obesity, improves glucose homeostasis, alleviates hepatic steatosis, and facilitates adaptive thermogenesis in vivo. Loss of DOT1L in primary preadipocytes significantly promotes brown and beige adipogenesis and thermogenesis in vitro. Mechanistically, DOT1L epigenetically regulates the brown adipose tissue-selective gene program by modulating H3K79 methylation, in particular H3K79me2 modification. Thus, our study demonstrates that DOT1L exerts an important role in energy homeostasis by regulating thermogenic adipocyte differentiation and function.

Synthesis and biological activity of selenopsammaplin A and its analogues as antitumor agents with DOT1L inhibitory activity

Disruptor of telomeric silencing-1 like (DOT1L) is a histone H3 methyltransferase which specifically catalyzes the methylation of histone H3 lysine-79 residue. Recent findings demonstrate that DOT1L is abnormally overexpressed and the upregulated DOT1L evokes the proliferation and metastasis in human breast cancer cells. Therefore, the DOT1L inhibitor is considered a promising strategy to treat breast cancers. Non-nucleoside DOT1L inhibitors, selenopsammaplin A and its analogues, were firstly reported in the present study. Selenopsammaplin A was newly designed and synthesized with 25% overall yield in 8 steps from 3-bromo-4-hydroxybenzaldahyde, and thirteen analogues of selenopsammaplin A were prepared for structure–activity relationship studies of their cytotoxicity against cancer cells and inhibitory activity toward DOT1L for antitumor potential. All synthetic selenopsammaplin A analogues exhibited the higher cytotoxicity compared to psammaplin A with up to 6 – 60 times depending on cancer cells, and most analogues showed significant inhibitory activities against DOT1L. Among the prepared analogues, the phenyl analogue (10) possessed the most potent activity with both cytotoxicity and inhibition of DOT1L. Compound 10 also exhibited the antitumor and antimetastatic activity in an orthotopic mouse metastasis model implanted with MDA-MB-231 human breast cancer cells. These biological findings suggest that analogue 10 is a promising candidate for development as a cancer chemotherapeutic agent in breast cancers.

Nucleoside and Non-Nucleoside DOT1L Inhibitors: Dawn of MLLrearranged Leukemia.

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.

Inhibition of DOT1L by Half-Selenopsammaplin A Analogs Suppresses Tumor Growth and EMT-Mediated Metastasis in Triple-Negative Breast Cancer.

Due to a lack of hormone receptors, current treatment strategies for triple-negative breast cancer (TNBC) are limited with frequent disease recurrence and metastasis. Recent findings have suggested that aberrant methylation of histone H3 lysine 79 residue (H3K79me) by the histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is a potential therapeutic target for TNBC clinical management. Therefore, we developed DOT1L inhibitors as potential antitumor agents against TNBC cells. We reveal that a synthetic half-selenopsammaplin A analog 9l (subsequently known as 9l) exhibited inhibitory activity against DOT1L-mediated H3K79 methylation, and showed antitumor activity in TNBC cells. The analog 9l also significantly inhibited TNBC invasion and migration via the modulation of epithelial-mesenchymal transition (EMT) markers, including N-cadherin and vimentin downregulation and E-cadherin upregulation. In an MDA-MB-231/Luc-implanted orthotopic mouse metastasis model, treatment with 9l effectively inhibited tumor growth and lung metastasis via DOT1L regulatory activity and EMT processes. Taken together, these findings highlight the potential of 9l as a novel therapeutic candidate for treating metastatic TNBC via DOT1L modulation.

Targeting the Histone Methyltransferase Disruptor of Telomeric Silencing 1-Like Restricts Avian Leukosis Virus Subgroup J Replication by Restoring the Innate Immune Response in Chicken Macrophages.

Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is known to cause immunosuppression and various types of cancer in chickens. Recent reports have shown that epigenetic changes in DNA and chromatin are widely implicated in the life cycle of diverse viruses, and reversal of these changes in host cells can lead to alterations in the propagation of viruses. In the present study, we found that disruptor of telomeric silencing 1-like (DOT1L), a histone H3 lysine79 (H3K79) methyltransferase, was upregulated during ALV-J infection in chicken macrophage HD11 cells. Subsequently, we show that targeting DOT1L with a specific inhibitor can significantly decrease the ALV-J replication and viral production. By generating of DOT1L-knockout (KO) HD11 cells using the CRISPR/Cas9 system, we show that deletion of the DOT1L led to an increase in the induction of IFNβ and interferon-stimulated genes (ISGs) in HD11 cells in response to ALV-J infection. Importantly, we confirmed that ALV-J infection impaired the activation of the melanoma differentiation-associated protein 5 (MDA5)-mediated-IFN pathway by suppressing the MDA5 expression, and knockout DOT1L rescued the expression of MDA5 and signal transducer and activator of transcription 1 (STAT1), both of which tightly control the antiviral innate immunity. Collectively, it can be deduced from the current data that blocking DOT1L activity or deletion of DOT1L can lead to ALV-J replication inhibition and restoration of the virally suppressed host innate immunity. Thus, we suggest that DOT1L might be a potential drug target for modulating host innate immune responses to combat ALV-J infection.

The Histone Methyltransferase DOT1L Is Essential for Humoral Immune Responses

Histone modifiers are essential for the ability of immune cells to reprogram their gene expression during differentiation. The recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like) induces oncogenic gene expression in a subset of B cell leukemias. Despite its importance, its role in the humoral immune system is unclear. Here, we demonstrate that DOT1L is a critical regulator of B cell biology. B cell development is defective in Dot1lf/fMb1Cre/+ mice, culminating in a reduction of peripheral mature B cells. Upon immunization or influenza infection of Dot1lf/fCd23Cre/+ mice, class-switched antibody-secreting cells are significantly attenuated and germinal centers fail to form. Consequently, DOT1L is essential for B cell memory formation. Transcriptome, pathway, and histological analyses identified a role for DOT1L in reprogramming gene expression for appropriate localization of B cells during the initial stage of the response. Together, these results demonstrate an essential role for DOT1L in generating an effective humoral immune response.

Characterizing Proteins That Mediate CALM-AF10 Leukemogenesis

Background: Leukemia is the most common type of childhood cancer. Although the prognosis for many pediatric leukemias has improved, leukemias associated with the t(10;11) CALM-AF10 translocation remain difficult to treat. CALM-AF10 leukemias account for ~5-10% of childhood T-cell acute lymphoid leukemia (T-ALL) as well as a subset of acute myeloid leukemia (AML). CALM-AF10 leukemias exhibit increased expression of proleukemic HOXA genes, but relatively little is known about the cellular mechanisms that drive CALM-AF10 leukemogenesis. Our laboratory has demonstrated that the CALM protein contains a nuclear export signal (NES) that is critical for CALM-AF10-dependent leukemogenesis. The NES interacts with the CRM1/XPO1 nuclear export receptor, which shuttles proteins from the nucleus to the cytoplasm through the nuclear pore complex. We have shown that transcriptional activation of HOXA genes by CALM-AF10 is critically dependent on its interaction with CRM1. Importantly, CRM1 does not contain a recognized DNA binding domain, and it is not currently understood how the CALM-AF10/CRM1 complex interacts with regulatory regions of HOXAgenes. In order to identify proteins that mediate the interaction between the CALM-AF10/CRM1 complex and DNA, we took advantage of a proximity-based labeling approach using BioID2, a second-generation biotin ligase. When fused to a protein of interest and in the presence of biotin, BioID2 biotinylates proteins in close proximity to the ligase. These biotinylated proteins can then be identified by mass spectrometry (MS). Methods: We prepared an expression plasmid in which BioID2 was cloned in-frame with CALM-AF10. We then transiently transfected Human Embryonic Kidney 293 (HEK293) cells with the BioID2-CALM-AF10 plasmid, grew them in the presence or absence of biotin, and performed streptavidin-pulldown followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to identify candidate interacting proteins. Proteins were considered candidates if they had a peptide spectrum match (PSM) score > 10 and at least a two-fold greater PSM score versus negative control. We validated direct interactions of candidate proteins with CALM-AF10 by performing co-immunoprecipitation experiments. Results: We first confirmed that the addition of BioID2 to CALM-AF10 does not affect the transcriptional activation of HOXA genes or CALM-AF10 mediated immortalization of hematopoietic stem cells. We carried out three independent transfections/LC-MS/MS experiments, which identified 71, 95 and 61 proteins, respectively. Of the proteins identified, 11 candidates were common to all three experiments.Of particular interest, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), a protein known to interact with AF10, and Nuclear pore complex protein 214 (NUP214), a protein that has been identified in leukemogenic translocations. The nine additional candidate proteins included: EPS15, DVL2, DVL3, and DDX3X -all known to play a role in leukemogenesis. We performed initial validation of direct interactions via co-immunoprecipitation and found that Epidermal Growth Factor Receptor Substrate 15(EPS15) co-precipitates with CALM-AF10. Conclusion: We used biotin ligase-dependent proximity-based labeling to identify candidate proteins that potentially interact with the CALM-AF10 fusion protein. Our identification of DOT1L validates the approach, since DOT1L is known to interact with CALM-AF10. We have started to investigate other candidate proteins, focusing on known translocation partners in various leukemias. Our screen identified EPS15, a protein involved in receptor-mediated endocytosis of epidermal growth factor and a known translocation partner for MLL/KMT2A. KMT2A-EPS15 translocations (t(1;11)(p32;q23)) have been identified in both AML and ALL, and KMT2A-EPS15 is among the eight most common KMT2A rearrangements. We have shown that EPS15 co-immunoprecipitates with CALM-AF10, suggesting that EPS15 may also play a role in CALM-AF10 leukemogenesis. Further evaluation of this interaction is underway, and may lead to identification of novel pathways involved in CALM-AF10 leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Permissive epigenomes endow reprogramming competence to transcriptional regulators.

Identifying molecular and cellular processes that regulate reprogramming competence of transcription factors broadens our understanding of reprogramming mechanisms. In the present study, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks including disruptor of telomeric silencing 1-like (DOT1L)-mediated H3K79/K27 methylation, but also other epigenetic pathways, catalyzed by lysine-specific histone demethylase 1A, DNA methyltransferases and histone deacetylases, allows induced pluripotent stem cell generation with almost all OCT factors. We found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors, but in fact enables dismantling of species-dependent reprogramming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these induced permissive epigenetic states, we performed an additional screen with 98 candidate genes. Thereby, we identified 25 transcriptional regulators (OTX2, SIX3, and so on) that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptual framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.

Nullifying epigenetic writer DOT1L attenuates neointimal hyperplasia

Background and aimsHistone methyltransferases are emerging targets for epigenetic therapy. DOT1L (disruptor of telomeric silencing 1-like) is the only known methylation writer at histone 3 lysine 79 (H3K79). It is little explored for intervention of cardiovascular disease. We investigated the role of DOT1L in neointimal hyperplasia (IH), a basic etiology of occlusive vascular diseases. Methods and resultsIH was induced via balloon angioplasty in rat carotid arteries. DOT1L and its catalytic products H3K79me2 and H3K79me3 (immunostaining) increased by 4.69 ± 0.34, 2.38 ± 0.052, and 3.07 ± 0.27 fold, respectively, in injured (versus uninjured) carotid arteries at post-injury day 7. Dot1l silencing via shRNA-lentivirus infusion in injured arteries reduced DOT1L, H3K79me2, and IH at day 14 by 54.5%, 37.1%, and 76.5%, respectively. Moreover, perivascular administration of a DOT1L-selective inhibitor (EPZ5676) reduced H3K79me2, H3K79me3, and IH by 56.1%, 58.6%, and 39.9%, respectively. In addition, Dot1l silencing and its inhibition (with EPZ5676) in vivo in injured arteries boosted smooth muscle α-actin immunostaining; pretreatment of smooth muscle cells with EPZ5676 in vitro reduced pro-proliferative marker proteins, including proliferating cell nuclear antigen (PCNA) and cyclin-D1. ConclusionsWhile DOT1L is upregulated in angioplasty-injured rat carotid arteries, either its genetic silencing or pharmacological inhibition diminishes injury-induced IH. As such, this study presents a strong rationale for continued mechanistic and translational investigation into DOT1L targeting for treatment of (re)stenotic vascular conditions.

Abstract A80: Targeting ovarian cancer stem cell by Dot1L inhibition

Abstract Cancer stem cells (CSCs) are a specific population of cancer cells that are responsible for tumor initiation, chemoresistance, and metastasis. Previously, our lab has demonstrated ovarian cancer cells with high aldehyde dehydrogenase (ALDH) activities have multiple stem cell-like characteristics, including self-renewal, high clonogenicity, and chemoresistance. Here we identify that H3K79 methyltransferase DOT1L (disruptor of telomeric silencing-1-like) is a critical factor in self-renewal and tumor initiation capabilities of ovarian cancer stem cells (OCSCs). Firstly, we found that DOT1L have higher expression in ALDH(+)/CD133(+) OCSCs compared to double negative cells. Ovarian cancer cells with DOT1L-knockdown have reduced CSC fractions in flow cytometry analysis. Besides, knockdown of DOT1L expression impairs clonogenicity characteristics of CSCs, according to in vitro colony-forming assay and spheroid formation assay results. In order to effectively inhibit the enzymatic activity of DOT1L, we applied specific DOT1L inhibitor (DOT1Li) EPZ5676 to specifically target OCSCs and confirmed that the treatment of DOT1Li was able to block H3K79 methylation in ovarian cancer cells, and effectively represses the expression of stemness-associated transcriptional factors. Results from RNA-seq analysis and gene set enrichment assay have indicated that multiple stem-related pathways are repressed in DOT1Li-treated OCSCs. Furthermore, DOT1Li treatment not only shows inhibitory effects on colony forming in vitro but also delays tumor initiation in vivo compared to control treatment, which validates the effects of DOT1Li on CSCs inhibition. Based on our result, we infer that DOT1L is critical for self-renewal and tumor initiation abilities in ovarian cancer stem cells, and DOT1L inhibition by specific inhibitor provides a potential therapeutic strategy to target CSCs in ovarian cancer treatment. Citation Format: Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Daniela Matei. Targeting ovarian cancer stem cell by Dot1L inhibition [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A80.

DOT1L-Mediated H3K79 Methylation Regulates Thermogenic Adipocyte Differentiation and Function

Brown and beige adipocytes are characterized as thermogenic adipocytes and have great potential for treating obesity and associated metabolic diseases. Here, we identify a conserved mammalian lysine 79 of histone H3 (H3K79) methyltransferase, disruptor of telomeric silencing -1 like (DOT1L), as a new epigenetic regulator that controls thermogenic adipocyte development and function. We show that loss of DOT1L in primary preadipocytes significantly promotes brown and beige adipogenesis and thermogenesis in vitro. Notably, deletion of DOT1L in thermogenic adipocytes potently protects mice from diet-induced obesity, improves glucose homeostasis, alleviates hepatic steatosis, and facilitates adaptive thermogenesis in vivo. Mechanistically, DOT1L negatively regulates the BAT-selective gene program through H3K79 methylation. Thus, our study demonstrates that DOT1L exerts an important role in energy homeostasis by regulating thermogenic adipocyte function in mice.

MAT2A as Key Regulator and Therapeutic Target in MLLr Leukemogenesis.

Epigenetic dysregulation plays a pivotal role in mixed-lineage leukemia (MLL) pathogenesis, therefore serving as a suitable therapeutic target. S-adenosylmethionine (SAM) is the universal methyl donor in human cells and is synthesized by methionine adenosyltransferase 2A (MAT2A), which is deregulated in different cancer types. Here, we used our human CRISPR/Cas9-MLL-rearranged (CRISPR/Cas9-MLLr) leukemia model, faithfully mimicking MLLr patients’ pathology with indefinite growth potential in vitro, to evaluate the unknown role of MAT2A. Comparable to publicly available patient data, we detected MAT2A to be significantly overexpressed in our CRISPR/Cas9-MLLr model compared to healthy controls. By using non-MLLr and MLLr cell lines and our model, we detected an MLLr-specific enhanced response to PF-9366, a new MAT2A inhibitor, and small interfering (si) RNA-mediated knockdown of MAT2A, by alteration of the proliferation, viability, differentiation, apoptosis, cell cycling, and histone methylation. Moreover, the combinational treatment of PF-9366 with chemotherapy or targeted therapies against the SAM-dependent methyltransferases, disruptor of telomeric silencing 1 like (DOT1L) and protein arginine methyltransferase 5 (PRMT5), revealed even more pronounced effects. In summary, we uncovered MAT2A as a key regulator in MLL leukemogenesis and its inhibition led to significant anti-leukemic effects. Therefore, our study paves the avenue for clinical application of PF-9366 to improve the treatment of poor prognosis MLLr leukemia.

Histone Methyltransferase DOT1L Is Involved in Larval Molting and Second Stage Nymphal Feeding in Ornithodoros moubata

Epigenetic mechanisms have not been characterized in ticks despite their importance as vectors of human and animal diseases worldwide. Our investigation identifies and functionally characterizes the orthologue of S-adenosylmethionine (SAM) binding methyltransferase enzyme, disruptor of telomeric silencing 1-like (DOT1L) in Ornithodoros moubata (OmDOT1L), a soft tick vector for the relapsing fever pathogen Borrelia duttonii and the African swine fever virus. The OmDOT1L tertiary structure was predicted and compared to the Homo sapiens DOT1L which had been co-crystalized with SGC0946, a DOT1L-specific inhibitor. The amino acid residues crucial for SAM and SGC0946 binding conserved in most DOT1L sequences available, are also conserved in OmDOT1L. Quantitative PCR of Omdot1l during O. moubata life stages showed that transcripts were significantly upregulated in first-stage nymphs. O. moubata larvae exposed to SGC0946 displayed high mortality during molting to first-stage nymphs. Furthermore, a significant decrease in weight was observed in second-stage nymphs fed on recombinant OmDOT1L-immunized rabbits. In contrast, artificial blood feeding supplemented with SGC0946 did not affect survival and reproductive performance of adult female ticks. We concluded that OmDOT1L plays an essential role in the regulation of larval molting and the feeding of O. moubata second-stage nymphs.

Antiproliferative and Antimigration Activities of Fluoro-Neplanocin A via Inhibition of Histone H3 Methylation in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is among the most aggressive and potentially metastatic malignancies. Most affected patients have poor clinical outcomes due to the lack of specific molecular targets on tumor cells. The upregulated expression of disruptor of telomeric silencing 1-like (DOT1L), a histone methyltransferase specific for the histone H3 lysine 79 residue (H3K79), is strongly correlated with TNBC cell aggressiveness. Therefore, DOT1L is considered a potential molecular target in TNBC. Fluoro-neplanocin A (F-NepA), an inhibitor of S-adenosylhomocysteine hydrolase, exhibited potent antiproliferative activity against various types of cancer cells, including breast cancers. However, the molecular mechanism underlying the anticancer activity of F-NepA in TNBC cells remains to be elucidated. We determined that F-NepA exhibited a higher growth-inhibitory activity against TNBC cells relative to non-TNBC breast cancer and normal breast epithelial cells. Moreover, F-NepA effectively downregulated the level of H3K79me2 in MDA-MB-231 TNBC cells by inhibiting DOT1L activity. F-NepA also significantly inhibited TNBC cell migration and invasion. These activities of F-NepA might be associated with the upregulation of E-cadherin and downregulation of N-cadherin and Vimentin in TNBC cells. Taken together, these data highlight F-NepA as a strong potential candidate for the targeted treatment of high-DOT1L-expressing TNBC.

Amplification of the Mutation-Carrying BRCA2 Allele Promotes RAD51 Loading and PARP Inhibitor Resistance in the Absence of Reversion Mutations.

Patients harboring germline breast cancer susceptibility genes 1 and 2 (BRCA1/2) mutations are predisposed to developing breast, pancreatic, and ovarian cancers. BRCA2 plays a critical role in homologous recombination (HR) DNA repair and deleterious mutations in BRCA2 confer sensitivity to PARP inhibition. Recently, the PARP inhibitors olaparib and rucaparib were FDA approved for the treatment of metastatic breast cancer and patients with recurrent ovarian cancer with mutations in BRCA1/2. Despite their initial antitumor activity, the development of resistance limits the clinical utility of PARP inhibitor therapy. Multiple resistance mechanisms have been described, including reversion mutations that restore the reading frame of the BRCA2 gene. In this study, we generated olaparib- and rucaparib-resistant BRCA2-mutant Capan1 cell lines. We did not detect secondary reversion mutations in the olaparib- or rucaparib-resistant clones. Several of the resistant clones had gene duplication and amplification of the mutant BRCA2 allele, with a corresponding increase in expression of a truncated BRCA2 protein. In addition, HR-mediated DNA repair was rescued, as evidenced by the restoration of RAD51 foci formation. Using mass spectrometry, we identified Disruptor Of Telomeric silencing 1-Like (DOT1L), as an interacting partner of truncated BRCA2. RNAi-mediated knockdown of BRCA2 or DOT1L was sufficient to resensitize cells to olaparib. The results demonstrate that independent of a BRCA2 reversion, mutation amplification of a mutant-carrying BRCA2 contributes to PARP inhibitor resistance.

The role of DOT1L in the proliferation and prognosis of gastric cancer.

Background: Disruptor of telomeric silencing-1-like (DOT1L), a methyltransferase of H3K79, was observed to be amplified and overexpressed in certain malignancies. This work was aimed at investigating the differences in DOT1L expression and its regulatory mechanism in gastric cancer (GC) and healthy samples.Methods: Immunohistochemistry was used to detect DOT1L levels in 101 cases of GC and marching adjacent normal tissues. DOT1L was inhibited by small interfering RNA (siRNA) and EPZ5676; a targeting drug. The ability of cells to proliferate were checked by cell counting kit-8 (CCK-8) and clone formation assays, with flow cytometry for observing the cell cycle. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot revealed the gene and protein profiles. Finally, the outcome of EPZ5676 administration was checked on a murine model.Results: The expression of DOT1L is significantly increased in gastric malignant tumors that is related to the degree of differentiation, lymph node metastasis and TNM staging. DOT1L serves as an independent marker for the prognosis of overall survival (OS) with high levels implying worse prognosis. In addition, DOT1L regulates cyclin-dependent kinase (CDK) 4 (CDK4) and CDK6 through H3K79me2, which leads to a change in the cell cycle at G1, thereby affecting the proliferation of tumors in vitro and in vivo.Conclusions: This is a first clinical demonstration of the applicability of DOT1L overexpression in gastric tumors. The work is suggestive of altered proliferation of cells by DOT1L via regulating cyclins and H3K79 methylation. This indicates the role of DOT1L in the prognosis and possible medical intervention of GC.