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Pharmacology-based methods that promote antitumor immunity have the potential to be highly efficacious while avoiding the systemic cytotoxicity associated with traditional chemotherapies. Activation of type I interferon (IFN) signaling in antigen-presenting cell types [e.g., macrophages and dendritic cells (DCs)] is critical, if not essential, for inducing a tumor-specific adaptive immune response, including the activation of cytolytic CD8 T cells. In the context of promoting antitumor immunity, the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway has emerged as a principal regulator of essential type I IFN signaling. As such, STING represents a highly attractive target for developing a first-in-class immunotherapy, albeit one with a potential for significant cell type- and downstream pathway-dependent on-target toxicities, as well as conceivable pharmacogenomic liabilities.

Significance We have completed a screen of ∼106 small molecules to identify compounds that induce cell death in multipotent glioblastoma multiforme (GBM) cancer stem cells (CSCs). This resulted in the identification of a hit class (RIPGBM) that was found to induce apoptosis in GBM CSCs in a cell type-selective manner. Metabolite profiling experiments led to the identification of a proapoptotic derivative of RIPGBM (cRIPGBM), which was found to be selectively formed in GBM CSCs. Mechanistic studies revealed that cRIPGBM induces apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) in a mode that results in the formation of a proapoptotic RIPK2/caspase 1 complex. In a physiologically relevant orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly inhibit in vivo tumor formation., Glioblastoma multiforme (GBM; grade IV astrocytoma) is the most prevalent and aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation, tumor maintenance, metastasis, drug resistance, and recurrence following surgery. Here we report the identification of a small molecule, termed RIPGBM, from a cell-based chemical screen that selectively induces apoptosis in multiple primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of this compound appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an orthotopic intracranial GBM CSC tumor xenograft mouse model, RIPGBM was found to significantly suppress tumor formation in vivo. Our chemical genetics-based approach has identified a drug candidate and a potential drug target that provide an approach to the development of treatments for this devastating disease.

INTRODUCTION Glioblastoma multiforme (GBM) is the most aggressive form of primary brain cancer. A subpopulation of multipotent cells termed GBM cancer stem cells (CSCs) play a critical role in tumor initiation and maintenance, drug resistance, and recurrence following surgery. New therapeutic strategies for the treatment of GBM have recently focused on targeting CSCs. Here we have used an unbiased large-scale screening approach to identify drug-like small molecules that induce apoptosis in GBM CSCs in a cell type-selective manner. METHODS A luciferase-based survival assay of patient-derived GBM CSC lines was established to perform a large-scale screen of ∼one million drug-like small molecules with the goal of identifying novel compounds that are selectively toxic to chemoresistant GBM CSCs. Compounds found to kill GBM CSC lines as compared to control cell types were further characterized. A caspase activation assay was used to evaluate the mechanism of induced cell death. A xenograft animal model using patient-derived GBM CSCs was employed to test the leading candidate for suppression of in vivo tumor formation. RESULTS We identified a small molecule, termed RIPGBM, from the cell-based chemical screen that induces apoptosis in primary patient-derived GBM CSC cultures. The cell type-dependent selectivity of RIPGBM appears to arise at least in part from redox-dependent formation of a proapoptotic derivative, termed cRIPGBM, in GBM CSCs. cRIPGBM induces caspase 1-dependent apoptosis by binding to receptor-interacting protein kinase 2 (RIPK2) and acting as a molecular switch, which reduces the formation of a prosurvival RIPK2/TAK1 complex and increases the formation of a proapoptotic RIPK2/caspase 1 complex. In an intracranial GBM xenograft mouse model, RIPGBM was found to significantly suppress tumor formation. CONCLUSIONS Our chemical genetics-based approach has identified a small molecule drug candidate and a potential drug target that selectively targets cancer stem cells and provides an approach for the treatment of GBMs.

Stimulator of interferon genes (STING) links innate immunity to biological processes ranging from antitumor immunity to microbiome homeostasis. Mechanistic understanding of the anticancer potential for STING receptor activation is currently limited by metabolic instability of the natural cyclic dinucleotide (CDN) ligands. From a pathway-targeted cell-based screen, we identified a non-nucleotide, small-molecule STING agonist, termed SR-717, that demonstrates broad interspecies and interallelic specificity. A 1.8-angstrom cocrystal structure revealed that SR-717 functions as a direct cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) mimetic that induces the same "closed" conformation of STING. SR-717 displayed antitumor activity; promoted the activation of CD8+ T, natural killer, and dendritic cells in relevant tissues; and facilitated antigen cross-priming. SR-717 also induced the expression of clinically relevant targets, including programmed cell death 1 ligand 1 (PD-L1), in a STING-dependent manner.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases.

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving direct functional perturbation and/or ligand-induced degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells, underscoring the potential of electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

Lrp5 is typically described as a Wnt signaling receptor, albeit a less effective Wnt signaling receptor than the better-studied sister isoform, Lrp6. Here we show that Lrp5 is only a minor player in the response to Wnt3a-type ligands in mammary epithelial cells; instead, Lrp5 is required for glucose uptake, and glucose uptake regulates the growth rate of mammary epithelial cells in culture. Thus, a loss of Lrp5 leads to profound growth suppression, whether growth is induced by serum or by specific growth factors, and this inhibition is not due to a loss of Wnt signaling. Depletion of Lrp5 decreases glucose uptake, lactate secretion, and oxygen consumption rates; inhibition of glucose consumption phenocopies the loss of Lrp5 function. Both Lrp5 knockdown and low external glucose induce mitochondrial stress, as revealed by the accumulation of reactive oxygen species (ROS) and the activation of the ROS-sensitive checkpoint, p38α. In contrast, loss of function of Lrp6 reduces Wnt responsiveness but has little impact on growth. This highlights the distinct functions of these two Lrp receptors and an important Wnt ligand-independent role of Lrp5 in glucose uptake in mammary epithelial cells.

Summary Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

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Fibrosis, a disease in which excessive amounts of connective tissue accumulate in response to physical damage and/or inflammatory insult, affects nearly every tissue in the body and can progress to a state of organ malfunction and death. A hallmark of fibrotic disease is the excessive accumulation of extracellular matrix-secreting activated myofibroblasts (MFBs) in place of functional parenchymal cells. As such, the identification of agents that selectively inhibit the transdifferentiation process leading to the formation of MFBs represents an attractive approach for the treatment of diverse fibrosis-related diseases. Herein we report the development of a high throughput image-based screen using primary hepatic stellate cells that identified the antifungal drug itraconazole (ITA) as an inhibitor of MFB cell fate in resident fibroblasts derived from multiple murine and human tissues (i.e., lung, liver, heart, and skin). Chemical optimization of ITA led to a molecule (CBR-096-4) devoid of antifungal and human cytochrome P450 inhibitory activity with excellent pharmacokinetics, safety, and efficacy in rodent models of lung, liver, and skin fibrosis. These findings may serve to provide a strategy for the safe and effective treatment of a broad range of fibrosis-related diseases.

Introduction Harnessing CD8 T cells to respond to tumorigenic antigens remains a supreme therapeutic strategy for sustained clearance of tumours and a resulting cancer-free life for patients. Efficient tumor-initiated T cell priming requires interferon-beta (IFN-b) production by dendritic cells and the expression of IFN-b has been demonstrated to be dependent upon activation of the Stimulator of Interferon Genes (STING) pathway. Indeed, intratumoral delivery of nucleotide-based STING agonists induces a profound regression of established tumours in syngeneic mouse models. Material and methods Here, we describe a high-throughput screening platform for identifying non-nucleotide small molecule STING agonists. This has been established using a primary assay involving a human THP-1 cell line carrying an IRF-inducible reporter with 5 copies of the IFN signalling response element. Counter screens, involving alternative reporter constructs, rodent cell-based assays, as well as cGAS and STING knockout cell lines, are used to eliminate luciferase artefacts and ensure human-rodent cross species reactivity, as well as pathway selectivity. Biochemical assays, involving cGAS enzymatic activity and STING protein binding assays, are used to identify the specific target of identified hits. Results and discussions To date, from an initial screen of ~1 00 000 compounds we have identified at least one novel highly tractable STING agonist scaffold (SRCB-0001, EC 50 ~1 µM), which induces expression of a type I interferon-stimulated gene signature in relevant cell types and IFN-b protein production in human peripheral blood mononuclear cells (PBMCs) with efficacy that is comparable to that observed for 2’3’-cGAMP, a natural dinucleotide STING ligand. Unlike nucleotide-based STING agonists, we have found that SRCB-0001 can be dosed orally in mice thereby lifting the restrictions of intratumoral injection as the route of administration. Conclusion Thus, we propose SRCB-0074 as a first-in-class small molecule STING agonist for immuno-oncology applications.

CRD-BP/IGF2BP1 has been characterized as an "oncofetal" RNA binding protein typically highly expressed in embryonic tissues, suppressed in normal adult tissues, but induced in many tumor types. In this study, we show that adult breast tissues express ubiquitous but low levels of CRD-BP protein and mRNA. Although CRD-BP mRNA expression is induced in breast tumor cells, levels remain ∼1000-fold lower than in embryonic tissues. Despite low expression levels, CRD-BP is required for clonogenic growth of breast cancer cells. We reveal that because the most common protein isoform in normal adult breast and breast tumors has an N-terminal deletion (lacking two RNA recognition motif (RRM) domains) and is therefore missing antibody epitopes, CRD-BP expression has been under-reported by previous studies. We show that a CRD-BP mutant mouse strain retains expression of the shorter transcript (ΔN-CRD-BP), which originates in intron 2, suggesting that the impact of complete ablation of this gene in mice is not yet known. Either the full-length CRD-BP or the N-terminally truncated version can rescue the clonogenicity of CRD-BP knockdown breast cancer cells, suggesting that clonogenic function is served by either CRD-BP isoform. In summary, although CRD-BP expression levels are low in breast cancer cells, this protein is necessary for clonogenic activity.