Chemical genomics with pyrvinium identifies C1orf115 as a regulator of drug efflux.

Pyrvinium is a quinoline-derived cyanine dye and an approved anti-helminthic drug reported to inhibit WNT signaling and have anti-proliferative effects in various cancer cell lines. To further understand the mechanism by which pyrvinium is cytotoxic, we conducted a pooled genome-wide CRISPR loss-of-function screen in the human HAP1 cell model. The top drug-gene sensitizer interactions implicated the malate-aspartate and glycerol-3-phosphate shuttles as mediators of cytotoxicity to mitochondrial complex I inhibition including pyrvinium. By contrast, perturbation of the poorly characterized gene C1orf115/RDD1 resulted in strong resistance to the cytotoxic effects of pyrvinium through dysregulation of the major drug efflux pump ABCB1/MDR1. Interestingly, C1orf115/RDD1 was found to physically associate with ABCB1/MDR1 through proximity-labeling experiments and perturbation of C1orf115 led to mis-localization of ABCB1/MDR1. Our results are consistent with a model whereby C1orf115 modulates drug efflux through regulation of the major drug exporter ABCB1/MDR1.

Glutaminase inhibition impairs CD8 T cell activation in STK11-/Lkb1-deficient lung cancer.

The tumor microenvironment (TME) contains a rich source of nutrients that sustains cell growth and facilitate tumor development. Glucose and glutamine in the TME are essential for the development and activation of effector T cells that exert antitumor function. Immunotherapy unleashes T cell antitumor function, and although many solid tumors respond well, a significant proportion of patients do not benefit. In patients with KRAS-mutant lung adenocarcinoma, KEAP1 and STK11/Lkb1 co-mutations are associated with impaired response to immunotherapy. To investigate the metabolic and immune microenvironment of KRAS-mutant lung adenocarcinoma, we generated murine models that reflect the KEAP1 and STK11/Lkb1 mutational landscape in these patients. Here, we show increased glutamate abundance in the Lkb1-deficient TME associated with CD8 T cell activation in response to anti-PD1. Combination treatment with the glutaminase inhibitor CB-839 inhibited clonal expansion and activation of CD8 T cells. Thus, glutaminase inhibition negatively impacts CD8 T cells activated by anti-PD1 immunotherapy.

Preclinical toxicology profile of squalene epoxidase inhibitors.

Small cell lung cancer (SCLC) is a particularly aggressive subset of lung cancer, and identification of new therapeutic options is of significant interest. We recently reported that SCLC cell lines display a specific vulnerability to inhibition of squalene epoxidase (SQLE), an enzyme in the cholesterol biosynthetic pathway that catalyzes the conversion of squalene to 2,3-oxidosqualene. Since it has been reported that SQLE inhibition can result in dermatitis in dogs, we conducted a series of experiments to determine if SQLE inhibitors would be tolerated at exposures predicted to drive maximal efficacy in SCLC tumors. Detailed profiling of the SQLE inhibitor NB-598 showed that dogs did not tolerate predicted efficacious exposures, with dose-limiting toxicity due to gastrointestinal clinical observations, although skin toxicities were also observed. To extend these studies, two SQLE inhibitors, NB-598 and Cmpd-4″, and their structurally inactive analogs, NB-598.ia and Cmpd-4″.ia, were profiled in monkeys. While both active SQLE inhibitors resulted in dose-limiting gastrointestinal toxicity, the structurally similar inactive analogs did not. Collectively, our data demonstrate that significant toxicities arise at exposures well below the predicted levels needed for anti-tumor activity. The on-target nature of the toxicities identified is likely to limit the potential therapeutic utility of SQLE inhibition for the treatment of SCLC.

Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

Structure and inhibition mechanism of the catalytic domain of human squalene epoxidase.

Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3(S)-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors.

A chemical biology screen identifies a vulnerability of neuroendocrine cancer cells to SQLE inhibition.

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.

Corrigendum: mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

This corrects the article DOI: 10.1038/nature22964.

EGFR signaling promotes inflammation and cancer stem-like activity in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most lethal and aggressive type of breast cancer, with a strong proclivity to metastasize, and IBC-specific targeted therapies have not yet been developed. Epidermal growth factor receptor (EGFR) has emerged as an important therapeutic target in IBC. However, the mechanism behind the therapeutic effect of EGFR targeted therapy is not well defined. Here, we report that EGFR regulates the IBC cell population that expresses cancer stem-like cell (CSC) markers through COX-2, a key mediator of inflammation whose expression correlates with worse outcome in IBC. The COX-2 pathway promoted IBC cell migration and invasion and the CSC marker-bearing population in vitro, and the inhibition of this pathway reduced IBC tumor growth in vivo. Mechanistically, we identified Nodal, a member of the TGFß superfamily, as a potential driver of COX-2-regulated invasive capacity and the CSC phenotype of IBC cells. Our data indicate that the EGFR pathway regulates the expression of COX-2, which in turn regulates the expression of Nodal and the activation of Nodal signaling. Together, our findings demonstrate a novel connection between the EGFR/COX-2/Nodal signaling axis and CSC regulation in IBC, which has potential implications for new combination approaches with EGFR targeted therapy for patients with IBC.

mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer.

Activation of the PTEN-PI3K-mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

Differential Aspartate Usage Identifies a Subset of Cancer Cells Particularly Dependent on OGDH.

Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.

Changes in cancer cell metabolism revealed by direct sample analysis with MALDI mass spectrometry.

Biomarker discovery using mass spectrometry (MS) has recently seen a significant increase in applications, mainly driven by the rapidly advancing field of metabolomics. Instrumental and data handling advancements have allowed for untargeted metabolite analyses which simultaneously interrogate multiple biochemical pathways to elucidate disease phenotypes and therapeutic mechanisms. Although most MS-based metabolomic approaches are coupled with liquid chromatography, a few recently published studies used matrix-assisted laser desorption (MALDI), allowing for rapid and direct sample analysis with minimal sample preparation. We and others have reported that prostaglandin E3 (PGE3), derived from COX-2 metabolism of the omega-3 fatty acid eicosapentaenoic acid (EPA), inhibited the proliferation of human lung, colon and pancreatic cancer cells. However, how PGE3 metabolism is regulated in cancer cells, particularly human non-small cell lung cancer (NSCLC) cells, is not fully understood. Here, we successfully used MALDI to identify differences in lipid metabolism between two human non-small-cell lung cancer (NSCLC) cell lines, A549 and H596, which could contribute to their differential response to EPA treatment. Analysis by MALDI-MS showed that the level of EPA incorporated into phospholipids in H596 cells was 4-fold higher than A549 cells. Intriguingly, H596 cells produced much less PGE3 than A549 cells even though the expression of COX-2 was similar in these two cell lines. This appears to be due to the relatively lower expression of cytosolic phospholipase A2 (cPLA2) in H596 cells than that of A549 cells. Additionally, the MALDI-MS approach was successfully used on tumor tissue extracts from a K-ras transgenic mouse model of lung cancer to enhance our understanding of the mechanism of action of EPA in the in vivo model. These results highlight the utility of combining a metabolomics workflow with MALDI-MS to identify the biomarkers that may regulate the metabolism of omega-3 fatty acids and ultimately affect their therapeutic potentials.

Quantitative MALDI tandem mass spectrometric imaging of cocaine from brain tissue with a deuterated internal standard.

Mass spectrometric imaging (MSI) is an analytical technique used to determine the distribution of individual analytes within a given sample. A wide array of analytes and samples can be investigated by MSI, including drug distribution in rats, lipid analysis from brain tissue, protein differentiation in tumors, and plant metabolite distributions. Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique capable of desorbing and ionizing a large range of compounds, and it is the most common ionization source used in MSI. MALDI mass spectrometry (MS) is generally considered to be a qualitative analytical technique because of significant ion-signal variability. Consequently, MSI is also thought to be a qualitative technique because of the quantitative limitations of MALDI coupled with the homogeneity of tissue sections inherent in an MSI experiment. Thus, conclusions based on MS images are often limited by the inability to correlate ion signal increases with actual concentration increases. Here, we report a quantitative MSI method for the analysis of cocaine (COC) from brain tissue using a deuterated internal standard (COC-d(3)) combined with wide-isolation MS/MS for analysis of the tissue extracts with scan-by-scan COC-to-COC-d(3) normalization. This resulted in significant improvements in signal reproducibility and calibration curve linearity. Quantitative results from the MSI experiments were compared with quantitative results from liquid chromatography (LC)-MS/MS results from brain tissue extracts. Two different quantitative MSI techniques (standard addition and external calibration) produced quantitative results comparable to LC-MS/MS data. Tissue extracts were also analyzed by MALDI wide-isolation MS/MS, and quantitative results were nearly identical to those from LC-MS/MS. These results clearly demonstrate the necessity for an internal standard for quantitative MSI experiments.