The metabolic cofactor Coenzyme A enhances alternative macrophage activation via MyD88-linked signaling.

Metabolites and metabolic co-factors can shape the innate immune response, though the pathways by which these molecules adjust inflammation remain incompletely understood. Here we show that the metabolic cofactor Coenzyme A (CoA) enhances IL-4 driven alternative macrophage activation [m(IL-4)] in vitro and in vivo. Unexpectedly, we found that perturbations in intracellular CoA metabolism did not influence m(IL-4) differentiation. Rather, we discovered that exogenous CoA provides a weak TLR4 signal which primes macrophages for increased receptivity to IL-4 signals and resolution of inflammation via MyD88. Mechanistic studies revealed MyD88-linked signals prime for IL-4 responsiveness, in part, by reshaping chromatin accessibility to enhance transcription of IL-4-linked genes. The results identify CoA as a host metabolic co-factor that influences macrophage function through an extrinsic TLR4-dependent mechanism, and suggests that damage-associated molecular patterns (DAMPs) can prime macrophages for alternative activation and resolution of inflammation.

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation.

Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.

A ketogenic diet can mitigate SARS-CoV-2 induced systemic reprogramming and inflammation.

The ketogenic diet (KD) has demonstrated benefits in numerous clinical studies and animal models of disease in modulating the immune response and promoting a systemic anti-inflammatory state. Here we investigate the effects of a KD on systemic toxicity in mice following SARS-CoV-2 infection. Our data indicate that under KD, SARS-CoV-2 reduces weight loss with overall improved animal survival. Muted multi-organ transcriptional reprogramming and metabolism rewiring suggest that a KD initiates and mitigates systemic changes induced by the virus. We observed reduced metalloproteases and increased inflammatory homeostatic protein transcription in the heart, with decreased serum pro-inflammatory cytokines (i.e., TNF-α, IL-15, IL-22, G-CSF, M-CSF, MCP-1), metabolic markers of inflammation (i.e., kynurenine/tryptophane ratio), and inflammatory prostaglandins, indicative of reduced systemic inflammation in animals infected under a KD. Taken together, these data suggest that a KD can alter the transcriptional and metabolic response in animals following SARS-CoV-2 infection with improved mice health, reduced inflammation, and restored amino acid, nucleotide, lipid, and energy currency metabolism.

MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species.

Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state. One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.

Telomerase reverse transcriptase induces targetable alterations in glutathione and nucleotide biosynthesis in glioblastomas.

Telomerase reverse transcriptase (TERT) is essential for glioblastoma (GBM) proliferation. Delineating metabolic vulnerabilities induced by TERT can lead to novel GBM therapies. We previously showed that TERT upregulates glutathione (GSH) pool size in GBMs. Here, we show that TERT acts via the FOXO1 transcription factor to upregulate expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme of de novo GSH synthesis. Inhibiting GCLC using siRNA or buthionine sulfoximine (BSO) reduces synthesis of 13 C-GSH from [U- 13 C]-glutamine and inhibits clonogenicity. However, GCLC inhibition does not induce cell death, an effect that is associated with elevated [U- 13 C]-glutamine metabolism to glutamate and pyrimidine nucleotide biosynthesis. Mechanistically, GCLC inhibition activates MYC and leads to compensatory upregulation of two key glutamine-utilizing enzymes i.e., glutaminase (GLS), which generates glutamate from glutamine, and CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotatase), the enzyme that converts glutamine to the pyrimidine nucleotide precursor dihydroorotate. We then examined the therapeutic potential of inhibiting GLS and CAD in combination with GCLC. 6-diazo-5-oxy-L-norleucin (DON) is a potent inhibitor of glutamine-utilizing enzymes including GLS and CAD. The combination of BSO and DON suppresses GSH and pyrimidine nucleotide biosynthesis and is synergistically lethal in GBM cells. Importantly, in vivo stable isotope tracing indicates that combined treatment with JHU-083 (a brain-penetrant prodrug of DON) and BSO abrogates synthesis of GSH and pyrimidine nucleotides from [U- 13 C]-glutamine and induces tumor shrinkage in mice bearing intracranial GBM xenografts. Collectively, our studies exploit a mechanistic understanding of TERT biology to identify synthetically lethal metabolic vulnerabilities in GBMs. SIGNIFICANCE: Using in vivo stable isotope tracing, metabolomics, and loss-of-function studies, we demonstrate that TERT expression is associated with metabolic alterations that can be synergistically targeted for therapy in glioblastomas.

Fibroblast Activation Protein Expression in Sarcomas.

Objectives: Fibroblast activation protein alpha (FAP) is highly expressed by cancer-associated fibroblasts in multiple epithelial cancers. The aim of this study was to characterize FAP expression in sarcomas to explore its potential utility as a diagnostic and therapeutic target and prognostic biomarker in sarcomas. Methods: Available tissue samples from patients with bone or soft tissue tumors were identified at the University of California, Los Angeles. FAP expression was evaluated via immunohistochemistry (IHC) in tumor samples (n = 63), adjacent normal tissues (n = 30), and positive controls (n = 2) using semiquantitative systems for intensity (0 = negative; 1 = weak; 2 = moderate; and 3 = strong) and density (none, <25%, 25-75%; >75%) in stromal and tumor/nonstromal cells and using a qualitative overall score (not detected, low, medium, and high). Additionally, RNA sequencing data in publicly available databases were utilized to compare FAP expression in samples (n = 10,626) from various cancer types and evaluate the association between FAP expression and overall survival (OS) in sarcoma (n = 168). Results: The majority of tumor samples had FAP IHC intensity scores ≥2 and density scores ≥25% for stromal cells (77.7%) and tumor cells (50.7%). All desmoid fibromatosis, myxofibrosarcoma, solitary fibrous tumor, and undifferentiated pleomorphic sarcoma samples had medium or high FAP overall scores. Sarcomas were among cancer types with the highest mean FAP expression by RNA sequencing. There was no significant difference in OS in patients with sarcoma with low versus high FAP expression. Conclusion: The majority of the sarcoma samples showed FAP expression by both stromal and tumor/nonstromal cells. Further investigation of FAP as a potential diagnostic and therapeutic target in sarcomas is warranted.

CDKN2A deletion remodels lipid metabolism to prime glioblastoma for ferroptosis.

Malignant tumors exhibit heterogeneous metabolic reprogramming, hindering the identification of translatable vulnerabilities for metabolism-targeted therapy. How molecular alterations in tumors promote metabolic diversity and distinct targetable dependencies remains poorly defined. Here we create a resource consisting of lipidomic, transcriptomic, and genomic data from 156 molecularly diverse glioblastoma (GBM) tumors and derivative models. Through integrated analysis of the GBM lipidome with molecular datasets, we identify CDKN2A deletion remodels the GBM lipidome, notably redistributing oxidizable polyunsaturated fatty acids into distinct lipid compartments. Consequently, CDKN2A-deleted GBMs display higher lipid peroxidation, selectively priming tumors for ferroptosis. Together, this study presents a molecular and lipidomic resource of clinical and preclinical GBM specimens, which we leverage to detect a therapeutically exploitable link between a recurring molecular lesion and altered lipid metabolism in GBM.

Tumor heterogeneity in VHL drives metastasis in clear cell renal cell carcinoma.

Loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark of clear cell renal cell carcinoma (ccRCC). The importance of heterogeneity in the loss of this tumor suppressor has been under reported. To study the impact of intratumoral VHL heterogeneity observed in human ccRCC, we engineered VHL gene deletion in four RCC models, including a new primary tumor cell line derived from an aggressive metastatic case. The VHL gene-deleted (VHL-KO) cells underwent epithelial-to-mesenchymal transition (EMT) and exhibited increased motility but diminished proliferation and tumorigenicity compared to the parental VHL-expressing (VHL+) cells. Renal tumors with either VHL+ or VHL-KO cells alone exhibit minimal metastatic potential. Combined tumors displayed rampant lung metastases, highlighting a novel cooperative metastatic mechanism. The poorly proliferative VHL-KO cells stimulated the proliferation, EMT, and motility of neighboring VHL+ cells. Periostin (POSTN), a soluble protein overexpressed and secreted by VHL non-expressing (VHL-) cells, promoted metastasis by enhancing the motility of VHL-WT cells and facilitating tumor cell vascular escape. Genetic deletion or antibody blockade of POSTN dramatically suppressed lung metastases in our preclinical models. This work supports a new strategy to halt the progression of ccRCC by disrupting the critical metastatic crosstalk between heterogeneous cell populations within a tumor.

Targeting PEA3 transcription factors to mitigate small cell lung cancer progression.

Small cell lung cancer (SCLC) remains a lethal disease with a dismal overall survival rate of 6% despite promising responses to upfront combination chemotherapy. The key drivers of such rapid mortality include early metastatic dissemination in the natural course of the disease and the near guaranteed emergence of chemoresistant disease. Here, we found that we could model the regression and relapse seen in clinical SCLC in vitro. We utilized time-course resolved RNA-sequencing to globally profile transcriptome changes as SCLC cells responded to a combination of cisplatin and etoposide-the standard-of-care in SCLC. Comparisons across time points demonstrated a distinct transient transcriptional state resembling embryonic diapause. Differential gene expression analysis revealed that expression of the PEA3 transcription factors ETV4 and ETV5 were transiently upregulated in the surviving fraction of cells which we determined to be necessary for efficient clonogenic expansion following chemotherapy. The FGFR-PEA3 signaling axis guided the identification of a pan-FGFR inhibitor demonstrating in vitro and in vivo efficacy in delaying progression following combination chemotherapy, observed inhibition of phosphorylation of the FGFR adaptor FRS2 and corresponding downstream MAPK and PI3K-Akt signaling pathways. Taken together, these data nominate PEA3 transcription factors as key mediators of relapse progression in SCLC and identify a clinically actionable small molecule candidate for delaying relapse of SCLC.

Primary and metastatic tumors exhibit systems-level differences in dependence on mitochondrial respiratory function.

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and processes such as tumor dissemination and metastatic growth remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines, we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells down-regulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial respiratory chain function impact in vivo tumor growth in a context-specific manner with differential impacts on primary and metastatic tumors.

Cardiomyocytes disrupt pyrimidine biosynthesis in nonmyocytes to regulate heart repair.

Various populations of cells are recruited to the heart after cardiac injury, but little is known about whether cardiomyocytes directly regulate heart repair. Using a murine model of ischemic cardiac injury, we demonstrate that cardiomyocytes play a pivotal role in heart repair by regulating nucleotide metabolism and fates of nonmyocytes. Cardiac injury induced the expression of the ectonucleotidase ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which hydrolyzes extracellular ATP to form AMP. In response to AMP, cardiomyocytes released adenine and specific ribonucleosides that disrupted pyrimidine biosynthesis at the orotidine monophosphate (OMP) synthesis step and induced genotoxic stress and p53-mediated cell death of cycling nonmyocytes. As nonmyocytes are critical for heart repair, we showed that rescue of pyrimidine biosynthesis by administration of uridine or by genetic targeting of the ENPP1/AMP pathway enhanced repair after cardiac injury. We identified ENPP1 inhibitors using small molecule screening and showed that systemic administration of an ENPP1 inhibitor after heart injury rescued pyrimidine biosynthesis in nonmyocyte cells and augmented cardiac repair and postinfarct heart function. These observations demonstrate that the cardiac muscle cell regulates pyrimidine metabolism in nonmuscle cells by releasing adenine and specific nucleosides after heart injury and provide insight into how intercellular regulation of pyrimidine biosynthesis can be targeted and monitored for augmenting tissue repair.

Plasticity of Extrachromosomal and Intrachromosomal BRAF Amplifications in Overcoming Targeted Therapy Dosage Challenges.

Focal amplifications (FA) can mediate targeted therapy resistance in cancer. Understanding the structure and dynamics of FAs is critical for designing treatments that overcome plasticity-mediated resistance. We developed a melanoma model of dual MAPK inhibitor (MAPKi) resistance that bears BRAFV600 amplifications through either extrachromosomal DNA (ecDNA)/double minutes (DM) or intrachromosomal homogenously staining regions (HSR). Cells harboring BRAFV600E FAs displayed mode switching between DMs and HSRs, from both de novo genetic changes and selection of preexisting subpopulations. Plasticity is not exclusive to ecDNAs, as cells harboring HSRs exhibit drug addiction-driven structural loss of BRAF amplicons upon dose reduction. FA mechanisms can couple with kinase domain duplications and alternative splicing to enhance resistance. Drug-responsive amplicon plasticity is observed in the clinic and can involve other MAPK pathway genes, such as RAF1 and NRAS. BRAF FA-mediated dual MAPKi-resistant cells are more sensitive to proferroptotic drugs, extending the spectrum of ferroptosis sensitivity in MAPKi resistance beyond cases of dedifferentiation. SIGNIFICANCE: Understanding the structure and dynamics of oncogene amplifications is critical for overcoming tumor relapse. BRAF amplifications are highly plastic under MAPKi dosage challenges in melanoma, through involvement of de novo genomic alterations, even in the HSR mode. Moreover, BRAF FA-driven, dual MAPKi-resistant cells extend the spectrum of resistance-linked ferroptosis sensitivity. This article is highlighted in the In This Issue feature, p. 873.

Melanoma dedifferentiation induced by IFN-γ epigenetic remodeling in response to anti-PD-1 therapy.

Melanoma dedifferentiation has been reported to be a state of cellular resistance to targeted therapies and immunotherapies as cancer cells revert to a more primitive cellular phenotype. Here, we show that, counterintuitively, the biopsies of patient tumors that responded to anti-programmed cell death 1 (anti-PD-1) therapy had decreased expression of melanocytic markers and increased neural crest markers, suggesting treatment-induced dedifferentiation. When modeling the effects in vitro, we documented that melanoma cell lines that were originally differentiated underwent a process of neural crest dedifferentiation when continuously exposed to IFN-γ, through global chromatin landscape changes that led to enrichment in specific hyperaccessible chromatin regions. The IFN-γ-induced dedifferentiation signature corresponded with improved outcomes in patients with melanoma, challenging the notion that neural crest dedifferentiation is entirely an adverse phenotype.

Metabolic reprogramming and epigenetic changes of vital organs in SARS-CoV-2-induced systemic toxicity.

Extrapulmonary manifestations of COVID-19 are associated with a much higher mortality rate than pulmonary manifestations. However, little is known about the pathogenesis of systemic complications of COVID-19. Here, we create a murine model of SARS-CoV-2-induced severe systemic toxicity and multiorgan involvement by expressing the human ACE2 transgene in multiple tissues via viral delivery, followed by systemic administration of SARS-CoV-2. The animals develop a profound phenotype within 7 days with severe weight loss, morbidity, and failure to thrive. We demonstrate that there is metabolic suppression of oxidative phosphorylation and the tricarboxylic acid (TCA) cycle in multiple organs with neutrophilia, lymphopenia, and splenic atrophy, mirroring human COVID-19 phenotypes. Animals had a significantly lower heart rate, and electron microscopy demonstrated myofibrillar disarray and myocardial edema, a common pathogenic cardiac phenotype in human COVID-19. We performed metabolomic profiling of peripheral blood and identified a panel of TCA cycle metabolites that served as biomarkers of depressed oxidative phosphorylation. Finally, we observed that SARS-CoV-2 induces epigenetic changes of DNA methylation, which affects expression of immune response genes and could, in part, contribute to COVID-19 pathogenesis. Our model suggests that SARS-CoV-2-induced metabolic reprogramming and epigenetic changes in internal organs could contribute to systemic toxicity and lethality in COVID-19.

Intracellular C4BPA Levels Regulate NF-κB-Dependent Apoptosis.

The importance of innate immunity in cancer is increasingly being recognized with recent reports suggesting tumor cell-intrinsic intracellular functions for innate immunity proteins. However, such functions are often poorly understood, and it is unclear whether these are affected by patient-specific mutations. Here, we show that C4b-binding protein alpha chain (C4BPA), typically thought to reside in the extracellular space, is expressed intracellularly in cancer cells, where it interacts with the NF-κB family member RelA and regulates apoptosis. Interestingly, intracellular C4BPA expression is regulated in a stress- and mutation-dependent manner and C4BPA mutations are associated with improved cancer survival outcome. Using cell lines harboring patient-specific C4BPA mutations, we show that increasing intracellular C4BPA levels correlate with sensitivity to oxaliplatin-induced apoptosis in vitro and in vivo. Mechanistically, sensitive C4BPA mutants display increased IκBα expression and increased inhibitory IκBα-RelA complex stability. These data suggest a non-canonical intracellular role for C4BPA in regulating NF-κB-dependent apoptosis.

Defining the ATPome reveals cross-optimization of metabolic pathways.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.

Thermodynamic energetics underlying genomic instability and whole-genome doubling in cancer.

Genomic instability contributes to tumorigenesis through the amplification and deletion of cancer driver genes. DNA copy number (CN) profiling of ensembles of tumors allows a thermodynamic analysis of the profile for each tumor. The free energy of the distribution of CNs is found to be a monotonically increasing function of the average chromosomal ploidy. The dependence is universal across several cancer types. Surprisal analysis distinguishes two main known subgroups: tumors with cells that have or have not undergone whole-genome duplication (WGD). The analysis uncovers that CN states having a narrower distribution are energetically more favorable toward the WGD transition. Surprisal analysis also determines the deviations from a fully stable-state distribution. These deviations reflect constraints imposed by tumor fitness selection pressures. The results point to CN changes that are more common in high-ploidy tumors and thus support altered selection pressures upon WGD.

Somatic copy number profiling from hepatocellular carcinoma circulating tumor cells.

Somatic copy number alterations (SCNAs) are important genetic drivers of many cancers. We investigated the feasibility of obtaining SCNA profiles from circulating tumor cells (CTCs) as a molecular liquid biopsy for hepatocellular carcinoma (HCC). CTCs from ten HCC patients underwent SCNA profiling. The Cancer Genome Atlas (TCGA) SCNA data were used to develop a cancer origin classification model, which was then evaluated for classifying 44 CTCs from multiple cancer types. Sequencing of 18 CTC samples (median: 4 CTCs/sample) from 10 HCC patients using a low-resolution whole-genome sequencing strategy (median: 0.88 million reads/sample) revealed frequent SCNAs in previously reported HCC regions such as 8q amplifications and 17p deletions. SCNA profiling revealed that CTCs share a median of 80% concordance with the primary tumor. CTCs had SCNAs not seen in the primary tumor, some with prognostic implications. Using a SCNA profiling model, the tissue of origin was correctly identified for 32/44 (73%) CTCs from 12/16 (75%) patients with different cancer types.

Integrin α6 mediates the drug resistance of acute lymphoblastic B-cell leukemia.

Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.