DOT1L Inhibitor Research Articles

Determination of nucleoside DOT1L inhibitors’ residence times by τRAMD simulations

Human epigenetic enzyme disruptor of telomeric silencing 1-like (DOT1L) is a key drug target for treating acute myeloid leukemia. Several nucleoside and non-nucleoside DOT1L inhibitors have been developed to inhibit its histone methyltransferase activity. Non-mechanism-based nucleoside DOT1L inhibitors have shown good inhibitory activity and high on-target residence times. Previous computational studies have explored the dynamic behavior of this group of molecules on DOT1L to design compounds with enhanced binding affinities. Nevertheless, it is well known that drug-target kinetics also plays a crucial role in the discovery of new drugs. Therefore, we performed τ-Random Acceleration Molecular Dynamics (τRAMD) simulations to estimate the residence times of nucleoside DOT1L inhibitors. The high correlation between the calculated and experimental residence times suggested that the method can reliably estimate the residence time of nucleoside DOT1L inhibitors when modifications are made to those substituents that occupy the buried hydrophobic pocket of the active site, exhibit hydrophobic interactions with F245 or that form H-bonds with D161 and G163. Overall, this study will be a step toward understanding the binding kinetics of nucleoside DOT1L inhibitors for the treatment of acute myeloid leukemia.

DOT1L Epigenetically Regulates Autophagy and Mitochondria Fusion in Cell Lines of Renal Cancer.

DOT1L, a histone methylase, is overexpression in renal cell cancer. However, the role and detailed molecular mechanism of DOT1L involved in renal cancer development remain unknown. The inhibition of DOT1L was used by SGC0946 and short hairpin RNA silencing. Monodansylcadaverine staining and transmission electron microscope were performed to detect autophagy changes as a result of the inhibition of DOT1L. MitoTracker Red assay was used to analyze mitochondrial morphology. The autophagy markers and mitochondria-related proteins were analyzed by Western blot, qPCR, or immunofluorescence. ChIP assay was performed to demonstrate H3K79me2 is involved in the direct regulation of Farnesoid X receptor transcription. DOT1L inhibition increased autophagy activity and promoted mito chondria fusion in cell lines of renal cancer. Inhibition of DOT1L upregulated levels of LC3α/β, P62, MFN1, and MFN2, which contributed to autophagy activity or mitochondria fusion. DOT1L knockdown showed a similar the above process. DOT1L inhibition or silencing resulted in AMP-activated protein kinase activation and mammalian target of rapamycin inhibition. Mechanistically, the DOT1L inhibitor and its short hairpin RNAs decreased the expression of Farnesoid X receptor in a histone methylase-dependent manner. We revealed the essential role of Farnesoid X receptor in regulating DOT1L-induced autophagy and mitochondrial fission through the AMP-activated protein kinase/mammalian target of rapamycin pathway in cell lines of renal cancer, which may provide new insights into the pathogenesis of renal cell cancer.

Identification of DOT1L inhibitor in a screen for factors that promote dopaminergic neuron survival.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra region of the midbrain. Diagnostic criteria for PD require that at least two of three motor signs are observed: tremor, rigidity, and/or bradykinesia. The most common and effective treatment for PD is Levodopa (L-DOPA) which is readily converted to DA and has been the primary treatment since the 1960's. Dopamine agonists have also been developed but are less effective than L-DOPA. Although the lack of a model system to study PD has hampered efforts to identify treatments, diverse screening strategies have been proposed for identification of new pharmaceutical candidates. Here, we describe a pilot screen to identify candidate molecules from a bioactive compound library, that might increase formation, maintenance and/or survival of DA neurons in vitro. The screen used a previously characterized reporter construct consisting of the luciferase gene inserted downstream of the endogenous tyrosine hydroxylase (TH) gene and neurons differentiated from human pluripotent stem cells for 18 days. The reporter mimics expression of TH and includes a secreted luciferase whose activity can be measured non-invasively over multiple timepoints. Screening of the bioactive compound library resulted in the identification of a single molecule, SGC0946, that is an inhibitor of DOT1L (Disruptor Of Telomeric silencing 1-Like) which encodes a widely-conserved histone H3K79 methyltransferase that is able to both activate and repress gene transcription. Our results indicate that SGC0946 increased reporter luciferase activity with a single treatment for 48-h post-plating being equivalent to continuous treatment. Moreover, data suggested that the total number of neurons differentiated in the assays was comparable from experiment to experiment under different SGC0946 treatments over time. In contrast, data suggested that the survival and/or maintenance of DA neurons might be specifically enhanced by SGC0946 treatment. These results document the feasibility of a set of tools for further exploration of small molecules that may impact DA neuron differentiation, maintenance and/or survival. Results provide evidence in support of other reports that indicate inhibition of DOT1L may play an important role in maintenance and survival of neural progenitor cells (NPCs) and their lineage-specific differentiation.

Epigenetic drug library screening reveals targeting DOT1L abrogates NAD+ synthesis by reprogramming H3K79 methylation in uveal melanoma

Uveal melanoma (UM) is the most frequent and life-threatening ocular malignancy in adults. Aberrant histone methylation contributes to the abnormal transcriptome during oncogenesis. However, a comprehensive understanding of histone methylation patterns and their therapeutic potential in UM remains enigmatic. Herein, using a systematic epi-drug screening and a high-throughput transcriptome profiling of histone methylation modifiers, we observed that disruptor of telomeric silencing-1-like (DOT1L), a methyltransferase of histone H3 lysine 79 (H3K79), was activated in UM, especially in the high-risk group. Concordantly, a systematic epi-drug library screening revealed that DOT1L inhibitors exhibited salient tumor-selective inhibitory effects on UM cells, both in vitro and in vivo. Combining Cleavage Under Targets and Tagmentation (CUT&Tag), RNA sequencing (RNA-seq), and bioinformatics analysis, we identified that DOT1L facilitated H3K79 methylation of nicotinate phosphoribosyltransferase (NAPRT) and epigenetically activated its expression. Importantly, NAPRT served as an oncogenic accelerator by enhancing nicotinamide adenine dinucleotide (NAD+) synthesis. Therapeutically, DOT1L inhibition epigenetically silenced NAPRT expression through the diminishment of dimethylation of H3K79 (H3K79me2) in the NAPRT promoter, thereby inhibiting the malignant behaviors of UM. Conclusively, our findings delineated an integrated picture of the histone methylation landscape in UM and unveiled a novel DOT1L/NAPRT oncogenic mechanism that bridges transcriptional addiction and metabolic reprogramming.

Treatment Outcomes of Childhood Picalm:MLLT10+ Acute Leukemias: An International Retrospective Study

Introduction: Acute leukemia with translocation t(10;11)(p12-13;q14-21) results in generation of the PICALM:MLLT10 fusion gene, and is distinct from t(10;11)(p11-13;q23) which gives rise to KMT2A (formerly MLL) gene rearrangement. Defective PICALM function leads to aberrant hematopoiesis, while defective MLLT10 function impairs chromatin-mediated gene regulation. The PICALM:MLLT10 gene has been reported in patients with Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL) predominantly T-cell, Acute Undifferentiated Leukemia (AUL) and Mixed Phenotype Acute Leukemia (MPAL). MLLT10 interacts directly with DOT1L in a complex that regulates the methylation of H3K79. MLLT10 fusions are thought to misdirect DOT1L to the promoters of HOXA genes, leading to hypermethylation of H3K79. This hypermethylation causes constitutive activation of HOXA activity and prevents cell maturation and differentiation. The clinico-biological characteristics, response to conventional treatments, and survival outcomes of childhood PICALM:MLLT10+ leukemias are poorly understood. Methods: This was an international, multicenter, retrospective cohort study. Data were submitted by ten International-BFM (I-BFM) study Group-AML and ALL committees and by the Children`s Oncology Group (COG)-AML Study committee. For study inclusions, patients were required to be ≤ 21 years of age at diagnosis, diagnosed between January 1995 and December 2019 with de novo ALL, AML, AUL or MPAL and PICALM:MLLT10 fusion-positive. Patients with KMT2A-rearranged leukemia or acute promyelocytic leukemia were excluded. Kaplan Meier curves were employed for survival analysis with Cox proportional hazard modelling to compare effect of AML vs ALL therapy, and receipt of hematopoietic stem cell transplant (HSCT) vs no HSCT, on survival. Pearson's Chi Squared was employed as a non-parametric measure to determine the effect of end of induction-1 (EIO-1) MRD positive vs negative status on incidence of relapse. P value cut-off for data to reach statistical significance was <0.05. Results: Ninety-eight children with acute leukemia were included, 60 were males. Median age at diagnosis was 12 years (IQR:9-14.8 years) and median initial white cell count was 40.5 x 109/L (IQR:13.8-168.9). Fifty-five (55.6%) patients were diagnosed with T-ALL, 39 (39.4%) with AML, while precursor-B ALL, AUL and MPAL were each diagnosed in single patients; 1 subtype diagnosis was unknown. Ten (10.1%) patients presented with CNS disease and 13 (13.1%) presented with non-CNS extramedullary manifestations. Of 66 with cytogenetic/molecular data available, 33 (50%) had additional cytogenetic-molecular aberrations; NOTCH1, WT1 and RAS pathway mutations predominated. Most patients received an initial chemotherapy regimen guided by their disease immunophenotype: 58/98 (58.6%) received an ALL-like chemotherapy regimen upfront, 38 (38.4%) received an AML-like regimen, 1 (1.0%) a hybrid regimen, 1 initial chemotherapy regimen was unknown. Five-year event-free survival (EFS) was 50.4% (CI 39.1-64.9). For AML patients, 5-year EFS was 22.5% (CI 9.75-52.0) and for ALL 67.7% (CI 54.2-84.7), p=<0.001. Five-year overall survival (OS) was 56.3% (CI 46.3-68.5). For AML patients, 5-year OS was 26.7% (CI 13.3-53.6) and for ALL 75.7% (CI 64.6-88.8), p=<0.001. Thirty-eight patients died, 22 (57.9%) deaths were disease-related, 4 (10.5%) treatment-related. Patients who received HSCT in CR1, demonstrated a trend towards an inferior 5-year EFS (23.7% vs 56.5%) and OS (42.6% vs 64.5%) than those who did not; however, analysis was limited by small sample size. Four (22.2%) HSCT recipients had ALL but most, 14 (77.8%), had AML, which may account for the unfavourable outcomes associated with HSCT. There was no statistically significant difference in rate of relapse in the EOI-1 MRD-positive compared with MRD-negative patients (p=0.49). Of those who relapsed, 25 (75.8%) had an isolated bone marrow relapse, 3 (9.1%) isolated CNS, 3 (9.1%) other sites and, for 2 patients, site of relapse was unknown. Conclusions: Children with PICALM:MLLT10+ AML have inferior 5-year EFS and OS outcomes compared to their T-ALL counterparts. MRD status at EOI-1 was not predictive of relapse. Targeted therapies with DOT1L inhibitors or DNA demethylating agents should be explored, particularly for patients with AML.

Establishment and Characterization of a Model of Acquired Resistance to DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic Leukemia Cells

KMT2A-rearranged acute lymphoblastic leukemia (ALL) in infants (<1 year of age) is an aggressive malignancy with event-free survival rates of only ~37%. KMT2A translocation represents the cytogenetic hallmark of this type of leukemia that is largely driven by inappropriate epigenetic events: oncogenic KMT2A fusion proteins recruit the histone methyltransferase DOT1L which leads to misplaced H3K79 methylation inducing an abnormal transcriptomic landscape that favors leukemia development. Therefore, the development and subsequent (pre-)clinical testing of the specific DOT1L inhibitor pinometostat was anticipated to be key to the successful treatment of KMT2A-rearranged acute leukemia. However, although clinical trials in adult KMT2A-rearranged acute leukemia patients did report initial responses (including complete remissions), disease progression due to treatment-related resistance appeared unavoidable. Nonetheless, while these observations urge the development of more effective inhibitors of DOT1L, the mechanisms underlying acquired resistance to DOT1L inhibition may well provide valuable insights into the unique biology of KMT2A-rearranged leukemia. To explore this, we provoked and established acquired pinometostat resistance in a pediatric KMT2A::AFF1+ ALL cell line model that stably became ~35-fold more resistant to DOT1L inhibition. Interestingly, pinometostat was still able to completely inhibit H3K79 di-methylation (H3K79me2) but failed to induce apoptosis, indicating that these cells largely became independent of DOT1L-mediated H3K79 methylation. Yet, shRNA-mediated knock-down of KMT2A::AFF1 revealed that these cells remained completely dependent on the presence of the KMT2A-fusion protein. In addition, pinometostat-resistant KMT2A-rearranged ALL cells proliferate more slowly and appeared to have switched their preference from oxidative phosphorylation to glycolysis to provide energy for metabolic activities, while remaining perfectly viable. As anticipated, pinometostat-resistant cells displayed a slight cross-resistance to most chemotherapeutic agents currently used in ALL treatments but became remarkably more sensitive toward the BCL-2 inhibitor venetoclax. To further characterize these cells and gain insights into the underlying mechanisms of resistance, we performed RNA-, ATAC-, and ChIP-sequencing analysis of KMT2A and AFF1 as well as of histone marks essential in KMT2A-rearranged ALL, including H3K79me2, H3K4 tri-methylation (H3K4me3) and H3K27 acetylation (H3K27ac). This revealed that acquired resistance to DOT1L inhibition leads to a selective loss of KMT2A-fusion driven epigenetic regulation and expression of genes such as PROM1 (encoding the hematopoietic/ leukemia stem cell marker CD133) and its enhancer TAPT1, as well as putative KMT2A::AFF1 target genes including RUNX2, GNAQ, SERPINB1, PRSS12, and ZC3H12C. In contrast, however, the expression of (and levels of H3K79 methylation at) other established KMT2A::AFF1 target genes like HOXA9, HOXA10, MEIS1, and CDK6 remained unaffected. Concomitantly, pinometostat-resistant KMT2A-rearranged ALL cells are characterized by significant enrichment of gene expression of various MYC targets, and specific up-regulation of genes associated with a myeloid immunophenotype, including CD33, CD38, LILRB4, MPEG1, LIMK1, and CCL5. Taken together, we here present an in vitro model of acquired resistance to DOT1L inhibition in KMT2A-rearranged ALL, instantly providing new insights and raising important questions regarding this elusive type of leukemia. For instance, KMT2A-rearranged ALL cells readily adapt to prolonged inhibition of H3K79 methylation but remain dependent on the KMT2A fusion, suggesting that recruitment of DOT1L may not be the only oncogenic action exerted by KMT2A fusion proteins. Also, our model demonstrates that under prolonged pressure of DOT1L inhibition, KMT2A-rearranged ALL cells seem to initiate a reprogramming process that involves the acquirement (or selection) of myeloid-like characteristics, an ability that may be connected to leukemic lineage switches which are not uncommon in KMT2A-rearranged acute leukemias. Hence, our model represents an important tool to study the complex biology of KMT2A-rearranged leukemia, and its existence and availability requires to be shared with the community.

Thrombopoietin Receptor Signaling Promotes the Competitive Advantage of TET2-Mutated Hematopoietic Stem and Progenitor Cells in Clonal Hematopoiesis

Clonal Hematopoiesis of Indeterminate Potential (CHIP) is an age-related condition that predisposes individuals to blood cancers. The time interval from acquisition of CHIP to overt neoplasia can span many years, providing an opportunity to delay or prevent malignant transformation. A key to achieving this goal is identifying differential dependencies between mutant and normal hematopoietic stem and progenitor cells (HSPCs). Targeting such dependencies may prevent against CHIP-related health risks. One of the most commonly mutated genes in CHIP is TET2, which encodes a methylcytosine dioxygenase. In mouse models, Tet2 knockout (KO) HSPCs have a competitive advantage over wild-type (WT) HSPCs. In this study, we aimed to identify differential dependencies between Tet2KO and Tet2WT HSPCs. We first generated isogenic murine cell lines by overexpressing HOXB4, a transcription factor involved in self-renewal, in HSPCs derived from a Tet2KO mouse and Tet2WT littermate. Using the isogenic cell lines termed HPCHOXB4, we conducted an epigenetic drug screen and found SGC0946, a selective inhibitor of H3K79 methyltransferase DOT1L, reduced the advantage of Tet2KO HPCHOXB4 cells in competition assays by preferentially reducing their proliferation and viability. SGC0946 also reduced the competitive advantage of unmodified Tet2KO HSPCs in competition assays, indicating that the dependency was not a consequence of HOXB4 overexpression. To decipher the mechanism behind this differential dependency, we performed RNA-seq and ChIP-seq for H3K79me2 on Tet2KO and Tet2WT HPCHOXB4 cells at baseline and after treatment with SGC0946. Expression of Mpl, which encodes the thrombopoietin receptor TPOR, was 20-fold higher in Tet2KO HPCHOXB4 than in Tet2WT cells at baseline and decreased after drug treatment. Mpl expression at the mRNA and protein level was also elevated in unmodified Tet2KO HSPCs compared with Tet2WT cells. Importantly, ChIP-seq analysis revealed that the level of H3K79me2 which associates with active transcription at the Mpl gene locus was higher in Tet2KO than in Tet2WT HPCHOXB4 cells at baseline, a difference that was erased with SGC0946 treatment. Based on the above findings, we hypothesized that Tet2KO HSPCs are more dependent on TPOR signaling than Tet2WT cells. Consistent with our hypothesis, a higher concentration of TPO in the culture medium increased the competitive advantage of Tet2KO HPCHOXB4 cells. Moreover, overexpression of Mpl in Tet2KO HPCHOXB4 cells rescued the effects of DOT1L inhibition, and shRNA-mediated suppression of Mpl expression was sufficient to decrease the competitive advantage of Tet2KO HPCHOXB4 cells and unmodified Tet2KO HSPCs over their corresponding Tet2WT counterparts. To explore the translational relevance of our findings, we tested if inhibition of JAK2, a critical mediator of TPOR intracellular signaling, would also reduce the competitive advantage of Tet2KO HSPCs. In line with our hypothesis, treatment with JAK2 inhibitors (fedratinib and AZ960), but not JAK1 inhibitors, suppressed the competitive advantage of Tet2KO HPCHOXB4 cells in vitro. Importantly, competitive repopulation studies showed that treatment with ruxolitinib, a clinically approved JAK1/2 inhibitor, also reduced the competitive advantage of untransformed Tet2KO HSPCs in vivo. To determine if our findings apply to humans, we generated two TET2KO cell pools from cord blood HSPCs using CRISPR/Cas9. The TET2KO cells expressed higher levels of TPOR and were more sensitive to treatment with SGC0946 and ruxolitinib compared with control cells. In addition, we expanded HSPCs from TET2-mutated mobilized peripheral blood samples in short-term cultures and monitored the size of the mutant clones by measuring the allele frequency of the sample-specific TET2 variants. In untreated cultures, the relative size of the mutant clones increased over time, an effect that was diminished by treatment with either SGC0946 or ruxolitinib. In summary, our findings demonstrate that TET2-mutated HSPCs are more dependent on TPOR signaling than TET2WT HSPCs. This differential dependency is mediated through increased H3K79 methylation at the MPL gene locus and enhanced expression of MPL. Furthermore, inhibition of the TPOR/JAK2 signaling axis is effective in suppressing the competitive advantage of TET2-mutated HSPCs, thus identifying a tractable therapeutic target for TET2-mutated CHIP.

Identification of Novel Cell Surface Therapeutic Targets for KMT2A-Rearranged Acute Myeloid Leukemia

INTRODUCTION. Acute myeloid leukemia (AML) is a highly heterogeneous disease that represents 80% of adult leukemias and is characterized by chromosomal rearrangements such as translocations and inversions, as well as genetic mutations. These genomic alterations affect the normal maturation process of myeloid precursor cells and lead to their clonal expansion in the bone marrow and peripheral blood. Genetic aberrations define AML subgroups, which are associated with risk stratification (favorable, intermediate or adverse risk) and prognosis. One AML subgroup, KMT2A-rearranged AML (KMT2Ar), is driven by chromosomal translocations involving the lysine methyltransferase 2A gene (KMT2A) located on chromosome band 11q23.3, leading to gene fusions with over 94 distinct partners. KMT2A rearrangements are present in 5 to 10% of adult AML. Patients with t(9;11)(p21.3;q23.3) translocation generating the MLLT3::KMT2A fusion are classified in the intermediate cytogenetic risk group according to the 2022 ELN genetic risk classification, but KMT2A fusions with other translocation partners are included in the adverse risk group with current therapies. Ongoing clinical trials for KMT2Ar AML focus mainly on chemical inhibitors (DOT1L and Menin inhibitors) that target and disrupt KMT2A fusion protein complexes, with encouraging results. Precision medicine, such as antibody-based immunotherapy, also represents a promising approach for the treatment of KMT2Ar AML, but is currently underexploited due to the limited availability of targetable surface antigens. To overcome this issue, we analyzed the cell surface proteome (surfaceome) of primary human KMT2Ar AML samples to identify novel cell surface antigens with high therapeutic potential. METHODS AND RESULTS. We used a proteomics-based approach to analyze the surfaceome of 100 primary human AML specimens from 13 different AML subgroups, including 13 KMT2Ar AML samples. Briefly, we combined cell surface biotinylation with streptavidin affinity purification to enrich cell extracts with cell surface proteins, which were then analyzed by LC-MS/MS to obtain an overview of primary AML sample surfaceomes. Differential surfaceome analyses were performed to identify surface proteins selectively expressed by KMT2Ar AML specimens, and proteins with low expression in normal hematopoietic cells and normal tissues, as determined by expression analyses using publicly available datasets, were further selected. Using this approach, we identified several potential KMT2Ar AML surface antigens, including CD93, reported to be expressed by KMT2Ar AML stem cells. We then analyzed surface expression of these antigens by flow cytometry using a collection of 112 primary human AML specimens including 10 KMT2Ar AML samples. Results indicate that in the majority of KMT2Ar AML samples analyzed, more than 90% of blasts express selected antigens, suggesting that targeting these antigens with immunotherapeutic approaches could potentially lead to the elimination of most AML cells. Furthermore, flow cytometry data showed specificity of antigen expression for KMT2Ar AML samples, opening new therapeutic opportunities for this AML subgroup. Finally, single cell RNA sequencing of 8 primary KMT2Ar AML specimens was performed to further analyze the expression profile of these antigens in patient samples. AML specimens harboring the MLLT3::KMT2A fusion expressed these antigens in the vast majority of blast cells, confirming the homogeneous expression profile of identified antigens and the high interest to target these antigens for KMT2Ar AML. CONCLUSIONS Using a cell surface proteomics-based approach for the analysis of KMT2Ar AML surfaceome, we identified several antigens for this AML subgroup with high therapeutic potential. These antigens are very specific for KMT2Ar AML and are expressed homogeneously in primary human KMT2Ar AML samples, suggesting that targeting these antigens represents a promising therapeutic strategy for these patients. Several immunotherapeutic approaches to target these antigens are currently being developed and tested by our group, with the hope of identifying novel therapeutic strategies for the treatment of KMT2Ar AML.

EPCO-28. DOT1L AND PRC2 REGULATE A SHARED EPIGENETIC MECHANISM IN GLIOBLASTOMA AND MIXED LINEAGE LEUKEMIA

Abstract Glioblastoma (GBM) is the most aggressive adult brain tumor, with a median survival of 15 months despite current treatments. We recently established that the epigenetic regulator Disruptor of Telomeric Silencing-1-Like (DOT1L) was essential for the growth of brain tumor stem cells (BTSCs), which are thought to underlie GBM tumor initiation and treatment resistance. Given the previously recognized importance of DOT1L histone methylation for the regulation of the similarly rare and aggressive childhood cancer, Mixed Lineage Leukemia (MLL), we interrogated for common mechanisms in both BTSCs and MLL cells, that overlap due to the epigenetic role of DOT1L. To gain a more detailed perspective of the importance of the DOT1L epigenetic mark in BTSCs, we performed a chemogenomic screen using the DOT1L inhibitor, EPZ-5676. Results from this screen revealed genes from transcriptional and epigenetic complexes required for a therapeutic response to DOT1L inhibition in BTSCs. Gene targeting approaches and growth assays further identified the Polycomb Repressive Complex 2 (PRC2) as a common determining factor for the growth response of both BTSCs and MLL cells following DOT1L inhibition. Furthermore, analysis of the chromatin accessibility changes regulated by DOT1L and PRC2 histone methylation identified both shared and unique epigenetic characteristics of GBM and MLL. The extent to which these shared mechanisms underpin the pathogenic process in these distinct diseases is being further investigated by assessing the divergence in transcriptional responses that emerge from this common epigenetic phenomenon. The findings from this study will provide insight into the importance of shared epigenetic mechanisms that underlie the tumorigenesis of unique cancers affecting the brain and blood.

DOT1L inhibition does not modify the sensitivity of cutaneous T cell lymphoma to pan-HDAC inhibitors in vitro.

Cutaneous T-cell lymphomas (CTCLs) are a subset of T-cell malignancies presenting in the skin. The treatment options for CTCL, in particular in advanced stages, are limited. One of the emerging therapies for CTCL is treatment with histone deacetylase (HDAC) inhibitors. We recently discovered an evolutionarily conserved crosstalk between HDAC1, one of the targets of HDAC inhibitors, and the histone methyltransferase DOT1L. HDAC1 negatively regulates DOT1L activity in yeast, mouse thymocytes, and mouse thymic lymphoma. Here we studied the functional relationship between HDAC inhibitors and DOT1L in two human CTCL cell lines, specifically addressing the question whether the crosstalk between DOT1L and HDAC1 observed in mouse T cells plays a role in the therapeutic effect of clinically relevant broad-acting HDAC inhibitors in the treatment of human CTCL. We confirmed that human CTCL cell lines were sensitive to treatment with pan-HDAC inhibitors. In contrast, the cell lines were not sensitive to DOT1L inhibitors. Combining both types of inhibitors did neither enhance nor suppress the inhibitory effect of HDAC inhibitors on CTCL cells. Thus our in vitro studies suggest that the effect of commonly used pan-HDAC inhibitors in CTCL cells relies on downstream effects other than DOT1L misregulation.

Combinatorial targeting of menin and the histone methyltransferase DOT1L as a novel therapeutic strategy for treatment of chemotherapy-resistant ovarian cancer

BackgroundOvarian cancer (OC) is characterized by a low response rate and high frequency of resistance development to currently available treatments. The therapeutic potential of histone methyltransferase DOT1L inhibitor in OC cells has been demonstrated, but optimal efficacy and safety of this targeted therapy approach still require improvement. We set forth to evaluate if this problem can be overcome by combinatorial targeting of this epigenetic modifier and menin, one of its functional partners in chromatin.MethodssiRNA-mediated gene knock-down and pharmacological inhibition of menin, a key component of the MLL/SET1 complex and a fitness gene in OC cells, coupled to cell proliferation assays on a panel of high grade serous OC cell lines, including chemotherapy-sensitive and -resistant clones, were applied in order to evaluate how depletion or blockade of this enzyme influences growth and viability of OC cells. RNA sequencing was applied to identify menin target genes and pathways, and the effects of combined inhibition of menin and DOT1L on growth and transcriptome of these OC models were evaluated.ResultsSilencing and pharmacological inhibition of menin exert antiproliferative effects in all OC cells tested and, in PEO1 and PEO4 cells, a profound impact on transcriptome via down-regulation of cell cycle regulatory pathways, aryl hydrocarbon receptor, MYC and KRAS signalling. We demonstrated association of menin and DOT1L in OC cells and identified a subset of genes co-regulated by the two factors. Interestingly, co-treatment with DOT1L and menin pharmacological inhibitors exerts an additive effect on growth inhibition on chemotherapy-sensitive and -refractory OC cells mediated by transcriptome changes controlled by menin and DOT1L activities.ConclusionThese results indicate that menin functionally cooperates with DOT1L in OC cells modulating transcription of genes involved in key cellular functions including, among others, cell proliferation and survival, that are strongly affected by combined inhibition of these two epigenetic regulators, suggesting that this may represent a novel therapeutic strategy for chemotherapy-resistant OCs.Trial registrationNA; The manuscript does not contain clinical trials.

DOT1L regulates lipid biosynthesis and inflammatory responses in macrophages and promotes atherosclerotic plaque stability.

Macrophages are critical immune cells in inflammatory diseases, and their differentiation and function are tightly regulated by histone modifications. H3K79 methylation is a histone modification associated with active gene expression, and DOT1L is the only histone methyltransferase for H3K79. Here we determine the role of DOT1L in macrophages by applying a selective DOT1L inhibitor in mouse and human macrophages and using myeloid-specific Dot1l-deficient mice. We found that DOT1L directly regulates macrophage function by controlling lipid biosynthesis gene programs including central lipid regulators like sterol regulatory element-binding proteins SREBP1 and SREBP2. DOT1L inhibition also leads to macrophage hyperactivation, which is associated with disrupted SREBP pathways. Invivo, myeloid Dot1l deficiency reduces atherosclerotic plaque stability and increases the activation of inflammatory plaque macrophages. Our data show that DOT1L is a crucial regulator of macrophage inflammatory responses and lipid regulatory pathways and suggest a high relevance of H3K79 methylation in inflammatory disease.

Targeting inflammatory macrophages rebuilds therapeutic efficacy of DOT1L inhibition in hepatocellular carcinoma

Epigenetic reprogramming is a promising therapeutic strategy for aggressive cancers, but its limitations invivo remain unclear. Here, we showed, in detailed studies of data regarding 410 patients with human hepatocellular carcinoma (HCC), that increased histone methyltransferase DOT1L triggered epithelial-mesenchymal transition-mediated metastasis and served as a therapeutic target for human HCC. Unexpectedly, although targeting DOT1L invitro abrogated the invasive potential of hepatoma cells, abrogation of DOT1L signals hardly affected the metastasis of hepatoma invivo. Macrophages, which constitute the major cellular component of the stroma, abrogated the anti-metastatic effect of DOT1L targeting. Mechanistically, NF-κB signal elicited by macrophage inflammatory response operated via a non-epigenetic machinery to eliminate the therapeutic efficacy of DOT1L targeting. Importantly, therapeutic strategy combining DOT1L-targeted therapy with macrophage depletion or NF-κB inhibition invivo effectively and successfully elicited cancer regression. Moreover, we found that the densities of macrophages in HCC determined malignant cell DOT1L-associated clinical outcome of the patients. Our results provide insight into the crosstalk between epigenetic reprogramming and cancer microenvironments and suggest that strategies to influence the functional activities of inflammatory cells may benefit epigenetic reprogramming therapy.

DOT1L regulates MTDH-mediated angiogenesis in triple-negative breast cancer: intermediacy of NF-κB-HIF1α axis.

DOT1L, a specific H3K79 methyltransferase, has a tumour-promoting role in various cancers, including triple-negative breast cancer (TNBC). However, the molecular mechanism by which the deregulated DOT1L promotes cancer progression is unclear. Herein, we show that a significantly higher basal level of DOTL1 strongly correlates with MTDH, an oncogene, in clinical TNBC patient cohorts and mediates TNBC progression by enhancing MTDH-induced angiogenesis. In parallel, severe combined immunodeficiency mice-bearing MDA-MB-231 cells with MTDH-Wt or MTDHΔ7 (spliced isoform of MTDH) overexpression constructs showed enhanced blood vessel formations at the tumour site in comparison with control groups. Selective inhibition of DOT1L by EPZ004777, a specific DOT1L inhibitor, or siDOT1L, significantly impaired MTDH-induced proliferation, invasion and angiogenic markers expression in TNBC cells. ChIP assay revealed that Dot1L promotes MTDH-Wt/Δ7 transcription by increasing H3K79me3 levels on its promoter. Dot1L depletion reversed this effect. Mechanistically, DOT1L-induced MTDH caused enhanced nuclear factor kappa B (NF-κB) occupancy on the hypoxia-inducible factor1α (HIF1α) promoter and increased its transcription, leading to elevated levels of proangiogenic mediators in TNBC cells. Moreover, the condition media obtained from MDA-MB-231 cells stably expressing either MTDH-Wt or MTDHΔ7 treated with EPZ004777 or Bay-11-7082 (NF-κB inhibitor) or FM19G11 (HIF1α inhibitor) significantly inhibited MTDH-induced tube formation in human umbilical vein endothelial cells, rat aortic ring sprouting and vessel formations by chick chorioallantoic membrane assay mimicking physiological angiogenic vasculature. Collectively, our findings reveal a novel epigenetic regulation of MTDH by DOTL1, which drives angiogenesis, and that the therapeutic disruption of the DOT1L-MTDH-NF-κB-HIF1α axis may have usefulness in the management of TNBC.

Inhibition of the cardiac fibroblast-enriched histone methyltransferase Dot1L prevents cardiac fibrosis and cardiac dysfunction

BackgroundCardiac fibrosis is characterized by excessive extracellular matrix deposition that contributes to compromised cardiac function and potentially heart failure. Disruptor of telomeric silencing 1-like (Dot1L) is the catalytic enzyme required for histone H3K79 methylation which has been demonstrated to play a role in transcriptional activation. However, the functions of Dot1L in the process of cardiac fibrosis still remain unknown.ResultsIn the present study, we found that endogenous Dot1L is upregulated in cardiac fibroblasts (CFs) treated with angiotensin II (Ang II) or transforming growth factor (TGF)-β1, along with elevated extracellular matrix (ECM) such as fibronectin, collagen I and III. Silencing or inhibiting Dot1L mitigated Ang II-induced myofibroblast generation and fibrogenesis. We identified the transcription factor-forkhead box O (FoxO) 3a as a novel substrate of Dot1L, the transcriptional activating mark H3K79me3 level on the promoter of FoxO3a was increase in activated-CFs, and inhibition of Dot1L markedly decreased FoxO3a transcription accompanied by a significant decrease in the expression of fibrogenic gene. Knockdown of FoxO3a could alleviate ECM deposition induced by Ang II, on the contrary, overexpression FoxO3a resulting in CFs activation. Consistently, in vivo Dot1L ablation rescued myocardial ischemia-induced cardiac fibrosis and improved cardiac function.ConclusionsOur findings conclude that upregulation of Dot1L results in activation of the cardiac fibroblasts to promote profibrotic gene, eventually causes cardiac fibrosis. Pharmacological targeting for Dot1L might represent a promising therapeutic approach for the treatment of human cardiac fibrosis and other fibrotic diseases.

Abstract 681: Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition

Abstract MLL1 (KMT2A) translocations lead to aberrant expression of stem cell associated gene programs in hematopoietic cells producing a particularly aggressive subtype of leukemia, namely MLL-rearranged acute myeloid and lymphoblastic leukemia (AML and ALL, respectively). In pediatric leukemia, the most common rearrangement is the t(9; 11) (p22; q23) reciprocal translocation, which results in the expression of the MLL-AF9 fusion gene. Since the MLL-AF9 rearrangement does not lead to direct activation of an enzyme, small molecule drug discovery remains challenging and despite decades of research on MLL-rearranged leukemia, we have been limited by the inability of acutely assessing the consequences of direct MLL-AF9 inactivation in relevant model systems. By coupling rapid PROTAC-mediated degradation of the oncogenic MLL-AF9 fusion protein with genome-wide gene expression and chromatin analyses we have now been able to identify the immediate transcriptional and chromatin state consequences of MLL-AF9 degradation. Specifically, we have established a core set of MLL-AF9 target genes whose expression changes within minutes of MLL-AF9 degradation. These rapid expression changes are mediated by changes in productive RNA Polymerase II elongation and increased RNA Polymerase II pausing. At later timepoints, degradation of the fusion protein also induces loss of an active chromatin landscape at MLL-AF9 target genes, characterized by loss of MLL-AF9 associated proteins and activating histone modifications. These insights improve our mechanistic understanding of how MLL-AF9 mediates chromatin remodeling and leukemogenesis, while also helping us evaluate small molecule inhibitors of epigenetic mechanisms that target the MLL-AF9 protein complex. Specifically, we investigated how degradation of MLL-AF9 compares to enzymatic inhibition of DOT1L or inhibition of the MENIN-MLL interaction. Interestingly, both DOT1L and MENIN inhibitors fell short when comparing their effects to MLL-AF9 degradation and only combined DOT1L/MENIN inhibition induced global MLL-AF9 destabilization on chromatin, thus disrupting the full MLL-AF9 directed epigenetic and transcriptional program. Ultimately, we were able to confirm these findings in a patient-derived xenograft model in vivo, where only the combination was able to significantly prolong leukemia free survival. In summary, our studies establish the cellular and molecular consequences of targeted degradation of the MLL-AF9 fusion oncoprotein and help improve our understanding of small molecule inhibitors of epigenetic mechanisms as cancer therapies. Citation Format: Sarah Naomi Olsen, Laura Godfrey, James P. Healy, Charles Hatton, Scott A. Armstrong. Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 681.

Histone methyltransferase DOT1L is essential for self-renewal of germline stem cells.

Self-renewal of spermatogonial stem cells is vital to lifelong production of male gametes and thus fertility. However, the underlying mechanisms remain enigmatic. Here, we show that DOT1L, the sole H3K79 methyltransferase, is required for spermatogonial stem cell self-renewal. Mice lacking DOT1L fail to maintain spermatogonial stem cells, characterized by a sequential loss of germ cells from spermatogonia to spermatids and ultimately a Sertoli cell only syndrome. Inhibition of DOT1L reduces the stem cell activity after transplantation. DOT1L promotes expression of the fate-determining HoxC transcription factors in spermatogonial stem cells. Furthermore, H3K79me2 accumulates at HoxC9 and HoxC10 genes. Our findings identify an essential function for DOT1L in adult stem cells and provide an epigenetic paradigm for regulation of spermatogonial stem cells.

The Role of DOT1L in Normal and Malignant Hematopoiesis.

Disruptor of telomeric silencing 1 (DOT1) was first identified in yeast (DOT1p) and is the sole methyltransferase responsible for histone three lysine 79 (H3K79) mono-, di-, and tri-methylation. Mammalian DOT1 (DOT1-like protein or DOT1L) has been implicated in many cellular processes, such as cell cycle progression, DNA damage response, and development. A notable developmental process reliant on DOT1L function is normal hematopoiesis, as DOT1L knockout leads to impairment in blood lineage formation. Aberrant activity of DOT1L has been implicated in hematopoietic malignancies as well, especially those with high expression of the homeobox (HOX) genes, as genetic or pharmacological DOT1L inhibition causes defects in leukemic transformation and maintenance. Recent studies have uncovered methyltransferase-independent functions and a novel mechanism of DOT1L function. Here, we summarize the roles of DOT1L in normal and malignant hematopoiesis and the potential mechanism behind DOT1L function in hematopoiesis, in light of recent discoveries.

Design, Synthesis, and Biological Evaluations of DOT1L Peptide Mimetics Targeting the Protein-Protein Interactions between DOT1L and MLL-AF9/MLL-ENL.

On the basis of a previously identified DOT1L peptide mimetic (compound 3), a series of novel peptide mimetics were designed and synthesized. These compounds can potently bind to AF9 and ENL either in cell-free binding assays or in leukemia cells, and selectively inhibit the growth of leukemia cells containing mixed lineage leukemia (MLL) fusion proteins. The most potent compound 12 exhibited comparable anticancer cellular activities to those of EPZ5676, a clinical stage enzymatic inhibitor of DOT1L in several leukemia cell lines containing MLL fusion proteins. Mechanism studies for compound 12 indicated that it did not affect the global methylation of H3K79 catalyzed by DOT1L but could effectively suppress the methylation of H3K79 at MLL fusion proteins targeted genes and inhibit the expressions of these genes. Our studies thus demonstrated that inhibiting the protein-protein interactions between DOT1L and MLL fusion proteins is a potentially effective strategy for the treatment of MLL rearranged leukemias.

BMP4 drives primed to naïve transition through PGC-like state

Multiple pluripotent states have been described in mouse and human stem cells. Here, we apply single-cell RNA-seq to a newly established BMP4 induced mouse primed to naïve transition (BiPNT) system and show that the reset is not a direct reversal of cell fate but goes through a primordial germ cell-like cells (PGCLCs) state. We first show that epiblast stem cells bifurcate into c-Kit+ naïve and c-Kit− trophoblast-like cells, among which, the naïve branch undergoes further transition through a PGCLCs intermediate capable of spermatogenesis in vivo. Mechanistically, we show that DOT1L inhibition permits the transition from primed pluripotency to PGCLCs in part by facilitating the loss of H3K79me2 from Gata3/6. In addition, Prdm1/Blimp1 is required for PGCLCs and naïve cells, while Gata2 inhibits PGC-like state by promoting trophoblast-like fate. Our work not only reveals an alternative route for primed to naïve transition, but also gains insight into germ cell development.