Targeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors.

Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.

Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

Regulation of MLL/COMPASS stability through its proteolytic cleavage by taspase1 as a possible approach for clinical therapy of leukemia.

Chromosomal translocations of the Mixed-lineage leukemia 1 (MLL1) gene generate MLL chimeras that drive the pathogenesis of acute myeloid and lymphoid leukemia. The untranslocated MLL1 is a substrate for proteolytic cleavage by the endopeptidase threonine aspartase 1 (taspase1); however, the biological significance of MLL1 cleavage by this endopeptidase remains unclear. Here, we demonstrate that taspase1-dependent cleavage of MLL1 results in the destabilization of MLL. Upon loss of taspase1, MLL1 association with chromatin is markedly increased due to the stabilization of its unprocessed version, and this stabilization of the uncleaved MLL1 can result in the displacement of MLL chimeras from chromatin in leukemic cells. Casein kinase II (CKII) phosphorylates MLL1 proximal to the taspase1 cleavage site, facilitating its cleavage, and pharmacological inhibition of CKII blocks taspase1-dependent MLL1 processing, increases MLL1 stability, and results in the displacement of the MLL chimeras from chromatin. Accordingly, inhibition of CKII in a MLL-AF9 mouse model of leukemia delayed leukemic progression in vivo. This study provides insights into the direct regulation of the stability of MLL1 through its cleavage by taspase1, which can be harnessed for targeted therapeutic approaches for the treatment of aggressive leukemia as the result of MLL translocations.

MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells.

Mixed lineage leukemia-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce MLL through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here, we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II in HEL, a human erythroleukemia cell line without MLL1 rearrangement. MLL1 and AF9 only coregulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely believed elongation.

Unraveling MLL1-fusion leukemia: Epigenetic revelations from an iPS cell point mutation.

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of the catalytic SET domain of MLL1 (known for H3K4 monomethylation and dimethylation) remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human-induced pluripotent stem cells. This mutation mimics the histone methylation, gene expression, and epithelial-mesenchymal transition phenotypes of MLL1F-LSCs-without requiring a translocation or functional WT MLL1. The mutation caused a genome-wide redistribution of the H3K4 trimethyl mark and upregulated LSC-maintenance genes like HoxA9-A13, Meis1, and the HOTTIP long noncoding RNA. Epithelial-mesenchymal transition markers such as ZEB1, SNAI2, and HIC-5 were also increased leading to enhanced cellular migration and invasiveness. These observations underscore the essential role of MLL1's enzymatic activity in restraining the cascade of epigenetic changes associated with the gene-activating H3K4 trimethylation mark, which we show may be catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1's enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1's histone methyltransferase activity. We propose targeting SETd1a in precision medicine as a new therapeutic approach for MLL1-associated leukemias.

Multistate structures of the MLL1-WRAD complex bound to H2B-ubiquitinated nucleosome.

The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.

The inflammatory and oxidative phenotype of gestational diabetes is epigenetically transmitted to the offspring: role of methyltransferase MLL1-induced H3K4me3.

BACKGROUND AND AIMS: Hyperglycaemia during gestational diabetes (GD) predisposes women and their offspring to later cardiometabolic disease. The hyperglycaemia-mediated epigenetic changes remain to be elucidated. Methyltransferase MLL1-induced trimethylation of histone 3 at lysine 4 (H3K4me3) activates inflammatory and oxidative phenotype. This epigenetic mark in GD women and its transmission to the offspring were investigated. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from GD and control (C) women and also from adolescents born to women of both groups. Endothelial human umbilical vein endothelial cells (HUVEC) and cord blood mononuclear cells (CBMC) were from umbilical cords. The NF-κBp65 and NOX4 expressions were investigated by reverse transcription quantitative polymerase chain reaction and immunofluorescence (IF). MLL1 and H3K4me3 were investigated by immunoblotting and IF. H3K4me3 on NF-κBp65 and NOX4 promoters was studied by chromatin immunoprecipitation. Superoxide anion generation was measured by electron spin resonance spectroscopy. Plasma cytokines were measured by enzyme-linked immunosorbent assay. To investigate the role of MLL1, HUVEC were exposed to inhibitor MM102 or siRNA transfection. RESULTS: PBMC, CBMC, and HUVEC showed an increase of NF-κBp65, IL-6, ICAM-1, MCP-1, and VCAM-1 mRNAs. These findings were associated with H3K4me3 enrichment in the promoter of NF-κBp65. Elevated H3K4me3 and cytokine levels were observed in GD adolescents. MLL1 drives H3K4me3 not only on NF-kB p65, but also on NOX4 promoter. Inhibition of MLL1 blunted NF-κBp65 and NOX4 by modulating inflammatory and oxidative phenotype. CONCLUSIONS: Such proof-of-concept study shows persistence of MLL1-dependent H3K4me3 in offspring born to GD women, suggesting an epigenetic-driven transmission of maternal phenotype. These findings may pave the way for pharmacological reprogramming of adverse histone modifications to mitigate abnormal phenotypes underlying early ASCVD.

NFIB-MLL1 complex is required for the stemness and Dlx5-dependent osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.

Despite significant progress in our understanding of the molecular mechanism of mesenchymal stem cell (MSC) differentiation, less is known about the factors maintaining the stemness and plasticity of MSCs. Here, we show that the NFIB-MLL1 complex plays key roles in osteogenic differentiation and stemness of C3H10T1/2 MSCs. We find that depletion of either NFIB or MLL1 results in a severely hampered osteogenic potential and failed activation of key osteogenic transcription factors, such as Dlx5, Runx2, and Osx, following osteogenic stimuli. In addition, the NFIB-MLL1 complex binds directly to the promoter of Dlx5, and exogenous expression of Myc-Dlx5, but not the activation of either the BMP- or the Wnt-signaling pathway, is sufficient to restore the osteogenic potential of cells depleted of NFIB or MLL1. Moreover, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis showed that the NFIB-MLL1 complex mediates the deposition of trimethylated histone H3K4 at both Dlx5 and Cebpa, key regulator genes that function at the early stages of osteogenic and adipogenic differentiation, respectively, in uncommitted C3H10T1/2 MSCs. Surprisingly, the depletion of either NFIB or MLL1 leads to decreased trimethylated histone H3K4 and results in elevated trimethylated histone H3K9 at those developmental genes. Furthermore, gene expression profiling and ChIP-sequencing analysis revealed lineage-specific changes in chromatin landscape and gene expression in response to osteogenic stimuli. Taken together, these data provide evidence for the hitherto unknown role of the NFIB-MLL1 complex in the maintenance and lineage-specific differentiation of C3H10T1/2 MSCs and support the epigenetic regulatory mechanism underlying the stemness and plasticity of MSCs.

Epigenetic drug screening for trophoblast syncytialization reveals a novel role for MLL1 in regulating fetoplacental growth.

BACKGROUND: Abnormal placental development is a significant factor contributing to perinatal morbidity and mortality, affecting approximately 5-7% of pregnant women. Trophoblast syncytialization plays a pivotal role in the establishment and maturation of the placenta, and its dysregulation is closely associated with several pregnancy-related disorders, including preeclampsia and intrauterine growth restriction. However, the underlying mechanisms and genetic determinants of syncytialization are largely unknown. METHODS: We conducted a systematic drug screen using an epigenetic compound library to systematically investigate the epigenetic mechanism essential for syncytialization, and identified mixed lineage leukemia 1 (MLL1), a histone 3 lysine 4 methyltransferase, as a crucial regulator of trophoblast syncytialization. BeWo cells were utilized to investigate the role of MLL1 during trophoblast syncytialization. RNA sequencing and CUT&Tag were further performed to search for potential target genes and the molecular pathways involved. Human placenta tissue was used to investigate the role of MLL1 in TEA domain transcription factor 4 (TEAD4) expression and the upstream signaling during syncytialization. A mouse model was used to examine whether inhibition of MLL1-mediated H3K4me3 regulated placental TEAD4 expression and fetoplacental growth. RESULTS: Genetic knockdown of MLL1 or pharmacological inhibition of the MLL1 methyltransferase complex (by MI-3454) markedly enhanced syncytialization, while overexpression of MLL1 inhibited forskolin (FSK)-induced syncytiotrophoblast formation. In human placental villous tissue, MLL1 was predominantly localized in the nuclei of cytotrophoblasts. Moreover, a notable upregulation in MLL1 expression was observed in the villus tissue of patients with preeclampsia compared with that in the control group. Based on RNA sequencing and CUT&Tag analyses, depletion of MLL1 inhibited the Hippo signaling pathway by suppressing TEAD4 expression by modulating H3K4me3 levels on the TEAD4 promoter region. TEAD4 overexpression significantly reversed the FSK-induced or MLL1 silencing-mediated trophoblast syncytialization. Additionally, decreased hypoxia-inducible factor 1A (HIF1A) enrichment at the MLL1 promoter was observed during syncytialization. Under hypoxic conditions, HIF1A could bind to and upregulate MLL1, leading to the activation of the MLL1/TEAD4 axis. In vivo studies demonstrated that the administration of MI-3454 significantly enhanced fetal vessel development and increased the thickness of the syncytial layer, thereby supporting fetoplacental growth. CONCLUSIONS: These results revealed a novel epigenetic mechanism underlying the progression of syncytialization with MLL1, and suggest potential avenues for identifying new therapeutic targets for pregnancy-related disorders.

Developmental reprogramming of myometrial stem cells by endocrine disruptor linking to risk of uterine fibroids.

BACKGROUND: The stage, when tissues and organs are growing, is very vulnerable to environmental influences, but it's not clear how exposure during this time causes changes to the epigenome and increases the risk of hormone-related illnesses like uterine fibroids (UFs). METHODS: Developmental reprogramming of myometrial stem cells (MMSCs), the putative origin from which UFs originate, was investigated in vitro and in the Eker rat model by RNA-seq, ChIP-seq, RRBS, gain/loss of function analysis, and luciferase activity assays. RESULTS: When exposed to the endocrine-disrupting chemical (EDC) diethylstilbestrol during Eker rat development, MMSCs undergo a reprogramming of their estrogen-responsive transcriptome. The reprogrammed genes in MMSCs are known as estrogen-responsive genes (ERGs) and are activated by mixed lineage leukemia protein-1 (MLL1) and DNA hypo-methylation mechanisms. Additionally, we observed a notable elevation in the expression of ERGs in MMSCs from Eker rats exposed to natural steroids after developmental exposure to EDC, thereby augmenting estrogen activity. CONCLUSION: Our studies identify epigenetic mechanisms of MLL1/DNA hypo-methylation-mediated MMSC reprogramming. EDC exposure epigenetically targets MMSCs and leads to persistent changes in the expression of a subset of ERGs, imparting a hormonal imprint on the ERGs, resulting in a "hyper-estrogenic" phenotype, and increasing the hormone-dependent risk of UFs.

MLL1 is essential for the senescence-associated secretory phenotype.

Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces "SASP-like" inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS- or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression.

The Wdr5-H3K4me3 Epigenetic Axis Regulates Pancreatic Tumor Immunogenicity and Immune Suppression.

The WDR5/MLL1-H3K4me3 epigenetic axis is often activated in both tumor cells and tumor-infiltrating immune cells to drive various cellular responses in the tumor microenvironment and has been extensively studied in hematopoietic cancer, but its respective functions in tumor cells and immune cells in the context of tumor growth regulation of solid tumor is still incompletely understood. We report here that WDR5 exhibits a higher expression level in human pancreatic tumor tissues compared with adjacent normal pancreas. Moreover, WDR5 expression is negatively correlated with patients' response to chemotherapy or immunotherapy in human colon cancer and melanoma. However, WDR5 expression is positively correlated with the HLA level in human cancer cells, and H3K4me3 enrichment is observed at the promoter region of the HLA-A, HLA-B, and HLA-C genes in pancreatic cancer cells. Using mouse tumor cell lines and in vivo tumor models, we determined that WDR5 deficiency or inhibition significantly represses MHC I expression in vitro and in vivo in pancreatic tumor cells. Mechanistically, we determine that WDR5 deficiency inhibits H3K4me3 deposition at the MHC I (H2K) promoter region to repress MHC I (H2K) transcription. On the other hand, WDR5 depletion leads to the effective downregulation of immune checkpoints and immunosuppressive cytokines, including TGFß and IL6, in the pancreatic tumor microenvironments. Our data determine that WDR5 not only regulates tumor cell immunogenicity to suppress tumor growth but also activates immune suppressive pathways to promote tumor immune evasion. Selective activation of the WDR5-MHC I pathway and/or selective inhibition of the WDR5-immune checkpoint and WDR5-cytokine pathways should be considered in WDR5-based epigenetic cancer immunotherapy.

Binding to the Other Side: The AT-Hook DNA-Binding Domain Allows Nuclear Factors to Exploit the DNA Minor Groove.

The "AT-hook" is a peculiar DNA-binding domain that interacts with DNA in the minor groove in correspondence to AT-rich sequences. This domain has been first described in the HMGA protein family of architectural factors and later in various transcription factors and chromatin proteins, often in association with major groove DNA-binding domains. In this review, using a literature search, we identified about one hundred AT-hook-containing proteins, mainly chromatin proteins and transcription factors. After considering the prototypes of AT-hook-containing proteins, the HMGA family, we review those that have been studied in more detail and that have been involved in various pathologies with a particular focus on cancer. This review shows that the AT-hook is a domain that gives proteins not only the ability to interact with DNA but also with RNA and proteins. This domain can have enzymatic activity and can influence the activity of the major groove DNA-binding domain and chromatin docking modules when present, and its activity can be modulated by post-translational modifications. Future research on the function of AT-hook-containing proteins will allow us to better decipher their function and contribution to the different pathologies and to eventually uncover their mutual influences.

A novel Menin-MLL1 inhibitor, DS-1594a, prevents the progression of acute leukemia with rearranged MLL1 or mutated NPM1.

BACKGROUND: Mixed lineage leukemia 1-rearranged (MLL1-r) acute leukemia patients respond poorly to currently available treatments and there is a need to develop more effective therapies directly disrupting the Menin‒MLL1 complex. Small-molecule-mediated inhibition of the protein‒protein interaction between Menin and MLL1 fusion proteins is a potential therapeutic strategy for patients with MLL1-r or mutated-nucleophosmin 1 (NPM1c) acute leukemia. In this study, we preclinically evaluated the new compound DS-1594a and its salts. METHODS: We evaluated the preclinical efficacy of DS-1594a as well as DS-1594a·HCl (the HCl salt of DS-1594a) and DS-1594a·succinate (the succinic acid salt of DS-1594a, DS-1594b) in vitro and in vivo using acute myeloid leukemia (AML)/acute lymphoblastic leukemia (ALL) models. RESULTS: Our results showed that MLL1-r or NPM1c human leukemic cell lines were selectively and highly sensitive to DS-1594a·HCl, with 50% growth inhibition values < 30 nM. Compared with cytrabine, the standard chemotherapy drug as AML therapy, both DS-1594a·HCl and DS-1594a·succinate mediated the eradication of potential leukemia-initiating cells by enhancing differentiation and reducing serial colony-forming potential in MLL1-r AML cells in vitro. The results were confirmed by flow cytometry, RNA sequencing, RT‒qPCR and chromatin immunoprecipitation sequencing analyses. DS-1594a·HCl and DS-1594a·succinate exhibited significant antitumor efficacy and survival benefit in MOLM-13 cell and patient-derived xenograft models of MLL1-r or NPM1c acute leukemia in vivo. CONCLUSION: We have generated a novel, potent, orally available small-molecule inhibitor of the Menin-MLL1 interaction, DS-1594a. Our results suggest that DS-1594a has medicinal properties distinct from those of cytarabine and that DS-1594a has the potential to be a new anticancer therapy and support oral dosing regimen for clinical studies (NCT04752163).

Menin-MLL1 Interaction Small Molecule Inhibitors: A Potential Therapeutic Strategy for Leukemia and Cancers.

Encoded by the MEN1 gene, menin protein is a fusion protein that is essential for the oncogenic transformation of mixed-lineage leukemia (MLL) and leads to acute leukemia (AL). Therefore, accumulating evidence has demonstrated that inhibition of the high-affinity relationship between menin and mixed-lineage leukemia 1 (MLL1 and KMT2A) is an effective treatment for MLL-rearranged (MLL-r) leukemia in vitro and in vivo. Meanwhile, recent studies found that menin-MLL1 interaction inhibitors exhibited a firm tumor suppressive ability in specific cancer cells, such as prostate cancer, breast cancer, liver cancer, and lung cancer. Overall, it seems to serve as a novel therapeutic means for cancers. Herein, we review the recent progress in exploring the inhibitors of small molecule menin-MLL1 interactions. The molecular mechanisms of these inhibitors' functions and their application prospects in the treatment of AL and cancers are explored.

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.

Mixed-lineage leukaemia 1 contributes to endometrial stromal cells progesterone responsiveness during decidualization.

Studies have reported that non-receptive endometrium or abnormal decidualization was closely related to recurrent implantation failure (RIF). MLL1 is a histone H3 lysine 4 trimethylation (H3K4me3) transferase that regulates the transcriptional activation of target genes. The role of MLL1 has been underexplored during decidualization. In our research, we found the expression of MLL1 was closely related to endometrial receptivity, and it was responsible to hormone stimulation. Inhibiting the function of MLL1 by MM102 reduced the transformation of HESCs. Furthermore, down-regulation of MLL1 by siRNA transfection significantly decreased PGR and its target genes expression. MLL1 act as a co-activator of ERα, and both of them were recruited to PGR regulatory regions, thus promote PGR transcription. Our study showed that MLL1 plays a key role in promoting progesterone signalling transmission.

FLII and MLL1 Cooperatively Regulate Aryl Hydrocarbon Receptor-Mediated Transcription in ARPE-19 Cells.

Aryl hydrocarbon receptors (AHRs), a class of ligand-dependent nuclear receptors that regulate cellular responses by inducing the expression of various target genes in response to external signals, are implicated in maintaining retinal tissue homeostasis. Previous studies have shown that the regulation of AHR-induced gene expression requires transcriptional co-regulators. However, it is not yet clear how chromatin remodelers, histone methyltransferases and coactivators interact during AHR-mediated gene expression in human retinal cells. In this study, we reveal that the histone methyltransferase MLL1 and the coactivator FLII are involved in AHR-mediated gene expression in retinal pigment epithelial cells. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly increased the expression of CYP1A1, CYP1B1 and AHRR in ARPE-19 cells, whereas FLII or MLL1 depletion significantly reduced the expression of these genes induced by TCDD. Mechanistically, FLII binds to AHR in a ligand-dependent manner in ARPE-19 cells. In particular, the binding of FLII to MLL1 occurs through the GelB domain of FLII. In addition, MLL1 binds to AHR in a ligand-independent manner. FLII is involved in the recruitment of the BRG1 chromatin remodeler and MLL1 histone methyltransferase to the AHR-regulated CYP1A1 gene region in ARPE-19 cells and consequently, plays an important role in RNA polymerase II binding and transcriptional activity by modulating chromatin accessibility. Our results identify the functions and mechanisms of action of FLII and MLL1 in AHR-induced gene expression in human retinal pigment epithelial cells.

Expanding the genotypic and phenotypic spectrum in a diverse cohort of 104 individuals with Wiedemann-Steiner syndrome.

Wiedemann-Steiner syndrome (WSS) is an autosomal dominant disorder caused by monoallelic variants in KMT2A and characterized by intellectual disability and hypertrichosis. We performed a retrospective, multicenter, observational study of 104 individuals with WSS from five continents to characterize the clinical and molecular spectrum of WSS in diverse populations, to identify physical features that may be more prevalent in White versus Black Indigenous People of Color individuals, to delineate genotype-phenotype correlations, to define developmental milestones, to describe the syndrome through adulthood, and to examine clinicians' differential diagnoses. Sixty-nine of the 82 variants (84%) observed in the study were not previously reported in the literature. Common clinical features identified in the cohort included: developmental delay or intellectual disability (97%), constipation (63.8%), failure to thrive (67.7%), feeding difficulties (66.3%), hypertrichosis cubiti (57%), short stature (57.8%), and vertebral anomalies (46.9%). The median ages at walking and first words were 20 months and 18 months, respectively. Hypotonia was associated with loss of function (LoF) variants, and seizures were associated with non-LoF variants. This study identifies genotype-phenotype correlations as well as race-facial feature associations in an ethnically diverse cohort, and accurately defines developmental trajectories, medical comorbidities, and long-term outcomes in individuals with WSS.

The Cks1/Cks2 axis fine-tunes Mll1 expression and is crucial for MLL-rearranged leukaemia cell viability.

The Cdc28 protein kinase subunits, Cks1 and Cks2, play dual roles in Cdk-substrate specificity and Cdk-independent protein degradation, in concert with the E3 ubiquitin ligase complexes SCFSkp2 and APCCdc20. Notable targets controlled by Cks include p27 and Cyclin A. Here, we demonstrate that Cks1 and Cks2 proteins interact with both the MllN and MllC subunits of Mll1 (Mixed-lineage leukaemia 1), and together, the Cks proteins define Mll1 levels throughout the cell cycle. Overexpression of CKS1B and CKS2 is observed in multiple human cancers, including various MLL-rearranged (MLLr) AML subtypes. To explore the importance of MLL-Fusion Protein regulation by CKS1/2, we used small molecule inhibitors (MLN4924 and C1) to modulate their protein degradation functions. These inhibitors specifically reduced the proliferation of MLLr cell lines compared to primary controls. Altogether, this study uncovers a novel regulatory pathway for MLL1, which may open a new therapeutic approach to MLLr leukaemia.