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Human cytomegalovirus (HCMV) modulates cellular metabolism to support productive infection, and the HCMV UL38 protein drives many aspects of this HCMV-induced metabolic program. However, it remains to be determined whether virally-induced metabolic alterations might induce novel therapeutic vulnerabilities in virally infected cells. Here, we explore how HCMV infection and the UL38 protein modulate cellular metabolism and how these changes alter the response to nutrient limitation. We find that expression of UL38, either in the context of HCMV infection or in isolation, sensitizes cells to glucose limitation resulting in cell death. This sensitivity is mediated through UL38’s inactivation of the TSC complex subunit 2 (TSC2) protein, a central metabolic regulator that possesses tumor-suppressive properties. Further, expression of UL38 or the inactivation of TSC2 results in anabolic rigidity in that the resulting increased levels of fatty acid biosynthesis are insensitive to glucose limitation. This failure to regulate fatty acid biosynthesis in response to glucose availability sensitizes cells to glucose limitation, resulting in cell death unless fatty acid biosynthesis is inhibited. These experiments identify a regulatory circuit between glycolysis and fatty acid biosynthesis that is critical for cell survival upon glucose limitation and highlight a metabolic vulnerability associated with viral infection and the inactivation of normal metabolic regulatory controls.ImportanceViruses modulate host cell metabolism to support the mass production of viral progeny. For Human Cytomegalovirus, we find that the viral UL38 protein is critical for driving these pro-viral metabolic changes. However, our results indicate that these changes come at a cost, as UL38 induces an anabolic rigidity that leads to a metabolic vulnerability. We find that UL38 decouples the link between glucose availability and fatty acid biosynthetic activity. Normal cells respond to glucose limitation by down-regulating fatty acid biosynthesis. Expression of UL38 results in the inability to modulate fatty acid biosynthesis in response to glucose limitation, which results in cell death. We find this vulnerability in the context of viral infection, but this linkage between fatty acid biosynthesis, glucose availability, and cell death could have broader implications in other contexts or pathologies that rely on glycolytic remodeling, for example, oncogenesis.

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All dose-response curves. Dose-response curves for all library compounds

Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type–specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity–promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell–specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.

SUMMARYCells rely on antioxidants to survive. The most abundant antioxidant is glutathione (GSH). The synthesis of GSH is non-redundantly controlled by the glutamate-cysteine ligase catalytic subunit (GCLC). GSH imbalance is implicated in many diseases, but the requirement for GSH in adult tissues is unclear. To interrogate this, we developed a series ofin vivomodels to induceGclcdeletion in adult animals. We find that GSH is essential to lipid abundancein vivo. GSH levels are reported to be highest in liver tissue, which is also a hub for lipid production. While the loss of GSH did not cause liver failure, it decreased lipogenic enzyme expression, circulating triglyceride levels, and fat stores. Mechanistically, we found that GSH promotes lipid abundance by repressing NRF2, a transcription factor induced by oxidative stress. These studies identify GSH as a fulcrum in the liver’s balance of redox buffering and triglyceride production.

Buffering oxidative stress is as a crucial requirement for tumorigenesis. Antioxidant is a term reserved for molecules that quench reactive oxygen species (ROS) and alleviate oxidative stress. The details regarding antioxidant synthesis, their utilization to eliminate ROS, and their ability to promote different stages of tumorigenesis are unclear. Here, we focus on glutathione (GSH), the most abundant antioxidant in the cell, and its precursor amino acids (cysteine, glutamate, and glycine). Even though GSH was discovered more than a century ago, continued research into this antioxidant has provided answers to longstanding questions while also posing new ones.

Control of cellular identity involves coordination of developmental programs with environmental factors such as nutrient availability, suggesting that modulating aspects of metabolism could enable therapeutically relevant changes in cell fate. We show that nucleotide depletion facilitates gene expression changes towards a new cell fate by perturbing DNA replication in models of acute myeloid leukemia, a cancer characterized by a differentiation blockade. This transition starts in S phase and is independent of replication stress signaling and DNA damage signaling pathways. Moreover, it occurs despite sustained oncogene-driven expression of the progenitor program and is accompanied by limited changes in chromatin accessibility. Altering lineage-determining transcription factor expression redirects cell fate progression towards an alternate fate upon replication stress, suggesting that perturbing DNA replication allows cells to mobilize primed maturation programs. Our work, along with other findings in diverse systems, suggests a conserved mechanism by which metabolic changes can orchestrate cell fate transitions.

Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type–specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity–promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell–specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.

Background: While new treatments and improved subtyping schemas are anticipated to improve treatment response in triple-negative breast cancer (TNBC) patients, therapeutic resistance remains a significant challenge. Moreover, there is an urgent need for additional research model systems to study resistance and residual disease in breast cancer, including aggressive subtypes of breast cancer. Organoid culture is a promising technology used for growing breast cancer cells with high efficiency; however, the extent to which treatment resistance can be modeled using this system is unknown. This research used patient-derived organoid cultures in the context of computational analyses of large molecular and clinical datasets to study resistance mechanisms, biomarkers, and alternative treatment strategies to overcome drug resistance in early-stage TNBC. Methods: Organoid cultures were derived from breast tumor samples (taken from lumpectomy, mastectomy, or core biopsy samples), digested to the organoid level using collagenase, and grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium (Dekkers et al. Nat Protoc 2021). An evaluation of all available I-SPY2 biomarker data (Wolf et al. Cancer Cell 2022) was performed focusing on genes, proteins, and pathways associated with resistance. These were then used to study whether resistance biomarkers identified in patient tumors are also present in organoids propagated from breast cancer post-treatment residual disease. To this end, bulk RNA sequencing data of organoids were normalized and merged with the TCGA dataset (Hoadley et al. Cell 2018) to enable analysis in a larger context, and immunofluorescence staining of organoids was performed. A high-throughput 386 anti-cancer drug compound screen and subsequent synergy testing with the most promising compounds were performed to identify and predict alternative treatment strategies. Additional assays to explore kinome activity in this organoid model are in progress. Results: A TNBC organoid biobank was established (n=31), which was enriched for inflammatory breast cancer (IBC; n=15), an aggressive form of breast cancer. Most organoids were derived from residual disease after neoadjuvant therapy. Bulk RNA sequencing analysis performed on 10 TNBC organoids revealed 3 subsets that were characterized predominantly by either normal-like/luminal androgen receptor or basal-like features or expressed distinct gene expression profiles, with IBC cases present in all 3 subsets. Intriguingly, the IBC organoids were characterized by higher expression of a number of immune-related signatures, despite an absence of clear immune cells in culture. A residual disease IBC/TNBC organoid resistant to chemotherapy was used to perform the 386-drug compound screen. The organoid model showed resistance to veliparib-cisplatin, consistent with the expression of gene/protein biomarkers predictive of drug resistance found in I-SPY2 (low PARPi7 levels and high pFOXO1 and pMEK1/2 expression). In addition, the screen identified multiple classes of inhibitors as promising synergistic/additive candidates for overcoming resistance to cisplatin. Conclusion: In this proof-of-principle study, we demonstrated the utility of matching I-SPY2 resistance biomarkers and signatures to residual disease tumor organoid cultures. We show that tumor organoid cultures modeling drug resistance states are a useful complement to existing research models of breast cancer and can be used for compound testing. We have developed a pipeline to propagate residual tumors from patients enrolled in I-SPY2 into organoid cultures to create a broader platform for preclinical drug testing informed by tumor biology with the ultimate goal of enabling faster, more successful translational studies and increased treatment options for resistant disease. Citation Format: Tam Binh V. Bui, Denise M. Wolf, Kaitlin Moore, Isaac S. Harris, Pravin Phadatare, Christina Yau, Lamorna A. Brown Swigart, Laura J. Esserman, Jean-Philippe Coppe, Julia Wulfkuhle, Emanuel F. Petricoin, Michael Campbell, Laura M. Selfors, Deborah A. Dillon, Beth Overmoyer, Filipa Lynce, Laura Van ’t Veer, Jennifer Rosenbluth. PD5-02 An Organoid Model System to Study Resistance Mechanisms, Predictive Biomarkers, and New Strategies to Overcome Therapeutic Resistance in Early-Stage Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-02.

SUMMARYMutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they appear less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1 mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH mutant gliomas. Mechanistically, this vulnerability selectively applies to de novo pyrimidine, but not purine, synthesis because glioma cells engage disparate programs to produce these nucleotide species and because IDH oncogenes increase DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

Summary A hallmark of acute myeloid leukemia (AML) is the inability of self-renewing malignant cells to mature into a non-dividing terminally differentiated state. This differentiation block has been linked to dysregulation of multiple cellular processes, including transcriptional, chromatin, and metabolic regulation. The transcription factor HOXA9 and the histone demethylase LSD1 are examples of such regulators that promote differentiation blockade in AML. To identify metabolic targets that interact with LSD1 inhibition to promote myeloid maturation, we screened a small molecule library to identify druggable substrates. We found that differentiation caused by LSD1 inhibition is enhanced by combined perturbation of purine nucleotide salvage and de novo lipogenesis pathways, and identified multiple lines of evidence to support the specificity of these pathways and suggest a potential basis of how perturbation of these pathways may interact synergistically to promote myeloid differentiation. In sum, these findings suggest potential drug combination strategies in the treatment of AML., Graphical abstract, Highlights • Combined epigenetic and metabolic perturbations induce myeloid differentiation • Combination treatment alters nucleotide, lipid, and gene expression profiles • Combination treatment induces differentiation of human acute myeloid leukemia cells, Molecular biology; Stem cells research; Systems biology

A new positive selection assay could enhance the discovery of compounds that degrade previously undruggable proteins., Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal (“down”) assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal (“up”) assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9–based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

Cancer cells have differential metabolic dependencies compared to their non-malignant counterparts. However, few metabolism-targeting compounds have been successful in clinical trials. Here, we investigated the metabolic vulnerabilities of triple-negative breast cancer (TNBC), in particular those metabolic perturbations that increased mitochondrial apoptotic priming and sensitivity to BH3 mimetics (drugs that antagonize anti-apoptotic proteins). We used high throughput-dynamic BH3 profiling (HT-DBP) to screen a library of metabolism-perturbing small molecules, which revealed inhibitors of the enzyme nicotinamide phosphoribosyltransferase (NAMPT) as top candidates. In some TNBC cells but not in non-malignant cells, NAMPT inhibitors increased overall apoptotic priming and induced dependencies on specific anti-apoptotic BCL-2 family members. Treatment of TNBC cells with NAMPT inhibitors sensitized them to subsequent treatment with BH3 mimetics. The combination of a NAMPT inhibitor (FK866) and an MCL-1 antagonist (S63845) reduced tumor growth in a TNBC patient-derived xenograft model in vivo. We found that NAMPT inhibition reduced NAD(+) concentrations below a critical threshold that resulted in depletion of adenine, which was the metabolic trigger that primed TNBC cells for apoptosis. These findings demonstrate a close interaction between metabolic and mitochondrial apoptotic signaling pathways and reveal that exploitation of a tumor-specific metabolic vulnerability can sensitize some TNBC to BH3 mimetics.

This talk will focus on the development of material platforms and physical mechanisms for high-fidelity control, storage, and transmission of quantum information. In particular, emerging quantum materials - novel diamond impurities - featuring long-lived, optically addressable quantum states are promising candidates fulfilling most of these requirements. We will discuss possible mechanisms for efficient interfacing of these novel solid-state qubits, in particular allowing for high-fidelity quantum-state transduction to superconducting qubits or to the long-lived nuclear spins. Finally, the physical phenomena enabling local quantum control in these materials, including strain-mediated or optically mediated state-transfer and two-qubit gates, will be discussed.

Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are uncharacterized. Here, we find that cystine starvation promotes ferroptosis not only through the inhibition of glutathione (GSH) synthesis, but also through the accumulation of glutamate. Surprisingly, we find that glutamate-cysteine ligase catalytic subunit (GCLC) prevents glutamate accumulation through the generation of alternative γ-glutamyl peptides. Further, inhibition of GCLC accelerates ferroptosis under cystine starvation in a GSH-independent manner. These results indicate that GCLC has an additional, non-canonical role in the protection against ferroptosis to maintain glutamate homeostasis under cystine starvation.

Metabolic constraints in the tumor microenvironment constitute a barrier to effective anti-tumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8(+) T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type–specific sensitivities of activated CD8(+) T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8(+) T cells. We illustrated the applicability of this screening platform by showing that CD8(+) T cells were more sensitive to ferroptosis than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8(+) T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity–promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8(+) T cells from ferroptosis, but impaired anti-tumor CD8(+) T cell responses. Our screen also revealed high T cell–specific vulnerabilities for compounds targeting NAD(+) metabolism or autophagy and ER stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of anti-tumor CD8(+) T-cell function and potential therapeutic targets.

Metastasis remains the leading cause of cancer mortality, and reactive oxygen species (ROS) signaling promotes the metastatic cascade. However, the molecular pathways that control ROS signaling relevant to metastasis are little studied. Here, we identify SIRT3, a mitochondrial deacetylase, as a regulator of cell migration via its control of ROS signaling. We find that, although mitochondria are present at the leading edge of migrating cells, SIRT3 expression is down-regulated during migration, resulting in elevated ROS levels. This SIRT3-mediated control of ROS represses Src oxidation and attenuates focal adhesion kinase (FAK) activation. SIRT3 overexpression inhibits migration and metastasis in breast cancer cells. Finally, in human breast cancers, SIRT3 expression is inversely correlated with metastatic outcome and Src/FAK signaling. Our results reveal a role for SIRT3 in cell migration, with important implications for breast cancer progression.

70–90% of lower-grade gliomas and secondary glioblastomas harbor gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1), causing overproduction of the oncometabolite (R)-2-hydroxyglutarate [(R)-2HG]. Although inhibitors of mutant IDH enzymes are effective in other cancers, including leukemia, they have shown guarded efficacy in preclinical and clinical brain tumor studies, thus underscoring the need to identify additional therapeutic targets in IDH mutant glioma. We sought to identify tumor-specific metabolic vulnerabilities induced by IDH1 mutations that could be exploited therapeutically. To uncover such vulnerabilities, we conducted a chemical synthetic lethality screen using isogenic IDH1 mutant and IDH1 wild-type (WT) glioma cell lines and a novel metabolic inhibitor screening platform. We discovered that IDH1 mutant cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). This vulnerability is specific because inhibitors of purine nucleotide metabolism did not score in our screen. We validated that the cytotoxicity of pyrimidine synthesis inhibitors is on-target and showed that IDH1 mutant patient-derived glioma stem-like cell lines are also hyperdependent on de novo pyrimidine nucleotide synthesis compared to IDH1 WT lines. To test pyrimidine synthesis dependence of IDH1 mutant gliomas in vivo, we used a brain-penetrent DHODH inhibitor currently undergoing evaluation in leukemia patients, BAY 2402234. We found that BAY 2402234 displays monotherapy activity against gliomas in an orthotopic xenograft model of IDH1 mutant glioma, with an effect size that compared favorably with radiotherapy. We also developed novel genetically engineered and allograft mouse models of mutant IDH1-driven anaplastic astrocytoma and showed that BAY 2402234 blocked growth of orthotopic astrocytoma allografts. Our findings bolster rationale to target DHODH in glioma, highlight BAY 2402234 as a clinical-stage drug that can be used to inhibit DHODH in brain tumors, and establish IDH1 mutations as predictive biomarkers of DHODH inhibitor efficacy.

Cancer genome sequencing studies have identified frequent driver mutations that alter the function of transcription factors (TFs). However, most TFs, are classically "undruggable". The discovery that IMiDs [immunomodulatory drugs e.g.Lenalidomide (LEN) and Pomalidomide (POM)] exert powerful antimyeloma effects by triggering the proteasomal degradation of the critical TFsIKZF1 and IKZF3has led togreat interest in the discovery of"degraders"in other cancers. Existing "down" assays to identify destabilizers (e.g.measuring the loss of the protein of interest [POI]) often have(1) poor signal/noise ratios and narrow dynamic ranges and (2) generate uninterestinghits e.g.drugs thatinhibit transcription or translation. We present a novel positive-selection "up" assay, compatible with high throughput screening, for identifying drugs or sgRNAs that destabilize a POI. We have used this platform to identify novel destabilizers of IKZF1. Our assay is based on a previously published "suicide gene", a variant of the nucleotide salvage gene deoxycytidine kinase (DCK). This variant (DCK*) has mutations that increase its specificity for the synthetic substrate 2-Bromovinyldeoxyuridine (BVdU). Cells that express DCK* are killed by BVdU. We made mammalian expression vectors co-expressing GFP and DCK*, IKZF1, or a DCK*-IKZF1 fusion protein as a single bicistronic mRNA and stably introduced each into 293T cells. In the absence of POM, cells expressing DCK* or DCK*-IKZF1 are killed by BVdU . However, in the presence of an IKZF1 destabilizer, e.g. POM, the DCK*-IKZF1 protein is degraded, making the cells resistant to BVdU. Cells expressing DCK* alone are unaffected by POM and serve as a specificity control for the assay (Panel A). We did a pilot chemical "up" screen with a ~ 2000 bioactive compound library (that included LEN and POM) using 293T cells expressing DCK*-IKZF1. In parallel, we conducted a "down" screen, using 293T cells co-expressing an IKZF1-Firefly Luciferase (Fluc) fusion protein and Renilla luciferase (Rluc) from a single bicistronic mRNA. Compounds that decreased the ratio of Fluc/Rluc activity were scored as hits. As expected, LEN and POM scored in both assays,but there was considerably more noise in the down assay. Lastly, we used the DCK*-IKZF1 cells to screen a library of uncharacterized IMiD derivatives. The screen correctly identified a novel IMiD derivative (MI-2-61) and a known next-generation IMiD (Avadomide) with greater potency against IKZF1 than POM. To identify novel degraders of IKZF1, we used the DCK*-IKZF1 cells and DCK* control cells to screena metabolic inhibitor/anticancer library of ~600 compounds. We identified Spautin-1 as a compound that rescues DCK*-IKZF1 cells, but not DCK* control cells from BVdU toxicity. Spautin-1 downregulates exogenous IKZF1 in 293T cells (Panels B and C) and endogenous IKZF1 in KMS11 myeloma cells. Spautin-1 reportedly suppresses autophagy through inhibition of USP10 and USP13. However, siRNA mediated knockdown of USP10 and USP10 neither altered IKZF1 protein levels, nor blocked downregulation of IKZF1 by Spautin-1. Moreover, Spautin-1 downregulated IKZF1 in 293FT cells in which autophagy was disabled by CRISPR/Cas9-mediated disruption of genes crucial to the autophagy pathway suggesting that the downregulation of IKZF1 by Spautin-1 occurs via a novel mechanism. Unlike IMiDs, the downregulation of IKZF1 by Spautin-1 does not require the CRBN E3 ligase. However, it is blocked by inhibitors of the E1 ubiquitin activating enzyme or the proteasome, but not by neddylation inhibitors required for Cullin-dependent E3 ligases. These data suggest that Spautin-1 triggers the proteasomal degradation of IKZF1 using a non-Cullen E3 ligase. The downregulation of exogenous IKZF1 by Spautin-1 requires the N-terminus of IKZF1, but not the zinc finger domain (ZF2) targeted by IMiDs. Preliminary structure-activity relationship (SAR) studies identified both active and inactive Spautin-1 derivatives, suggesting that downregulation of IKZF1 by Spautin-1 reflects a specific protein binding event and that Spautin-1's potency and specificity can be optimized further. We are undertaking studies to identify the mechanism by which Spautin-1 degrades IKZF1 in the hope that it may represent a novel druggable pathway to therapeutically degrade IKZF1, and validate the use of this platform to discover degraders of "undruggable" targets in other cancers. Figure Disclosures Koduri: Cedilla Therapeutics: Consultancy. Paulk:Novartis: Current Employment. Harris:ONO Pharmaceutical: Consultancy. Buhrlage:Adenoid Cystic Carcinoma Foundation: Other: Scientific Advisory Board. Kaelin:Cedilla Therapeutics: Other: Scientific Founder; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; Fibrogen: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Tango Therapeutics: Other: Founder; Tracon Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Reactive oxygen species (ROS) play important roles in tissue homeostasis, cellular signaling, differentiation, and survival. In this review, we discuss the types of ROS, their impact on cellular processes, and their pro- and antitumorigenic effects. Further, we discuss recent advances in our understanding of both endogenous and exogenous antioxidants in tumorigenic processes. Finally, we discuss how aberrant activation of antioxidant programs by the transcription factor NFE2-related factor 2 (NRF2) influences tumorigenesis and metastasis, and where the current gaps in our knowledge remain.

Cysteine is required for maintaining cellular redox homeostasis in both normal and transformed cells. Deprivation of cysteine induces the iron-dependent form of cell death known as ferroptosis; however, the metabolic consequences of cysteine starvation beyond impairment of glutathione synthesis are poorly characterized. Here, we find that cystine starvation of non-small cell lung cancer cell lines induces an unexpected accumulation of γ-glutamyl-peptides, which are produced due to a non-canonical activity of glutamate-cysteine ligase catalytic subunit (GCLC). This activity is enriched in cell lines with high levels of NRF2, a key transcriptional regulator of GCLC, but is also inducible in healthy murine tissues following cysteine limitation. γ-glutamyl-peptide synthesis limits the accumulation of glutamate, thereby protecting against ferroptosis. These results indicate that GCLC has a glutathione-independent, non-canonical role in the protection against ferroptosis by maintaining glutamate homeostasis under cystine starvation.

Cancer-associated mutations that stabilize NRF2, an oxidant defense transcription factor, are predicted to promote tumor development. Here, utilizing 3D cancer spheroid models coupled with CRISPR-Cas9 screens, we investigate the molecular pathogenesis mediated by NRF2 hyperactivation. NRF2 hyperactivation was necessary for proliferation and survival in lung tumor spheroids. Antioxidant treatment rescued survival but not proliferation, suggesting the presence of distinct mechanisms. CRISPR screens revealed that spheroids are differentially dependent on the mammalian target of rapamycin (mTOR) for proliferation and the lipid peroxidase GPX4 for protection from ferroptosis of inner, matrix-deprived cells. Ferroptosis inhibitors blocked death from NRF2 downregulation, demonstrating a critical role of NRF2 in protecting matrix-deprived cells from ferroptosis. Interestingly, proteomics analyses show global enrichment of selenoproteins, including GPX4, by NRF2 downregulation, and targeting NRF2 and GPX4 killed spheroids overall. These results illustrate the value of spheroid culture in revealing environmental or spatial differential dependencies on NRF2 and reveal exploitable vulnerabilities of NRF2-hyperactivated tumors.

BET bromodomain inhibitors (BBDIs) are candidate therapeutic agents for triple-negative breast cancer (TNBC) and other cancer types, but inherent and acquired resistance to BBDIs limits their potential clinical use. Using CRISPR and small-molecule inhibitor screens combined with comprehensive molecular profiling of BBDI response and resistance, we identified synthetic lethal interactions with BBDIs and genes that, when deleted, confer resistance. We observed synergy with regulators of cell cycle progression, YAP, AXL, and SRC signaling, and chemotherapeutic agents. We also uncovered functional similarities and differences among BRD2, BRD4, and BRD7. Although deletion of BRD2 enhances sensitivity to BBDIs, BRD7 loss leads to gain of TEAD-YAP chromatin binding and luminal features associated with BBDI resistance. Single-cell RNA-seq, ATAC-seq, and cellular barcoding analysis of BBDI responses in sensitive and resistant cell lines highlight significant heterogeneity among samples and demonstrate that BBDI resistance can be pre-existing or acquired.

Regulatory T cells (Tregs) maintain immune homeostasis and prevent autoimmunity. Serine stimulates glutathione (GSH) synthesis and feeds into the one-carbon metabolic network (1CMet) essential for effector T cell (Teff) responses. However, serine's functions, linkage to GSH, and role in stress responses in Tregs are unknown. Here, we show, using mice with Treg-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that GSH loss in Tregs alters serine import and synthesis and that the integrity of this feedback loop is critical for Treg suppressive capacity. Although Gclc ablation does not impair Treg differentiation, mutant mice exhibit severe autoimmunity and enhanced anti-tumor responses. Gclc-deficient Tregs show increased serine metabolism, mTOR activation, and proliferation but downregulated FoxP3. Limitation of cellular serine in vitro and in vivo restores FoxP3 expression and suppressive capacity of Gclc-deficient Tregs. Our work reveals an unexpected role for GSH in restricting serine availability to preserve Treg functionality. Regulatory T cells (Tregs) rely on oxidative metabolism, which triggers the generation of reactive oxygen species (ROS). Accumulating ROS are controlled by the antioxidant glutathione (GSH). Kurniawan et al. reveal an unexpected subset-specific role of GSH in serine metabolism and Treg function.

Color centers in diamond have emerged as leading solid-state artificial atoms for a range of quantum technologies, from quantum sensing to quantum networks. Concerted research activities are now underway to identify new color centers that combine stable spin and optical properties of the nitrogen vacancy (NV$^-$) with the spectral stability of the silicon vacancy (SiV$^-$) centers in diamond, with recent research identifying other group IV color centers with superior properties. In this Letter, we investigate a new class of diamond quantum emitters from first principles, the group III color centers, which we show to be thermodynamically stable in a spin-1, electric-field insensitive structure. From ab initio electronic structure methods, we characterize the product Jahn-Teller (pJT) effect present in the excited state manifold of these group III color centers, where we capture symmetry-breaking distortions associated with strong electron-phonon coupling. These predictions can guide experimental identification of group III vacancy centers and their use in applications in quantum information science and technology., Comment: 6 pages, 3 figures

Cells are subjected to oxidative stress during the initiation and progression of tumors, and this imposes selective pressure for cancer cells to adapt mechanisms to tolerate these conditions. Here, we examined the dependency of cancer cells on glutathione (GSH), the most abundant cellular antioxidant. While cancer cell lines displayed a broad range of sensitivities to inhibition of GSH synthesis, the majority were resistant to GSH depletion. To identify cellular pathways required for this resistance, we carried out genetic and pharmacologic screens. Both approaches revealed that inhibition of deubiquitinating enzymes (DUBs) sensitizes cancer cells to GSH depletion. Inhibition of GSH synthesis, in combination with DUB inhibition, led to an accumulation of polyubiquitinated proteins, induction of proteotoxic stress, and cell death. These results indicate that depletion of GSH renders cancer cells dependent on DUB activity to maintain protein homeostasis and cell viability and reveal a potentially exploitable vulnerability for cancer therapy.

Summary Cancer metastasis depends on cell survival following loss of extracellular matrix attachment and dissemination through the circulation. The metastatic spread can be enhanced by the clustering of detached cancer cells and increased antioxidant defense. Here, we link these responses by describing how cell clustering limits reactive oxygen species (ROS). Loss of attachment causes mitochondrial perturbations and increased ROS production. The formation of cell clusters induces a hypoxic environment that drives hypoxia-inducible factor 1-alpha (Hif1α)-mediated mitophagy, clearing damaged mitochondria and limiting ROS. However, hypoxia and reduced mitochondrial capacity promote dependence on glycolysis for ATP production that is supported by cytosolic reductive metabolism. Preventing this metabolic adaptation or disruption of cell clusters results in ROS accumulation, cell death, and a reduction of metastatic capacity in vivo. Our results provide a mechanistic explanation for the role of cell clustering in supporting survival during extracellular matrix detachment and metastatic spread and may point to targetable vulnerabilities., Graphical Abstract, Highlights • Loss of attachment causes mitochondrial perturbations and ROS production • Detached cancer cells cluster, inducing hypoxia, mitophagy, and reductive metabolism • This response clears damaged mitochondria and reduces mitochondrial ROS production • The clustering-induced metabolic switch promotes metastatic capacity, Labuschagne et al. show that the clustering of cancer cells following detachment from the extracellular matrix (ECM) results in hypoxia, which activates mitophagy to remove damaged mitochondria and reductive metabolism to support glycolysis. These responses limit mitochondrial ROS production, allowing cell survival and metastasis.

Isocitrate dehydrogenase-1 (Idh1) is an important metabolic enzyme that produces NADPH by converting isocitrate to α-ketoglutarate. Idh1 is known to reduce reactive oxygen species (ROS) induced in cells by treatment with lipopolysaccharide (LPS) in vitro. Here, we used Idh1-deficient knockout (Idh1 KO) mice to investigate the role of Idh1 in antioxidant defense in vivo. Idh1 KO mice showed heightened susceptibility to death induced by LPS and exhibited increased serum levels of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6. The serum of LPS-injected Idh1 KO mice also contained elevated levels of AST, a marker of inflammatory liver damage. Furthermore, after LPS injection, livers of Idh1 KO mice showed histological evidence of elevated oxidative DNA damage compared with livers of wild-type (WT) mice. Idh1 KO livers showed a faster and more pronounced oxidative stress than WT livers. In line with that, Idh1 KO hepatocytes showed higher ROS levels and an increase in the NADP(+)/NADPH ratio when compared with hepatocytes isolated from WT mice. These results suggest that Idh1 has a physiological function in protecting cells from oxidative stress by regulating the intracellular NADP(+)/NADPH ratio. Our findings suggest that stimulation of Idh1 activity may be an effective therapeutic strategy for reducing oxidative stress during inflammatory responses, including the early stages of septic shock.

Significance Large, multidimensional “landscaping” projects have provided datasets that can be mined to identify potential targets for subgroups of tumors. Here, we analyzed genomic and transcriptomic data from human breast tumors to identify genes whose expression is enriched in tumors harboring specific genetic alterations. However, this analysis revealed that two other factors, proliferation rate and tumor lineage, are more dominant factors in shaping tumor transcriptional programs than genetic alterations. This discovery shifted our attention to identifying genes that are independent of the dominant proliferation and lineage programs. A small subset of these genes represents candidate targets for combination cancer therapies because they are druggable, maintained after treatment with chemotherapy, essential for cell line survival, and elevated in drug-resistant stem-like cancer cells., Large, multidimensional cancer datasets provide a resource that can be mined to identify candidate therapeutic targets for specific subgroups of tumors. Here, we analyzed human breast cancer data to identify transcriptional programs associated with tumors bearing specific genetic driver alterations. Using an unbiased approach, we identified thousands of genes whose expression was enriched in tumors with specific genetic alterations. However, expression of the vast majority of these genes was not enriched if associations were analyzed within individual breast tumor molecular subtypes, across multiple tumor types, or after gene expression was normalized to account for differences in proliferation or tumor lineage. Together with linear modeling results, these findings suggest that most transcriptional programs associated with specific genetic alterations in oncogenes and tumor suppressors are highly context-dependent and are predominantly linked to differences in proliferation programs between distinct breast cancer subtypes. We demonstrate that such proliferation-dependent gene expression dominates tumor transcriptional programs relative to matched normal tissues. However, we also identified a relatively small group of cancer-associated genes that are both proliferation- and lineage-independent. A subset of these genes are attractive candidate targets for combination therapy because they are essential in breast cancer cell lines, druggable, enriched in stem-like breast cancer cells, and resistant to chemotherapy-induced down-regulation.

Cancer cell survival is dependent on oxidative-stress defenses against reactive oxygen species (ROS) that accumulate during tumorigenesis. Here, we show a non-canonical oxidative-stress defense mechanism through TRPA1, a neuronal redox-sensing Ca2+-influx channel. In TRPA1-enriched breast and lung cancer spheroids, TRPA1 is critical for survival of inner cells that exhibit ROS accumulation. Moreover, TRPA1 promotes resistance to ROS-producing chemotherapies, and TRPA1 inhibition suppresses xenograft tumor growth and enhances chemosensitivity. TRPA1 does not affect redox status but upregulates Ca2+-dependent anti-apoptotic pathways. NRF2, an oxidant-defense transcription factor, directly controls TRPA1 expression, thus providing an orthogonal mechanism for protection against oxidative stress together with canonical ROS-neutralizing mechanisms. These findings reveal an oxidative-stress defense program involving TRPA1 that could be exploited for targeted cancer therapies.

Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis.

The generation of viable sperm proceeds through a series of coordinated steps, including germ cell self-renewal, meiotic recombination, and terminal differentiation into functional spermatozoa. The p53 family of transcription factors, including p53, p63, and p73, are critical for many physiological processes, including female fertility, but little is known about their functions in spermatogenesis. Here, we report that deficiency of the TAp73 isoform, but not p53 or ΔNp73, results in male infertility because of severe impairment of spermatogenesis. Mice lacking TAp73 exhibited increased DNA damage and cell death in spermatogonia, disorganized apical ectoplasmic specialization, malformed spermatids, and marked hyperspermia. We demonstrated that TAp73 regulates the mRNA levels of crucial genes involved in germ stem/progenitor cells (CDKN2B), spermatid maturation/spermiogenesis (metalloproteinase and serine proteinase inhibitors), and steroidogenesis (CYP21A2 and progesterone receptor). These alterations of testicular histology and gene expression patterns were specific to TAp73 null mice and not features of mice lacking p53. Our work provides previously unidentified in vivo evidence that TAp73 has a unique role in spermatogenesis that ensures the maintenance of mitotic cells and normal spermiogenesis. These results may have implications for the diagnosis and management of human male infertility. published

Oxidative stress plays an important role in cancer development and treatment. Recent data implicate the tumor suppressor BRCA1 in regulating oxidative stress, but the molecular mechanism and the impact in BRCA1-associated tumorigenesis remain unclear. Here, we show that BRCA1 regulates Nrf2-dependent antioxidant signaling by physically interacting with Nrf2 and promoting its stability and activation. BRCA1-deficient mouse primary mammary epithelial cells show low expression of Nrf2-regulated antioxidant enzymes and accumulate reactive oxygen species (ROS) that impair survival in vivo. Increased Nrf2 activation rescues survival and ROS levels in BRCA1-null cells. Interestingly, 53BP1 inactivation, which has been shown to alleviate several defects associated with BRCA1 loss, rescues survival of BRCA1-null cells without restoring ROS levels. We demonstrate that estrogen treatment partially restores Nrf2 levels in the absence of BRCA1. Our data suggest that Nrf2-regulated antioxidant response plays a crucial role in controlling survival downstream of BRCA1 loss. The ability of estrogen to induce Nrf2 posits an involvement of an estrogen-Nrf2 connection in BRCA1 tumor suppression. Lastly, BRCA1-mutated tumors retain a defective antioxidant response that increases the sensitivity to oxidative stress. In conclusion, the role of BRCA1 in regulating Nrf2 activity suggests important implications for both the etiology and treatment of BRCA1-related cancers.

Erythrocytes endure constant exposure to oxidative stress. The major oxidative stress scavenger in erythrocytes is glutathione. The rate-limiting enzyme for glutathione synthesis is glutamate-cysteine ligase, which consists of a catalytic subunit (GCLC) and a modifier subunit (GCLM). Here, we examined erythrocyte survival in GCLM-deficient (gclm(-/-)) mice. Erythrocytes from gclm(-/-) mice showed greatly reduced intracellular glutathione. Prolonged incubation resulted in complete lysis of gclm(-/-) erythrocytes, which could be reversed by exogenous delivery of the antioxidant Trolox. To test the importance of GCLM in vivo, mice were treated with phenylhydrazine (PHZ; 0.07 mg/g b.w.) to induce oxidative stress. Gclm(-/-) mice showed dramatically increased hemolysis compared with gclm(+/+) controls. In addition, PHZ-treated gclm(-/-) mice displayed markedly larger accumulations of injured erythrocytes in the spleen than gclm(+/+) mice within 24 h of treatment. Iron staining indicated precipitations of the erythrocyte-derived pigment hemosiderin in kidney tubules of gclm(-/-) mice and none in gclm(+/+) controls. In fact, 24 h after treatment, kidney function began to diminish in gclm(-/-) mice as evident from increased serum creatinine and urea. Consequently, while all PHZ-treated gclm(+/+) mice survived, 90% of PHZ-treated gclm(-/-) mice died within 5 days of treatment. In vitro, upon incubation in the absence or presence of additional oxidative stress, gclm(-/-) erythrocytes exposed significantly more phosphatidylserine, a cell death marker, than gclm(+/+) erythrocytes, an effect at least partially due to increased cytosolic Ca(2+) concentration. Under resting conditions, gclm(-/-) mice exhibited reticulocytosis, indicating that the enhanced erythrocyte death was offset by accelerated erythrocyte generation. GCLM is thus indispensable for erythrocyte survival, in vitro and in vivo, during oxidative stress.

It is well known that protein tyrosine phosphatases (PTPs) that become oxidized due to exposure to reactive oxygen species (ROS) undergo a conformational change and are inactivated. However, whether PTPs can actively regulate ROS levels in order to prevent PTP inhibition has yet to be investigated. Here, we demonstrate that PTP non-receptor type 12 (PTPN12) protects cells against aberrant ROS accumulation and death induced by oxidative stress. Murine embryonic fibroblasts (MEFs) deficient in PTPN12 underwent increased ROS-induced apoptosis under conditions of antioxidant depletion. Cells lacking PTPN12 also showed defective activation of FOXO1/3a, transcription factors required for the upregulation of several antioxidant genes. PTPN12-mediated regulation of ROS appeared to be mediated by phosphoinositide-dependent kinase-1 (PDK1), which was hyperstimulated in the absence of PTPN12. As tight regulation of ROS to sustain survival is a key feature of cancer cells, we examined PTPN12 levels in tumors from a cohort of breast cancer patients. Patients whose tumors showed high levels of PTPN12 transcripts had a significantly poorer prognosis. Analysis of tissues from patients with various breast cancer subtypes revealed that more triple-negative breast cancers, the most aggressive breast cancer subtype, showed high PTPN12 expression than any other subtype. Furthermore, both human breast cancer cells and mouse mammary epithelial tumor cells engineered to lack PTPN12 exhibited reduced tumorigenic and metastatic potential in vivo that correlated with their elevated ROS levels. The involvement of PTPN12 in the antioxidant response of breast cancer cells suggests that PTPN12 may represent a novel therapeutic target for this disease.

Cluster of differentiation (CD)8(+) T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8(+) T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1(-/-)) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage.

Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.

SummaryProtein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H2O2. Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.

Interest in the topic of tumour metabolism has waxed and waned over the past century of cancer research. The early observations of Warburg and his contemporaries established that there are fundamental differences in the central metabolic pathways operating in malignant tissue. However, the initial hypotheses that were based on these observations proved inadequate to explain tumorigenesis, and the oncogene revolution pushed tumour metabolism to the margins of cancer research. In recent years, interest has been renewed as it has become clear that many of the signalling pathways that are affected by genetic mutations and the tumour microenvironment have a profound effect on core metabolism, making this topic once again one of the most intense areas of research in cancer biology.

As single agents, small molecule inhibitors of poly-ADP-ribose polymerase (PARP) have shown remarkable efficacy in BRCA1/2-mutant tumors due to their synthetic lethality in DNA damage response-deficient cells. While PARP inhibitors, such as olaparib, have gained FDA-approval for gynecological malignancies, recurrent disease remains a challenge. To identify drug combinations with the potential to improve the initial response to PARP inhibition, we carried out a high-throughput drug screen in which we examined the ability of olaparib to synergize with more than 500 small molecules in BRCA1-mutant breast cancer cells. The compounds investigated included not only conventional “anti-cancer” agents but also lesser-studied metabolic and stress-inducing drugs. All compounds were tested across 10-point dose curves using an image-based readout to avoid false positives/negatives associated with more traditional metabolic readouts of cell viability (i.e. Cell Titer Glo). This screening approach revealed that inhibition of the unfolded protein response (UPR) sensitizes breast cancer cells to PARP inhibition. We have further characterized interaction of one class of inhibitors that target IRE1 (inositol-requiring enzyme 1), which initiates adaptive responses to ER stress through unconventional mRNA splicing of the transcription factor X-box binding protein 1 (XBP1). Combining olaparib with an inhibitor of IRE1-mediated XBP1 splicing (STF-0831010) synergistically reduced cancer cell viability in BRCA1-mutant SUM149 breast cancer cells. This combination led to increased DNA damage, indicated by increased γ-H2AX staining. This study provides evidence that UPR inhibition sensitizes cells to PARP inhibition, suggesting a previously un-recognized connection between PARP inhibition and protein homeostasis. Furthermore, it identifies a potential new drug combination to increase the efficacy of PARP inhibitors in the clinic. Citation Format: Jennifer E. Endress, Isaac S. Harris, Jacob Stewart-Ornstein, Galit Lahav, Joan S. Brugge. Combined inhibition of PARP and the unfolded protein response synergistically reduces viability in BRCA1-mutant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1851.

Summary FOXO transcription factors are important regulators of cell survival in response to a variety of stress stimuli, among which are oxidative stress, DNA damage, and nutrient deprivation. Here we report a role for FOXO3a under conditions of hypoxic stress. In response to hypoxia, FOXO3a transcript levels accumulate in an HIF1-dependent way, resulting in enhanced FOXO3a activity. We show that transcription of CITED2, a transcriptional cofactor that functions in a negative feedback loop to control HIF1 activity, is induced by FOXO3a during hypoxia. In fibroblasts as well as in breast cancer cells, FOXO3a inhibits HIF1-induced apoptosis by stimulating the transcription of CITED2, which results in reduced expression of the proapoptotic HIF1 target genes NIX and RTP801. Thus, by fine-tuning HIF1 activity, FOXO3a plays an important role in the survival response of normal and cancer cells in response to hypoxic stress.