Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP.

Metabolic alterations in cancer precipitate in associated dependencies that can be therapeutically exploited. To meet this goal, natural product-inspired small molecules can provide a resource of invaluable chemotypes. Here, we identify orpinolide, a synthetic withanolide analog with pronounced antileukemic properties, via orthogonal chemical screening. Through multiomics profiling and genome-scale CRISPR-Cas9 screens, we identify that orpinolide disrupts Golgi homeostasis via a mechanism that requires active phosphatidylinositol 4-phosphate signaling at the endoplasmic reticulum-Golgi membrane interface. Thermal proteome profiling and genetic validation studies reveal the oxysterol-binding protein OSBP as the direct and phenotypically relevant target of orpinolide. Collectively, these data reaffirm sterol transport as a therapeutically actionable dependency in leukemia and motivate ensuing translational investigation via the probe-like compound orpinolide.

Discovery of a DCAF11-dependent cyanoacrylamide-containing covalent degrader of BET-proteins.

Targeted protein degradation is mediated by small molecules that induce or stabilize protein-protein interactions between targets and the ubiquitin-proteasome machinery. Currently, there remains a need to expand the repertoire of viable E3 ligases available for hijacking. Notably, covalent chemistry has been employed to engage a handful of E3 ligases, including DCAF11. Here, we disclose a covalent PROTAC that enables DCAF11-dependent degradation, featuring a cyanoacrylamide warhead. Our findings underscore DCAF11 as an interesting candidate with a capacity to accommodate diverse electrophilic chemistries compatible with targeted protein degradation.

Targeted protein degradation via intramolecular bivalent glues.

Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.

Discovery of a Drug-like, Natural Product-Inspired DCAF11 Ligand Chemotype.

Targeted proteasomal and autophagic protein degradation, often employing bifunctional modalities, is a new paradigm for modulation of protein function. In an attempt to explore protein degradation by means of autophagy we combine arylidene-indolinones reported to bind the autophagy-related LC3B-protein and ligands of the PDEδ lipoprotein chaperone, the BRD2/3/4-bromodomain containing proteins and the BTK- and BLK kinases. Unexpectedly, the resulting bifunctional degraders do not induce protein degradation by means of macroautophagy, but instead direct their targets to the ubiquitin-proteasome system. Target and mechanism identification reveal that the arylidene-indolinones covalently bind DCAF11, a substrate receptor in the CUL4A/B-RBX1-DDB1-DCAF11 E3 ligase. The tempered α, ß-unsaturated indolinone electrophiles define a drug-like DCAF11-ligand class that enables exploration of this E3 ligase in chemical biology and medicinal chemistry programs. The arylidene-indolinone scaffold frequently occurs in natural products which raises the question whether E3 ligand classes can be found more widely among natural products and related compounds.

Immunoediting instructs tumor metabolic reprogramming to support immune evasion.

Immunoediting sculpts immunogenicity and thwarts host anti-tumor responses in tumor cells during tumorigenesis; however, it remains unknown whether metabolic programming of tumor cells can be guided by immunosurveillance. Here, we report that T cell-mediated immunosurveillance in early-stage tumorigenesis instructs c-Myc upregulation and metabolic reprogramming in tumor cells. This previously unexplored tumor-immune interaction is controlled by non-canonical interferon gamma (IFNγ)-STAT3 signaling and supports tumor immune evasion. Our findings uncover that immunoediting instructs deregulated bioenergetic programs in tumor cells to empower them to disarm the T cell-mediated immunosurveillance by imposing metabolic tug-of-war between tumor and infiltrating T cells and forming the suppressive tumor microenvironment.

Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders.

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined 'functional hotspots'. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.

Acute BAF perturbation causes immediate changes in chromatin accessibility.

Cancer-associated, loss-of-function mutations in genes encoding subunits of the BRG1/BRM-associated factor (BAF) chromatin-remodeling complexes1-8 often cause drastic chromatin accessibility changes, especially in important regulatory regions9-19. However, it remains unknown how these changes are established over time (for example, immediate consequences or long-term adaptations), and whether they are causative for intracomplex synthetic lethalities, abrogating the formation or activity of BAF complexes9,20-24. In the present study, we use the dTAG system to induce acute degradation of BAF subunits and show that chromatin alterations are established faster than the duration of one cell cycle. Using a pharmacological inhibitor and a chemical degrader of the BAF complex ATPase subunits25,26, we show that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in an almost complete loss of chromatin accessibility at BAF-controlled sites, especially also at superenhancers, providing a mechanism for intracomplex synthetic lethalities.

Disturbed mitochondrial dynamics in CD8+ TILs reinforce T cell exhaustion.

The metabolic challenges present in tumors attenuate the metabolic fitness and antitumor activity of tumor-infiltrating T lymphocytes (TILs). However, it remains unclear whether persistent metabolic insufficiency can imprint permanent T cell dysfunction. We found that TILs accumulated depolarized mitochondria as a result of decreased mitophagy activity and displayed functional, transcriptomic and epigenetic characteristics of terminally exhausted T cells. Mechanistically, reduced mitochondrial fitness in TILs was induced by the coordination of T cell receptor stimulation, microenvironmental stressors and PD-1 signaling. Enforced accumulation of depolarized mitochondria with pharmacological inhibitors induced epigenetic reprogramming toward terminal exhaustion, indicating that mitochondrial deregulation caused T cell exhaustion. Furthermore, supplementation with nicotinamide riboside enhanced T cell mitochondrial fitness and improved responsiveness to anti-PD-1 treatment. Together, our results reveal insights into how mitochondrial dynamics and quality orchestrate T cell antitumor responses and commitment to the exhaustion program.

Rational discovery of molecular glue degraders via scalable chemical profiling.

Targeted protein degradation is a new therapeutic modality based on drugs that destabilize proteins by inducing their proximity to E3 ubiquitin ligases. Of particular interest are molecular glues that can degrade otherwise unligandable proteins by orchestrating direct interactions between target and ligase. However, their discovery has so far been serendipitous, thus hampering broad translational efforts. Here, we describe a scalable strategy toward glue degrader discovery that is based on chemical screening in hyponeddylated cells coupled to a multi-omics target deconvolution campaign. This approach led us to identify compounds that induce ubiquitination and degradation of cyclin K by prompting an interaction of CDK12-cyclin K with a CRL4B ligase complex. Notably, this interaction is independent of a dedicated substrate receptor, thus functionally segregating this mechanism from all described degraders. Collectively, our data outline a versatile and broadly applicable strategy to identify degraders with nonobvious mechanisms and thus empower future drug discovery efforts.

Selective Mediator dependence of cell-type-specifying transcription.

The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.

Breast Cancer Classification Based on Proteotypes Obtained by SWATH Mass Spectrometry.

Accurate classification of breast tumors is vital for patient management decisions and enables more precise cancer treatment. Here, we present a quantitative proteotyping approach based on sequential windowed acquisition of all theoretical fragment ion spectra (SWATH) mass spectrometry and establish key proteins for breast tumor classification. The study is based on 96 tissue samples representing five conventional breast cancer subtypes. SWATH proteotype patterns largely recapitulate these subtypes; however, they also reveal varying heterogeneity within the conventional subtypes, with triple negative tumors being the most heterogeneous. Proteins that contribute most strongly to the proteotype-based classification include INPP4B, CDK1, and ERBB2 and are associated with estrogen receptor (ER) status, tumor grade status, and HER2 status. Although these three key proteins exhibit high levels of correlation with transcript levels (R > 0.67), general correlation did not exceed R = 0.29, indicating the value of protein-level measurements of disease-regulated genes. Overall, this study highlights how cancer tissue proteotyping can lead to more accurate patient stratification.

The transcription factor Grainy head primes epithelial enhancers for spatiotemporal activation by displacing nucleosomes.

Transcriptional enhancers function as docking platforms for combinations of transcription factors (TFs) to control gene expression. How enhancer sequences determine nucleosome occupancy, TF recruitment and transcriptional activation in vivo remains unclear. Using ATAC-seq across a panel of Drosophila inbred strains, we found that SNPs affecting binding sites of the TF Grainy head (Grh) causally determine the accessibility of epithelial enhancers. We show that deletion and ectopic expression of Grh cause loss and gain of DNA accessibility, respectively. However, although Grh binding is necessary for enhancer accessibility, it is insufficient to activate enhancers. Finally, we show that human Grh homologs-GRHL1, GRHL2 and GRHL3-function similarly. We conclude that Grh binding is necessary and sufficient for the opening of epithelial enhancers but not for their activation. Our data support a model positing that complex spatiotemporal expression patterns are controlled by regulatory hierarchies in which pioneer factors, such as Grh, establish tissue-specific accessible chromatin landscapes upon which other factors can act.

SCENIC: single-cell regulatory network inference and clustering.

We present SCENIC, a computational method for simultaneous gene regulatory network reconstruction and cell-state identification from single-cell RNA-seq data (http://scenic.aertslab.org). On a compendium of single-cell data from tumors and brain, we demonstrate that cis-regulatory analysis can be exploited to guide the identification of transcription factors and cell states. SCENIC provides critical biological insights into the mechanisms driving cellular heterogeneity.

Identification of cis-regulatory mutations generating de novo edges in personalized cancer gene regulatory networks.

The identification of functional non-coding mutations is a key challenge in the field of genomics. Here we introduce µ-cisTarget to filter, annotate and prioritize cis-regulatory mutations based on their putative effect on the underlying "personal" gene regulatory network. We validated µ-cisTarget by re-analyzing the TAL1 and LMO1 enhancer mutations in T-ALL, and the TERT promoter mutation in melanoma. Next, we re-sequenced the full genomes of ten cancer cell lines and used matched transcriptome data and motif discovery to identify master regulators with de novo binding sites that result in the up-regulation of nearby oncogenic drivers. µ-cisTarget is available from http://mucistarget.aertslab.org .

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic.

Transcription factors regulate their target genes by binding to regulatory regions in the genome. Although the binding preferences of TP53 are known, it remains unclear what distinguishes functional enhancers from nonfunctional binding. In addition, the genome is scattered with recognition sequences that remain unoccupied. Using two complementary techniques of multiplex enhancer-reporter assays, we discovered that functional enhancers could be discriminated from nonfunctional binding events by the occurrence of a single TP53 canonical motif. By combining machine learning with a meta-analysis of TP53 ChIP-seq data sets, we identified a core set of more than 1000 responsive enhancers in the human genome. This TP53 cistrome is invariably used between cell types and experimental conditions, whereas differences among experiments can be attributed to indirect nonfunctional binding events. Our data suggest that TP53 enhancers represent a class of unsophisticated cell-autonomous enhancers containing a single TP53 binding site, distinct from complex developmental enhancers that integrate signals from multiple transcription factors.

iRegulon and i-cisTarget: Reconstructing Regulatory Networks Using Motif and Track Enrichment.

Gene expression profiling is often used to identify genes that are co-expressed in a biological process or disease. Downstream analyses of co-expressed gene sets using bioinformatics methods can reveal candidate transcription factors (TF) that co-regulate these genes, based on the presence of shared TF binding sites. Drawing gene regulatory networks that connect TFs to their predicted target genes can uncover gene modules that implement a particular function. Here, we describe several protocols to analyze any set of co-expressed genes using iRegulon and i-cisTarget. These tools perform regulatory sequence analysis (motif discovery) and integrate and mine large collections of existing regulatory data, such as ChIP-Seq, DHS-seq, and FAIRE-seq (track discovery). While iRegulon focuses on sets of co-expressed genes, i-cisTarget also analyses genomic regions as input. The following protocols describe how to install and use these tools, how to interpret the obtained results, and will thus help to create meaningful regulatory networks.

Identification of High-Impact cis-Regulatory Mutations Using Transcription Factor Specific Random Forest Models.

Cancer genomes contain vast amounts of somatic mutations, many of which are passenger mutations not involved in oncogenesis. Whereas driver mutations in protein-coding genes can be distinguished from passenger mutations based on their recurrence, non-coding mutations are usually not recurrent at the same position. Therefore, it is still unclear how to identify cis-regulatory driver mutations, particularly when chromatin data from the same patient is not available, thus relying only on sequence and expression information. Here we use machine-learning methods to predict functional regulatory regions using sequence information alone, and compare the predicted activity of the mutated region with the reference sequence. This way we define the Predicted Regulatory Impact of a Mutation in an Enhancer (PRIME). We find that the recently identified driver mutation in the TAL1 enhancer has a high PRIME score, representing a "gain-of-target" for MYB, whereas the highly recurrent TERT promoter mutation has a surprisingly low PRIME score. We trained Random Forest models for 45 cancer-related transcription factors, and used these to score variations in the HeLa genome and somatic mutations across more than five hundred cancer genomes. Each model predicts only a small fraction of non-coding mutations with a potential impact on the function of the encompassing regulatory region. Nevertheless, as these few candidate driver mutations are often linked to gains in chromatin activity and gene expression, they may contribute to the oncogenic program by altering the expression levels of specific oncogenes and tumor suppressor genes.

i-cisTarget 2015 update: generalized cis-regulatory enrichment analysis in human, mouse and fly.

i-cisTarget is a web tool to predict regulators of a set of genomic regions, such as ChIP-seq peaks or co-regulated/similar enhancers. i-cisTarget can also be used to identify upstream regulators and their target enhancers starting from a set of co-expressed genes. Whereas the original version of i-cisTarget was focused on Drosophila data, the 2015 update also provides support for human and mouse data. i-cisTarget detects transcription factor motifs (position weight matrices) and experimental data tracks (e.g. from ENCODE, Roadmap Epigenomics) that are enriched in the input set of regions. As experimental data tracks we include transcription factor ChIP-seq data, histone modification ChIP-seq data and open chromatin data. The underlying processing method is based on a ranking-and-recovery procedure, allowing accurate determination of enrichment across heterogeneous datasets, while also discriminating direct from indirect target regions through a 'leading edge' analysis. We illustrate i-cisTarget on various Ewing sarcoma datasets to identify EWS-FLI1 targets starting from ChIP-seq, differential ATAC-seq, differential H3K27ac and differential gene expression data. Use of i-cisTarget is free and open to all, and there is no login requirement. Address: http://gbiomed.kuleuven.be/apps/lcb/i-cisTarget.

Combined Proteomics and Transcriptomics Identifies Carboxypeptidase B1 and Nuclear Factor κB (NF-κB) Associated Proteins as Putative Biomarkers of Metastasis in Low Grade Breast Cancer.

Current prognostic factors are insufficient for precise risk-discrimination in breast cancer patients with low grade breast tumors, which, in disagreement with theoretical prognosis, occasionally form early lymph node metastasis. To identify markers for this group of patients, we employed iTRAQ-2DLC-MS/MS proteomics to 24 lymph node positive and 24 lymph node negative grade 1 luminal A primary breast tumors. Another group of 48 high-grade tumors (luminal B, triple negative, Her-2 subtypes) was also analyzed to investigate marker specificity for grade 1 luminal A tumors. From the total of 4405 proteins identified (FDR < 5%), the top 65 differentially expressed together with 30 previously identified and control markers were analyzed also at transcript level. Increased levels of carboxypeptidase B1 (CPB1), PDZ and LIM domain protein 2 (PDLIM2), and ring finger protein 25 (RNF25) were associated specifically with lymph node positive grade 1 tumors, whereas stathmin 1 (STMN1) and thymosin beta 10 (TMSB10) associated with aggressive tumor phenotype also in high grade tumors at both protein and transcript level. For CPB1, these differences were also observed by immunohistochemical analysis on tissue microarrays. Up-regulation of putative biomarkers in lymph node positive (versus negative) luminal A tumors was validated by gene expression analysis of an independent published data set (n = 343) for CPB1 (p = 0.00155), PDLIM2 (p = 0.02027) and RELA (p = 0.00015). Moreover, statistically significant connections with patient survival were identified in another public data set (n = 1678). Our findings indicate unique pro-metastatic mechanisms in grade 1 tumors that can include up-regulation of CPB1, activation of NF-κB pathway and changes in cell survival and cytoskeleton. These putative biomarkers have potential to identify the specific minor subpopulation of breast cancer patients with low grade tumors who are at higher than expected risk of recurrence and who would benefit from more intensive follow-up and may require more personalized therapy.