Imaging the master regulator of the antioxidant response in non-small cell lung cancer with positron emission tomography

Abstract

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. The cystine/glutamate antiporter, system x_c^-, is one of the >200 cytoprotective proteins controlled by NRF2, which can be non-invasively imaged by (S)-4-(3-¹⁸F-fluoropropyl)-l-glutamate ([¹⁸F]FSPG) positron emission tomography (PET). Through genetic and pharmacologic manipulation, we show that [¹⁸F]FSPG provides a sensitive and specific marker of NRF2 activation in advanced preclinical models of NSCLC. We validate imaging readouts with metabolomic measurements of system x_c^- activity and their coupling to intracellular glutathione concentration. A redox gene signature was measured in patients from the TRACERx 421 cohort, suggesting an opportunity for patient stratification prior to imaging. Furthermore, we reveal that system x_c^- is a metabolic vulnerability that can be therapeutically targeted for sustained tumour growth suppression in aggressive NSCLC. Our results establish [¹⁸F]FSPG as predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.

Figure 1.

Elevated NRF2 increases xCT expression, system x_c⁻ activity, and downstream antioxidant capacity, detectable by [¹⁸F]FSPG. a, Schematic of system x_c⁻ with its natural substrates cystine and glutamate, and the radiotracer [¹⁸F]FSPG (structure shown in insert). b, Protein expression of NRF2, xCT and NQO1 in a panel of NSCLC lines and corresponding KEAP1 mutations. Actin was used as a loading control. c, Cystine consumption in NSCLC lines following media replenishment. Cys₂, cystine. Intracellular glutamate (d) and GSH (e) in NSCLC lines. Flow cytometric measurement of total ROS levels using CellROX Green with representative histograms (f) and median fluorescent intensity (MFI; g) shown. h, Intracellular retention of [¹⁸F]FSPG. i, Correlation between intracellular GSH and intracellular [¹⁸F]FSPG accumulation. Broken lines represent the 95% confidence interval of the best fit line. Data are presented as mean ± SD. *, p < 0.05; ***, p < 0.001.

Figure 2.

[¹⁸F]FSPG retention is altered following pharmacological and genetic manipulation of NRF2. a, Chemical structure of KI696. b, Representative western blot of NRF2 and xCT expression in NRF2-low cell lines 24 h post treatment with vehicle control or 200 μM KI696. Actin was used as a loading control. c-f, Analysis of cystine (Cys₂) consumption (c), intracellular glutamate (d) and intracellular GSH (e) in NRF2-low lines following KI696 treatment compared to vehicle control. f, Intracellular [¹⁸F]FSPG retention in NRF2-low cells after KI696 treatment compared to vehicle control. g-j. Intracellular GSH (g,i) and [¹⁸F]FSPG retention (h,j) in genetically modified NSCLC cells. Data are presented as mean ± SD. *, p < 0.05; **; p < 0.01; ***, p < 0.001.

Figure 3.

[¹⁸F]FSPG PET can differentiate NRF2-high from NRF2-low tumours when grown orthotopically in the lungs of mice. a, Single slice CT axial images (top) and ex vivo H&E images (middle; scale bar = 5 mm) of lungs containing H1299 or H460 tumours, with xCT staining of corresponding tumours (bottom; scale bar = 50 μm). b, Representative in vivo [¹⁸F]FSPG PET/CT maximum intensity projections (MIPs; top) and axial single-slice PET/CT (bottom) of mice bearing H1299 or H460 orthotopic lung tumours. Dashed lines represent the tumour outline. c, Quantified [¹⁸F]FSPG retention in individual tumour lesions from orthotopic tumour-bearing mice. d, Representative western blot for xCT and NRF2 expression in H1299 and H460 orthotopically grown tumours (n = 4 per tumour). Actin was used as a loading control.

Figure 4.

[¹⁸F]FSPG retention is increased in Nrf2 mutant mice. a, Scheme depicting tumour formation in KP and KPN mice. KP mice conditionally express oncogenic Kras and have loss of p53 function. KPN mice conditionally express oncogenic Kras, have loss of p53 function and express a mutant Nrf2 which increases Nrf2 protein levels. b, CT MIP representing individual 3D tumour regions of interest. c, Total tumour volumes in the lungs of KP and KPN mice. Scatter plots represent individual animals. d, Representative coronal [¹⁸F]FSPG PET/CT images of 40–60 min summed activity in KP and KPN tumour-bearing mice. Dashed white lines indicate the lung. B, bladder; P, pancreas. e, Violin plots of [¹⁸F]FSPG tumour retention from individual lesions. n = 63–105 lesions from 5–6 mice per cohort. f, Nrf2 expression in KP and KPN tumour lesions. Actin was used as a loading control. g, Representative IHC staining of xCT from lesions taken from KP and KPN mice (scale bars = 20 μM). h, H-scores for xCT IHC staining. i, Heatmap depicting the H-scores for xCT IHC staining by tumour grade. AAH, adenomatous atypical hyperplasia; BH, bronchiolar hyperplasia. ***, p < 0.001.

Figure 5.

An antioxidant gene signature accompanies NRF2 mutations in patient tumours and patient derived xenograft (PDX) models, which is detectable by [¹⁸F]FSPG PET. a, Expression of NRF2-regulated genes in the TRACERx 421 patient cohort. b, Representative [¹⁸F]FSPG MIP of mice bearing PDXs either with (CRUK0772 R1) or without (CRUK0640 R8) a NRF2 mutation. c, Quantification of [¹⁸F]FSPG tumour retention (n = 9–11 mice/group). d, Post-imaging autoradiograms (ARG) from PDXs, illustrating the intratumoural heterogeneity of [¹⁸F]FSPG retention. Scale bar = 5 mm. e, xCT and NRF2 protein expression in PDX xenografts (n = 4 per tumour type). Actin was used as a loading control. f, GSH measurements from PDX tumours (n = 4). *, p < 0.05; ***, p < 0.001.

Figure 6.

An xCT-ADC controls tumour growth and prolongs survival of mice bearing H460 tumours. a, Structure of the anti-xCT tesirine conjugate, HM30-tesirine. b, Western blot using HM30-tesirine as the primary antibody in H1299 and H460 cell lysates. Actin was used as a loading control. c, HM30-tesirine MTT dose-response in H460 and H1299 cells. d, Schematic representation of HM30-tesirine treatment course in balb/c nu/nu mice. Antitumour activity (e) and survival benefit (f) of control (saline treated), cisplatin treated, and HM30-tesirine treated mice. For statistical analysis, a two-tailed t-test (for tumour volume) and a log-rank test (for survival curve) were used. To control the family-wise error rate in multiple comparisons, crude p-values were adjusted by the Holm–Bonferroni method. h, IHC for Ki67 and cleaved caspase 3 from FFPE tumours taken at endpoint. Scale bar, 50 μm. h, Corresponding quantification of tissue staining. Data are presented as the mean values ± SD (n = 3).