WDR5 Interaction Research Articles

Computational Studies of Novel Aniline Pyrimidine WDR5‐MLL1 Inhibitors: QSAR, Molecular Docking, and Molecular Dynamics Simulation

AbstractWD repeat‐containing protein 5 (WDR5) and mixed lineage leukemia (MLL) are critical for maintaining tumorigenesis in human cancers. Disruption of the MLL1–WDR5 interaction has been viewed as a promising therapeutic strategy for the treatment of leukemia. Here, we report a series of protein–protein binding interaction modes targeting MLL1‐WDR5 inhibitors using 3D‐QSAR, and molecular docking. CoMFA with q2 = 0.724, r2 = 0.957, rpred2 = 0.874 and CoMSIA with q2 = 0.826, r2 = 0.995, rpred2 = 0.827. Topomer CoMFA results show q2 = 0.807, r2 = 0.976, rpred2 = 0.789, and HQSAR results show q2 = 0.873, r2 = 0.982, rpred2 = 0.794. The 3D‐QSAR model with high validation and prediction performance is successfully constructed. Contour maps and molecular docking results according to the four models, 26 new compounds are finally designed on the computer after molecular screening. Further molecular dynamics simulations (MD) of compounds G01, G04, G09, G43, and G44 with high predicted activity are carried out to explore their possible binding modes. ILE305, PHE133, CYS261, and ALA176 are found to play crucial roles in stabilizing the inhibitor. ADMET predictions are also performed for these 26 new compounds. These results serve as references for the design of effective WDR5‐MLL1 inhibitors in the future.

Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5

The conserved WD40-repeat protein WDR5 interacts with multiple proteins both inside and outside the nucleus. However, it is currently unclear whether and how the distribution of WDR5 between complexes is regulated. Here, we show that an unannotated microprotein EMBOW (endogenous microprotein binder of WDR5) dually encoded in the human SCRIB gene interacts with WDR5 and regulates its binding to multiple interaction partners, including KMT2A and KIF2A. EMBOW is cell cycle regulated, with two expression maxima at late G1 phase and G2/M phase. Loss of EMBOW decreases WDR5 interaction with KIF2A, aberrantly shortens mitotic spindle length, prolongs G2/M phase, and delays cell proliferation. In contrast, loss of EMBOW increases WDR5 interaction with KMT2A, leading to WDR5 binding to off-target genes, erroneously increasing H3K4me3 levels, and activating transcription of these genes. Together, these results implicate EMBOW as a regulator of WDR5 that regulates its interactions and prevents its off-target binding in multiple contexts.

Structure-based discovery of potent WD repeat domain 5 inhibitors that demonstrate efficacy and safety in preclinical animal models

WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust invitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant invivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent invivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these invivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.

Impact of WIN site inhibitor on the WDR5 interactome

SUMMARYThe chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.

The Development of Inhibitors Targeting the Mixed Lineage Leukemia 1 (MLL1)-WD Repeat Domain 5 Protein (WDR5) Protein- Protein Interaction.

Mixed Lineage Leukemia 1 (MLL1), an important member of Histone Methyltransferases (HMT) family, is capable of catalyzing mono-, di-, and trimethylation of Histone 3 lysine 4 (H3K4). The optimal catalytic activity of MLL1 requires the formation of a core complex consisting of MLL1, WDR5, RbBP5, and ASH2L. The Protein-Protein Interaction (PPI) between WDR5 and MLL1 plays an important role in abnormal gene expression during tumorigenesis, and disturbing this interaction may have a potential for the treatment of leukemia harboring MLL1 fusion proteins. In this review, we will summarize recent progress in the development of inhibitors targeting MLL1- WDR5 interaction.

Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance

The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacologically inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MYC-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt's lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents.

Targeting WDR5: A WINning Anti-Cancer Strategy?

WDR5 is a component of multiple epigenetic regulatory complexes, including the mixed lineage leukemia (MLL)/SET complexes that deposit histone H3 lysine 4 methylation. Inhibitors of an arginine-binding cavity in WDR5, known as the WDR5-interaction (WIN) site, have been proposed to selectively kill MLL-rearranged malignancies via an epigenetic mechanism. We discovered potent WIN site inhibitors and found that they kill MLL cancer cells not through changes in histone methylation, but by displacing WDR5 from chromatin at protein synthesis genes, choking the translational capacity of these cells, and inducing death via a nucleolar stress response. The mechanism of action of WIN site inhibitors reveals new aspects of WDR5 function and forecasts broad therapeutic utility as anti-cancer agents.

Abstract 4994: Understanding the MYC and WDR5 interaction at chromatin

Abstract MYC is an oncoprotein that is overexpressed in the majority of malignancies and contributes to an estimated 70,000-100,000 cancer deaths in the United States every year. The broad pro-tumorigenic functions of MYC stem from its role as a sequence-specific transcriptional regulator, controlling the expression of thousands of genes linked to cell cycle control, growth, and metabolism. Key to understanding how MYC causes cancer, therefore, is understanding the mechanisms through which it selects its target genes. Histone modifications, DNA sequence, and interactions with other transcription factors have all been suggested to influence where MYC binds chromatin, but a consistent signature has not been defined. Our laboratory recently discovered that the chromatin regulatory protein WDR5—a core component of histone modifying complexes—interacts directly with MYC and co-localizes with MYC at a majority of its target genes in human cells (Thomas et al., Mol. Cell, 58: 440-452, 2015). Point mutations in MYC that disable interaction with WDR5 do not impact the ability of MYC to bind naked DNA, but they do prevent MYC from recognizing target genes in the context of chromatin and from driving tumorigenesis in mice. These studies led us to propose that the MYC-WDR5 interaction is a critical determinant in MYC target gene recognition, in a process we refer to as “facilitated recruitment.” In the facilitated recruitment model, the presence of WDR5 at chromatin promotes MYC binding at certain genomic loci over others. The role of WDR5 in target gene selection by MYC can explain much of the plasticity in genome-wide binding patterns of MYC that have been reported, and may provide a new avenue for therapeutically targeting MYC in cancer. Two important questions are raised by these studies, however. How does WDR5 recognize and select its target genes? And what other functions, if any, does WDR5 play in regulating MYC target genes? To answer these questions, we are employing traditional and quantitative proteomics, together with biochemical approaches, to characterize the composition and stoichiometry of the WDR5-containing complex that associates with MYC. These studies, still ongoing, have demonstrated that this complex is devoid of canonical WDR5-interaction partners such as RBBP5 and HCF-1, revealing that the function of WDR5 in this setting is distinct from its well-characterized roles in histone modifications. We propose that the WDR5 complex that associates with MYC on chromatin is either entirely novel, or is a ‘ghost’ complex in which select protein components have been excluded by the direct interaction of MYC with WDR5. We continue to define the biochemical constituents of the MYC-WDR5 complex, and will interrogate their role in facilitated recruitment using a combination of genetic, biochemical, and genomic, approaches. Citation Format: Alissa D. Guarnaccia, April M. Weissmiller, Lance R. Thomas, William P. Tansey. Understanding the MYC and WDR5 interaction at chromatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4994. doi:10.1158/1538-7445.AM2017-4994

MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis

Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer.

Abstract PR09: Targeting the MYC-WDR5 nexus in colorectal cancer

Abstract The overexpression of c-MYC is one of the most prominent alterations in colorectal cancer (CRC), featuring in roughly 70% of colorectal adenocarcinomas. Induction of MYC is a critical event in the development and progression of CRC, as it causes a variety of biological responses, including cell cycle progression, cellular growth, and differentiation. The contribution of the MYC oncoprotein to the pathogenesis of CRC is evidenced by its widespread overexpression and its correlation with poor patient survival, and by observations that deletion of c-Myc—or mutation of the β-catenin/TCF/LEF enhancer upstream of c-Myc—dramatically reduces intestinal tumor burden in ApcMin mice. Despite the pervasive involvement of MYC in CRC, and a wealth of studies in other systems demonstrating that genetic inhibition of MYC promotes frank tumor regression, MYC is generally considered undruggable—as of yet, no drug-like molecules have been discovered that are capable of blocking MYC function in cancer cells. Recently, however, we devised a novel approach to target MYC, based on our observations that binding of MYC to its target genes in the context of chromatin is dependent on interaction with the chromatin-scaffolding protein WDR5. We have proposed that target gene recognition by MYC is an avidity-based mechanism that involves two critical sets of interactions: one between MYC/MAX heterodimers and DNA, and another between MYC and chromatin-bound WDR5. We showed that interaction with WDR5 is necessary for the ability of MYC to function as a transcription factor and oncoprotein. Importantly, structural analysis revealed that MYC directly binds WDR5 by engaging a shallow, hydrophobic, cleft on the surface of WDR5 that is well-suited for drug discovery. The tractability of the MYC–WDR5 interface as a vehicle for drug discovery should make it possible to identify drug-like molecules that disrupt interaction of MYC with chromatin and diminish its tumorigenic potential. Fueled by the potential of this discovery, the Fesik and Tansey laboratories collaborate to identify, refine, and validate drug-like molecules that disrupt the MYC–WDR5 interaction, and propose to explore their efficacy as anti-cancer agents. Since the overexpression of MYC is featured in the majority of colorectal adenomas, we will investigate the requirement of the MYC–WDR5 interaction in colorectal carcinoma cell lines and in mice. During the small-molecule discovery phase, an inducible mutant version of MYC will be utilized to determine the effects of loss of the MYC–WDR5 interaction in CRC cell lines and in mice with adenomatous polyps, both further elucidating the biological basis of this interaction, as well as establishing a baseline for effects we should see from on-target small-molecule inhibitors. Through cellular, molecular, and genomic comparison of the effects of genetic versus chemical disruption of the MYC–WDR5 interaction, we will validate on-target action of probe compounds, providing critical information needed to select candidate molecules that will be refined for drug-like properties. Successful completion of this work will generate novel insights into MYC biology, its role in CRC, as well as first-in-class MYC–WDR5 inhibitors validated for their efficacy in CRC. This abstract is also being presented as Poster B18. Citation Format: Audra M. Foshage, Lance R. Thomas, Shaun R. Stauffer, Stephen W. Fesik, William P. Tansey. Targeting the MYC-WDR5 nexus in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr PR09.

Abstract IA15: Novel approaches for targeting MYC

Abstract The MYC family (c-, L-, and N-MYC) is one of the most important groups of proteins in human cancer. More than 50% of all malignancies overexpress at least one MYC family member, and estimates suggest that upwards of 100,000 Americans die from cancer every year as a result of inappropriate MYC expression or activity. Preclinical mouse models have demonstrated that perturbing MYC function in preexisting cancers promotes frank tumor regression, establishing MYC proteins as bonafide and high-value targets for discovery of broadly effective anti-cancer therapies. Despite the potential of MYC inhibitors to impact cancer therapy, efforts to discover MYC inhibitors are stymied by numerous factors, including an absence of MYC interaction partners with characteristics that make them amenable to drug discovery efforts. The best understood MYC interaction partner, MAX, is required for MYC to assemble a competent DNA-binding module and would theoretically be a superb target for anti-MYC therapies. The MYC–MAX interaction, however, encompasses more that 1800 Å2 of buried protein surface, making it difficult to effectively disrupt by a small molecule with drug-like properties. Breaking the stalemate in drugging MYC requires a fresh perspective on how MYC functions and where the vulnerabilities in its molecular mechanisms of action lie. Structure-function studies have delineated a potent activation domain at the amino-terminus of MYC and a DNA-binding domain at the carboxy-terminus, but the central portion between these domains has not been studied in detail. To rectify this deficit, we recently initiated screens to identify proteins that bind to the central portion of MYC, with the idea that these proteins would reveal new aspects of MYC regulation or function; some of which may expose new ways to target MYC activity. One novel MYC-interaction partner to emerge from this screen was WDR5. WDR5 is a chromatin-associated protein that co-binds with MYC at a majority of its target genes. MYC binds weakly to WDR5 by engaging a structured hydrophobic cleft on one side of the WDR5 protein. Mutations in MYC that disrupt interaction with WDR5 block the ability of MYC to bind chromatin and disable its tumorigenic properties in mice, demonstrating that WDR5 is a critical co-factor for MYC. These studies led to a revised model for target gene recognition by MYC in which WDR5 facilitates the recruitment of MYC/MAX complexes to chromatin, and revealed that the MYC–WDR5 interface is a tractable surface for small molecule inhibition. In collaboration with the laboratory of Dr. Stephen Fesik we seek to discover and validate drug-like molecules that disrupt the MYC–WDR5 interaction and explore their utility in cell and animal-based models of cancer. Citation Format: William Tansey. Novel approaches for targeting MYC. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA15.

Structure-based design of ester compounds to inhibit MLL complex catalytic activity by targeting mixed lineage leukemia 1 (MLL1)–WDR5 interaction

WDR5 is an essential protein for enzymatic activity of MLL1. Targeting the protein–protein interaction (PPI) between MLL1 and WDR5 represents a new potential therapeutic strategy for MLL leukemia. Based on the structure of reported inhibitor WDR5-0103, a class of ester compounds were designed and synthetized to disturb MLL1–WDR5 PPI. These inhibitors efficiently inhibited the histone methyltransferase activity in vitro. Especially, WL-15 was one of the most potent inhibitors, blocking the interaction of MLL1–WDR5 with IC50 value of 26.4nM in competitive binding assay and inhibiting the catalytic activity of MLL1 complex with IC50 value of 5.4μM. Docking model indicated that ester compounds suitably occupied the central cavity of WDR5 protein and recapitulated the interactions of WDR5-0103 and the hydrophobic groups and key amino greatly increased the activity in blocking MLL1–WDR5 PPI.

High-affinity small molecular blockers of mixed lineage leukemia 1 (MLL1)-WDR5 interaction inhibit MLL1 complex H3K4 methyltransferase activity

MLL1-WDR5 protein-protein interaction is essential for MLL1 H3K4 methyltransferase activity. Targeting MLL1 enzymatic activity to regulate expression level of MLL-dependent genes represents a therapeutic strategy for acute leukemia harboring MLL fusion proteins. Herein we reported a series of biphenyl compounds disturbed MLL1-WDR5 interaction. These compounds effectively inhibited MLL1 histone methyltransferase (HMT) activity in vitro and in MV4-11 cell line. The representative compound 30 (DDO-2084) inhibited proliferation and induced apoptosis of MV4-11 cells through deregulating expression level of Hoxa9 and Meis-1 genes, which emphasized our compounds were on-target. Optimization of compound 30 led to high-affinity inhibitors. Especially, compound 42 (DDO-2117, IC50 = 7.6 nM) bearing an amino and a 4-aminobutanamido group was the most potent inhibitor reported to-date, and showed the most potent inhibitory activity (IC50 = 0.19 μM) in HMT assay.

The MYC-WDR5 Nexus and Cancer.

The MYC oncogenes encode a family of transcription factors that feature prominently in cancer. MYC proteins are overexpressed or deregulated in a majority of malignancies and drive tumorigenesis by inducing widespread transcriptional reprogramming that promotes cell proliferation, metabolism, and genomic instability. The ability of MYC to regulate transcription depends on its dimerization with MAX, which creates a DNA-binding domain that recognizes specific sequences in the regulatory elements of MYC target genes. Recently, we discovered that recognition of target genes by MYC also depends on its interaction with WDR5, a WD40-repeat protein that exists as part of several chromatin-regulatory complexes. Here, we discuss how interaction of MYC with WDR5 could create an avidity-based chromatin recognition mechanism that allows MYC to select its target genes in response to both genetic and epigenetic determinants. We rationalize how the MYC-WDR5 interaction provides plasticity in target gene selection by MYC and speculate on the biochemical and genomic contexts in which this interaction occurs. Finally, we discuss how properties of the MYC-WDR5 interface make it an attractive point for discovery of small-molecule inhibitors of MYC function in cancer cells.

Structural analysis of the KANSL1/WDR5/KANSL2 complex reveals that WDR5 is required for efficient assembly and chromatin targeting of the NSL complex.

The subunits of the nonspecific lethal (NSL) complex, which include the histone acetyltransferase MOF (males absent on the first), play important roles in various cellular functions, including transcription regulation and stem cell identity maintenance and reprogramming, and are frequently misregulated in disease. Here, we provide the first biochemical and structural insights into the molecular architecture of this large multiprotein assembly. We identified several direct interactions within the complex and show that KANSL1 acts as a scaffold protein interacting with four other subunits, including WDR5, which in turn binds KANSL2. Structural analysis of the KANSL1/WDR5/KANSL2 subcomplex reveals how WDR5 is recruited into the NSL complex via conserved linear motifs of KANSL1 and KANSL2. Using structure-based KANSL1 mutants in transgenic flies, we show that the KANSL1-WDR5 interaction is required for proper assembly, efficient recruitment of the NSL complex to target promoters, and fly viability. Our data clearly show that the interactions of WDR5 with the MOF-containing NSL complex and MLL/COMPASS histone methyltransferase complexes are mutually exclusive. We propose that rather than being a shared subunit, WDR5 plays an important role in assembling distinct histone-modifying complexes with different epigenetic regulatory roles.

Synthesis, Optimization, and Evaluation of Novel Small Molecules as Antagonists of WDR5-MLL Interaction.

The WD40-repeat protein WDR5 plays a critical role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small molecules has recently been presented as a practical way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compound 2 (K dis = 7 μM), leading to identification of more potent antagonist 47 (K dis = 0.3 μM).

Structural Basis for WDR5 Interaction (Win) Motif Recognition in Human SET1 Family Histone Methyltransferases

Translocations and amplifications of the mixed lineage leukemia-1 (MLL1) gene are associated with aggressive myeloid and lymphocytic leukemias in humans. MLL1 is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases, which are required for transcription of genes involved in hematopoiesis and development. MLL1 associates with a subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD), which together form the MLL1 core complex that is required for sequential mono- and dimethylation of H3K4. We previously demonstrated that WDR5 binds the conserved WDR5 interaction (Win) motif of MLL1 in vitro, an interaction that is required for the H3K4 dimethylation activity of the MLL1 core complex. In this investigation, we demonstrate that arginine 3765 of the MLL1 Win motif is required to co-immunoprecipitate WRAD from mammalian cells, suggesting that the WDR5-Win motif interaction is important for the assembly of the MLL1 core complex in vivo. We also demonstrate that peptides that mimic SET1 family Win motif sequences inhibit H3K4 dimethylation by the MLL1 core complex with varying degrees of efficiency. To understand the structural basis for these differences, we determined structures of WDR5 bound to six different naturally occurring Win motif sequences at resolutions ranging from 1.9 to 1.2 Å. Our results reveal that binding energy differences result from interactions between non-conserved residues C-terminal to the Win motif and to a lesser extent from subtle variation of residues within the Win motif. These results highlight a new class of methylation inhibitors that may be useful for the treatment of MLL1-related malignancies.

The role of WDR5 in silencing human fetal globin gene expression

Histone H3 lysine 4 (K4) methylation has been linked with transcriptional activity in mammalian cells. The WD40-repeat protein, WDR5, is an essential component of the MLL complex that induces histone H3 K4 methylation, but the role of WDR5 in human globin gene regulation has not yet been established. To study the role of WDR5 in human globin gene regulation, we performed knockdown experiments in both K562 cells and primary human bone marrow erythroid progenitor cells (BMC). The effects of WDR5 knockdown on γ-globin gene expression were determined. Biochemical approaches were also employed to investigate WDR5 interaction molecules. Chromosomal marks in the globin locus were analyzed by ChIP. We found that WDR5 interacted with protein arginine methyltransferase 5 (PRMT5), a known repressor of γ-globin gene expression, and was essential for generating tri-methylated H3K4 (H3K4me3) at the γ-globin promoter in K562 cells. Enforced expression of WDR5 in K562 cells reduced γ-globin gene expression, whereas knockdown of WDR5 increased γ-globin gene expression in both K562 cells and primary human bone marrow erythroid progenitor cells. Consistent with this, both histone H3 and H4 acetylation at the γ-globin promoter were increased, while histone H4R3 and H3K9 methylation were decreased, in WDR5 knockdown cells compared to controls. We found that WDR5 interacted with HDAC1 and a PHD domaincontaining protein, ING2 (inhibitor of growth), an H3K4me3 mark reader, to enhance γ-globin gene transcriptional repression. In human BMC, levels of WDR5 were highly enriched on the γ-promoter relative to levels on other globin promoters and compared to the γ-promoter in cord blood erythroid progenitors, suggesting that WDR5 is important in the developmental globin gene expression program. Our data are consistent with a model in which WDR5 binds the γ-globin promoter in a PRMT5-dependent manner; H3K4me3 induced at the γ-globin promoter by WDR5 may result in the recruitment of the ING2-associated HDAC1 component and consequent silencing of γ-globin gene expression.

Exploring the role of the MLL1 complex in leukemia by small molecule targeting of an essential protein‐protein interaction

MLL1 is a histone H3 lysine 4 (H3K4) specific methyltransferase that has essential functions in embryonic development, hematopoiesis and leukemia. Dysregulation of MLL1 activity, by mutation, translocation or amplification of one copy of the MLL1 gene, has been found to play an essential role in many cases of acute leukemia with poor prognosis. In most cases of MLL1‐associated leukemia, MLL1 wild‐type functions coordinately with the mutant copy of MLL1 to aberrantly overexpress MLL1 target genes, specifically HoxA9 and Meis1. Overexpression of these genes is essential for leukemogenesis. In the cell, MLL1 activity is upregulated by stable association of MLL1 with the proteins WDR5, RbBP5 and Ash2L. In this stable enzyme complex, WDR5 serves as the structural platform to mediate complex assembly and facilitate the interaction of RbBP5 and Ash2L with MLL1. We hypothesize that small molecule targeting of the MLL1:WDR5 interaction will downregulate MLL1 activity and provide a therapeutic avenue to treat MLL1‐involved leukemias. To this end, we have developed a potent, peptidomimetic inhibitor of the MLL1:WDR5 interaction. We have found that treatment of leukemia cell lines with this inhibitor downregulates expression of MLL1 target genes and has moderate inhibitory effects on cell viability in leukemia‐derived cell lines.

Structural basis for the recognition of Set1 family Win motifs by WDR5

The Mixed Lineage Leukemia protein‐1 (MLL1) associates with WDR5, RbBP5 and Ash2L to form the MLL1 core complex that catalyzes mono‐ and dimethylation of histone H3 Lysine 4 (H3K4). We have recently established that WDR5 interacts with MLL1 through a highly conserved WDR5 interaction (Win) motif in MLL1 and a peptide derived from the Win motif in MLL1 has been shown to disrupt this interaction and specifically inhibit the H3K4 dimethylation activity of the MLL1 core complex. The Win motif is highly conserved among the metazoan Set1 family members, however, little is known about how the different Set1 family Win motifs are recognized by WDR5. To understand the molecular details of these interactions, here we compare 3‐dimensional structures and thermodynamic binding properties of different human Set1 family Win motif based peptides bound to WDR5. We show that Win motif peptides derived from human Set1 family members (MLL2, MLL3, MLL4, SET1a and SET1b) interact with WDR5 with dissociation constants (Kd) ranging from 50–470 nM. These binding affinities are 4 to 30‐fold tighter than the interaction between MLL1 Win motif and WDR5. The crystal structure of WDR5 bound to the human SET1a Win motif peptide reveals more favorable interactions outside the Arginine binding pocket in WDR5 that contributes to the increased affinity as compared to that of MLL1 Win motif peptide. We also demonstrate that the human SET1a Win motif peptide is a four‐fold better inhibitor of the H3K4 dimethylation activity of the MLL1 core complex with an IC50 value of 70 μM. These studies establish the structural basis for the differential interaction affinities between WDR5 and Set1 family Win motifs and suggest a framework for the design of Win motif based peptide inhibitors of Set1 family complexes.