Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

The NTE domain of PTENα/ß promotes cancer progression by interacting with WDR5 via its SSSRRSS motif.

PTENα/ß, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/ß to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/ß associated cancers. These findings not only shed light on the important role of the PTENα/ß-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.

KMT2 Family of H3K4 Methyltransferases: Enzymatic Activity-dependent and -independent Functions.

Histone-lysine N-methyltransferase 2 (KMT2) methyltransferases are critical for gene regulation, cell differentiation, animal development, and human diseases. KMT2 biological roles are often attributed to their methyltransferase activities on lysine 4 of histone H3 (H3K4). However, recent data indicate that KMT2 proteins also possess non-enzymatic functions. In this review, we discuss the current understanding of KMT2 family, with a focus on their enzymatic activity-dependent and -independent functions. Six mammalian KMT2 proteins of three subgroups, KMT2A/B (MLL1/2), KMT2C/D (MLL3/4), and KMT2F/G (SETD1A/B or SET1A/B), have shared and distinct protein domains, catalytic substrates, genomic localizations, and associated complex subunits. Recent studies have revealed the importance of KMT2C/D in enhancer regulation, differentiation, development, tumor suppression and highlighted KMT2C/D enzymatic activity-dependent and -independent roles in mouse embryonic development and cell differentiation. Catalytic dependent and independent functions for KMT2A/B and KMT2F/G in gene regulation, differentiation, and development are less understood. Finally, we provide our perspectives and lay out future research directions that may help advance the investigation on enzymatic activity-dependent and -independent biological roles and working mechanisms of KMT2 methyltransferases.

Histone H4K16ac Binding Function of the Triple PHD Finger Cassette of MLL4.

The human methyltransferase MLL4 plays a critical role in embryogenesis and development, and aberrant activity of MLL4 is linked to neurodegenerative and developmental disorders and cancer. MLL4 contains the catalytic SET domain that catalyzes mono methylation of lysine 4 of histone H3 (H3K4me1) and seven plant homeodomain (PHD) fingers, six of which have not been structurally and functionally characterized. Here, we demonstrate that the triple PHD finger cassette of MLL4, harboring its fourth, fifth and sixth PHD fingers (MLL4PHD456) forms an integrated module, maintains the binding selectivity of the PHD6 finger toward acetylated lysine 16 of histone H4 (H4K16ac), and is capable of binding to DNA. Our findings highlight functional correlation between H4K16ac and H3K4me1, two major histone modifications that are recognized and written, respectively, by MLL4.

Defining Biological and Biochemical Functions of Noncanonical SET Domain Proteins.

Within the SET domain superfamily of lysine methyltransferases, there is a well-conserved subfamily, frequently referred to as the Set3 SET domain subfamily, which contain noncanonical SET domains carrying divergent amino acid sequences. These proteins are implicated in diverse biological processes including stress responses, cell differentiation, and development, and their disruption is linked to diseases including cancer and neurodevelopmental disorders. Interestingly, biochemical and structural analysis indicates that they do not possess catalytic methyltransferase activity. At the molecular level, Set3 SET domain proteins appear to play critical roles in the regulation of gene expression, particularly repression and heterochromatin maintenance, and in some cases, via scaffolding other histone modifying activities at chromatin. Here, we explore the common and unique functions among Set3 SET domain subfamily proteins and analyze what is known about the specific contribution of the conserved SET domain to functional roles of these proteins, as well as propose areas of investigation to improve understanding of this important, noncanonical subfamily of proteins.

Overlapping characteristics of weak interactions of two transcriptional regulators with WDR5.

The WD40 repeat protein 5 (WDR5) is a nuclear hub that critically influences gene expression by interacting with transcriptional regulators. Utilizing the WDR5 binding motif (WBM) site, WDR5 interacts with the myelocytomatosis (MYC), an oncoprotein transcription factor, and the retinoblastoma-binding protein 5 (RbBP5), a scaffolding element of an epigenetic complex. Given the clinical significance of these protein-protein interactions (PPIs), there is a pressing necessity for a quantitative assessment of these processes. Here, we use biolayer interferometry (BLI) to examine interactions of WDR5 with consensus peptide ligands of MYC and RbBP5. We found that both interactions exhibit relatively weak affinities arising from a fast dissociation process. Remarkably, live-cell imaging identified distinctive WDR5 localizations in the absence and presence of full-length binding partners. Although WDR5 tends to accumulate within nucleoli, WBM-mediated interactions with MYC and RbBP5 require their localization outside nucleoli. We utilize fluorescence resonance energy transfer (FRET) microscopy to confirm these weak interactions through a low FRET efficiency of the MYC-WDR5 and RbBP5-WDR5 complexes in living cells. In addition, we evaluate the impact of peptide and small-molecule inhibitors on these interactions. These outcomes form a fundamental basis for further developments to clarify the multitasking role of the WBM binding site of WDR5.

MRG15 activates histone methyltransferase activity of ASH1L by recruiting it to the nucleosomes.

ASH1L is a histone methyltransferase that regulates gene expression through methylation of histone H3 on lysine K36. While the catalytic SET domain of ASH1L has low intrinsic activity, several studies found that it can be vastly enhanced by the interaction with MRG15 protein and proposed allosteric mechanism of releasing its autoinhibited conformation. Here, we found that full-length MRG15, but not the MRG domain alone, can enhance the activity of the ASH1L SET domain. In addition, we showed that catalytic activity of MRG15-ASH1L depends on nucleosome binding mediated by MRG15 chromodomain. We found that in solution MRG15 binds to ASH1L, but has no impact on the conformation of the SET domain autoinhibitory loop or the S-adenosylmethionine cofactor binding site. Moreover, MRG15 binding did not impair the potency of small molecule inhibitors of ASH1L. These findings suggest that MRG15 functions as an adapter that enhances ASH1L catalytic activity by recruiting nucleosome substrate.

Lysine Methyltransferase SMYD1 Regulates Myogenesis via skNAC Methylation.

The SMYD family is a unique class of lysine methyltransferases (KMTases) whose catalytic SET domain is split by a MYND domain. Among these, Smyd1 was identified as a heart- and skeletal muscle-specific KMTase and is essential for cardiogenesis and skeletal muscle development. SMYD1 has been characterized as a histone methyltransferase (HMTase). Here we demonstrated that SMYD1 methylates is the Skeletal muscle-specific splice variant of the Nascent polypeptide-Associated Complex (skNAC) transcription factor. SMYD1-mediated methylation of skNAC targets K1975 within the carboxy-terminus region of skNAC. Catalysis requires physical interaction of SMYD1 and skNAC via the conserved MYND domain of SMYD1 and the PXLXP motif of skNAC. Our data indicated that skNAC methylation is required for the direct transcriptional activation of myoglobin (Mb), a heart- and skeletal muscle-specific hemoprotein that facilitates oxygen transport. Our study revealed that the skNAC, as a methylation target of SMYD1, illuminates the molecular mechanism by which SMYD1 cooperates with skNAC to regulate transcriptional activation of genes crucial for muscle functions and implicates the MYND domain of the SMYD-family KMTases as an adaptor to target substrates for methylation.

The engagement of histone lysine methyltransferases with nucleosomes: structural basis, regulatory mechanisms, and therapeutic implications.

Histone lysine methyltransferases (HKMTs) deposit methyl groups onto lysine residues on histones and play important roles in regulating chromatin structure and gene expression. The structures and functions of HKMTs have been extensively investigated in recent decades, significantly advancing our understanding of the dynamic regulation of histone methylation. Here, we review the recent progress in structural studies of representative HKMTs in complex with nucleosomes (H3K4, H3K27, H3K36, H3K79, and H4K20 methyltransferases), with emphasis on the molecular mechanisms of nucleosome recognition and trans-histone crosstalk by these HKMTs. These structural studies inform HKMTs' roles in tumorigenesis and provide the foundations for developing new therapeutic approaches targeting HKMTs in cancers.

The menin inhibitor revumenib in KMT2A-rearranged or NPM1-mutant leukaemia.

Targeting critical epigenetic regulators reverses aberrant transcription in cancer, thereby restoring normal tissue function1-3. The interaction of menin with lysine methyltransferase 2A (KMT2A), an epigenetic regulator, is a dependence in acute leukaemia caused by either rearrangement of KMT2A or mutation of the nucleophosmin 1 gene (NPM1)4-6. KMT2A rearrangements occur in up to 10% of acute leukaemias and have an adverse prognosis, whereas NPM1 mutations occur in up to 30%, forming the most common genetic alteration in acute myeloid leukaemia7,8. Here, we describe the results of the first-in-human phase 1 clinical trial investigating revumenib (SNDX-5613), a potent and selective oral inhibitor of the menin-KMT2A interaction, in patients with relapsed or refractory acute leukaemia (ClinicalTrials.gov, NCT04065399). We show that therapy with revumenib was associated with a low frequency of grade 3 or higher treatment-related adverse events and a 30% rate of complete remission or complete remission with partial haematologic recovery (CR/CRh) in the efficacy analysis population. Asymptomatic prolongation of the QT interval on electrocardiography was identified as the only dose-limiting toxicity. Remissions occurred in leukaemias refractory to multiple previous lines of therapy. We demonstrate clearance of residual disease using sensitive clinical assays and identify hallmarks of differentiation into normal haematopoietic cells, including differentiation syndrome. These data establish menin inhibition as a therapeutic strategy for susceptible acute leukaemia subtypes.

Structural and Energetic Origin of Different Product Specificities and Activities for SETD3 and Its Mutants on the Methylation of the ß-Actin H73K Peptide: Insights from a QM/MM Study.

The methylation of the lysine residue can affect some fundamental biological processes, and specific biological effects of the methylations are often related to product specificity of methyltransferases. The question remains concerning how active-site structural features and dynamics control the activity as well as the number (1, 2, or 3) of methyl groups on methyl lysine products. SET domain containing protein 3 (SETD3) has been identified recently as the ß-actin histidine73-N3 methyltransferase, and also, it has a weak methylation activity on the H73K ß-actin peptide for which the target H73 residue is mutated into K73. Interestingly, the K73 methylation activity of SETD3 increases significantly as a result of the N255 → A or N255 → F/W273 → A mutation, and the N255A product specificity also differs from that of wild-type. Here, we performed QM/MM molecular dynamics and potential of mean force (PMF) simulations for SETD3 and its mutants (N255A and N255F/W273A) to study how SETD3 and its mutants could have different product specificities and activities for the K73 methylation. The PMF simulations show that the barrier for the first methylation of K73 is higher compared to the barrier of the H73 methylation in SETD3. Moreover, the second methylation of K73 has been found to have a barrier from the free energy simulation that is higher by 2.2 kcal/mol compared to the barrier of the first methyl transfer to K73, agreeing with the suggestion that SETD3 is a monomethylase. For the first, second, and third methylations of K73 in the N255A mutant, the barriers obtained from the PMF simulations for transferring the second and third methyl groups are found to be lower relative to the barrier for the first methyl transfer. Thus, N255A can be considered as a trimethyl lysine methyltransferase. In addition, for the first K73 methylation, the activities from the PMF simulations follow the order of N255F/W273A > N255A > WT, in agreement with experiments. The examination of the structural and dynamic results at the active sites provides better understanding of different product specificities and activities for the K73 methylations in SETD3 and its mutants. It is demonstrated that the existence of well-balanced interactions at the active site leading to the near attack conformation is of crucial importance for the efficient methyl transfers. Moreover, the presence of potential interactions (e.g., the C-H···O and cation-π interactions) that are strengthening at the transition state can also be important. Furthermore, the activity as well as product specificity of the K73 methylation also seems to be controlled by certain active-site water molecules which may be released to provide extra space for the addition of more methyl groups on K73.

Targeting SMYD2 inhibits angiogenesis and increases the efficiency of apatinib by suppressing EGFL7 in colorectal cancer.

Angiogenesis is an essential factor affecting the occurrence and development of solid tumors. SET And MYND Domain Containing 2 (SMYD2) serves as an oncogene in various cancers. However, whether SMYD2 is involved in tumor angiogenesis remains unclear. Here, we report that SMYD2 expression is associated with microvessel density in colorectal cancer (CRC) tissues. SMYD2 promotes CRC angiogenesis in vitro and in vivo. Mechanistically, SMYD2 physically interacts with HNRNPK and mediates lysine monomethylation at K422 of HNRNPK, which substantially increases RNA binding activity. HNRNPK acts by binding and stabilizing EGFL7 mRNA. As an angiogenic stimulant, EGFL7 enhances CRC angiogenesis. H3K4me3 maintained by PHF8 mediates the abnormal overexpression of SMYD2 in CRC. Moreover, targeting SMYD2 blocks CRC angiogenesis in tumor xenografts. Treatment with BAY-598, a functional inhibitor of SMYD2, can also synergize with apatinib in patient-derived xenografts. Overall, our findings reveal a new regulatory axis of CRC angiogenesis and provide a potential strategy for antiangiogenic therapy.

Protein Lysine Methyltransferases Inhibitors.

Protein lysine methylation is a significant protein post-translational modification (PTMs) and has a key function in epigenetic regulation. Protein lysine methyltransferase (PKMTs) mainly catalyzes the lysine methylation of various core histones and a few non-histone proteins. It has been observed that aberrant activity of PKMTs has been found in many cancers and other diseases, and some PKMT inhibitors have been discovered and progressed to clinical trials. This field developed rapidly and has aroused great interest. In this paper, we reviewed the biochemical and biological activities of PKMTs and their association with various cancers. Selective small-molecule inhibitors, including their chemical structure, structure-activity relationship, and in vitro/vivo studies, are also described to provide ideas for the discovery of highly potent, selective PKMT inhibitors.

Dissecting the Kinetic Mechanism of Human Lysine Methyltransferase 2D and Its Interactions with the WRAD2 Complex.

Human lysine methyltransferase 2D (hKMT2D) is an epigenetic writer catalyzing the methylation of histone 3 lysine 4. hKMT2D by itself has little catalytic activity and reaches full activation as part of the WRAD2 complex, additionally comprising binding partners WDR5, RbBP5, Ash2L, and DPY30. Here, a detailed mechanistic study of the hKMT2D SET domain and its WRAD2 interactions is described. We characterized the WRAD2 subcomplexes containing full-length components and the hKMT2D SET domain. By performing steady-state analysis as a function of WRAD2 concentration, we identified the inner stoichiometry and determined the binding affinities for complex formation. Ash2L and RbBP5 were identified as the binding partners critical for the full catalytic activity of the SET domain. Contrary to a previous report, product and dead-end inhibitor studies identified hKMT2D as a rapid equilibrium random Bi-Bi mechanism with EAP and EBQ dead-end complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) analysis showed that hKMT2D uses a distributive mechanism and gives further insights into how the WRAD2 components affect mono-, di-, and trimethylation. We also conclude that the Win motif of hKMT2D is not essential in complex formation, unlike other hKMT2 proteins.

Histone-lysine N-methyltransferase 2 (KMT2) complexes - a new perspective.

Histone H3 Lys4 (H3K4) methylation is catalyzed by the Histone-Lysine N-Methyltransferase 2 (KMT2) protein family, and its members are required for gene expression control. In vertebrates, the KMT2s function in large multisubunit complexes known as COMPASS or COMPASS-like complexes (COMplex of Proteins ASsociated with Set1). The activity of these complexes is critical for proper development, and mutation-induced defects in their functioning have frequently been found in human cancers. Moreover, inherited or de novo mutations in KMT2 genes are among the etiological factors in neurodevelopmental disorders such as Kabuki and Kleefstra syndromes. The canonical role of KMT2s is to catalyze H3K4 methylation, which results in a permissive chromatin environment that drives gene expression. However, current findings described in this review demonstrate that these enzymes can regulate processes that are not dependent on methylation: noncatalytic functions of KMT2s include DNA damage response, cell division, and metabolic activities. Moreover, these enzymes may also methylate non-histone substrates and play a methylation-dependent function in the DNA damage response. In this review, we present an overview of the new, noncanonical activities of KMT2 complexes in a variety of cellular processes. These discoveries may have crucial implications for understanding the functions of these methyltransferases in developmental processes, disease, and epigenome-targeting therapeutic strategies in the future.

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.

Mechanistic and functional extrapolation of SET and MYND domain-containing protein 2 to pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal neoplasms worldwide and represents the vast majority of pancreatic cancer cases. Understanding the molecular pathogenesis and the underlying mechanisms involved in the initiation, maintenance, and progression of PDAC is an urgent need, which may lead to the development of novel therapeutic strategies against this deadly cancer. Here, we review the role of SET and MYND domain-containing protein 2 (SMYD2) in initiating and maintaining PDAC development through methylating multiple tumor suppressors and oncogenic proteins. Given the broad substrate specificity of SMYD2 and its involvement in diverse oncogenic signaling pathways in many other cancers, the mechanistic extrapolation of SMYD2 from these cancers to PDAC may allow for developing new hypotheses about the mechanisms driving PDAC tumor growth and metastasis, supporting a proposition that targeting SMYD2 could be a powerful strategy for the prevention and treatment of PDAC.

Detection and Quantification of Histone Methyltransferase Activity In Vitro.

Histone methyltransferases (HMTs) catalyze the methylation of lysine and arginine residues in histone as well as nonhistone substrates. In vitro histone methyltransferase assays have been instrumental in identifying HMTs, and they continue to be invaluable tools for the study of these important enzymes, revealing novel substrates and modes of regulation.Here we describe a universal protocol to examine HMT activity in vitro that can be adapted to a range of HMTs, substrates, and experimental objectives. We provide protocols for the detection of activity based on incorporation of 3H-labeled methyl groups from S-adenosylmethionine (SAM), methylation-specific antibodies, and quantification of the reaction product S-adenosylhomocysteine (SAH).

Characterization of SET-Domain Histone Lysine Methyltransferase Substrates Using a Cofactor S-Adenosyl-L-Methionine Surrogate.

Identification of histone lysine methyltransferase (HKMT) substrates has recently benefited from chemical-biology-based strategies in which artificial S-adenosyl-L-methionine (SAM) cofactors are engineered to allow substrate labeling using either the wild-type target enzyme or designed mutants. Once labeled, substrates can be selectively functionalized with an affinity tag, using a bioorthogonal ligation reaction, to allow their recovery from cell extracts and subsequent identification. In this chapter, we describe steps on how to proceed to set up such an approach to characterize substrates of specific HKMTs of the SET domain superfamily, from the characterization of the HKMT able to accommodate a SAM surrogate containing a bioorthogonal moiety, to the proteomic analysis conducted on a cell extract. We focus in particular on the controls that are necessary to ensure reliable proteomic data analysis. The example of PR-Set7 on which we have implemented this approach is shown.

Ensemble Simulations and Experimental Free Energy Distributions: Evaluation and Characterization of Isoxazole Amides as SMYD3 Inhibitors.

Optimization of binding affinities for ligands to their target protein is a primary objective in rational drug discovery. Herein, we report on a collaborative study that evaluates various compounds designed to bind to the SET and MYND domain-containing protein 3 (SMYD3). SMYD3 is a histone methyltransferase and plays an important role in transcriptional regulation in cell proliferation, cell cycle, and human carcinogenesis. Experimental measurements using the scintillation proximity assay show that the distributions of binding free energies from a large number of independent measurements exhibit non-normal properties. We use ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) and TIES (thermodynamic integration with enhanced sampling) protocols to predict the binding free energies and to provide a detailed chemical insight into the nature of ligand-protein binding. Our results show that the 1-trajectory ESMACS protocol works well for the set of ligands studied here. Although one unexplained outlier exists, we obtain excellent statistical ranking across the set of compounds from the ESMACS protocol and good agreement between calculations and experiments for the relative binding free energies from the TIES protocol. ESMACS and TIES are again found to be powerful protocols for the accurate comparison of the binding free energies.