Histone Methyltransferase DOT1L Research Articles

LSD1 Inhibition Exerts Its Anti-Leukemic Effect By Recommissioning PU.1- and C/Ebpα-Dependent Enhancers in AML

Epigenetic regulators are recurrently mutated and aberrantly expressed in acute myeloid leukemia (AML). Targeted therapies designed to inhibit these chromatin-modifying enzymes, such as the histone demethylase lysine specific demethylase 1 (LSD1) and the histone methyltransferase DOT1L, have been developed as novel treatment modalities for these often refractory diseases. We aimed to investigate the chromatin changes induced by an irreversible inhibitor of LSD1 (GSK-LSD1) on rMLL+ leukemias. For this, we treated rMLL+ murine AML and patient-derived xenografts with the GSK-LSD1 inhibitor in vivo and assessed chromatin accessibility changes using the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq). Treated mice exhibited a lower proportion of MLL-AF9+ leukemia cells detected in the bone marrow (BM) and signs of myeloid differentiation. Treated mice also had markedly improved survival with a median survival of 78 days vs. control mice (39 days). After 6 weeks of treatment with GSK-LSD1, mice xenotransplanted with human rMLL+ AML samples showed a significant decrease in engraftment and myeloid differentiation. Serial transplantation of murine AML revealed eradication of LSC upon GSK-LSD1 treatment of donor mice. Most importantly, LSD1 inhibition resulted in gain in chromatin accessibility in both murine and PDX-AML cells in vitro as well as in vivo . We then wanted to compare the effect of another anti-leukemic epigenetic drug, also known to induce myeloid differentiation of MLL-AF9+ cells, such as the DOT1L inhibitor EPZ4777. In contrast to GSK-LSD1, EPZ4777 treatment caused a global decrease in chromatin accessibility. The intersection of sites with dynamic accessibility upon DOT1L and LSD1 inhibition showed minimal overlap between the 2 treatments. The distribution of genomic regions with dynamic accessibility upon these drugs indicated that DOT1L inhibition affects a greater number of sites in promoter regions, whereas LSD1 inhibition affected a higher proportion of loci occupied within the intergenic and intronic regions. Furthermore, we sought to identify TF motif signatures at GSK-LSD1-induced dynamic sites. The most highly enriched motifs captured were those for myeloid TFs PU.1 and C/EBPα-β. We then performed ChIP-seq to identify sites of C/EBPα and PU.1 occupancy in the setting of GSK-LSD1 treatment. Interestingly, we observed a redistribution of PU.1 and C/EBPα occupancy upon treatment, consistent with the role of LSD1 in regulating the binding and activity of TFs. While PU.1 ChIPseq signal increased at dynamic ATAC-seq peaks in response to LSD1 inhibition, no increase in C/EBPα occupancy was observed. These dynamic sites also demonstrates GSK-LSD1-dependent co-occupancy of PU.1 and C/EBPα. We next sought to delineate whether PU.1 and C/EBPα are critical for these dynamic changes in chromatin accessibility in response to LSD1 inhibition. We performed ATAC-seq on PU.1 URE KO MLL-AF9 leukemias and C/EBPα KO MLL-AF9 leukemias treated with vehicle or GSK-LSD1. The increase in chromatin accessibility in TF-WT MLL-AF9 leukemias upon GSK-LSD1 was abrogated in both PU.1 URE KO and C/EBPα KO MLL-AF9 leukemia cells. We next assess the impact of TF-depletion on sensitivity to LSD1 inhibition. We treated PU.1 URE KO leukemias cells with GSK-LSD1 for 48 hours in vitro . These cells were more resistant to LSD1 inhibition when compared to PU.1 URE heterozygous and WT AML cells. In vivo, we reported loss of survival benefit upon GSK-LSD1 treatment in PU.1 URE KO MLL-AF9+ mice vs. WT mice. Similarly, C/EBPα KO leukemia cells were more resistant to LSD1 inhibition when compared to WT leukemia cells. After 2 weeks of treatment in vivo, C/EBPα KO leukemic mice also showed no decrease in MLL-AF9 allele burden. Taken together, our comparative investigations into the chromatin-modifying effects of two distinct epigenetic therapies targeting either LSD1 or DOT1L is illustrative of the concept that multiple paths toward myeloid differentiation can potentially be engaged to achieve therapeutic efficacy in AML. Our findings reveal that pharmacologic inhibition of LSD1 depends on balanced expression of the TFs PU.1 and C/EBPα, which can be used as predictors of response in AML aberrantly expressing these factors. DisclosuresMohammad:GSK: Employment. Ye:Incyte: Employment. Will:Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl:Celgene: Consultancy; Bayer Healthcare: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding. Kruger:GSK: Employment. Armstrong:Imago: Consultancy; Cyteir: Consultancy; C4 Therapeutics: Consultancy; Epizyme: Consultancy.

Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation.

Differentiation of naïve peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B‐cell fate remain unclear. Here, we identified a role for the histone H3K79 methyltransferase DOT1L in controlling B‐cell differentiation. Mouse B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells in which Dot1L was deleted showed aberrant differentiation and prematurely acquired plasma cell characteristics. Similar results were obtained when DOT1L was chemically inhibited in mature B cells in vitro. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro‐proliferative, pro‐GC program. In addition, DOT1L indirectly supports the repression of an anti‐proliferative plasma cell differentiation program by maintaining the repression of Polycomb Repressor Complex 2 (PRC2) targets. Our findings show that DOT1L is a key modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B‐cell naivety and GC B‐cell differentiation.

Mesenchyme-specific loss of Dot1L histone methyltransferase leads to skeletal dysplasia phenotype in mice.

Chromatin modifying enzymes play essential roles in skeletal development and bone maintenance, and deregulation of epigenetic mechanisms can lead to skeletal growth and malformation disorders. Here, we report a novel skeletal dysplasia phenotype in mice with conditional loss of Disruptor of telomeric silencing 1-like (Dot1L) histone methyltransferase in limb mesenchymal progenitors and downstream descendants. Phenotypic characterizations of mice with Dot1L inactivation by Prrx1-Cre (Dot1L-cKOPrrx1) revealed limb shortening, abnormal bone morphologies, and forelimb dislocations. Our in vivo and in vitro data support a crucial role for Dot1L in regulating growth plate chondrocyte proliferation and differentiation, extracellular matrix production, and secondary ossification center formation. Micro-computed tomography analysis of femurs revealed that partial loss of Dot1L expression is sufficient to impair trabecular bone formation and microarchitecture in young mice. Moreover, RNAseq analysis of Dot1L deficient chondrocytes implicated Dot1L in the regulation of key genes and pathways necessary to promote cell cycle regulation and skeletal growth. Collectively, our data show that early expression of Dot1L in limb mesenchyme provides essential regulatory control of endochondral bone morphology, growth, and stability.

Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer

The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and is involved in Mixed Lineage Leukemia (MLL) fusion leukemogenesis; however, its role in prostate cancer (PCa) is undefined. Here we show that DOT1L is overexpressed in PCa and is associated with poor outcome. Genetic and chemical inhibition of DOT1L selectively impaired the viability of androgen receptor (AR)-positive PCa cells and organoids, including castration-resistant and enzalutamide-resistant cells. The sensitivity of AR-positive cells is due to a distal K79 methylation-marked enhancer in the MYC gene bound by AR and DOT1L not present in AR-negative cells. DOT1L inhibition leads to reduced MYC expression and upregulation of MYC-regulated E3 ubiquitin ligases HECTD4 and MYCBP2, which promote AR and MYC degradation. This leads to further repression of MYC in a negative feed forward manner. Thus DOT1L selectively regulates the tumorigenicity of AR-positive prostate cancer cells and is a promising therapeutic target for PCa.

The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells

Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T cell-specific ablation of Dot1L resulted in loss of naïve CD8+ T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8+ T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of Dot1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity.

Inhibition of Methyltransferase DOT1L Sensitizes to Sorafenib Treatment AML Cells Irrespective of MLL-Rearrangements: A Novel Therapeutic Strategy for Pediatric AML.

Pediatric acute myeloid leukemia (AML) is an aggressive malignancy with poor prognosis for which there are few effective targeted approaches, despite the numerous genetic alterations, including MLL gene rearrangements (MLL-r). The histone methyltransferase DOT1L is involved in supporting the proliferation of MLL-r cells, for which a target inhibitor, Pinometostat, has been evaluated in a clinical trial recruiting pediatric MLL-r leukemic patients. However, modest clinical effects have been observed. Recent studies have reported that additional leukemia subtypes lacking MLL-r are sensitive to DOT1L inhibition. Here, we report that targeting DOT1L with Pinometostat sensitizes pediatric AML cells to further treatment with the multi-kinase inhibitor Sorafenib, irrespectively of MLL-r. DOT1L pharmacologic inhibition induces AML cell differentiation and modulates the expression of genes with relevant roles in cancer development. Such modifications in the transcriptional program increase the apoptosis and growth suppression of both AML cell lines and primary pediatric AML cells with diverse genotypes. Through ChIP-seq analysis, we identified the genes regulated by DOT1L irrespective of MLL-r, including the Sorafenib target BRAF, providing mechanistic insights into the drug combination activity. Our results highlight a novel therapeutic strategy for pediatric AML patients.

Specific patterns of H3K79 methylation influence genetic interaction of oncogenes in AML

AbstractUnderstanding mechanisms of cooperation between oncogenes is critical for the development of novel therapies and rational combinations. Acute myeloid leukemia (AML) cells with KMT2A-fusions and KMT2A partial tandem duplications (KMT2APTD) are known to depend on the histone methyltransferase DOT1L, which methylates histone 3 lysine 79 (H3K79). About 30% of KMT2APTD AMLs carry mutations in IDH1/2 (mIDH1/2). Previous studies showed that 2-hydroxyglutarate produced by mIDH1/2 increases H3K79 methylation, and mIDH1/2 patient samples are sensitive to DOT1L inhibition. Together, these findings suggested that stabilization or increases in H3K79 methylation associated with IDH mutations support the proliferation of leukemias dependent on this mark. However, we found that mIDH1/2 and KMT2A alterations failed to cooperate in an experimental model. Instead, mIDH1/2 and 2-hydroxyglutarate exert toxic effects, specifically on KMT2A-rearranged AML cells (fusions/partial tandem duplications). Mechanistically, we uncover an epigenetic barrier to efficient cooperation; mIDH1/2 expression is associated with high global histone 3 lysine 79 dimethylation (H3K79me2) levels, whereas global H3K79me2 is obligate low in KMT2A-rearranged AML. Increasing H3K79me2 levels, specifically in KMT2A-rearrangement leukemias, resulted in transcriptional downregulation of KMT2A target genes and impaired leukemia cell growth. Our study details a complex genetic and epigenetic interaction of 2 classes of oncogenes, IDH1/2 mutations and KMT2A rearrangements, that is unexpected based on the high percentage of IDH mutations in KMT2APTD AML. KMT2A rearrangements are associated with a trend toward lower response rates to mIDH1/2 inhibitors. The substantial adaptation that has to occur for 2 initially counteracting mutations to be tolerated within the same leukemic cell may provide at least a partial explanation for this observation.

Chromatin Complexes Maintain Self-Renewal of Myeloid Progenitors in AML: Opportunities for Therapeutic Intervention

Specific subgroups of acute myeloid leukemia (AML), including those containing MLL rearrangements and NPM1c mutations, possess characteristic stem cell-like gene expression profiles. These expression programs are highly dependent on components of the MLL histone methyltransferase complex, including Menin and DOT1L. Understanding the chromatin-based mechanisms through which cancer cells subvert certain aspects of normal stem cell biology helped identify specific vulnerabilities and translate them into targeted therapy approaches. Exciting progress has been made in the development of small-molecule inhibitors targeting this epigenetic machinery in leukemia cells and prompted the development of clinical trials in patients with hematologic malignancies.

Epigenetic regulation of protein translation in KMT2A-rearranged AML

Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.

NANOG and LIN28 dramatically improve human cell reprogramming by modulating LIN41 and canonical WNT activities.

ABSTRACTHuman cell reprogramming remains extremely inefficient and the underlying mechanisms by different reprogramming factors are elusive. We found that NANOG and LIN28 (NL) synergize to improve OCT4, SOX2, KLF4 and MYC (OSKM)-mediated reprogramming by ∼76-fold and shorten reprogramming latency by at least 1 week. This synergy is inhibited by GLIS1 but reinforced by an inhibitor of the histone methyltransferase DOT1L (iDOT1L) to a ∼127-fold increase in TRA-1-60-positive (+) iPSC colonies. Mechanistically, NL serve as the main drivers of reprogramming in cell epithelialization, the expression of Let-7 miRNA target LIN41, and the activation of canonical WNT/β-CATENIN signaling, which can be further enhanced by iDOT1L treatment. LIN41 overexpression in addition to OSKM similarly promoted cell epithelialization and WNT activation in reprogramming, and a dominant-negative LIN41 mutation significantly blocked NL- and iDOT1L-enhanced reprogramming. We also found that NL- and iDOT1L-induced canonical WNT activation facilitates the initial development kinetics of iPSCs. However, a substantial increase in more mature, homogeneous TRA-1-60+ colony formation was achieved by inhibiting WNT activity at the middle-to-late-reprogramming stage. We further found that LIN41 can replace LIN28 to synergize with NANOG, and that the coexpression of LIN41 with NL further enhanced the formation of mature iPSCs under WNT inhibition. Our study established LIN41 and canonical WNT signaling as the key downstream effectors of NL for the dramatic improvement in reprogramming efficiency and kinetics, and optimized a condition for the robust formation of mature human iPSC colonies from primary cells.This article has an associated First Person interview with the first author of the paper.

The DOT1L inhibitor Pinometostat decreases the host-response against infections: Considerations about its use in human therapy

Patients with acute myeloid leukemia frequently present translocations of MLL gene. Rearrangements of MLL protein (MLL-r) in complexes that contain the histone methyltransferase DOT1L are common, which elicit abnormal methylation of lysine 79 of histone H3 at MLL target genes. Phase 1 clinical studies with pinometostat (EPZ-5676), an inhibitor of DOT1L activity, demonstrated the therapeutic potential for targeting DOT1L in MLL-r leukemia patients. We previously reported that down-regulation of DOT1L increases influenza and vesicular stomatitis virus replication and decreases the antiviral response. Here we show that DOT1L inhibition also reduces Sendai virus-induced innate response and its overexpression decreases influenza virus multiplication, reinforcing the notion of DOT1L controlling viral replication. Accordingly, genes involved in the host innate response against pathogens (RUBICON, TRIM25, BCL3) are deregulated in human lung epithelial cells treated with pinometostat. Concomitantly, deregulation of some of these genes together with that of the MicroRNA let-7B, may account for the beneficial effects of pinometostat treatment in patients with MLL-r involving DOT1L. These results support a possible increased vulnerability to infection in MLL-r leukemia patients undergoing pinometostat treatment. Close follow up of infection should be considered in pinometostat therapy to reduce some severe side effects during the treatment.

A higher-order configuration of the heterodimeric DOT1L–AF10 coiled-coil domains potentiates their leukemogenenic activity

Chromosomal translocations of MLL1 (Mixed Lineage Leukemia 1) yield oncogenic chimeric proteins containing the N-terminal portion of MLL1 fused with distinct partners. The MLL1-AF10 fusion causes leukemia through recruiting the H3K79 histone methyltransferase DOT1L via AF10's octapeptide and leucine zipper (OM-LZ) motifs. Yet, the precise interaction sites in DOT1L, detailed interaction modes between AF10 and DOT1L, and the functional configuration of MLL1-AF10 in leukeomogenesis remain unknown. Through a combined approach of structural and functional analyses, we found that the LZ domain of AF10 interacts with the coiled-coil domains of DOT1L through a conserved binding mode and discovered that the C-terminal end of the LZ domain and the OM domain of AF10 mediate the formation of a DOT1L-AF10 octamer via tetramerization of the binary complex. We reveal that the oligomerization ability of the DOT1L-AF10 complex is essential for MLL1-AF10's leukemogenic function. These findings provide insights into the molecular basis of pathogenesis by MLL1 rearrangements.

2044-P: Histone Methyltransferase DOT1L Regulates Brown Adipocyte Differentiation and Thermogenesis

Brown fat has been extensively studied due to its potential in therapies against obesity. Various epigenetic regulators have been shown to modulate brown fat development and function. Here we show that histone methyltransferase DOT1L is a key regulator of brown adipocyte differentiation and thermogenesis. In vitro, we demonstrate that inhibition by DOT1L inhibitor or knockdown of DOT1L markedly promotes brown adipocyte differentiation of C3H10T1/2 cells. Moreover, depletion of DOT1L enhances differentiation and thermogenesis of primary brown adipocytes which separated from DOT1LFlox/Flox mice by transfection with Cre-expressing lentiviruses ex vivo. Mechanistically, knockdown of DOT1L results in increased expression of brown-selective genes, and the expression of brown preadipocyte maker EBF2 is significantly increased due to knockdown of DOT1L at early stage. Furthermore, ChIP assays suggest that DOT1L-mediated H3K79 methylations are negatively associated with transcription of UCP1 and PRDM16. Finally, we observe that the skin surface temperature in interscapular area of DOT1LMyf5 knockout mice is significantly higher. Notably, energy expenditure and the oxygen consumption rate are also increased in knockout mice. These in vivo data suggest that DOT1L depletion leads to enhanced thermogenic function of brown fat. Collectively, our work indicates that DOT1L plays an important role in regulating brown fat differentiation and thermogenesis, and provides another linkage between epigenetics and brown fat development and function. Disclosure L. Shuai: None. J. Li: None. J. Li: None. Funding China Postdoctoral Science Foundation

Activation of the Lysosome-Associated Membrane Protein LAMP5 by DOT1L Serves as a Bodyguard for MLL Fusion Oncoproteins to Evade Degradation in Leukemia.

Despite many attempts to understand mixed-lineage leukemia (MLL leukemia), effective therapies for this disease remain limited. We identified a lysosome-associated membrane protein (LAMP) family member, LAMP5, that is specifically and highly expressed in patients with MLL leukemia. The purpose of the study was to demonstrate the functional relevance and clinical value of LAMP5 in the disease. We first recruited a large cohort of leukemia patients to validate LAMP5 expression and evaluate its clinical value. We then performed in vitro and in vivo experiments to investigate the functional relevance of LAMP5 in MLL leukemia progression or maintenance. LAMP5 was validated as being specifically and highly expressed in patients with MLL leukemia and was associated with a poor outcome. Functional studies showed that LAMP5 is a novel autophagic suppressor and protects MLL fusion proteins from autophagic degradation. Specifically targeting LAMP5 significantly promoted degradation of MLL fusion proteins and inhibited MLL leukemia progression in both an animal model and primary cells. We further revealed that LAMP5 is a direct target of the H3K79 histone methyltransferase DOT1L. Downregulating LAMP5 with a DOT1L inhibitor enhanced the selective autophagic degradation of MLL oncoproteins and extended survival in vivo; this observation was especially significant when combining DOT1L inhibitors with LAMP5 knockdown. This study demonstrates that LAMP5 serves as a "bodyguard" for MLL fusions to evade degradation and is the first to link H3K79 methylation to autophagy regulation, highlighting the potential of LAMP5 as a therapeutic target for MLL leukemia.

Discovery of a Selective Inhibitor for the YEATS Domains of ENL/AF9

Eleven-nineteen leukemia (ENL) contains an epigenetic reader domain (YEATS domain) that recognizes lysine acylation on histone 3 and facilitates transcription initiation and elongation through its interactions with the super elongation complex (SEC) and the histone methyl transferase DOT1L. Although it has been known for its role as a fusion protein in mixed lineage leukemia (MLL), overexpression of native ENL, and thus dysregulation of downstream genes in acute myeloid leukemia (AML), has recently been implicated as a driver of disease that is reliant on the epigenetic reader activity of the YEATS domain. We developed a peptide displacement assay (histone 3 tail with acylated lysine) and screened a small-molecule library totaling more than 24,000 compounds for their propensity to disrupt the YEATS domain–histone peptide binding. Among these, we identified a first-in-class dual inhibitor of ENL (Kd = 745 ± 45 nM) and its paralog AF9 (Kd = 523 ± 53 nM) and performed “SAR by catalog” with the aim of starting the development of a chemical probe for ENL.

Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells.

Breast cancer (BC) resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor α (ERα) signaling, and ways to block ERα pathway in these tumors are sought after. We identified the H3K79 methyltransferase DOT1L as a novel cofactor of ERα in BC cell chromatin, where the two proteins colocalize to regulate estrogen target gene transcription. DOT1L blockade reduces proliferation of hormone-responsive BC cells in vivo and in vitro, consequent to cell cycle arrest and apoptotic cell death, with widespread effects on ER-dependent gene transcription, including ERα and FOXA1 gene silencing. Antiestrogen-resistant BC cells respond to DOT1L inhibition also in mouse xenografts, with reduction in ERα levels, H3K79 methylation, and tumor growth. These results indicate that DOT1L is an exploitable epigenetic target for treatment of endocrine therapy-resistant ERα-positive BCs.

The histone methyltransferase DOT1L is required for proper DNA damage response, DNA repair, and modulates chemotherapy responsiveness

BackgroundDisruptor of telomeric silencing 1-like (DOT1L) is a non-SET domain containing methyltransferase known to catalyze mono-, di-, and tri-methylation of histone 3 on lysine 79 (H3K79me). DOT1L-mediated H3K79me has been implicated in chromatin-associated functions including gene transcription, heterochromatin formation, and DNA repair. Recent studies have uncovered a role for DOT1L in the initiation and progression of leukemia and other solid tumors. The development and availability of small molecule inhibitors of DOT1L may provide new and unique therapeutic options for certain types or subgroups of cancer.MethodsIn this study, we examined the role of DOT1L in DNA double-strand break (DSB) response and repair by depleting DOT1L using siRNA or inhibiting its methyltransferase activity using small molecule inhibitors in colorectal cancer cells. Cells were treated with different agents to induce DNA damage in DOT1L-depleted or -inhibited cells and analyzed for DNA repair efficiency and survival. Further, rectal cancer patient samples were analyzed for H3K79me3 levels in order to determine whether it may serve as a potential marker for personalized therapy.ResultsOur results indicate that DOT1L is required for a proper DNA damage response following DNA double-strand breaks by regulating the phosphorylation of the variant histone H2AX (γH2AX) and repair via homologous recombination (HR). Importantly, we show that small molecule inhibitors of DOT1L combined with chemotherapeutic agents that are used to treat colorectal cancers show additive effects. Furthermore, examination of H3K79me3 levels in rectal cancer patients demonstrates that lower levels correlate with a poorer prognosis.ConclusionsIn this study, we conclude that DOT1L plays an important role in an early DNA damage response and repair of DNA double-strand breaks via the HR pathway. Moreover, DOT1L inhibition leads to increased sensitivity to chemotherapeutic agents and PARP inhibition, which further highlights its potential clinical utility. Our results further suggest that H3K79me3 can be useful as a predictive and or prognostic marker for rectal cancer patients.

Graft-Versus-Host Disease Impairs the Histone Methyltransferase Dot1l-Regulated Reconstitution of Plasmacytoid Dendritic Cells in Mice Undergoing Allo-HSCT

Plasmacytoid dendritic cells (pDCs) derived either from adoptive transfer from the donor graft or stem cell reconstitution can attenuate and prevent graft-versus-host disease (GVHD) in both pre-clinical and clinical settings. However, the reconstitution of donor pDCs is severely impaired during GVHD via an unknown mechanism. Here we demonstrate that the histone methyltransferase Dot1l, which specifically catalyzes methylation of histone H3 at lysine 79 (H3K79me), is critical for regulating the commitment and differentiation of pDCs from hematopoietic stem cells (HSCs) and we observed its function was severely impaired in GVHD mice. We have previously demonstrated that Flt3L-induced DCs can program allogeneic T cells to reduce their GVHD toxicity (Blood 2016). Using this platform, we explored candidate histone methyltransferase(s) that affected pDC development. We found the inhibitor specific to Dot1l dramatically decreased the frequency and number of pDCs in cultures compared to other chemical probes that inhibit Ezh2, MLL1, G9a and Jmjd3, respectively. Under steady-state condition, pDCs develop from HSCs through successive steps of lineage commitment and differentiation: multiple potent progenitors (MPP) → macrophage and DC progenitors (MDP) → common DC progenitors (CDP). Upon culturing in the presence of Flt3L+SCF, MPP produced 2-fold and 5-fold more pDCs than MDP and HSCs, respectively, over 6 days' incubation. For this reason, we focused on evaluating the effect of Dot1l deletion on pDC development from MPP and CDP, which represent the early and later stage of pDC progenitors, respectively. To examine the specific role of Dot1l in DC development, we crossed Dot1l conditional knock mice (Dot1lf/f) to ER-Cre B6 mice to generate ER-Cre.Dot1lf/f B6 mice. We administered tamoxifen to ER-Cre.Dot1lf/f B6 mice at day 0 and day +1 to delete Dot1l, highly purified MPP and CDP, and cultured them in the presence of Flt3L+SCF. Deletion of Dot1l led to significant decrease of (3- to 5-fold) pDCs from MPP but marginally decreased CDP production of pDCs. These results suggest that Dot1l is required for the initial commitment and differentiation of MPP into pDCs or the expansion of selected pDCs. To test it, we added Dot1l inhibitor SGC to the cultures containing wild-type (WT) mouse-derived MPP, CDP and pDCs. Inhibiting Dot1l with SGC decreased both the frequency (3-fold) and number (5-fold) of pDCs in the MPP culture compared to control, however, it did not affect the generation of pDCs from the cultures of either CDP or pDCs. Thus, Dot1l regulates the commitment and differentiation of pDCs during the MPP stage. To understand the molecular mechanism by which Dot1l regulates pDC commitment and differentiation, we examined the expression of transcription factor Tcf4, which promotes pDC development, and Id2, which antagonizes Tcf4 effects on pDCs. Dot1l inhibition led to 2-fold reduction of Tcf4 expression and 5-fold increase of Id2 in Flt3L-induced bone marrow cells. ChIP assays confirmed the presence of high amount of Dot1l-catalyzed H3K79me2 at the promoter regions of Id2 and Tcf4 loci. Retroviral introduction of Tcf4 into hematopoietic progenitors lacking Dot1l restored their capacity to produce pDCs in cultures. These findings clearly establish that Tcf4 is the down-stream effector of Dot1l to control pDC development. Building on these observations, we finally examined whether GVHD may impair the reconstitution of donor pDCs through a mechanism of reducing Dot1l expression and function. Using the C57BL/6 (B6) into Balb/c mouse GVHD model, we confirmed the loss of donor pDCs in GVHD recipients at day 14 and day 21 after transplantation and, observed that these GVHD mice had 3- to 12-fold fewer MPP and CDP compared to normal donor mice. Additionally, both HSCs and MPP derived from GVHD mice showed significant reduction of H3K79me2 compared to their normal counterparts. Finally, adoptive transfer of donor BM-derived pDCs attenuated the incidence and severity of GVHD in Balb/c mice receiving B6 T cells. Collectively, these data suggest that GVHD-mediated inflammation profoundly decreases Dot1l function in engrafted donor DC progenitors, leading to damaged reconstitution of donor pDCs. Novel strategies that target Dot1l and its-regulated transcriptional programs may improve the reconstitution of donor pDCs to inhibit GVHD while also promoting anti-leukemia activity in recipients undergoing allo-HSCT. DisclosuresNo relevant conflicts of interest to declare.

Targeting MYC-Driven B-Cell Lymphoma By Inhibition of the Histone Methyltransferase DOT1L

Aberrant activation of the MYC proto-oncogene is a recurrent feature in human B-cell lymphomas of diverse sub-types, correlating with adverse prognosis and therapy resistance. Direct pharmacological MYC-targeting has proved difficult, but recent studies have shown that targeting chromatin regulators critical for MYC-driven oncogenesis may provide alternative avenues for therapeutic intervention. Recently, it has been demonstrated that MYC-driven oncogenesis in certain solid tumors is dependent on the histone 3 lysine 79 (H3K79) methyltransferase DOT1L. We hypothesized that B-cell lymphomas with hyperactive MYC-signaling might be responsive to DOT1L inhibition. In order to test this hypothesis, we tested the effect of the DOT1L inhibitor Pinometostat (EPZ-5676) on a panel of human B-cell lymphoma cell lines featuring elevated MYC. Pinometostat treatment reduced global H3K79 methylation levels, accompanied by a time and dose-dependent decrease in proliferation of several Burkitt's lymphoma cell lines including P493-6, Daudi and Raji. We observed that key MYC-target genes including CDK4, PPAT and NPM1 were significantly downregulated upon Pinometostat treatment, suggesting that DOT1L is required for the transcriptional activation of MYC-target genes in these cells. Pinometostat-treated B-lymphoma cells showed a significant decrease of cells in S-phase compared to controls as assessed by BrdU-labeling assays. Similar results were also obtained in a panel of B-cell lymphoma cell lines with MYC-rearrangements including mantle cell lymphoma (MCL) cell lines Jeko-1, JVM2, Mino-1 and Maver-1 and the diffused large B-cell lymphoma (DLBCL) cell line Karpas 422.Next, we sought to investigate whether the DOT1L-dependence of MYC-driven B-cell lymphoma could be reproduced in a well-defined model of MYC-driven B-cell lymphoma. Towards this end, we utilized a mouse model in which expression of the Cre recombinase from a B cell specific promoter leads to ectopic expression of a transgenic human MYC allele and concomitant deletion of the tumor suppressor Pten in B cells. Similar to our in vitro studies, Pinometostat treatment led to a significant reduction in proliferation of B-cell lymphoma cells from these mice with an IC50 of 0.5 µM.Furthermore, we sought to ascertain whether these findings reflected on-target effects related to DOT1L inhibition. Therefore, we deleted DOT1L using CRISPR/Cas9 in B-cell lymphoma cell lines and assessed the effect on proliferation using competitive-proliferation assays. We observed that DOT1L-deletion progressively diminished the relative growth of anti-DOT1L sgRNA-expressing P493-6 and Jeko1 cells compared to non-targeted cells invitro. In order to test the requirement for DOT1L in lymphoma propagation in vivo, we performed intravenous injections of equal number of Jeko-1 cells with either anti-DOT1L or anti-Renilla control sgRNAs into sub-lethally irradiated non-obese diabetic/severe combined immunodeficiency mice (NOD/SCID) mice. Mice injected with control anti-Renilla sgRNAs succumbed to disease with a median latency of 34 days while the latency of disease in the anti-DOT1L sgRNA cohort was 45 days. In summary, DOT1L depletion significantly delayed disease latency in this invivo disseminated model of B-cell lymphoma (P=0.02).We then performed transcriptomic analyses of Pinometostat-treated B-cell lymphoma cell lines compared to DMSO-treated counterparts using RNA-seq. Gene-set enrichment analysis (GSEA) of RNA-seq data from three different B-cell lymphoma cell lines demonstrated that Pinometostat treatment significantly decreased the expression of MYC-target genes. In order to investigate the intriguing role of DOT1L in regulating MYC-target gene expression, we used ChIP-seq to assess the genome-wide occupancy of MYC following DOT1L inhibitor treatment. Strikingly, our studies demonstrated that DOT1L inhibition significantly reduced the chromatin occupancy of MYC.Taken together, our experiments demonstrate the role of DOT1L in MYC-driven B-cell lymphoma pathogenesis invitro and invivo. Furthermore, our genome-wide studies demonstrate the importance of DOT1L for genomic MYC occupancy. Based on these findings, we propose that therapeutic DOT1L targeting may be a viable strategy in MYC-driven B-cell lymphoma. DisclosuresWeigert:Roche: Research Funding; Novartis: Research Funding. Rickert:Pfizer: Employment. Ren:Elli Lilly: Consultancy, Membership on an entity's Board of Directors or advisory committees; Arima Genomics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Deshpande:Salgomed Therapeutics: Membership on an entity's Board of Directors or advisory committees; A2A Pharma: Membership on an entity's Board of Directors or advisory committees.

Abstract 3687: Dot1l inhibition selectively impairs androgen receptor dependent prostate cancer growth through loss of telomere integrity

Abstract The histone methyltransferase DOT1L methylates lysine 79 (K79) on histone H3 and this histone mark is associated with activation of gene expression. DOT1L has a well-known role in MLL fusion leukemogenesis, however, its role in prostate cancer is yet to be elucidated. Here we show that DOT1L is overexpressed in many solid tumors including prostate cancer and is associated with a poor outcome. Genetic and chemical targeting of DOT1L showed a preferential loss in cell viability and colony formation in androgen receptor (AR)-signaling competent prostate cancer cells, including castration-resistant and enzalutamide-resistant cells. DOT1L inhibition reduced AR levels and upregulated enzymes involved in testosterone catabolism but only affected the expression of a subset of AR target genes. More importantly, loss of DOT1L selectively impaired telomere integrity by promoting telomere uncapping and shortening in AR-signaling competent cells. DOT1L inhibition also reduced the expression of telomerase and Shelterin complex genes including TRF2 and POT1. Recruitment of AR to telomeres and K79 methylation at sub-telomeric regions was also diminished. Consequently, loss of DOT1L leads to induction of Telomere dysfunction induced foci (TIF) and activates a cellular senescence program in these cells. Hence, these results provide compelling evidence for DOT1L inhibition as a novel therapeutic approach in advanced prostate cancers. Citation Format: Rajita Vatapalli, Yara Rodriguez, Changsheng Zhao, Vinay Sagar, Jonathan Anker, Sahithi Pamarthi, Kenji Unno, Benedito Carneiro, Debabrata Chakravarti, Sarki Abdulkadir. Dot1l inhibition selectively impairs androgen receptor dependent prostate cancer growth through loss of telomere integrity [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 3687.