S100A8/A9 predicts response to PIM kinase and PD-1/PD-L1 inhibition in triple-negative breast cancer mouse models.

BACKGROUND: Understanding why some triple-negative breast cancer (TNBC) patients respond poorly to existing therapies while others respond well remains a challenge. This study aims to understand the potential underlying mechanisms distinguishing early-stage TNBC tumors that respond to clinical intervention from non-responders, as well as to identify clinically viable therapeutic strategies, specifically for TNBC patients who may not benefit from existing therapies. METHODS: We conducted retrospective bioinformatics analysis of historical gene expression datasets to identify a group of genes whose expression levels in early-stage tumors predict poor clinical outcomes in TNBC. In vitro small-molecule screening, genetic manipulation, and drug treatment in syngeneic mouse models of TNBC were utilized to investigate potential therapeutic strategies and elucidate mechanisms of drug action. RESULTS: Our bioinformatics analysis reveals a robust association between increased expression of immunosuppressive cytokine S100A8/A9 in early-stage tumors and subsequent disease progression in TNBC. A targeted small-molecule screen identifies PIM kinase inhibitors as capable of decreasing S100A8/A9 expression in multiple cell types, including TNBC and immunosuppressive myeloid cells. Combining PIM inhibition and immune checkpoint blockade induces significant antitumor responses, especially in otherwise resistant S100A8/A9-high PD-1/PD-L1-positive tumors. Notably, serum S100A8/A9 levels mirror those of tumor S100A8/A9 in a syngeneic mouse model of TNBC. CONCLUSIONS: Our data propose S100A8/A9 as a potential predictive and pharmacodynamic biomarker in clinical trials evaluating combination therapy targeting PIM and immune checkpoints in TNBC. This work encourages the development of S100A8/A9-based liquid biopsy tests for treatment guidance.

Breast cancer is a complex disease, and not all patients respond well to existing treatments. In this study, we sought to understand why some patients with a specific type of breast cancer called triple-negative breast cancer respond poorly to current therapies. We also aimed to identify new treatments for these patients. We analyzed genetic data from breast cancer patients and identified a factor called S100A8/A9, which is linked to poor outcomes in early-stage cancer. We tested drugs that can reduce the levels of this factor in tumors and found promising results, especially when combined with another treatment called immunotherapy. Our findings suggest that S100A8/A9 could help predict how patients will respond to treatments, potentially leading to better therapies in the future.

S100A8/A9 predicts triple-negative breast cancer response to PIM kinase and PD-1/PD-L1 inhibition.

It remains elusive why some triple-negative breast cancer (TNBC) patients respond poorly to existing therapies while others respond well. Our retrospective analysis of historical gene expression datasets reveals that increased expression of immunosuppressive cytokine S100A8/A9 in early-stage tumors is robustly associated with subsequent disease progression in TNBC. Although it has recently gained recognition as a potential anticancer target, S100A8/A9 has not been integrated into clinical study designs evaluating molecularly targeted therapies. Our small molecule screen has identified PIM kinase inhibitors as capable of decreasing S100A8/A9 expression in multiple cell types, including TNBC and immunosuppressive myeloid cells. Furthermore, combining PIM inhibition and immune checkpoint blockade induces significant antitumor responses, especially in otherwise resistant S100A8/A9-high PD-1/PD-L1-positive tumors. Importantly, serum S100A8/A9 levels mirror those of tumor S100A8/A9 in a syngeneic mouse model of TNBC. Thus, our data suggest that S100A8/A9 could be a predictive and pharmacodynamic biomarker in clinical trials evaluating combination therapy targeting PIM and immune checkpoints in TNBC and encourage the development of S100A8/A9-based liquid biopsy tests.

Oncogene-regulated release of extracellular vesicles.

Oncogenes can alter metabolism by changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic MCF10A cells transformed with nine different oncogenes, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle (EV) release. While MYC and AURKB elicited the highest number of EVs, each oncogene selectively altered the protein composition of released EVs. Likewise, oncogenes alter secreted miRNAs. MYC-overexpressing cells require ceramide, whereas AURKB requires ESCRT to release high levels of EVs. We identify an inverse relationship between MYC upregulation and activation of the RAS/MEK/ERK signaling pathway for regulating EV release in some tumor cells. Finally, lysosome genes and activity are downregulated in the context of MYC and AURKB, suggesting that cellular contents, instead of being degraded, were released via EVs. Thus, oncogene-mediated biomass regulation via differential EV release is a new metabolic phenotype.

A risk-associated Active transcriptome phenotype expressed by histologically normal human breast tissue and linked to a pro-tumorigenic adipocyte population.

BACKGROUND: Previous studies have identified and validated a risk-associated Active transcriptome phenotype commonly expressed in the cancer-adjacent and histologically normal epithelium, stroma, and adipose containing peritumor microenvironment of clinically established invasive breast cancers, conferring a 2.5- to 3-fold later risk of dying from recurrent breast cancer. Expression of this Active transcriptome phenotype has not yet been evaluated in normal breast tissue samples unassociated with any benign or malignant lesions; however, it has been associated with increased peritumor adipocyte composition. METHODS: Detailed histologic and transcriptomic (RNAseq) analyses were performed on normal breast biopsy samples from 151 healthy, parous, non-obese (mean BMI = 29.60 ± 7.92) women, ages 27-66 who donated core breast biopsy samples to the Komen Tissue Bank, and whose average breast cancer risk estimate (Gail score) at the time of biopsy (1.27 ± 1.34) would not qualify them for endocrine prevention therapy. RESULTS: Full genome RNA sequencing (RNAseq) identified 52% (78/151) of these normal breast samples as expressing the Active breast phenotype. While Active signature genes were found to be most variably expressed in mammary adipocytes, donors with the Active phenotype had no difference in BMI but significantly higher Gail scores (1.46 vs. 1.18; p = 0.007). Active breast samples possessed 1.6-fold more (~ 80%) adipocyte nuclei, larger cross-sectional adipocyte areas (p < 0.01), and 0.5-fold fewer stromal and epithelial cell nuclei (p < 1e-6). Infrequent low-level expression of cancer gene hotspot mutations was detected but not enriched in the Active breast samples. Active samples were enriched in gene sets associated with adipogenesis and fat metabolism (FDR q ≤ 10%), higher signature scores for cAMP-dependent lipolysis known to drive breast cancer progression, white adipose tissue browning (Wilcoxon p < 0.01), and genes associated with adipocyte activation (leptin, adiponectin) and remodeling (CAV1, BNIP3), adipokine growth factors (IGF-1, FGF2), and pro-inflammatory fat signaling (IKBKG, CCL13). CONCLUSIONS: The risk-associated Active transcriptome phenotype first identified in cancer-adjacent breast tissues also occurs commonly in healthy women without breast disease who do not qualify for breast cancer chemoprevention, and independently of breast expressed cancer-associated mutations. The risk-associated Active phenotype appears driven by a pro-tumorigenic adipocyte microenvironment that can predate breast cancer development.

MYC Dysregulates Mitosis, Revealing Cancer Vulnerabilities.

Tumors that overexpress the MYC oncogene are frequently aneuploid, a state associated with highly aggressive cancers and tumor evolution. However, how MYC causes aneuploidy is not well understood. Here, we show that MYC overexpression induces mitotic spindle assembly defects and chromosomal instability (CIN) through effects on microtubule nucleation and organization. Attenuating MYC expression reverses mitotic defects, even in established tumor cell lines, indicating an ongoing role for MYC in CIN. MYC reprograms mitotic gene expression, and we identify TPX2 to be permissive for spindle assembly in MYC-high cells. TPX2 depletion blocks mitotic progression, induces cell death, and prevents tumor growth. Further elevating TPX2 expression reduces mitotic defects in MYC-high cells. MYC and TPX2 expression may be useful biomarkers to stratify patients for anti-mitotic therapies. Our studies implicate MYC as a regulator of mitosis and suggest that blocking MYC activity can attenuate the emergence of CIN and tumor evolution.

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer.

Comprehensive analysis of normal adjacent to tumor transcriptomes.

Histologically normal tissue adjacent to the tumor (NAT) is commonly used as a control in cancer studies. However, little is known about the transcriptomic profile of NAT, how it is influenced by the tumor, and how the profile compares with non-tumor-bearing tissues. Here, we integrate data from the Genotype-Tissue Expression project and The Cancer Genome Atlas to comprehensively analyze the transcriptomes of healthy, NAT, and tumor tissues in 6506 samples across eight tissues and corresponding tumor types. Our analysis shows that NAT presents a unique intermediate state between healthy and tumor. Differential gene expression and protein-protein interaction analyses reveal altered pathways shared among NATs across tissue types. We characterize a set of 18 genes that are specifically activated in NATs. By applying pathway and tissue composition analyses, we suggest a pan-cancer mechanism of pro-inflammatory signals from the tumor stimulates an inflammatory response in the adjacent endothelium.

Cancer recurrence monitoring using hyperpolarized [1-13C]pyruvate metabolic imaging in murine breast cancer model.

The purpose of this work was to study the anatomic and metabolic changes that occur with tumor progression, regression and recurrence in a switchable MYC-driven murine breast cancer model. Serial 1H MRI and hyperpolarized [1-13C]pyruvate metabolic imaging were used to investigate the changes in tumor volume and glycolytic metabolism over time during the multistage tumorigenesis. We show that acute de-induction of MYC expression in established tumors results in rapid tumor regression and significantly reduced glycolytic metabolism as measured by pyruvate-to-lactate conversion. Moreover, cancer recurrences occurring at the tumor sites independently of MYC expression were observed to accompany markedly increased lactate production.

In vivo Reprogramming of Cancer Metabolism by MYC.

The past few decades have welcomed tremendous advancements toward understanding the functional significance of altered metabolism during tumorigenesis. However, many conclusions drawn from studies of cancer cells in a dish (i.e., in vitro) have been put into question as multiple lines of evidence have demonstrated that the metabolism of cells can differ significantly from that of primary tumors (in vivo). This realization, along with the need to identify tissue-specific vulnerabilities of driver oncogenes, has led to an increased focus on oncogene-dependent metabolic programming in vivo. The oncogene c-MYC (MYC) is overexpressed in a wide variety of human cancers, and while its ability to alter cellular metabolism is well-established, translating the metabolic requirements, and vulnerabilities of MYC-driven cancers to the clinic has been hindered by disparate findings from in vitro and in vivo models. This review will provide an overview of the in vivo strategies, mechanisms, and conclusions generated thus far by studying MYC's regulation of metabolism in various cancer models.

MYC-driven inhibition of the glutamate-cysteine ligase promotes glutathione depletion in liver cancer.

How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC-driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC-driven tumors. We find that fewer glutamine-derived carbons are incorporated into GSH in tumor tissue relative to non-tumor tissue. Expression of GCLC, the rate-limiting enzyme of GSH synthesis, is attenuated by the MYC-induced microRNA miR-18a. Inhibition of miR-18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC-driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC-dependent attenuation of GCLC by miR-18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine.

PIM1 kinase inhibition as a targeted therapy against triple-negative breast tumors with elevated MYC expression.

Triple-negative breast cancer (TNBC), in which cells lack expression of the estrogen receptor (ER), the progesterone receptor (PR) and the ERBB2 (also known as HER2) receptor, is the breast cancer subtype with the poorest outcome. No targeted therapy is available against this subtype of cancer owing to a lack of validated molecular targets. We previously reported that signaling involving MYC-an essential, pleiotropic transcription factor that regulates the expression of hundreds of genes-is disproportionally higher in triple-negative (TN) tumors than in receptor-positive (RP) tumors. Direct inhibition of the oncogenic transcriptional activity of MYC has been challenging to achieve. Here, by conducting a shRNA screen targeting the kinome, we identified PIM1, a non-essential serine-threonine kinase, in a synthetic lethal interaction with MYC. PIM1 expression was higher in TN tumors than in RP tumors and was associated with poor prognosis in patients with hormone- and HER2-negative tumors. Small-molecule PIM kinase inhibitors halted the growth of human TN tumors with elevated MYC expression in patient-derived tumor xenograft (PDX) and MYC-driven transgenic mouse models of breast cancer by inhibiting the oncogenic transcriptional activity of MYC and restoring the function of the endogenous cell cycle inhibitor, p27. Our findings warrant clinical evaluation of PIM kinase inhibitors in patients with TN tumors that have elevated MYC expression.

GSTP1 Is a Driver of Triple-Negative Breast Cancer Cell Metabolism and Pathogenicity.

Breast cancers possess fundamentally altered metabolism that fuels their pathogenicity. While many metabolic drivers of breast cancers have been identified, the metabolic pathways that mediate breast cancer malignancy and poor prognosis are less well understood. Here, we used a reactivity-based chemoproteomic platform to profile metabolic enzymes that are enriched in breast cancer cell types linked to poor prognosis, including triple-negative breast cancer (TNBC) cells and breast cancer cells that have undergone an epithelial-mesenchymal transition-like state of heightened malignancy. We identified glutathione S-transferase Pi 1 (GSTP1) as a novel TNBC target that controls cancer pathogenicity by regulating glycolytic and lipid metabolism, energetics, and oncogenic signaling pathways through a protein interaction that activates glyceraldehyde-3-phosphate dehydrogenase activity. We show that genetic or pharmacological inactivation of GSTP1 impairs cell survival and tumorigenesis in TNBC cells. We put forth GSTP1 inhibitors as a novel therapeutic strategy for combatting TNBCs through impairing key cancer metabolism and signaling pathways.

Inhibition of fatty acid oxidation as a therapy for MYC-overexpressing triple-negative breast cancer.

Expression of the oncogenic transcription factor MYC is disproportionately elevated in triple-negative breast cancer (TNBC), as compared to estrogen receptor-, progesterone receptor- or human epidermal growth factor 2 receptor-positive (RP) breast cancer. We and others have shown that MYC alters metabolism during tumorigenesis. However, the role of MYC in TNBC metabolism remains mostly unexplored. We hypothesized that MYC-dependent metabolic dysregulation is essential for the growth of MYC-overexpressing TNBC cells and may identify new therapeutic targets for this clinically challenging subset of breast cancer. Using a targeted metabolomics approach, we identified fatty acid oxidation (FAO) intermediates as being dramatically upregulated in a MYC-driven model of TNBC. We also identified a lipid metabolism gene signature in patients with TNBC that were identified from The Cancer Genome Atlas database and from multiple other clinical data sets, implicating FAO as a dysregulated pathway that is critical for TNBC cell metabolism. We found that pharmacologic inhibition of FAO catastrophically decreased energy metabolism in MYC-overexpressing TNBC cells and blocked tumor growth in a MYC-driven transgenic TNBC model and in a MYC-overexpressing TNBC patient-derived xenograft. These findings demonstrate that MYC-overexpressing TNBC shows an increased bioenergetic reliance on FAO and identify the inhibition of FAO as a potential therapeutic strategy for this subset of breast cancer.

Multiple breast cancer risk variants are associated with differential transcript isoform expression in tumors.

Genome-wide association studies have identified over 70 single-nucleotide polymorphisms (SNPs) associated with breast cancer. A subset of these SNPs are associated with quantitative expression of nearby genes, but the functional effects of the majority remain unknown. We hypothesized that some risk SNPs may regulate alternative splicing. Using RNA-sequencing data from breast tumors and germline genotypes from The Cancer Genome Atlas, we tested the association between each risk SNP genotype and exon-, exon-exon junction- or transcript-specific expression of nearby genes. Six SNPs were associated with differential transcript expression of seven nearby genes at FDR < 0.05 (BABAM1, DCLRE1B/PHTF1, PEX14, RAD51L1, SRGAP2D and STXBP4). We next developed a Bayesian approach to evaluate, for each SNP, the overlap between the signal of association with breast cancer and the signal of association with alternative splicing. At one locus (SRGAP2D), this method eliminated the possibility that the breast cancer risk and the alternate splicing event were due to the same causal SNP. Lastly, at two loci, we identified the likely causal SNP for the alternative splicing event, and at one, functionally validated the effect of that SNP on alternative splicing using a minigene reporter assay. Our results suggest that the regulation of differential transcript isoform expression is the functional mechanism of some breast cancer risk SNPs and that we can use these associations to identify causal SNPs, target genes and the specific transcripts that may mediate breast cancer risk.

Dengue virus induces and requires glycolysis for optimal replication.

UNLABELLED: Viruses rely on host cellular metabolism to provide the energy and biosynthetic building blocks required for their replication. Dengue virus (DENV), a member of the Flaviviridae family, is one of the most important arthropod-borne human pathogens worldwide. We analyzed global intracellular metabolic changes associated with DENV infection of primary human cells. Our metabolic profiling data suggested that central carbon metabolism, particularly glycolysis, is strikingly altered during a time course of DENV infection. Glucose consumption is increased during DENV infection and depriving DENV-infected cells of exogenous glucose had a pronounced impact on viral replication. Furthermore, the expression of both glucose transporter 1 and hexokinase 2, the first enzyme of glycolysis, is upregulated in DENV-infected cells. Pharmacologically inhibiting the glycolytic pathway dramatically reduced DENV RNA synthesis and infectious virion production, revealing a requirement for glycolysis during DENV infection. Thus, these experiments suggest that DENV induces the glycolytic pathway to support efficient viral replication. This study raises the possibility that metabolic inhibitors, such as those that target glycolysis, could be used to treat DENV infection in the future. IMPORTANCE: Approximately 400 million people are infected with dengue virus (DENV) annually, and more than one-third of the global population is at risk of infection. As there are currently no effective vaccines or specific antiviral therapies for DENV, we investigated the impact DENV has on the host cellular metabolome to identify metabolic pathways that are critical for the virus life cycle. We report an essential role for glycolysis during DENV infection. DENV activates the glycolytic pathway, and inhibition of glycolysis significantly blocks infectious DENV production. This study provides further evidence that viral metabolomic analyses can lead to the discovery of novel therapeutic targets to block the replication of medically important human pathogens.

Vaccinia virus requires glutamine but not glucose for efficient replication.

UNLABELLED: Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for successful viral replication. Vaccinia virus (VACV) is a member of the Poxviridae family, and its use as a vaccine enabled the eradication of variola virus, the etiologic agent of smallpox. A global metabolic screen of VACV-infected primary human foreskin fibroblasts suggested that glutamine metabolism is altered during infection. Glutamine and glucose represent the two main carbon sources for mammalian cells. Depriving VACV-infected cells of exogenous glutamine led to a substantial decrease in infectious virus production, whereas starving infected cells of exogenous glucose had no significant impact on replication. Viral yield in glutamine-deprived cells or in cells treated with an inhibitor of glutaminolysis, the pathway of glutamine catabolism, could be rescued by the addition of multiple tricarboxylic acid (TCA) cycle intermediates. Thus, VACV infection induces a metabolic alteration to fully rely on glutamine to anaplerotically maintain the TCA cycle. VACV protein synthesis, but not viral transcription, was decreased in glutamine-deprived cells, which corresponded with a dramatic reduction in all VACV morphogenetic intermediates. This study reveals the unique carbon utilization program implemented during poxvirus infection and provides a potential metabolic pathway to target viral replication. IMPORTANCE: Viruses are dependent on the metabolic machinery of the host cell to supply the energy and molecular building blocks needed for critical processes including genome replication, viral protein synthesis, and membrane production. This study investigates how vaccinia virus (VACV) infection alters global cellular metabolism, providing the first metabolomic analysis for a member of the poxvirus family. Unlike most viruses examined to date, VACV does not activate glycolysis, and exogenous glucose is not required for maximal virus production. Instead, VACV requires exogenous glutamine for efficient replication, and inhibition of glutamine metabolism effectively blocks VACV protein synthesis. This study defines a major metabolic perturbation essential for the replication of a poxvirus and may lead to the discovery of novel antiviral therapies based on metabolic inhibitors.

Global metabolic profiling of infection by an oncogenic virus: KSHV induces and requires lipogenesis for survival of latent infection.

Like cancer cells, virally infected cells have dramatically altered metabolic requirements. We analyzed global metabolic changes induced by latent infection with an oncogenic virus, Kaposi's Sarcoma-associated herpesvirus (KSHV). KSHV is the etiologic agent of Kaposi's Sarcoma (KS), the most common tumor of AIDS patients. Approximately one-third of the nearly 200 measured metabolites were altered following latent infection of endothelial cells by KSHV, including many metabolites of anabolic pathways common to most cancer cells. KSHV induced pathways that are commonly altered in cancer cells including glycolysis, the pentose phosphate pathway, amino acid production and fatty acid synthesis. Interestingly, over half of the detectable long chain fatty acids detected in our screen were significantly increased by latent KSHV infection. KSHV infection leads to the elevation of metabolites involved in the synthesis of fatty acids, not degradation from phospholipids, and leads to increased lipid droplet organelle formation in the infected cells. Fatty acid synthesis is required for the survival of latently infected endothelial cells, as inhibition of key enzymes in this pathway led to apoptosis of infected cells. Addition of palmitic acid to latently infected cells treated with a fatty acid synthesis inhibitor protected the cells from death indicating that the products of this pathway are essential. Our metabolomic analysis of KSHV-infected cells provides insight as to how oncogenic viruses can induce metabolic alterations common to cancer cells. Furthermore, this analysis raises the possibility that metabolic pathways may provide novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors.