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

JAK2/IDH-mutant–driven myeloproliferative neoplasm is sensitive to combined targeted inhibition

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617Fand mutant IDH1R132Hor Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617FIdh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mutand IDH2mutmutations. Taken together, these data suggest that combined JAK and IDH inhibition May offer a therapeutic advantage in this high-risk MPN subtype.Damon Runyon Cancer Research Foundation (DRG-2241-15)Howard Hughes Medical Institute (Faculty Scholars Award)Stand Up To CancerNational Cancer Institute (U.S.) (P50CA165962)National Cancer Institute (U.S.) (P30CA14051)Koch Institute for Integrative Cancer Research ( Dana-Farber Harvard Cancer Center Bridge Project)Leukemia & Lymphoma Society of America. Specialized Center of Research (SCOR) ProgramNational Institutes of Health (U.S.) (grant U54OD020355-01)National Institutes of Health (U.S.) (grant NCI R01CA172636)National Institutes of Health (U.S.) (grant R35CA197594)National Cancer Institute (U.S.) (Cancer Center Support Grant (P30 CA008747)

Association between EMT-related markers and BPTES sensitivity in NSCLC lines.

<p>(A) GeneGo analysis indicating four top-ranking mesenchymal signatures correlating with BPTES sensitivity. P-values are calculated within the GeneGo software using the hypergeometric distribution. (B) Anti-correlation between high E-cadherin low vimentin RNA expression levels and sensitivity to BPTES. (C) Mu/mumax values depicted following 72 hr treatment of NSCLC lines with 10 µM BPTES. Equal protein amounts of tumor cell line extracts were electrophoresed, with the group of more sensitive lines on the left and least sensitive on the right, and immunoblotted for the indicated proteins. Histone H3 was used as a loading control. (D) Quantitation of average GAC protein levels relative to PC protein expression (+/−SEM) in BPTES sensitive (n = 7), intermediate (n = 7), and insensitive (n = 7) lines. <i>P</i> = 0.03 comparing GAC/PC protein levels in sensitive compared to insensitive lines; <i>P</i> = 0.06 comparing sensitive to intermediate groups (unpaired 2-tail t-test).</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

EMT drives GLS1 dependence in two independent NSCLC models.

<p>(<b>A</b>) A427 cells were transfected with non-targeting (NT) or β-catenin targeting siRNAs and treated with the indicated concentrations of BPTES (left panel). Growth rates were normalized to DMSO treated cells transfected with NT siRNAs. The right panel depicts the effects of the β-catenin siRNAs on growth of DMSO treated cells. <b>(B</b>) Immunoblot of A427 protein extracts collected from cells 72 hrs post transfection. (<b>C</b>) NCI-H358 cells were treated with 25ng/ml TGF-ß for 4 wks. Induction of EMT was confirmed by the loss of E-cadherin and gain of vimentin. EMT was accompanied by an increase in GAC relative to PC protein levels (results representative of 2 independent experiments). Actin levels indicated for protein normalization. (<b>D</b>) NCI-H358 parental and 6wk TGF-ß treated cells were treated in triplicate with BPTES for 72 hrs and cell growth assessed by CTG. Results are representative of 3 independent experiments and are plotted as average growth rates (+/−SD) compared to DMSO treated cells.</p

Oxygen consumption rates differentially altered upon drug perturbation in sensitive compared to insensitive cells.

<p>(A) NCI-H358 epithelial and mesenchymal lines or (B) NCI-H1703 (BPTES sensitive) and NCI-H1563 (BPTES insensitive) cells were treated with DMSO or 8 µM BPTES and oxygen consumption rate (OCR) was monitored over a 20 hr treatment. Data normalized to OCR at time zero (100%) and presented as mean values +/−SEM. <i>P</i> = 0.024 comparing changes in oxygen consumption with BPTES in the epithelial vs mesenchymal line; <i>P</i> = 0.00017 comparing NCI-H1703 and NCI-H1563 cells. (C) OCR following treatment with the indicated drugs to perturb mitochondrial respiration. Data normalized to OCR measurement prior to oligomycin treatment. (D) Addition of pyruvate to media restores the impaired FCCP response in the mesenchymal line. Average values +/−SEM indicated. Results representative of 2–3 independent experiments.</p

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