Urea Cycle Sustains Cellular Energetics upon EGFR Inhibition in EGFR-Mutant NSCLC

Abstract

Mutations in oncogenes and tumor suppressor genes engender unique metabolic phenotypes crucial to the survival of tumor cells. EGFR signaling has been linked to the rewiring of tumor metabolism in non-small cell lung cancer (NSCLC). We have integrated the use of a functional genomics screen and metabolomics to identify metabolic vulnerabilities induced by EGFR inhibition. These studies reveal that following EGFR inhibition, EGFR-driven NSCLC cells become dependent on the urea cycle and, in particular, the urea cycle enzyme CPS1. Combining knockdown of CPS1 with EGFR inhibition further reduces cell proliferation and impedes cell-cycle progression. Profiling of the metabolome demonstrates that suppression of CPS1 potentiates the effects of EGFR inhibition on central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism, coinciding with reduced glycolysis and mitochondrial respiration. We show that EGFR inhibition and CPS1 knockdown lead to a decrease in arginine levels and pyrimidine derivatives, and the addition of exogenous pyrimidines partially rescues the impairment in cell growth. Finally, we show that high expression of CPS1 in lung adenocarcinomas correlated with worse patient prognosis in publicly available databases. These data collectively reveal that NSCLC cells have a greater dependency on the urea cycle to sustain central carbon metabolism, pyrimidine biosynthesis, and arginine metabolism to meet cellular energetics upon inhibition of EGFR. IMPLICATIONS: Our results reveal that the urea cycle may be a novel metabolic vulnerability in the context of EGFR inhibition, providing an opportunity to develop rational combination therapies with EGFR inhibitors for the treatment of EGFR-driven NSCLC.

Figure 1:. Metabolic screen uncovers CPS1 as a synthetic lethal hit with EGFR inhibition in NSCLC cells.

A. Experimental schematic of metabolic shRNA screen and data analyses. B. Diagram of urea cycle with synthetic lethal hits highlighted. NSCLC cells transduced with shRNA library were treated in replicates of 5 with vehicle (DMSO) or increasing concentrations of erlotinib (10 nM, 30 nM, 90 nM, 270 nM). Normalized shRNA counts represented across multiple doses of erlotinib are shown. C. Linear regression analysis of shRNA(s) targeting CPS1 in H1650, H322C, and PC9 NSCLC cell lines with p-values indicated. D. Linear regression analysis of shRNA(s) targeting ASS1 in H1650, H322C, and PC9 NSCLC cell lines with p-values indicated.

Figure 2:. Inhibition of EGFR and CPS1 knockdown leads to combinatorial inhibition of NSCLC cells.

A. HCC4006 and PC9 cells expressing either non-targeting control shRNA (shNT) or shRNAs targeting CPS1 (CPS1–4, CPS1–5) were treated in triplicate with vehicle (DMSO) or increasing concentrations (30, 60, 90 nM) of erlotinib (ERL) for 3 days, followed by replating without the presence of drug for 3 additional days. The number of viable cells was determined by flow cytometry using PI-exclusion. Statistical comparison of shNT to shCPS1 with and without each erlotinib dose is shown (similar comparisons are made for other drug treatments below). B. HCC4006 and PC9 cells expressing shNT or shCPS1 were treated in triplicate with DMSO or erlotinib (50 nM and 100 nM) for 3 days, and then replated without drug for colony forming assays. C. HFF cells expressing shNT or shCPS1 were plated for 3 days. Viable cells were counted by flow cytometry. D. H3122 cells transduced with shNT or shCPS1 were treated in triplicate with vehicle (DMS0) or crizotinib (100, 500, 1000 nM) for 3 days, followed by replating without the presence of drug for 3 more days. Viable cells were determined by flow cytometry. E. H3122 cells expressing shNT or shCPS1 were treated with vehicle or 500 nM crizotinib for 3 days in triplicate and then replated without drug for colony forming assays. F. HCC4006 expressing shNT or shCPS1 were treated with vehicle (DMSO) or increasing concentrations of doxorubicin (250 nM, 500 nM, 1000 nM) for 3 days, followed by replating without drug for 3 more days. Viable cells were counted by flow cytometry. (ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001)

Figure 3:. Combined EGFR inhibition and CPS1 knockdown leads to a decrease in central carbon metabolism and to G1 arrest.

A. HCC4006 and PC9 expressing shNT or shCPS1 cells were treated with vehicle (DMSO) or 100 nM erlotinib for 3 days in triplicate and apoptosis was measured by Annexin V staining by flow cytometry. B. HCC4006 and PC9 expressing shNT or shCPS1 cells were treated with vehicle (DMSO) or 100 nM erlotinib for 24h in triplicate and stained with PI for cell cycle analysis. Full cell cycle panels are shown with smaller panels depicting G and S phases below. HCC4006 and PC9 expressing shNT or shCPS1 cells were plated on microplates and treated with vehicle (DMSO) or 100 nM erlotinib for 24 hours. C. Extracellular acidification rate or glycolysis rate was measured at indicated time points and 95% confidence intervals were calculated for the area under the curve for each condition. D. Oligomycin-A (OA), FCCP, rotenone (Rot)/ antimycin A (Anti) were sequentially added and the oxygen consumption rate measured with Agilent Seahorse instrument. The 95% confidence intervals were calculated for the area under the curve for each condition across the course of the experiment. E. Basal respiration, maximal respiration and ATP production were determined for each condition. (ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Figure 4.. Metabolites involved in carbon metabolism are decreased by CPS1 knockdown with further exacerbation upon EGFR inhibition.

HCC4006 cells were treated with vehicle (DMSO) or 100 nM erlotinib for 22 hours in replicates of three. Cell extracts were processed for metabolic profiling using UPLC-MS/MS. A. Levels of glycolysis metabolites B. Levels of TCA cycle intermediates. ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 for comparisons of all samples to vehicle-treated control shNT cells, and pound signs indicate statistical significance (#P ≤ 0.05, ##P ≤ 0.01, ###P ≤ 0.001) for comparisons of CPS1 knockdown cells to control shNT cells under the same treatment conditions.

Figure 5:. Pyrimidine biosynthesis metabolites are reduced by combined CPS1 knockdown cells with erlotinib treatment and exogenous pyrimidines can partially rescue cell proliferation.

HCC4006 cells were treated with erlotinib for vehicle (DMSO) or 100 nM erlotinib for 22 and were processed using UPLC-MS/MS for metabolic profiling as in figure 4 A. Table showing the TOP ten metabolites identified using ANOVA. B. Pathway analysis performed showing the most affected metabolic pathways based on the top 25 significantly changed metabolites. C. Pyrimidine metabolite levels in HCC4006 cell treated with 100 nM erlotinib for 22 hours. D. HCC4006 shNT control or shCPS1 cells treated in triplicate with vehicle (DMSO), 100 nM erlotinib, 1 mM thymidine or thymidine and erlotinib for 6 days. Cell viability was determined by flow cytometry using PI. E. As in D, but with vehicle (DMSO), 100 nM erlotinib, 1 mM uridine or uridine and erlotinib for 6 days. Cell viability was determined by flow cytometry using PI. (ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Figure 6:. Arginine metabolites are decreased with combined CPS1 knockdown and EGFR inhibition.

A. Arginine metabolite levels in HCC4006 cells treated with 100 nM erlotinib for 22 hours B. HCC4006 expressing shNT control or shASL1 treated with vehicle (DMSO) or increasing dose of erlotinib (50 nM and 100 nM) in triplicate for 3 days, followed by 3 days without drug. Cell viability was determined by flow cytometry. C. PC9 expressing shNT control or shASL1 were treated with vehicle (DMSO) or increasing dose of erlotinib (50 nM and 100 nM) in triplicate for 3 days, followed by 3 days without drug. Cell viability was determined by flow cytometry. (ANOVA; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001).

Figure 7:. CPS1 expression is high in LADC and correlated with worse overall patient survival.

A. Oncomine analysis of CPS1 expression in fetal lung, normal lung, and lung adenocarcinoma. B. Oncomine analysis of CPS1 expression across cancers (Legend in Supplemental Table 5) with LADC highlighted (Group 17). C. High and low CPS1 expression correlated with overall survival probability in LADC patients across all stages. D. High and low CPS1 expression correlated with overall survival probability in LADC patients in stages 1/2. E. High and low CPS1 expression correlated with overall survival probability in LADC patients in stages 3/4. F. Overall survival probability for smoker and never smoker patients with LADC exhibiting high or low CPS1 expression.