A murine lung cancer co-clinical trial identifies genetic modifiers of therapeutic response

Abstract

Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors, has not been fully explored. Here we use genetically engineered mouse models to conduct a 'co-clinical' trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244) increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

Figure 1. Docetaxel and selumetinib combination therapy is more efficacious than docetaxel monotherapy in Kras and Kras/p53 lung cancers

a, Waterfall plot showing tumour response after 2 weeks of docetaxel treatment at 16 mg kg⁻¹ every 2 days. Each column represents one individual mouse, with data expressed relative to the pre-treatment tumour volume. b, Waterfall plot showing tumour response after 2 weeks of docetaxel treatment at 16 mg kg⁻¹ every 2 days in combination with daily selumetinib at 25 mg kg⁻¹. c, Response rate of docetaxel and selumetinib combination therapy and docetaxel only in mice bearing tumours with different genotypes. d, Box plot showing tumour response for different genotypes with either docetaxel monotherapy (D) or combination treatment (DS). Lines depict median response, small circles indicate outliers. Estimated magnitude of difference between single and combination treatment within each genotype and corresponding one-sided P values obtained by likelihood ratio test. e, f, Number of activated caspase-3 (e) and Ki-67 (f) positive cells per microscopic field in mice of different genotypes after short-term treatment. Data represent the average of 5 different fields ± standard deviation (s.d.) from 1–3 different mice (see Supplementary Table 6 for detailed information).

Figure 2. FDG-PET predicts treatment response

a, FDG-PET signal intensity (SUV_max) in Kras, Kras/p53 and Kras/Lkb1 mutant mice. Statistical significance determined by rank sum test, with *P <0.05 for Kras compared to Kras/p53 mutant mice (P =0.019), and Kras compared to Kras/Lkb1 mutant mice (P =0.014). b, FDG-PET signal intensity in patients with KRAS or KRAS/LKB1 mutant tumours. Statistical significance determined by two-sided Wilcoxon with *P = 0.048. c, Comparisons of changes in FDG uptake by PET imaging after 1 day of treatment. Data are represented as mean ± s.d. d, Representative FDG-PET/CT images of mice from different genotypes at baseline and 1 day after initiation of treatment. For each animal, the baseline and post-treatment (post-Rx) PET images are depicted with identical scales. The pseudocoloured FDG-PET images are fused with the grey-scale cross-sectional CT images.

Figure 3. Modulation of the MEK–ERK pathway in response to treatment is different across the three genotypes

a, Immunostaining of phospho-ERK before and after treatment with docetaxel alone or in combination with selumetinib. Scale bar, 50 μm. b, Pathology score of phospho-ERK staining of mouse tumours shown in a. Both percentage of phospho-ERK positive cells and average intensity of phospho-ERK staining were scored for individual nodules, with a composite score derived by multiplying phospho-ERK positive percentage and average intensity. All samples were stained in the same batch. D, docetaxel; DS, docetaxel plus selumetinib; No Rx, untreated. c, Mice were subjected to treatment (two doses in 24 h) as indicated, and killed 3 h after the second dose. Western blot was used to analyse tumour lysates with the indicated antibodies. d, Human NSCLC patients grouped by mutation status as indicated in the first column. Mean phospho-ERK (pERK) score from immunostaining is shown for each subset.

Figure 4. Long-term treatment outcome in Kras and Kras/p53 mice

a, Tumour volume was longitudinally assessed by MRI imaging in Kras and Kras/p53 mice treated with either docetaxel or docetaxel plus selumetinib. Data points represent median tumour volume relative to start of treatment for all available data at the indicated time point. b, Progression-free survival for Kras mice treated with either docetaxel or docetaxel plus selumetinib. Median survival for single and combination treatments was 6 weeks and 12 weeks respectively, with ***P =0.0003 by log-rank test. c, Progression-free survival for Kras/p53 mice treated with either docetaxel or docetaxel plus selumetinib. Median survival for single and combination treatments was 2 weeks and 4 weeks, respectively, with ***P <0.0001 by log-rank test. Progression was defined as the time point when total tumour volume exceeded the baseline volume. d, Immunostaining of activation-specific phospho-ERK of tumours from Kras/p53 and Kras mice with acquired resistance to docetaxel and selumetinib treatment.