Mesenchymal phenotype predisposes lung cancer cells to impaired proliferation and redox stress in response to glutaminase inhibition

Recent work has highlighted glutaminase (GLS) as a key player in cancer cell metabolism, providing glutamine-derived carbon and nitrogen to pathways that support proliferation. There is significant interest in targeting GLS for cancer therapy, although the gene is not known to be mutated or amplified in tumors. As a result, identification of tractable markers that predict GLS dependence is needed for translation of GLS inhibitors to the clinic. Herein we validate a small molecule inhibitor of GLS and show that non-small cell lung cancer cells marked by low E-cadherin and high vimentin expression, hallmarks of a mesenchymal phenotype, are particularly sensitive to inhibition of the enzyme. Furthermore, lung cancer cells induced to undergo epithelial to mesenchymal transition (EMT) acquire sensitivity to the GLS inhibitor. Metabolic studies suggest that the mesenchymal cells have a reduced capacity for oxidative phosphorylation and increased susceptibility to oxidative stress, rendering them unable to cope with the perturbations induced by GLS inhibition. These findings elucidate selective metabolic dependencies of mesenchymal lung cancer cells and suggest novel pathways as potential targets in this aggressive cancer type

Loss of Par-1a/MARK3/C-TAK1 Kinase Leads to Reduced Adiposity, Resistance to Hepatic Steatosis, and Defective Gluconeogenesis ▿

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice

Cell line panel screen and validation of BPTES as an on-target tool compound.

<p>(A) Sensitivity of a panel of NSCLC lines to inhibition of growth by 10 µM BPTES in a 72 hr proliferation assay. Growth rates (mu) plotted relative to DMSO control for each cell line (mu/max). (B,C) A427 parent cells or cells stably expressing an empty vector control (Con), wild-type GAC (GAC), or a BPTES-resistant GAC enzyme (GAC-BR) were treated with the indicated concentrations of BPTES (A) or the inactive BPTES analogue, AGX-4769 (C), in a 72 hr proliferation assay. Results are representative of three independent experiments with mean and standard deviation indicated. (D,E) Measurement of isotopomer labeled ¹³C(5)-Glu (D) or ¹³C(4)-Asp (E) from cells treated for 4 hr with BPTES or with inactive analogue AGX-4769. Results are the mean of three replicates with the standard deviation indicated. Calculated p-values from student t-test, ^*(3×10⁻⁸), ^**(10⁻⁹), ^#(10⁻⁷), ^##(2×10⁻⁶).</p

Metabolic profiling of epithelial and mesenchymal cells and effects of GLS inhibition.

<p>NCI-H358 epithelial (E) and mesenchymal (M) lines were treated with DMSO or 8 µM BPTES for 20 hrs. Cells were either unlabeled or U-¹³C-Glc or U-¹³C-Gln labels were included for the last 4 hrs of drug treatment and levels of central carbon metabolites were measured by LCMS. (A) Inhibition of GLS activity by BPTES. (B) Percentage of citrate pool that is either unlabeled or containing carbon derived from U-¹³C-Gln (DMSO treated cells). (C) TCA metabolite and glutathione pool sizes +/− BPTES. Data was collected in triplicate or quadruplicate from 3 independent experiments (for α-KG and citrate) and depicted as mean levels +/−SD relative to NCI-H358 DMSO treated cells; values were normalized for cell number; <i>P</i> =  *1×10⁻⁵; **4×10⁻⁶; ***9×10⁻⁸ (D) Citrate isotopomer percentages from ¹³C(6)-Glc labeled cells treated +/− BPTES. *<i>P</i> = 0.001, **<i>P</i> = 0.002 comparing % citrate m+2 in DMSO or BPTES treated E vs M cells, respectively. (E) Alterations in relative ratios of DHAP and 3PG levels in EvsM cells +/−BPTES. <i>P</i> values for comparison of metabolite levels +/−BPTES: *0.1, **0.57, ^#0.04, ^##0.07. Results representative of 2 independent experiments.</p