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.

Discovery of LLC0424 as a Potent and Selective in Vivo NSD2 PROTAC Degrader.

Nuclear receptor-binding SET domain-containing 2 (NSD2), a methyltransferase that primarily installs the dimethyl mark on lysine 36 of histone 3 (H3K36me2), has been recognized as a promising therapeutic target against cancer. However, existing NSD2 inhibitors suffer from low activity or inferior selectivity, and none of them can simultaneously remove the methyltransferase activity and chromatin binding function of NSD2. Herein we report the discovery of a novel NSD2 degrader LLC0424 by leveraging the proteolysis-targeting chimera technology. LLC0424 potently degraded NSD2 protein with a DC50 value of 20 nM and a Dmax value of 96% in acute lymphoblastic leukemia (ALL) RPMI-8402 cells. Mechanistic studies revealed LLC0424 to selectively induce NSD2 degradation in a cereblon- and proteasome-dependent fashion. LLC0424 also caused continuous downregulation of H3K36me2 and growth inhibition of ALL cell lines with NSD2 mutation. Importantly, intravenous or intraperitoneal injection of LLC0424 showed potent NSD2 degradation in vivo.

Discovery of the First-in-Class G9a/GLP PROTAC Degrader.

Aberrantly expressed lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9), have been implicated in numerous cancers. Recent studies have uncovered both catalytic and noncatalytic oncogenic functions of G9a/GLP. As such, G9a/GLP catalytic inhibitors have displayed limited anticancer activity. Here, we report the discovery of the first-in-class G9a/GLP proteolysis targeting chimera (PROTAC) degrader 10 (MS8709), as a potential anticancer therapeutic. 10 induces G9a/GLP degradation in a concentration-, time-, and ubiquitin-proteasome system (UPS)-dependent manner. Futhermore, 10 does not alter the mRNA expression of G9a/GLP and is selective for G9a/GLP over other methyltransferases. Moreover, 10 displays superior cell growth inhibition to the parent G9a/GLP inhibitor UNC0642 in prostate, leukemia, and lung cancer cells and has suitable mouse pharmacokinetic properties for in vivo efficacy studies. Overall, 10 is a valuable chemical biology tool to further investigate the functions of G9a/GLP and a potential therapeutic for treating G9a/GLP-dependent cancers.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Targeting G9a/DNMT1 methyltransferase activity impedes IGF2-mediated survival in hepatoblastoma.

BACKGROUND: As the variable clinical outcome of patients with hepatoblastoma (HB) cannot be explained by genetics alone, the identification of drugs with the potential to effectively reverse epigenetic alterations is a promising approach to overcome poor therapy response. The gene ubiquitin like with PHD and ring finger domains 1 (UHRF1) represents an encouraging epigenetic target due to its regulatory function in both DNA methylation and histone modifications and its clinical relevance in HB. METHODS: Patient-derived xenograft in vitro and in vivo models were used to study drug response. The mechanistic basis of CM-272 treatment was elucidated using RNA sequencing and western blot experiments. RESULTS: We validated in comprehensive data sets that UHRF1 is highly expressed in HB and associated with poor outcomes. The simultaneous pharmacological targeting of UHRF1-dependent DNA methylation and histone H3 methylation by the dual inhibitor CM-272 identified a selective impact on HB patient-derived xenograft cell viability while leaving healthy fibroblasts unaffected. RNA sequencing revealed downregulation of the IGF2-activated survival pathway as the main mode of action of CM-272 treatment, subsequently leading to loss of proliferation, hindered colony formation capability, reduced spheroid growth, decreased migration potential, and ultimately, induction of apoptosis in HB cells. Importantly, drug response depended on the level of IGF2 expression, and combination assays showed a strong synergistic effect of CM-272 with cisplatin. Preclinical testing of CM-272 in a transplanted patient-derived xenograft model proved its efficacy but also uncovered side effects presumably caused by its strong antitumor effect in IGF2-driven tumors. CONCLUSIONS: The inhibition of UHRF1-associated epigenetic traces, such as IGF2-mediated survival, is an attractive approach to treat high-risk HB, especially when combined with the standard-of-care therapeutic cisplatin.

Positioning of an unprecedented 1,5-oxaza spiroquinone scaffold into SMYD2 inhibitors in epigenetic space.

Lysine methyltransferases are important regulators of epigenetic signaling and are emerging as a novel drug target for drug discovery. This work demonstrates the positioning of novel 1,5-oxaza spiroquinone scaffold into selective SET and MYND domain-containing proteins 2 methyltransferases inhibitors. Selectivity of the scaffold was identified by epigenetic target screening followed by SAR study for the scaffold. The optimization was performed iteratively by two-step optimization consisting of iterative synthesis and computational studies (docking, metadynamics simulations). Computational binding studies guided the important interactions of the spiro[5.5]undeca scaffold in pocket 1 and Lysine channel and suggested extension of tail length for the improvement of potency (IC50: up to 399 nM). The effective performance of cell proliferation assay for chosen compounds (IC50: up to 11.9 nM) led to further evaluation in xenograft assay. The potent compound 24 demonstrated desirable in vivo efficacy with growth inhibition rate of 77.7% (4 fold decrease of tumor weight and 3 fold decrease of tumor volume). Moreover, mirosomal assay and pharmacokinetic profile suggested further developability of this scaffold through the identification of major metabolites (dealkylation at silyl group, reversible hydration product, the absence of toxic quinone fragments) and enough exposure of the testing compound 24 in plasma. Such spiro[5.5]undeca framework or ring system was neither been reported nor suggested as a modulator of methyltransferases. The chemo-centric target positioning and structural novelty can lead to potential pharmacological benefit.

EHMT2/G9a as an Epigenetic Target in Pediatric and Adult Brain Tumors.

Epigenetic mechanisms, including post-translational modifications of DNA and histones that influence chromatin structure, regulate gene expression during normal development and are also involved in carcinogenesis and cancer progression. The histone methyltransferase G9a (euchromatic histone lysine methyltransferase 2, EHMT2), which mostly mediates mono- and dimethylation by histone H3 lysine 9 (H3K9), influences gene expression involved in embryonic development and tissue differentiation. Overexpression of G9a has been observed in several cancer types, and different classes of G9a inhibitors have been developed as potential anticancer agents. Here, we review the emerging evidence suggesting the involvement of changes in G9a activity in brain tumors, namely glioblastoma (GBM), the main type of primary malignant brain cancer in adults, and medulloblastoma (MB), the most common type of malignant brain cancer in children. We also discuss the role of G9a in neuroblastoma (NB) and the drug development of G9a inhibitors.

Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia.

Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.

Targeting INMT and interrupting its methylation pathway for the treatment of castration resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) is associated with a very poor prognosis, and the treatment of which remains a serious clinical challenge. METHODS: RNA-seq, qPCR, western blot and immunohistochemistry were employed to identify and confirm the high expression of indolethylamine N-methyltransferase (INMT) in CRPC and the clinical relevance. Chip assay was used to identify Histone-Lysine N-Methyltransferase (SMYD3) as a major epigenetic regulator of INMT. LC-MS/MS were used to identify new substrates of INMT methylation in CRPC tissues. Gene knockdown/overexpression, MTT and mouse cancer models were used to examine the role of INMT as well as the anticancer efficacy of INMT inhibitor N,N-dimethyltryptamine (DMT), the SMYD3 inhibitor BCl-12, the selenium compounds methaneseleninic acid (MSA) and Se-(Methyl)selenocysteine hydrochloride (MSC), and the newly identified endogenous INMT substrate Bis(7)-tacrine. RESULTS: We found that the expression of INMT was highly increased in CRPC and was correlated with poor prognosis of clinical prostate cancer (PCa). INMT promoted PCa castration resistance via detoxification of anticancer metabolites. Knockdown of INMT or treatment with INMT inhibitor N,N-dimethyltryptamine (DMT) significantly suppressed CRPC development. Histone-Lysine N-Methyltransferase SMYD3 was a major epigenetic regulator of INMT expression, treatment with SMYD3 inhibitor BCl-121 suppressed INMT expression and inhibits CRPC development. Importantly, INMT knockdown significantly increased the anticancer effect of the exogenous selenium compounds methaneseleninic acid (MSA) and Se-(Methyl)selenocysteine hydrochloride (MSC) as well as the endogenous metabolite Bis(7)-tacrine. CONCLUSIONS: Our study suggests that INMT drives PCa castration resistance through detoxification of anticancer metabolites, targeting INMT or its regulator SMYD3 or/and its methylation metabolites represents an effective therapeutic avenue for CRPC treatment.

Targeting the Nuclear Receptor-Binding SET Domain Family of Histone Lysine Methyltransferases for Cancer Therapy: Recent Progress and Perspectives.

Nuclear receptor-binding SET domain (NSD) proteins are a class of histone lysine methyltransferases (HKMTases) that are amplified, mutated, translocated, or overexpressed in various types of cancers. Several campaigns to develop NSD inhibitors for cancer treatment have begun following recent advances in knowledge of NSD1, NSD2, and NSD3 structures and functions as well as the U.S. FDA approval of the first HKMTase inhibitor (tazemetostat, an EZH2 inhibitor) to treat follicular lymphoma and epithelioid sarcoma. This perspective highlights recent findings on the structures of catalytic su(var), enhancer-of-zeste, trithorax (SET) domains and other functional domains of NSD methyltransferases. In addition, recent progress and efforts to discover NSD-specific small molecule inhibitors against cancer-targeting catalytic SET domains, plant homeodomains, and proline-tryptophan-tryptophan-proline domains are summarized.

Novel insights into SMYD2 and SMYD3 inhibitors: from potential anti-tumoural therapy to a variety of new applications.

The revelance of the epigenetic regulation of cancer led to the design and testing of many drugs targeting epigenetic modifiers. The Su(Var)3-9, Enhancer-of-zeste and Trithorax (SET) and myeloid, Nervy, and DEAF-1 (MYND) domain-containing protein 2 (SMYD2) and 3 (SMYD3) are methyltransferases which act on histone and non-histone proteins to promote tumorigenesis in many cancer types. In addition to their oncogenic roles, SMYD2 and SMYD3 are involved in many other physiopathological conditions. In this review we will focus on the advances made in the last five years in the field of pharmacology regarding drugs targeting SMYD2 (such as LLY-507 or AZ505) and SMYD3 (such as BCI-121 or EPZ031686) and their potential cellular and molecular mechanisms of action and application in anti-tumoural therapy and/or against other diseases.

Role of NSD1 as potential therapeutic target in tumor.

Nuclear receptor binding SET Domain Protein 1 (NSD1) is a bifunctional transcriptional regulatory protein that encodes histone methyltransferase. Mono- and di-methylation of H3K36 by NSD1 is mainly primarily involved in the regulation of gene expression, DNA repair, alternative splicing, and other important biological processes. Many types of cancers, including acute myelogenous leukemia (AML), liver cancer, lung cancer, endometrial carcinoma, colorectal cancer, and pancreatic cancer, are associated with NSD1 fusion, missense mutation, nonsense mutation, silent mutation, deletion, and insertion of frameshift, and deletion in a frame. Therefore, targeting NSD1 may be a potential strategy for tumor therapy. An in-depth study of the structure and biological activities of NSD1 sets the groundwork for improving tumor therapy and creating NSD1 inhibitors. This article emphasizes the role of NSD1 in tumorigenesis and the development of NSD1 targeted small-molecule inhibitors.

Discovery of a Non-Nucleoside SETD2 Methyltransferase Inhibitor against Acute Myeloid Leukemia.

Histone methyltransferases (HMTs) have attracted considerable attention as potential targets for pharmaceutical intervention in various malignant diseases. These enzymes are known for introducing methyl marks at specific locations of histone proteins, creating a complex system that regulates epigenetic control of gene expression and cell differentiation. Here, we describe the identification of first-generation cell-permeable non-nucleoside type inhibitors of SETD2, the only mammalian HMT that is able to tri-methylate the K36 residue of histone H3. By generating the epigenetic mark H3K36me3, SETD2 is involved in the progression of acute myeloid leukemia. We developed a structure-based virtual screening protocol that was first validated in retrospective studies. Next, prospective screening was performed on a large library of commercially available compounds. Experimental validation of 22 virtual hits led to the discovery of three compounds that showed dose-dependent inhibition of the enzymatic activity of SETD2. Compound C13 effectively blocked the proliferation of two acute myeloid leukemia (AML) cell lines with MLL rearrangements and led to decreased H3K36me3 levels, prioritizing this chemotype as a viable chemical starting point for drug discovery projects.

A proteomic and phosphoproteomic landscape of KRAS mutant cancers identifies combination therapies.

KRAS mutant cancer, characterized by the activation of a plethora of phosphorylation signaling pathways, remains a major challenge for cancer therapy. Despite recent advancements, a comprehensive profile of the proteome and phosphoproteome is lacking. This study provides a proteomic and phosphoproteomic landscape of 43 KRAS mutant cancer cell lines across different tissue origins. By integrating transcriptomics, proteomics, and phosphoproteomics, we identify three subsets with distinct biological, clinical, and therapeutic characteristics. The integrative analysis of phosphoproteome and drug sensitivity information facilitates the identification of a set of drug combinations with therapeutic potentials. Among them, we demonstrate that the combination of DOT1L and SHP2 inhibitors is an effective treatment specific for subset 2 of KRAS mutant cancers, corresponding to a set of TCGA clinical tumors with the poorest prognosis. Together, this study provides a resource to better understand KRAS mutant cancer heterogeneity and identify new therapeutic possibilities.

Set7/9 controls proliferation and genotoxic drug resistance of NSCLC cells.

The SET domain containing lysine-specific methyltransferase, Set7/9, covalently attaches methyl moieties to a variety of histone and non-histone substrates. Among the substrates of Set7/9 are: p53, NF-kB, PARP1, E2F1, and other transcription factors that regulate many vital processes in the cell. Through the post-translational regulation of these critical master-regulators Set7/9 is involved in regulation of cell proliferation, cancer progression, and DNA damage response. Noteworthy, the role of Set7/9 in tumorigenesis is contradictory and apparently depends on the cellular context. In this study, we investigated the effect of Set7/9 on tumorigenic characteristics of lung cancer cells. We showed that CRISPR/Cas9-mediated knock-out of Set7/9 in A549 and its shRNA-mediated knock-down in H1299 NSCLC cell lines both augment the proliferation rate of tumor cells compared to the matching wild-type cells. Mechanistically, ablation of Set7/9 increased the expression of cyclin A2 and D1 genes thereby promoting the accumulation of cells in S phase. Furthermore, knockout of Set7/9 decreased the expression of E-cadherin, whose product is critical for cell-cell interactions. Accordingly, this led to the increased migration of lung cancer cells. Finally, both ablation or pharmacological inhibition of Set7/9 enzymatic methyltransferase activity by the selective inhibitor (R)-PFI-2 sensitized NSCLC cells to genotoxic drug, doxorubicin. This effect was also recapitulated on patients-derived NSCLC cell lines. Taken together, our results suggest that Set7/9 plays anti-proliferative and DNA damage-protective roles in NSCLC cells and hence represents an attractive target for anti-cancer chemotherapy.

Inhibition of WHSC1 Allows for Reprogramming of the Immune Compartment in Prostate Cancer.

Immunotherapy initially demonstrated promising results in prostate cancer (PCa), but the modest or negative results of many recent trials highlight the need to overcome the poor immunogenicity of this cancer. The design of effective therapies for PCa is challenged by the limited understanding of the interface between PCa cells and the immune system in mediating therapeutic resistance. Prompted by our recent observations that elevated WHSC1, a histone methyltransferase known to promote progression of numerous cancers, can silence antigen processing and presentation in PCa, we performed a single-cell analysis of the intratumoral immune dynamics following in vivo pharmacological inhibition of WHSC1 in mice grafted with TRAMP C2 cells. We observed an increase in cytotoxic T and NK cells accumulation and effector function, accompanied by a parallel remodeling of the myeloid compartment, as well as abundant shifts in key ligand-receptor signaling pathways highlighting changes in cell-to-cell communication driven by WHSC1 inhibition. This comprehensive profiling of both immune and molecular changes during the course of WHSC1 blockade deepens our fundamental understanding of how anti-tumor immune responses develop and can be enhanced therapeutically for PCa.

The Benzene Hematotoxic and Reactive Metabolite 1,4-Benzoquinone Impairs the Activity of the Histone Methyltransferase SET Domain Containing 2 (SETD2) and Causes Aberrant Histone H3 Lysine 36 Trimethylation (H3K36me3).

Human SETD2 is the unique histone methyltransferase that generates H3K36 trimethylation (H3K36me3), an epigenetic mark that plays a key role in normal hematopoiesis. Interestingly, recurrent inactivating mutations of SETD2 and aberrant H3K36me3 are increasingly reported to be involved in hematopoietic malignancies. Benzene (BZ) is a ubiquitous environmental pollutant and carcinogen that causes leukemia. The leukemogenic properties of BZ depend on its biotransformation in the bone marrow into oxidative metabolites, in particular 1,4-benzoquinone (BQ). This hematotoxic metabolite can form DNA and protein adducts that result in the damage and the alteration of cellular processes. Recent studies suggest that BZ-dependent leukemogenesis could depend on epigenetic perturbations, notably aberrant histone methylation. We investigated whether H3K36 trimethylation by SETD2 could be impacted by BZ and its hematotoxic metabolites. Herein, we show that BQ, the major leukemogenic metabolite of BZ, inhibits irreversibly the human histone methyltransferase SETD2, resulting in decreased H3K36me3. Our mechanistic studies further indicate that the BQ-dependent inactivation of SETD2 is due to covalent binding of BQ to reactive Zn-finger cysteines within the catalytic domain of the enzyme. The formation of these quinoprotein adducts results in loss of enzyme activity and protein crosslinks/oligomers. Experiments conducted in hematopoietic cells confirm that exposure to BQ results in the formation of SETD2 crosslinks/oligomers and concomitant loss of H3K36me3 in cells. Taken together, our data indicate that BQ, a major hematotoxic metabolite of BZ, could contribute to BZ-dependent leukemogenesis by perturbing the functions of SETD2, a histone lysine methyltransferase of hematopoietic relevance. SIGNIFICANCE STATEMENT: Benzoquinone is a major leukemogenic metabolite of benzene. Dysregulation of histone methyltransferase is involved in hematopoietic malignancies. This study found that benzoquinone irreversibly impairs SET domain containing 2, a histone H3K36 methyltransferase that plays a key role in hematopoiesis. Benzoquinone forms covalent adducts on Zn-finger cysteines within the catalytic site, leading to loss of activity, protein crosslinks/oligomers, and concomitant decrease of H3K36me3 histone mark. These data provide evidence that a leukemogenic metabolite of benzene can impair a key epigenetic enzyme.

Discovery of a potent MLL1 and WDR5 protein-protein interaction inhibitor with in vivo antitumor activity.

MLL1-WDR5 interaction is essential for the formation of MLL core complex and its H3K4 methyltransferase activity. Disrupting MLL1-WDR5 interaction has been proposed as a potential therapeutic approach in the treatment of leukemia. A "toolkit" of well-characterized chemical probe will allow exploring animal studies. Based on a specific MLL1-WDR5 PPI inhibitor (DDO-2117), which was previously reported by our group, we conducted a bioisosterism approach by click chemistry to discover novel phenyltriazole scaffold MLL1-WDR5 interaction blockers. Here, our efforts resulted in the best inhibitor 24 (DDO-2093) with high binding affinity (Kd = 11.6 nM) and with improved drug-like properties. Both in vitro and in vivo assays revealed 24 could efficiently block the MLL1-WDR5 interaction. Furthermore, 24 significantly suppressed tumor growth in the MV4-11 xenograft mouse model and showed a favorable safety profile. We propose 24 as a chemical probe that is suitable for in vivo pharmacodynamic and biological studies of MLL1-WDR5 interaction.

Discovery of DDO-2213 as a Potent and Orally Bioavailable Inhibitor of the WDR5-Mixed Lineage Leukemia 1 Protein-Protein Interaction for the Treatment of MLL Fusion Leukemia.

WD repeat-containing protein 5 (WDR5) is essential for the stability and methyltransferase activity of the mixed lineage leukemia 1 (MLL1) complex. Dysregulation of the MLL1 gene is associated with human acute leukemias, and the direct disruption of the WDR5-MLL1 protein-protein interaction (PPI) is emerging as an alternative strategy for MLL-rearranged cancers. Here, we represent a new aniline pyrimidine scaffold for WDR5-MLL1 inhibitors. A comprehensive structure-activity analysis identified a potent inhibitor 63 (DDO-2213), with an IC50 of 29 nM in a competitive fluorescence polarization assay and a Kd value of 72.9 nM for the WDR5 protein. Compound 63 selectively inhibited MLL histone methyltransferase activity and the proliferation of MLL translocation-harboring cells. Furthermore, 63 displayed good pharmacokinetic properties and suppressed the growth of MV4-11 xenograft tumors in mice after oral administration, first verifying the in vivo efficacy of targeting the WDR5-MLL1 PPI by small molecules.

5-Aminonaphthalene derivatives as selective nonnucleoside nuclear receptor binding SET domain-protein 2 (NSD2) inhibitors for the treatment of multiple myeloma.

Approximately 20% of multiple myeloma (MM) are caused by a chromosomal translocation t (4; 14) that leads to the overexpression of the nuclear receptor binding SET domain-protein 2 (NSD2) histone methyltransferase. NSD2 catalyzes the methylation of lysine 36 on histone H3 (H3K36me2) and is associated with transcriptionally active regions. Using high-throughput screening (HTS) with biological analyses, a series of 5-aminonaphthalene derivatives were designed and synthesized as novel NSD2 inhibitors. Among all the prepared compounds, 9c displayed a good NSD2 inhibitory activity (IC50 = 2.7 µM) and selectivity against both SET-domain-containing and non-SET-domain-containing methyltransferases. Preliminary research indicates the inhibition mechanism of compound 9c by significantly suppressed the methylation of H3K36me2. Compound 9c specifically inhibits the proliferation of the human B cell precursor leukemia cell line RS4:11 and the human myeloma cell line KMS11 by inducing cell cycle arrest and apoptosis with little cytotoxicity. It has been reported that the anti-cancer effect of compound 9c is partly achieved by completely suppressing the transcriptional activation of NSD2-targeted genes. When administered intraperitoneally at 25 mg/kg, compound 9c suppressed the tumor growth of RS4:11 xenografts in vivo and no body weight loss was detected in the tested SCID mice.