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Background: Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease in women, while the oncogenic mechanisms which contribute to TNBC development, progression, and metastasis are poorly investigated and therefore warrant the critical need to identify novel oncogenic signalings and develop new therapeutic targets. Results: We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α protein level and activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggesting that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles in controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC. Conclusion: ZHX2 has been highlighted as one critical hypoxia-related factors regulator contributing to triple-negative breast cancer in this work, which has enriched the upstream regulatory network of HIF-1α signaling. Moreover, identification of key residuals determining biological function of ZHX2 provides novel approaches for treating TNBC via targeting the hypoxia pathway. Citation Format: Chengheng Liao, Wentong Fang, Rachel Shi, Jeremy Simon, Travis Ptacek, Giada Zurlo, Youqiong Ye, Leng Han, Cheng Fan, Lei Bao, Christopher Llynard Ortiz, Hong-Rui Lin, Ujjawal Manocha, Weibo Luo, Yan Peng, William Y Kim, Lee-Wei Yang, Qing Zhang. ZHX2 promotes HIF1α oncogenic signaling in 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 P4-08-23.

Quantification of IHC staining

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. Because of its heterogeneity and lack of hormone receptors or HER2 expression, targeted therapy is limited. Here, by performing a functional siRNA screening for 2-OG–dependent enzymes, we identified gamma-butyrobetaine hydroxylase 1 (BBOX1) as an essential gene for TNBC tumorigenesis. BBOX1 depletion inhibits TNBC cell growth while not affecting normal breast cells. Mechanistically, BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3) in an enzymatic-dependent manner and prevents its ubiquitination and proteasomal degradation. BBOX1 depletion suppresses IP3R3-mediated endoplasmic reticulum calcium release, therefore impairing calcium-dependent energy-generating processes including mitochondrial respiration and mTORC1-mediated glycolysis, which leads to apoptosis and impaired cell-cycle progression in TNBC cells. Therapeutically, genetic depletion or pharmacologic inhibition of BBOX1 inhibits TNBC tumor growth in vitro and in vivo. Our study highlights the importance of targeting the previously uncharacterized BBOX1–IP3R3–calcium oncogenic signaling axis in TNBC.Significance:We provide evidence from unbiased screens that BBOX1 is a potential therapeutic target in TNBC and that genetic knockdown or pharmacologic inhibition of BBOX1 leads to decreased TNBC cell fitness. This study lays the foundation for developing effective BBOX1 inhibitors for treatment of this lethal disease.This article is highlighted in the In This Issue feature, p. 1611

Table S1. List of 65 2-OG dioxygenases

Extended methods and supplementary figures

Supplementary Data

TANK binding kinase 1 (TBK1) is an important kinase involved in the innate immune response. Here we discover that TBK1 is hyperactivated by von Hippel-Lindau (VHL) loss or hypoxia in cancer cells. Tumors from patients with kidney cancer with VHL loss display elevated TBK1 phosphorylation. Loss of TBK1 via genetic ablation, pharmacologic inhibition, or a new cereblon-based proteolysis targeting chimera specifically inhibits VHL-deficient kidney cancer cell growth, while leaving VHL wild-type cells intact. TBK1 depletion also significantly blunts kidney tumorigenesis in an orthotopic xenograft model in vivo. Mechanistically, TBK1 hydroxylation on Proline 48 triggers VHL as well as the phosphatase PPM1B binding that leads to decreased TBK1 phosphorylation. We identify that TBK1 phosphorylates p62/SQSTM1 on Ser366, which is essential for p62 stability and kidney cancer cell proliferation. Our results establish that TBK1, distinct from its role in innate immune signaling, is a synthetic lethal target in cancer with VHL loss.Significance:The mechanisms that lead to TBK1 activation in cancer and whether this activation is connected to its role in innate immunity remain unclear. Here, we discover that TBK1, distinct from its role in innate immunity, is activated by VHL loss or hypoxia in cancer.See related commentary by Bakouny and Barbie, p. 348.This article is highlighted in the In This Issue feature, p. 327

Supplementary Figure from Integrated Metabolic Profiling and Transcriptional Analysis Reveals Therapeutic Modalities for Targeting Rapidly Proliferating Breast Cancers

Metabolic dysregulation is a prominent feature in breast cancer, but it remains poorly characterized in patient tumors. In this study, untargeted metabolomics analysis of triple-negative breast cancer (TNBC) and patient with estrogen receptor (ER)–positive breast cancer samples, as well as TNBC patient-derived xenografts (PDX), revealed two major metabolic groups independent of breast cancer histologic subtypes: a “Nucleotide/Carbohydrate-Enriched” group and a “Lipid/Fatty Acid-Enriched” group. Cell lines grown in vivo more faithfully recapitulated the metabolic profiles of patient tumors compared with those grown in vitro. Integrated metabolic and gene expression analyses identified genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof of principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell lines or PDX xenografts with a pyrimidine biosynthesis inhibitor or a glutaminase inhibitor led to therapeutic efficacy. In multiple in vivo models of TNBC, treatment with the pyrimidine biosynthesis inhibitor conferred better therapeutic outcomes than chemotherapeutic agents. This study provides a metabolic stratification of breast tumor samples that can guide the selection of effective therapeutic strategies targeting breast cancer subsets. In addition, we have developed a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study to facilitate future research.Significance:A multiomics strategy that integrates metabolic and gene expression profiling in patient tumor samples and animal models identifies effective pharmacologic approaches to target rapidly proliferating breast tumor subtypes.

Supplementary Table from Integrated Metabolic Profiling and Transcriptional Analysis Reveals Therapeutic Modalities for Targeting Rapidly Proliferating Breast Cancers

Metabolic dysregulation is a prominent feature in breast cancer, but it remains poorly characterized in patient tumors. In this study, untargeted metabolomics analysis of triple-negative breast cancer (TNBC) and patient with estrogen receptor (ER)–positive breast cancer samples, as well as TNBC patient-derived xenografts (PDX), revealed two major metabolic groups independent of breast cancer histologic subtypes: a “Nucleotide/Carbohydrate-Enriched” group and a “Lipid/Fatty Acid-Enriched” group. Cell lines grown in vivo more faithfully recapitulated the metabolic profiles of patient tumors compared with those grown in vitro. Integrated metabolic and gene expression analyses identified genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof of principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell lines or PDX xenografts with a pyrimidine biosynthesis inhibitor or a glutaminase inhibitor led to therapeutic efficacy. In multiple in vivo models of TNBC, treatment with the pyrimidine biosynthesis inhibitor conferred better therapeutic outcomes than chemotherapeutic agents. This study provides a metabolic stratification of breast tumor samples that can guide the selection of effective therapeutic strategies targeting breast cancer subsets. In addition, we have developed a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study to facilitate future research. Significance: A multiomics strategy that integrates metabolic and gene expression profiling in patient tumor samples and animal models identifies effective pharmacologic approaches to target rapidly proliferating breast tumor subtypes.

Histone post-translational modifications (PTMs) are important forregulating various DNA-templated processes. Here, we report theexistence of a histone PTM in mammalian cells, namely histone H3 withhydroxylation of proline at residue 16 (H3P16oh), which is catalyzed by theproline hydroxylase EGLN2. We show that H3P16oh enhances direct bindingof KDM5A to its substrate, histone H3 with trimethylation at the fourthlysine residue (H3K4me3), resulting in enhanced chromatin recruitmentof KDM5A and a corresponding decrease of H3K4me3 at target genes.Genome- and transcriptome-wide analyses show that the EGLN2–KDM5Aaxis regulates target gene expression in mammalian cells. Specifically, ourdata demonstrate repression of the WNT pathway negative regulator DKK1through the EGLN2-H3P16oh-KDM5A pathway to promote WNT/β-cateninsignaling in triple-negative breast cancer (TNBC). This study characterizesa regulatory mark in the histone code and reveals a role for H3P16oh inregulating mammalian gene expression

Diminished oxygen availability, termed hypoxia, within solid tumors is one of the most common characteristics of cancer. Hypoxia shapes the landscape of the tumor microenvironment (TME) into a pro-tumorigenic and pro-metastatic niche through arrays of pathological alterations such as abnormal vasculature, altered metabolism, immune-suppressive phenotype, etc. In addition, emerging evidence suggests that limited efficacy or the development of resistance towards antitumor therapy may be largely due to the hypoxic TME. This review will focus on summarizing the knowledge about the molecular machinery that mediates the hypoxic cellular responses and adaptations, as well as highlighting the effects and consequences of hypoxia, especially for angiogenesis regulation, cellular metabolism alteration, and immunosuppressive response within the TME. We also outline the current advances in novel therapeutic implications through targeting hypoxia in TME. A deep understanding of the basics and the role of hypoxia in the tumor will help develop better therapeutic avenues in cancer treatment.

Characterized by intracellular lipid droplet accumulation, clear cell renal cell carcinoma (ccRCC) is resistant to cytotoxic chemotherapy and is a lethal disease. Through an unbiased siRNA screen of 2-oxoglutarate (2-OG)-dependent enzymes, which play a critical role in tumorigenesis, we identified Jumonji domain-containing 6 (JMJD6) as an essential gene for ccRCC tumor development. The downregulation of JMJD6 abolished ccRCC colony formation in vitro and inhibited orthotopic tumor growth in vivo. Integrated ChIP-seq and RNA-seq analyses uncovered diacylglycerol O-acyltransferase 1 (DGAT1) as a critical JMJD6 effector. Mechanistically, JMJD6 interacted with RBM39 and co-occupied DGAT1 gene promoter with H3K4me3 to induce DGAT1 expression. JMJD6 silencing reduced DGAT1, leading to decreased lipid droplet formation and tumorigenesis. The pharmacological inhibition (or depletion) of DGAT1 inhibited lipid droplet formation in vitro and ccRCC tumorigenesis in vivo. Thus, the JMJD6-DGAT1 axis represents a potential new therapeutic target for ccRCC.

Metabolic dysregulation, although a prominent feature in breast cancer, remains undercharacterized in patient tumors. By performing untargeted metabolomics analyses on triple-negative breast cancer (TNBC) and Estrogen Receptor (ER) positive patient breast tumors, as well as TNBC patient-derived xenografts (PDXs), we identified two major metabolic groups independent of breast cancer histological subtypes: a “Nucleotide/Carbohydrate-Enriched” group and a “Lipid/Fatty Acid-Enriched” group. Cell lines grown in vivo more faithfully recapitulate the metabolic profiles of patient tumors. Integrated metabolic and gene expression analyses reveal genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof-of-principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell line or PDX xenografts with a pyrimidine biosynthesis inhibitor, and/or a glutaminase inhibitor, led to therapeutic efficacy. In addition, the pyrimidine biosynthesis inhibitor presents better therapeutic outcomes than chemotherapy agents in multiple murine TNBC models. Our study provides a new stratification of breast tumor samples based on integrated metabolic and gene expression profiling, which guides the selection of newly effective therapeutic strategies targeting rapidly proliferating breast cancer subsets. In addition, we develop a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease, which warrants the critical need to identify new therapeutic targets. We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggested that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles on controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC.

Liver malignancies are among the tumor types that are resistant to immune checkpoint inhibition therapy. Tumor-associated macrophages (TAMs) are highly enriched and play a major role in inducing immunosuppression in liver malignancies. Herein, CCL2 and CCL5 are screened as two major chemokines responsible for attracting TAM infiltration and inducing their polarization towards cancer-promoting M2-phenotype. To reverse this immunosuppressive process, we directly evolve an innovative single-domain antibody that bispecifically binds and neutralizes CCL2 and CCL5 (BisCCL2/5i) with high potency and specificity. mRNA encoding BisCCL2/5i was encapsulated in a clinically approved lipid nanoparticle platform, resulting in a liver-homing biomaterial that allows transient yet efficient expression of BisCCL2/5i in the diseased organ in a multiple dosage manner. This BisCCL2/5i mRNA nanoplatform significantly induces the polarization of TAMs toward the antitumoral M1 phenotype and reduces immunosuppression in the tumor microenvironment. The combination of BisCCL2/5i with PD-1 ligand inhibitor (PD-Li) achieves long-term survival in mouse models of primary liver cancer and liver metastasis of colorectal and pancreatic cancers. Our work provides an effective bispecific targeting strategy that could broaden the PD-Li therapy to multiple types of malignancies in the human liver.

Metabolic dysregulation is one of the distinctive features in breast cancer. However, examining the metabolic features in various subtypes of breast cancer in their relationship to gene expression features in a physiologically relevant setting remains understudied. By performing metabolic profiling on triple-negative breast cancer (TNBC) and ER+ breast cancers from patients, TNBC patient-derived xenografts (PDXs), and representative breast cancer cell lines grown as tumors in vivo, we identify two distinctive groups defined by metabolites; a “Nucleotide-Enriched” group that shows high levels of pyrimidine pathway metabolites and biosynthetic enzymes, and a “Arginine Biosynthesis-Enriched” group that shows high levels of arginine biosynthesis intermediates. We reveal different metabolic enrichment profiles between cell lines grown in vitro versus in vivo, where cell lines grown in vivo more faithfually recapitulate patient tumors metabolic profiles. In addition, with integrated metabolic and gene expression profiling we identify a subset of genes that strongly correlates with the Nucleotide-Enriched metabolic profile, and which strongly predicts patient prognosis. As a proof-of-principle, when we target Nucleotide-Enriched metabolic dysregulation with a pyrimidine biosynthesis inhibitor (Brequinar), and/or a glutaminase inhibitor (CB-839), we observe therapeutic efficacy and decreased tumor growth in representative TNBC cell lines and an in vivo PDX upon combinatorial drug treatment. Our study reveals new therapeutic opportunities in breast cancer guided by a genomic biomarker, which could prove highly impactful for rapidly proliferating breast cancers specifically.

Maintaining oxygen homeostasis is a most basic cellular process for adapting physiological oxygen variations, and its abnormality typically leads to various disorders in the human body. The key molecules of the oxygen-sensing system include the transcriptional regulator hypoxia-inducible factor (HIF), which controls a wide range of oxygen responsive target genes (eg, EPO and VEGF), certain members of the oxygen/2-oxoglutarate-dependent dioxygenase family, including the HIF proline hydroxylase (PHD, alias EGLN), and an E3 ubiquitin ligase component for HIF destruction called von Hippel-Lindau. In this review, we summarize the physiological role and highlight the pathologic function for each protein of the oxygen-sensing system. A better understanding of their molecular mechanisms of action will help uncover novel therapeutic targets and develop more effective treatment approaches for related human diseases, including cancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. Because of its heterogeneity and lack of hormone receptors or HER2 expression, targeted therapy is limited. Here, by performing a functional siRNA screening for 2-OG–dependent enzymes, we identified gamma-butyrobetaine hydroxylase 1 (BBOX1) as an essential gene for TNBC tumorigenesis. BBOX1 depletion inhibits TNBC cell growth while not affecting normal breast cells. Mechanistically, BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3) in an enzymatic-dependent manner and prevents its ubiquitination and proteasomal degradation. BBOX1 depletion suppresses IP3R3-mediated endoplasmic reticulum calcium release, therefore impairing calcium-dependent energy-generating processes including mitochondrial respiration and mTORC1-mediated glycolysis, which leads to apoptosis and impaired cell-cycle progression in TNBC cells. Therapeutically, genetic depletion or pharmacologic inhibition of BBOX1 inhibits TNBC tumor growth in vitro and in vivo. Our study highlights the importance of targeting the previously uncharacterized BBOX1–IP3R3–calcium oncogenic signaling axis in TNBC. Significance: We provide evidence from unbiased screens that BBOX1 is a potential therapeutic target in TNBC and that genetic knockdown or pharmacologic inhibition of BBOX1 leads to decreased TNBC cell fitness. This study lays the foundation for developing effective BBOX1 inhibitors for treatment of this lethal disease. This article is highlighted in the In This Issue feature, p. 1611

Metabolic dysregulation is one of the distinctive features of breast cancer, yet remains under-characterized in different subtypes of breast cancer. In this study, we performed full metabolome profiling and RNA-seq gene expression analyses on patient samples comprised of Triple Negative Breast Cancer (TNBC) and Estrogen Receptor (ER) positive breast cancers, as well as TNBC patient-derived xenografts (PDX). We identified two major metabolic groups using hierarchical clustering analysis of global metabolite levels: a Nucleotide/Carbohydrate-Enriched group and a Lipid/Fatty Acid-Enriched group. The Nucleotide/Carbohydrate-Enriched group is populated by the majority of TNBCs and all TNBC PDX samples, and shows high levels of energy consumption and nucleotide biosynthesis related metabolites, while the Lipid/Fatty Acid-Enriched group contained all ER+ cancers (and normal breast tissues) and showed high levels of lipids and fatty acids. Using these two metabolite-defined groups, we compared metabolic signatures to RNA-seq expression data and identified gene expression signatures that correlated with each metabolic group. This novel integrated signature identified classic proliferation-associated genes as highly expressed in the Nucleotide/Carbohydrate-Enriched metabolomics group. We next sought to therapeutically target the Nucleotide/Carbohydrate-Enriched group through targeting of nucleotide metabolism using a pyrimidine biosynthesis inhibitor (DHODH inhibitor, Brequinar), and/or a glutaminase inhibitor (CB-839), and observed tumor volume reductions and improved survival times using the TNBC PDX models. Notably,. when the DHODH inhibitor Brequinar was tested on four different immune-competent TNBC mouse models, it showed significant single agent activity, and in 3/4 cases had better activity than carboplatin/paclitaxel combination, and with less toxicity. Our study reveals a new molecularly targeted therapy for rapidly proliferating TNBCs, guided by a novel set of metabolic-genomic biomarkers, which might be translated quickly as DHODH inhibitors are already FDA approved for use in other diseases. Citation Format: Cherise R Glodowski, Chengheng Liao, Cheng Fan, Juan Liu, Kevin R Mott, Akash Kaushik, Hieu Vu, Jason W Locasale, Samuel K McBrayer, Ralph J DeBerardinis, Charles M Perou, Qing Zhang. Metabolite profiling and RNA-seq identifies novel metabolomic-genomic biomarker and therapeutic options for rapidly proliferating breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-01.

TANK binding kinase 1 (TBK1) is an important kinase involved in the innate immune response. Here we discover that TBK1 is hyperactivated by von Hippel-Lindau (VHL) loss or hypoxia in cancer cells. Tumors from patients with kidney cancer with VHL loss display elevated TBK1 phosphorylation. Loss of TBK1 via genetic ablation, pharmacologic inhibition, or a new cereblon-based proteolysis targeting chimera specifically inhibits VHL-deficient kidney cancer cell growth, while leaving VHL wild-type cells intact. TBK1 depletion also significantly blunts kidney tumorigenesis in an orthotopic xenograft model in vivo. Mechanistically, TBK1 hydroxylation on Proline 48 triggers VHL as well as the phosphatase PPM1B binding that leads to decreased TBK1 phosphorylation. We identify that TBK1 phosphorylates p62/SQSTM1 on Ser366, which is essential for p62 stability and kidney cancer cell proliferation. Our results establish that TBK1, distinct from its role in innate immune signaling, is a synthetic lethal target in cancer with VHL loss. Significance: The mechanisms that lead to TBK1 activation in cancer and whether this activation is connected to its role in innate immunity remain unclear. Here, we discover that TBK1, distinct from its role in innate immunity, is activated by VHL loss or hypoxia in cancer. See related commentary by Bakouny and Barbie, p. 348. This article is highlighted in the In This Issue feature, p. 327

Hypoxia, a common feature of solid tumors, greatly hinders the efficacy of conventional cancer treatments such as chemo-, radio-, and immunotherapy. The depletion of oxygen in proliferating and advanced tumors causes an array of genetic, transcriptional, and metabolic adaptations that promote survival, metastasis, and a clinically malignant phenotype. At the nexus of these interconnected pathways are hypoxia-inducible factors (HIFs) which orchestrate transcriptional responses under hypoxia. The following review summarizes current literature regarding effects of hypoxia on DNA repair, metastasis, epithelial-to-mesenchymal transition, the cancer stem cell phenotype, and therapy resistance. We also discuss mechanisms and pathways, such as HIF signaling, mitochondrial dynamics, exosomes, and the unfolded protein response, that contribute to hypoxia-induced phenotypic changes. Finally, novel therapeutics that target the hypoxic tumor microenvironment or interfere with hypoxia-induced pathways are reviewed.

Triple-negative breast cancer (TNBC), which accounts for 15-20% of breast cancers, causes the highest mortality rate among all breast cancer subtypes. Due to its heterogeneity and lack of estrogen and progesterone receptor or human epidermal growth factor receptor 2 expression, valuable targeted therapy is limited. Therefore, it is critical to identify novel therapeutic targets in TNBC. 2-oxoglutarate (2-OG)-dependent enzymes, including prolyl hydroxylases (EglN1-3), histone demethylases (for example, KDM5A) and DNA hydroxylases (such as TET1-3), are associated with cancer progression. However, the role of these enzymes in TNBC has never been systematically studied. Here, by performing a functional siRNA screening for 2-OG-dependent enzymes, we identified gamma-butyrobetaine hydroxylase 1 (BBOX1) as an essential gene for TNBC tumorigenesis. BBOX1 depletion inhibits TNBC cell growth, while not affecting normal breast cells. Mechanistically, BBOX1 binds with the calcium channel inositol-1,4,5-trisphosphate receptor type 3 (IP3R3) in an enzymatic-dependent manner and prevents its ubiquitination and proteasomal degradation. BBOX1 depletion suppresses IP3R3 mediated endoplasmic reticulum calcium release, therefore impairing calcium-dependent energy-generating processes including mitochondrial respiration and mTORC1 mediated glycolysis, which leads to apoptosis and impaired cell cycle progression in TNBC cells. Therapeutically, genetic depletion or pharmacological inhibition of BBOX1 inhibits TNBC tumor growth in vitro and in vivo. Our study highlights the importance of targeting previously uncharacterized BBOX1-IP3R3-calcium oncogenic signaling axis in TNBC. Citation Format: Chengheng Liao, Yang Zhang, Cheng Fan, Laura E. Herring, Juan Liu, Jason W. Locasale, Mamoru Takada, Jin Zhou, Giada Zurlo, Lianxin Hu, Jeremy M. Simon, Travis S. Ptacek, Victor G. Andrianov, Einars Loza, Yan Peng, Huanghe Yang, Charles M. Perou, Qing Zhang. Identification of BBOX1 as a therapeutic target in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-10.

The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.

Mechanistic insights into kidney cancer Many clear cell renal cell carcinomas (ccRCCs) have alterations to the gene encoding the von Hippel-Lindau protein (VHL). VHL is a ubiquitin ligase that degrades target proteins when they are prolyl-hydroxylated. Zhang et al. performed a genome-wide search for VHL target (see the Perspective by Sanchez and Simon). They identified ZHX2, a protein with structural motifs that indicate DNA binding. ZHX2 has been implicated in tumor suppression. Loss of ZHX2 inhibited signaling through the transcription factor NF-κB, and ZHX2 bound to many NF-κB target genes. Depletion of ZHX2 slowed growth of ccRCC cells in vitro and in a mouse model. Science , this issue p. 290 ; see also p. 226

Summary Saccharopolyspora erythraea has three citrate synthases encoded by gltA-2, citA, and citA4. Here, we characterized and identified the expression and regulatory properties of these synthases. Three pleiotropic global regulatory proteins of S. erythraea – CRP, GlnR, and DasR – are involved in carbon metabolism, nitrogen metabolism, and amino-sugar (chitin and GlcNAc) metabolism. Using electrophoretic mobility shift assays (EMSAs), we identified these regulators as proteins that bind directly to the promoter regions of all citrate synthase genes (gltA-2, citA, and citA4). Footprinting assays indicated the exact protect sequences of CRP, GlnR, and DasR on the promoter region of gltA-2, revealing binding competition between GlnR and DasR. Moreover, by comparing the transcription levels of citrate synthase genes between parental and glnR mutant or dasR mutant strains, or by comparing the transcription response of citrate synthases under various nutrient conditions, we found that GlnR and DasR negatively regulated citA and citA4 transcription but had no regulatory effects on the gltA-2 gene. Although no CRP mutant was available, the results indicated that CRP was a cAMP-binding receptor affecting gltA-2 transcription when the intracellular cAMP concentration increased. Thus, an overall model of CS regulation by C and/or N metabolism regulators and cAMP receptor protein was proposed.

Starch-degrading enzymes hydrolyze starch- and starch-derived oligosaccharides to yield glucose. We investigated the transcriptional regulation of genes encoding starch-degrading enzymes in the industrial actinobacterium Saccharopolyspora erythraea . We observed that most genes encoding amylolytic enzymes (one α-amylase, one glucoamylase, and four α-glucosidases) were regulated by GlnR and PhoP, which are global regulators of nitrogen and phosphate metabolism, respectively. Electrophoretic mobility shift assays and reverse transcription-PCR (RT-PCR) analyses demonstrated that GlnR and PhoP directly interact with their promoter regions and collaboratively or competitively activate their transcription. Deletion of glnR caused poor growth on starch, maltodextrin, and maltose, whereas overexpression of glnR and phoP increased the total activity of α-glucosidase, resulting in enhanced carbohydrate utilization. Additionally, transcript levels of amylolytic genes and total glucosidase activity were induced in response to nitrogen and phosphate limitation. Furthermore, regulatory effects of GlnR and PhoP on starch-degrading enzymes were conserved in Streptomyces coelicolor A3(2). These results demonstrate that GlnR and PhoP are involved in polysaccharide degradation by mediating the interplay among carbon, nitrogen, and phosphate metabolism in response to cellular nutritional states. Our study reveals a novel regulatory mechanism underlying carbohydrate metabolism, and suggests new possibilities for designing genetic engineering approaches to improve the rate of utilization of starch in actinobacteria. IMPORTANCE The development of efficient strategies for utilization of biomass-derived sugars, such as starch and cellulose, remains a major technical challenge due to the weak activity of associated enzymes. Here, we found that GlnR and PhoP directly regulate the transcription of genes encoding amylolytic enzymes and present insights into the regulatory mechanisms of degradation and utilization of starch in actinobacteria. Two nutrient-sensing regulators may play important roles in creating a direct association between nitrogen/phosphate metabolisms and carbohydrate utilization, as well as modulate the C:N:P balance in response to cellular nutritional states. These findings highlight the interesting possibilities for designing genetic engineering approaches and optimizing the fermentation process to improve the utilization efficiency of sugars in actinobacteria via overexpression of the glnR and phoP genes and nutrient signal stimulation.

The GntR-family transcription regulator, DasR, was previously identified as pleiotropic, controlling the primary amino sugar N-acetylglucosamine (GlcNAc) and chitin metabolism in Saccharopolyspora erythraea and Streptomyces coelicolor. Due to the remarkable regulatory impact of DasR on antibiotic production and development in the model strain of S. coelicolor, we here identified and characterized the role of DasR to secondary metabolite production and morphological development in industrial erythromycin-producing S. erythraea. The physiological studies have shown that a constructed deletion of dasR in S. erythraea resulted in antibiotic, pigment, and aerial hyphae production deficit in a nutrient-rich condition. DNA microarray assay, combined with quantitative real-time reverse transcription PCR (qRT-PCR), confirmed these results by showing the downregulation of the genes relating to secondary metabolite production in the dasR null mutant. Notably, electrophoretic mobility shift assays (EMSA) showed DasR as being the first identified regulator that directly regulates the pigment biosynthesis rpp gene cluster. In addition, further studies indicated that GlcNAc, the major nutrient signal of DasR-responsed regulation, blocked secondary metabolite production and morphological development. The effects of GlcNAc were shown to be caused by DasR mediation. These findings demonstrated that DasR is an important pleiotropic regulator for both secondary metabolism and morphological development in S. erythraea, providing new insights for the genetic engineering of S. erythraea with increased erythromycin production.

Chitin degradation and subsequentN-acetylglucosamine (GlcNAc) catabolism is thought to be a common trait of a large majority of actinomycetes. Utilization of aminosugars had been poorly investigated outside the model strainStreptomyces coelicolorA3(2), and we examined here the genetic setting of the erythromycin producerSaccharopolyspora erythraeafor GlcNAc and chitin utilization, as well as the transcriptional control thereof.Sacch. erythraeaefficiently utilize GlcNAc most likely via the phosphotransferase system (PTSGlcNAc); however, this strain is not able to grow when chitin orN,N′-diacetylchitobiose [(GlcNAc)2] is the sole nutrient source, despite a predicted extensive chitinolytic system (chigenes). The inability ofSacch. erythraeato utilize chitin and (GlcNAc)2is probably because of the loss of genes encoding the DasABC transporter for (GlcNAc)2import, and genes for intracellular degradation of (GlcNAc)2by β-N-acetylglucosaminidases. Transcription analyses revealed that inSacch. erythraeaall putativechiand GlcNAc utilization genes are repressed by DasR, whereas inStrep. coelicolorDasR displayed either activating or repressing functions whether it targets genes involved in the polymer degradation or genes for GlcNAc dimer and monomer utilization, respectively. A transcriptomic analysis further showed that GlcNAc not only activates the transcription of GlcNAc catabolism genes but also activateschigene expression, as opposed to the previously reported GlcNAc-mediated catabolite repression inStrep. coelicolor. Finally, synteny exploration revealed an identical genetic background for chitin utilization in other rare actinomycetes, which suggests that screening procedures that used only the chitin-based protocol for selective isolation of antibiotic-producing actinomycetes could have missed the isolation of many industrially promising strains.