Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus.

BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the 'WIN' site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Super-Enhancer Dysregulation in Rhabdoid Tumor Cells Is Regulated by the SWI/SNF ATPase BRG1.

Mutations in the SWI/SNF chromatin remodeling complex occur in ~20% of cancers. In rhabdoid tumors defined by loss of the SWI/SNF subunit SMARCB1, dysregulation of enhancer-mediated gene expression is pivotal in driving oncogenesis. Enhancer dysregulation in this setting is tied to retention of the SWI/SNF ATPase BRG1-which becomes essential in the absence of SMARCB1-but precisely how BRG1 contributes to this process remains unknown. To characterize how BRG1 participates in chromatin remodeling and gene expression in SMARCB1-deficient cells, we performed a genome-wide characterization of the impact of BRG1 depletion in multiple rhabdoid tumor cell lines. We find that although BRG1-regulated open chromatin sites are distinct at the locus level, the biological characteristics of the loci are very similar, converging on a set of thematically related genes and pointing to the involvement of the AP-1 transcription factor. The open chromatin sites regulated by BRG1 colocalize with histone-marked enhancers and intriguingly include almost all super-enhancers, revealing that BRG1 plays a critical role in maintaining super-enhancer function in this setting. These studies can explain the essentiality of BRG1 to rhabdoid tumor cell identity and survival and implicate the involvement of AP-1 as a critical downstream effector of rhabdoid tumor cell transcriptional programs.

WD Repeat Domain 5 Inhibitors for Cancer Therapy: Not What You Think.

WDR5 is a conserved nuclear protein that scaffolds the assembly of epigenetic regulatory complexes and moonlights in functions ranging from recruiting MYC oncoproteins to chromatin to facilitating the integrity of mitosis. It is also a high-value target for anti-cancer therapies, with small molecule WDR5 inhibitors and degraders undergoing extensive preclinical assessment. WDR5 inhibitors were originally conceived as epigenetic modulators, proposed to inhibit cancer cells by reversing oncogenic patterns of histone H3 lysine 4 methylation-a notion that persists to this day. This premise, however, does not withstand contemporary inspection and establishes expectations for the mechanisms and utility of WDR5 inhibitors that can likely never be met. Here, we highlight salient misconceptions regarding WDR5 inhibitors as epigenetic modulators and provide a unified model for their action as a ribosome-directed anti-cancer therapy that helps focus understanding of when and how the tumor-inhibiting properties of these agents can best be understood and exploited.

Ribosome subunit attrition and activation of the p53-MDM4 axis dominate the response of MLL-rearranged cancer cells to WDR5 WIN site inhibition.

The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the "WIN" site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small molecule WIN site inhibitors, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anti-cancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anti-cancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.

Structure-Based Discovery of Potent, Orally Bioavailable Benzoxazepinone-Based WD Repeat Domain 5 Inhibitors.

The chromatin-associated protein WDR5 (WD repeat domain 5) is an essential cofactor for MYC and a conserved regulator of ribosome protein gene transcription. It is also a high-profile target for anti-cancer drug discovery, with proposed utility against both solid and hematological malignancies. We have previously discovered potent dihydroisoquinolinone-based WDR5 WIN-site inhibitors with demonstrated efficacy and safety in animal models. In this study, we sought to optimize the bicyclic core to discover a novel series of WDR5 WIN-site inhibitors with improved potency and physicochemical properties. We identified the 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one core as an alternative scaffold for potent WDR5 inhibitors. Additionally, we used X-ray structural analysis to design partially saturated bicyclic P7 units. These benzoxazepinone-based inhibitors exhibited increased cellular potency and selectivity and favorable physicochemical properties compared to our best-in-class dihydroisoquinolinone-based counterparts. This study opens avenues to discover more advanced WDR5 WIN-site inhibitors and supports their development as novel anti-cancer therapeutics.

WDR5 facilitates recruitment of N-MYC to conserved WDR5 gene targets in neuroblastoma cell lines.

Collectively, the MYC family of oncoprotein transcription factors is overexpressed in more than half of all malignancies. The ability of MYC proteins to access chromatin is fundamental to their role in promoting oncogenic gene expression programs in cancer and this function depends on MYC-cofactor interactions. One such cofactor is the chromatin regulator WDR5, which in models of Burkitt lymphoma facilitates recruitment of the c-MYC protein to chromatin at genes associated with protein synthesis, allowing for tumor progression and maintenance. However, beyond Burkitt lymphoma, it is unknown whether these observations extend to other cancers or MYC family members, and whether WDR5 can be deemed as a "universal" MYC recruiter. Here, we focus on N-MYC amplified neuroblastoma to determine the extent of colocalization between N-MYC and WDR5 on chromatin while also demonstrating that like c-MYC, WDR5 can facilitate the recruitment of N-MYC to conserved WDR5-bound genes. We conclude based on this analysis that N-MYC and WDR5 colocalize invariantly across cell lines at predicted sites of facilitated recruitment associated with protein synthesis genes. Surprisingly, we also identify N-MYC-WDR5 cobound genes that are associated with DNA repair and cell cycle processes. Dissection of chromatin binding characteristics for N-MYC and WDR5 at all cobound genes reveals that sites of facilitated recruitment are inherently different than most N-MYC-WDR5 cobound sites. Our data reveals that WDR5 acts as a universal MYC recruiter at a small cohort of previously identified genes and highlights novel biological functions that may be coregulated by N-MYC and WDR5 to sustain the neuroblastoma state.

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 in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo 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 in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo 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.

Emerging Themes in Mechanisms of Tumorigenesis by SWI/SNF Subunit Mutation.

The SWI/SNF chromatin remodeling complex uses the energy of ATP hydrolysis to alter contacts between DNA and nucleosomes, allowing regions of the genome to become accessible for biological processes such as transcription. The SWI/SNF chromatin remodeler is also one of the most frequently altered protein complexes in cancer, with upwards of 20% of all cancers carrying mutations in a SWI/SNF subunit. Intense studies over the last decade have probed the molecular events associated with SWI/SNF dysfunction in cancer and common themes are beginning to emerge in how tumor-associated SWI/SNF mutations promote malignancy. In this review, we summarize current understanding of SWI/SNF complexes, their alterations in cancer, and what is known about the impact of these mutations on tumor-relevant transcriptional events. We discuss how enhancer dysregulation is a common theme in SWI/SNF mutant cancers and describe how resultant alterations in enhancer and super-enhancer activity conspire to block development and differentiation while promoting stemness and self-renewal. We also identify a second emerging theme in which SWI/SNF perturbations intersect with potent oncoprotein transcription factors AP-1 and MYC to drive malignant transcriptional programs.

The SWI/SNF ATPase BRG1 facilitates multiple pro-tumorigenic gene expression programs in SMARCB1-deficient cancer cells.

Malignant rhabdoid tumor (MRT) is driven by the loss of the SNF5 subunit of the SWI/SNF chromatin remodeling complex and then thought to be maintained by residual SWI/SNF (rSWI/SNF) complexes that remain present in the absence of SNF5. rSWI/SNF subunits colocalize extensively on chromatin with the transcription factor MYC, an oncogene identified as a novel driver of MRT. Currently, the role of rSWI/SNF in modulating MYC activity has neither been delineated nor has a direct link between rSWI/SNF and other oncogenes been uncovered. Here, we expose the connection between rSWI/SNF and oncogenic processes using a well-characterized chemical degrader to deplete the SWI/SNF ATPase, BRG1. Using a combination of gene expression and chromatin accessibility assays we show that rSWI/SNF complexes facilitate MYC target gene expression. We also find that rSWI/SNF maintains open chromatin at sites associated with hallmark cancer genes linked to the AP-1 transcription factor, suggesting that AP-1 may drive oncogenesis in MRT. Interestingly, changes in MYC target gene expression are not overtly connected to the chromatin remodeling function of rSWI/SNF, revealing multiple mechanisms used by rSWI/SNF to control transcription. This work provides an understanding of how residual SWI/SNF complexes may converge on multiple oncogenic processes when normal SWI/SNF function is impaired.

Discovery of Potent Orally Bioavailable WD Repeat Domain 5 (WDR5) Inhibitors Using a Pharmacophore-Based Optimization.

WD repeat domain 5 (WDR5) is a nuclear scaffolding protein that forms many biologically important multiprotein complexes. The WIN site of WDR5 represents a promising pharmacological target in a variety of human cancers. Here, we describe the optimization of our initial WDR5 WIN-site inhibitor using a structure-guided pharmacophore-based convergent strategy to improve its druglike properties and pharmacokinetic profile. The core of the previous lead remained constant while a focused SAR effort on the three pharmacophore units was combined to generate a new in vivo lead series. Importantly, this new series of compounds has picomolar binding affinity, improved cellular antiproliferative activity and selectivity, and increased kinetic aqueous solubility. They also exhibit a desirable oral pharmacokinetic profile with manageable intravenous clearance and high oral bioavailability. Thus, these new leads are useful probes toward studying the effects of WDR5 inhibition.

Elevating SOX2 Downregulates MYC through a SOX2:MYC Signaling Axis and Induces a Slowly Cycling Proliferative State in Human Tumor Cells.

Slowly cycling/infrequently proliferating tumor cells present a clinical challenge due to their ability to evade treatment. Previous studies established that high levels of SOX2 in both fetal and tumor cells restrict cell proliferation and induce a slowly cycling state. However, the mechanisms through which elevated SOX2 levels inhibit tumor cell proliferation have not been identified. To identify common mechanisms through which SOX2 elevation restricts tumor cell proliferation, we initially performed RNA-seq using two diverse tumor cell types. SOX2 elevation in both cell types downregulated MYC target genes. Consistent with these findings, elevating SOX2 in five cell lines representing three different human cancer types decreased MYC expression. Importantly, the expression of a dominant-negative MYC variant, omomyc, recapitulated many of the effects of SOX2 on proliferation, cell cycle, gene expression, and biosynthetic activity. We also demonstrated that rescuing MYC activity in the context of elevated SOX2 induces cell death, indicating that the downregulation of MYC is a critical mechanistic step necessary to maintain survival in the slowly cycling state induced by elevated SOX2. Altogether, our findings uncover a novel SOX2:MYC signaling axis and provide important insights into the molecular mechanisms through which SOX2 elevation induces a slowly cycling proliferative state.

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of SMARCB1, which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of SMARCB1 is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the 'WIN' site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation in vitro. These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.

WIN site inhibition disrupts a subset of WDR5 function.

WDR5 nucleates the assembly of histone-modifying complexes and acts outside this context in a range of chromatin-centric processes. WDR5 is also a prominent target for pharmacological inhibition in cancer. Small-molecule degraders of WDR5 have been described, but most drug discovery efforts center on blocking the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes that deposit histone H3 lysine 4 methylation (H3K4me). Therapeutic application of WIN site inhibitors is complicated by the disparate functions of WDR5, but is generally guided by two assumptions-that WIN site inhibitors disable all functions of WDR5, and that changes in H3K4me drive the transcriptional response of cancer cells to WIN site blockade. Here, we test these assumptions by comparing the impact of WIN site inhibition versus WDR5 degradation on H3K4me and transcriptional processes. We show that WIN site inhibition disables only a specific subset of WDR5 activity, and that H3K4me changes induced by WDR5 depletion do not explain accompanying transcriptional responses. These data recast WIN site inhibitors as selective loss-of-function agents, contradict H3K4me as a relevant mechanism of action for WDR5 inhibitors, and indicate distinct clinical applications of WIN site inhibitors and WDR5 degraders.

Gene-specific quantification of nascent transcription following targeted degradation of endogenous proteins in cultured cells.

Genome-wide nuclear run-ons are a powerful way to determine the impact of a perturbation such as transcription factor degradation on transcriptional patterns. But often investigators are interested in monitoring transcriptional effects at specific sets of genes, rather than the entire genome. Here we describe an approach that couples genome engineering to tag endogenous proteins for degradation with a streamlined nuclear run-on assay to yield gene-specific information on primary transcriptional changes elicited by factor depletion. For complete details on the use and execution of this protocol, please refer to Guarnaccia et al. (2021).

Multiple interactions of the oncoprotein transcription factor MYC with the SWI/SNF chromatin remodeler.

The SNF5 subunit of the SWI/SNF chromatin remodeling complex has been shown to act as a tumor suppressor through multiple mechanisms, including impairing the ability of the oncoprotein transcription factor MYC to bind chromatin. Beyond SNF5, however, it is unknown to what extent MYC can access additional SWI/SNF subunits or how these interactions affect the ability of MYC to drive transcription, particularly in SNF5-null cancers. Here, we report that MYC interacts with multiple SWI/SNF components independent of SNF5. We show that MYC binds the pan-SWI/SNF subunit BAF155 through the BAF155 SWIRM domain, an interaction that is inhibited by the presence of SNF5. In SNF5-null cells, MYC binds with remaining SWI/SNF components to essential genes, although for a purpose that is distinct from chromatin remodeling. Analysis of MYC-SWI/SNF target genes in SNF5-null cells reveals that they are associated with core biological functions of MYC linked to protein synthesis. These data reveal that MYC can bind SWI/SNF in an SNF5-independent manner and that SNF5 modulates access of MYC to core SWI/SNF complexes. This work provides a framework in which to interrogate the influence of SWI/SNF on MYC function in cancers in which SWI/SNF or MYC are altered.

Impact of WIN site inhibitor on the WDR5 interactome.

The 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.

MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with host cell factor-1.

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.

Tumours form when cells lose control of their growth. Usually, cells produce signals that control how much and how often they divide. But if these signals become faulty, cells may grow too quickly or multiply too often. For example, a group of proteins known as MYC proteins activate growth genes in a cell, but too much of these proteins causes cells to grow uncontrollably. With one third of all cancer deaths linked to excess MYC proteins, these molecules could be key targets for anti-cancer drugs. However, current treatments fail to target these proteins. One option for treating cancers linked to MYC proteins could be to target proteins that work alongside MYC proteins, such as the protein HCF-1, which can attach to MYC proteins. To test if HCF-1 could be a potential drug target, Popay et al. first studied how HCF-1 and MYC proteins interacted using specific cancer cells grown in the laboratory. This revealed that when the two proteins connected, they activated genes that trigger rapid cell growth. When these cancer cells were then injected into mice, tumours quickly grew. However, when the MYC and HCF-1 attachments in the cancer cells were disrupted, the tumours shrunk. This suggests that if anti-cancer drugs were able to target HCF-1 proteins, they could potentially reduce or even reverse the growth of tumours. While further research is needed to identify drug candidates, these findings reveal a promising target for treating tumours that stem from over-abundant MYC proteins.

Drugging the "Undruggable" MYCN Oncogenic Transcription Factor: Overcoming Previous Obstacles to Impact Childhood Cancers.

Effective treatment of pediatric solid tumors has been hampered by the predominance of currently "undruggable" driver transcription factors. Improving outcomes while decreasing the toxicity of treatment necessitates the development of novel agents that can directly inhibit or degrade these elusive targets. MYCN in pediatric neural-derived tumors, including neuroblastoma and medulloblastoma, is a paradigmatic example of this problem. Attempts to directly and specifically target MYCN have failed due to its similarity to MYC, the unstructured nature of MYC family proteins in their monomeric form, the lack of an understanding of MYCN-interacting proteins and ability to test their relevance in vivo, the inability to obtain structural information on MYCN protein complexes, and the challenges of using traditional small molecules to inhibit protein-protein or protein-DNA interactions. However, there is now promise for directly targeting MYCN based on scientific and technological advances on all of these fronts. Here, we discuss prior challenges and the reasons for renewed optimism in directly targeting this "undruggable" transcription factor, which we hope will lead to improved outcomes for patients with pediatric cancer and create a framework for targeting driver oncoproteins regulating gene transcription.