SuFEx-based chemical diversification for the systematic discovery of CRBN molecular glues.

Molecular glues are small molecules that stabilize protein-protein interactions, enabling new molecular pharmacologies, such as targeted protein degradation. They offer advantages over proteolysis targeting chimeras (PROTACs), which present challenges associated with the size and properties of heterobifunctional constructions, but glues lack the rational design principles analogous to PROTACs. One notable exception is the ability to alter the structure of Cereblon (CRBN)-based molecular glues and redirect their activity toward new neo-substrate proteins. We took a focused approach toward modifying the CRBN ligand, 5'-amino lenalidomide, to alter its neo-substrate specificity using high-throughput chemical diversification by parallelized sulfur(VI)-fluoride exchange (SuFEx) transformations. We synthesized over 3,000 analogs of 5'-amino lenalidomide using this approach and screened the crude products using a phenotypic screen for cell viability, identifying dozens of analogs with differentiated activity. We characterized four compounds that degrade G-to-S phase transition 1 (GSPT1) protein, providing a proof-of-concept model for SuFEx-based discovery of CRBN molecular glues.

Redirecting the pioneering function of FOXA1 with covalent small molecules.

Pioneer transcription factors (TFs) exhibit a specialized ability to bind to and open closed chromatin, facilitating engagement by other regulatory factors involved in gene activation or repression. Chemical probes are lacking for pioneer TFs, which has hindered their mechanistic investigation in cells. Here, we report the chemical proteomic discovery of electrophilic small molecules that stereoselectively and site-specifically bind the pioneer TF, FOXA1, at a cysteine (C258) within the forkhead DNA-binding domain. We show that these covalent ligands react with FOXA1 in a DNA-dependent manner and rapidly remodel its pioneer activity in prostate cancer cells reflected in redistribution of FOXA1 binding across the genome and directionally correlated changes in chromatin accessibility. Motif analysis supports a mechanism where the covalent ligands relax the canonical DNA binding preference of FOXA1 by strengthening interactions with suboptimal ancillary sequences in predicted proximity to C258. Our findings reveal a striking plasticity underpinning the pioneering function of FOXA1 that can be controlled by small molecules.

Inherited blood cancer predisposition through altered transcription elongation.

Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.

Small-molecule tools for YEATS domain proteins.

Chromatin reader domains are protein folds that bind to post-translational modifications of histones and other chromatin-associated proteins. Compared to other families of reader domains, the discovery that YEATS domains bind to acylated lysines is relatively recent. Four human proteins harbor a YEATS domain, and each is present in protein complexes that regulate chromatin and transcription (ENL, AF9, YEATS2, and YEATS4). Without chemical tools to enable temporally resolved perturbations, it is often unclear how reader domains contribute to protein function. Here, we will discuss recent progress in developing small-molecule tools for YEATS domains and highlight their usefulness for making biological discoveries.

Collateral lethality between HDAC1 and HDAC2 exploits cancer-specific NuRD complex vulnerabilities.

Transcriptional co-regulators have been widely pursued as targets for disrupting oncogenic gene regulatory programs. However, many proteins in this target class are universally essential for cell survival, which limits their therapeutic window. Here we unveil a genetic interaction between histone deacetylase 1 (HDAC1) and HDAC2, wherein each paralog is synthetically lethal with hemizygous deletion of the other. This collateral synthetic lethality is caused by recurrent chromosomal deletions that occur in diverse solid and hematological malignancies, including neuroblastoma and multiple myeloma. Using genetic disruption or dTAG-mediated degradation, we show that targeting HDAC2 suppresses the growth of HDAC1-deficient neuroblastoma in vitro and in vivo. Mechanistically, we find that targeted degradation of HDAC2 in these cells prompts the degradation of several members of the nucleosome remodeling and deacetylase (NuRD) complex, leading to diminished chromatin accessibility at HDAC2-NuRD-bound sites of the genome and impaired control of enhancer-associated transcription. Furthermore, we reveal that several of the degraded NuRD complex subunits are dependencies in neuroblastoma and multiple myeloma, providing motivation to develop paralog-selective HDAC1 or HDAC2 degraders that could leverage HDAC1/2 synthetic lethality to target NuRD vulnerabilities. Altogether, we identify HDAC1/2 collateral synthetic lethality as a potential therapeutic target and reveal an unexplored mechanism for targeting NuRD-associated cancer dependencies.

Acetyl-CoA biosynthesis drives resistance to histone acetyltransferase inhibition.

Histone acetyltransferases (HATs) are implicated as both oncogene and nononcogene dependencies in diverse human cancers. Acetyl-CoA-competitive HAT inhibitors have emerged as potential cancer therapeutics and the first clinical trial for this class of drugs is ongoing (NCT04606446). Despite these developments, the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Having discovered that multiple regulators of de novo coenzyme A (CoA) biosynthesis can modulate sensitivity to CBP/p300 HAT inhibition (PANK3, PANK4 and SLC5A6), we determined that elevated acetyl-CoA concentrations can outcompete drug-target engagement to elicit acquired drug resistance. This not only affects structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B HAT inhibitor that is currently in Phase 1 clinical trials. Altogether, this work uncovers CoA metabolism as an unexpected liability of anticancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.

Chemical Inhibition of ENL/AF9 YEATS Domains in Acute Leukemia.

Transcriptional coregulators, which mediate chromatin-dependent transcriptional signaling, represent tractable targets to modulate tumorigenic gene expression programs with small molecules. Genetic loss-of-function studies have recently implicated the transcriptional coactivator, ENL, as a selective requirement for the survival of acute leukemia and highlighted an essential role for its chromatin reader YEATS domain. Motivated by these discoveries, we executed a screen of nearly 300,000 small molecules and identified an amido-imidazopyridine inhibitor of the ENL YEATS domain (IC50 = 7 µM). Improvements to the initial screening hit were enabled by adopting and expanding upon a SuFEx-based approach to high-throughput medicinal chemistry, ultimately demonstrating that it is compatible with cell-based drug discovery. Through these efforts, we discovered SR-0813, a potent and selective ENL/AF9 YEATS domain inhibitor (IC50 = 25 nM). Armed with this tool and a first-in-class ENL PROTAC, SR-1114, we detailed the biological response of AML cells to pharmacological ENL disruption for the first time. Most notably, we discovered that ENL YEATS inhibition is sufficient to selectively suppress ENL target genes, including HOXA9/10, MYB, MYC, and a number of other leukemia proto-oncogenes. Cumulatively, our study establishes YEATS domain inhibition as a viable approach to disrupt the pathogenic function of ENL in acute leukemia and provides the first thoroughly characterized chemical probe for the ENL YEATS domain.

A short ORF-encoded transcriptional regulator.

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

Cell-Based Ligand Discovery for the ENL YEATS Domain.

ENL is a transcriptional coactivator that recruits elongation machinery to active cis-regulatory elements upon binding of its YEATS domain-a chromatin reader module-to acylated lysine side chains. Discovery chemistry for the ENL YEATS domain is highly motivated by its significance in acute leukemia pathophysiology, but cell-based assays able to support large-scale screening or hit validation efforts do not presently exist. Here, we report on the discovery of a target engagement assay that allows for high-throughput ligand discovery in living cells. This assay is based on the cellular thermal shift assay (CETSA) but does not require exposing cells to elevated temperatures, as small-molecule ligands are able to stabilize the ENL YEATS domain at 37 °C. By eliminating temperature shifts, we developed a simplified target engagement assay that requires just two steps: drug treatment and luminescence detection. To demonstrate its value for higher throughput applications, we miniaturized the assay to a 1536-well format and screened 37 120 small molecules, ultimately identifying an acyl-lysine-competitive ENL/AF9 YEATS domain inhibitor.

Pharmacological Modulation of Transcriptional Coregulators in Cancer.

Upon binding of transcription factors to cis-regulatory DNA sequences, transcriptional coregulators are required for the activation or suppression of chromatin-dependent transcriptional signaling. These coregulators are frequently implicated in oncogenesis via causal roles in dysregulated, malignant transcriptional control and represent one of the fastest-growing target classes in small-molecule drug discovery. However, challenges in targeting coregulators include identifying evidence of cancer-specific genetic dependency, matching the pharmacologically addressable protein fold to a functional role in disease pathology, and achieving the necessary selectivity to exploit a given genetic dependency. We discuss here how recent trends in cancer pharmacology have confronted these challenges, positioning coregulators as tractable targets in the development of new cancer therapies.

Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands.

The addressable pocket of a protein is often not functionally relevant in disease. This is true for the multidomain, bromodomain-containing transcriptional regulator TRIM24. TRIM24 has been posited as a dependency in numerous cancers, yet potent and selective ligands for the TRIM24 bromodomain do not exert effective anti-proliferative responses. We therefore repositioned these probes as targeting features for heterobifunctional protein degraders. Recruitment of the VHL E3 ubiquitin ligase by dTRIM24 elicits potent and selective degradation of TRIM24. Using dTRIM24 to probe TRIM24 function, we characterize the dynamic genome-wide consequences of TRIM24 loss on chromatin localization and gene control. Further, we identify TRIM24 as a novel dependency in acute leukemia. Pairwise study of TRIM24 degradation versus bromodomain inhibition reveals enhanced anti-proliferative response from degradation. We offer dTRIM24 as a chemical probe of an emerging cancer dependency, and establish a path forward for numerous selective yet ineffectual ligands for proteins of therapeutic interest.

The dTAG system for immediate and target-specific protein degradation.

Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12F36V with expression of FKBP12F36V in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRASG12V loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.

Enhancer invasion shapes MYCN-dependent transcriptional amplification in neuroblastoma.

Amplification of the locus encoding the oncogenic transcription factor MYCN is a defining feature of high-risk neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of MYCN perturbation in neuroblastoma. At oncogenic levels, MYCN associates with E-box binding motifs in an affinity-dependent manner, binding to strong canonical E-boxes at promoters and invading abundant weaker non-canonical E-boxes clustered at enhancers. Loss of MYCN leads to a global reduction in transcription, which is most pronounced at MYCN target genes with the greatest enhancer occupancy. These highly occupied MYCN target genes show tissue-specific expression and are linked to poor patient survival. The activity of genes with MYCN-occupied enhancers is dependent on the tissue-specific transcription factor TWIST1, which co-occupies enhancers with MYCN and is required for MYCN-dependent proliferation. These data implicate tissue-specific enhancers in defining often highly tumor-specific 'MYC target gene signatures' and identify disruption of the MYCN enhancer regulatory axis as a promising therapeutic strategy in neuroblastoma.

Translation Termination Factor GSPT1 Is a Phenotypically Relevant Off-Target of Heterobifunctional Phthalimide Degraders.

Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. While both series failed to induce degradation of their consensus targets, individual molecules displayed pronounced efficacy in leukemia cell lines. Orthogonal target identification supported by molecular docking led us to identify the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target, resulting from inadvertent E3 ligase modulation. This research highlights the importance of monitoring degradation events that are independent of the respective targeting ligand as a unique feature of small-molecule degraders.

Pharmacological perturbation of CDK9 using selective CDK9 inhibition or degradation.

Cyclin-dependent kinase 9 (CDK9), an important regulator of transcriptional elongation, is a promising target for cancer therapy, particularly for cancers driven by transcriptional dysregulation. We characterized NVP-2, a selective ATP-competitive CDK9 inhibitor, and THAL-SNS-032, a selective CDK9 degrader consisting of a CDK-binding SNS-032 ligand linked to a thalidomide derivative that binds the E3 ubiquitin ligase Cereblon (CRBN). To our surprise, THAL-SNS-032 induced rapid degradation of CDK9 without affecting the levels of other SNS-032 targets. Moreover, the transcriptional changes elicited by THAL-SNS-032 were more like those caused by NVP-2 than those induced by SNS-032. Notably, compound washout did not significantly reduce levels of THAL-SNS-032-induced apoptosis, suggesting that CDK9 degradation had prolonged cytotoxic effects compared with CDK9 inhibition. Thus, our findings suggest that thalidomide conjugation represents a promising strategy for converting multi-targeted inhibitors into selective degraders and reveal that kinase degradation can induce distinct pharmacological effects compared with inhibition.

BET Bromodomain Proteins Function as Master Transcription Elongation Factors Independent of CDK9 Recruitment.

Processive elongation of RNA Polymerase II from a proximal promoter paused state is a rate-limiting event in human gene control. A small number of regulatory factors influence transcription elongation on a global scale. Prior research using small-molecule BET bromodomain inhibitors, such as JQ1, linked BRD4 to context-specific elongation at a limited number of genes associated with massive enhancer regions. Here, the mechanistic characterization of an optimized chemical degrader of BET bromodomain proteins, dBET6, led to the unexpected identification of BET proteins as master regulators of global transcription elongation. In contrast to the selective effect of bromodomain inhibition on transcription, BET degradation prompts a collapse of global elongation that phenocopies CDK9 inhibition. Notably, BRD4 loss does not directly affect CDK9 localization. These studies, performed in translational models of T cell leukemia, establish a mechanism-based rationale for the development of BET bromodomain degradation as cancer therapy.

Transcription control by the ENL YEATS domain in acute leukaemia.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.

ENL links histone acetylation to oncogenic gene expression in acute myeloid leukaemia.

Cancer cells are characterized by aberrant epigenetic landscapes and often exploit chromatin machinery to activate oncogenic gene expression programs. Recognition of modified histones by 'reader' proteins constitutes a key mechanism underlying these processes; therefore, targeting such pathways holds clinical promise, as exemplified by the development of bromodomain and extra-terminal (BET) inhibitors. We recently identified the YEATS domain as an acetyl-lysine-binding module, but its functional importance in human cancer remains unknown. Here we show that the YEATS domain-containing protein ENL, but not its paralogue AF9, is required for disease maintenance in acute myeloid leukaemia. CRISPR-Cas9-mediated depletion of ENL led to anti-leukaemic effects, including increased terminal myeloid differentiation and suppression of leukaemia growth in vitro and in vivo. Biochemical and crystal structural studies and chromatin-immunoprecipitation followed by sequencing analyses revealed that ENL binds to acetylated histone H3, and co-localizes with H3K27ac and H3K9ac on the promoters of actively transcribed genes that are essential for leukaemia. Disrupting the interaction between the YEATS domain and histone acetylation via structure-based mutagenesis reduced the recruitment of RNA polymerase II to ENL-target genes, leading to the suppression of oncogenic gene expression programs. Notably, disrupting the functionality of ENL further sensitized leukaemia cells to BET inhibitors. Together, our data identify ENL as a histone acetylation reader that regulates oncogenic transcriptional programs in acute myeloid leukaemia, and suggest that displacement of ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukaemia.

Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1.

We previously developed an antibody-avidin fusion protein (ch128.1Av) that targets the human transferrin receptor 1 (TfR1) and exhibits direct cytotoxicity against malignant B cells in an iron-dependent manner. ch128.1Av is also a delivery system and its conjugation with biotinylated saporin (b-SO6), a plant ribosome-inactivating toxin, results in a dramatic iron-independent cytotoxicity, both in malignant cells that are sensitive or resistant to ch128.1Av alone, in which the toxin effectively inhibits protein synthesis and triggers caspase activation. We have now found that the ch128.1Av/b-SO6 complex induces a transcriptional response consistent with oxidative stress and DNA damage, a response that is not observed with ch128.1Av alone. Furthermore, we show that the antioxidant N-acetylcysteine partially blocks saporin-induced apoptosis suggesting that oxidative stress contributes to DNA damage and ultimately saporin-induced cell death. Interestingly, the toxin was detected in nuclear extracts by immunoblotting, suggesting the possibility that saporin might induce direct DNA damage. However, confocal microscopy did not show a clear and consistent pattern of intranuclear localization. Finally, using the long-term culture-initiating cell assay we found that ch128.1Av/b-SO6 is not toxic to normal human hematopoietic stem cells suggesting that this critical cell population would be preserved in therapeutic interventions using this immunotoxin.