Naproxen chemoprevention promotes immune activation in Lynch syndrome colorectal mucosa

Abstract

Objective: Patients with Lynch syndrome (LS) are at markedly increased risk for colorectal cancer. It is being increasingly recognised that the immune system plays an essential role in LS tumour development, thus making an ideal target for cancer prevention. Our objective was to evaluate the safety, assess the activity and discover novel molecular pathways involved in the activity of naproxen as primary and secondary chemoprevention in patients with LS.

Design: We conducted a Phase Ib, placebo-controlled, randomised clinical trial of two dose levels of naproxen sodium (440 and 220 mg) administered daily for 6 months to 80 participants with LS, and a co-clinical trial using a genetically engineered mouse model of LS and patient-derived organoids (PDOs).

Results: Overall, the total number of adverse events was not different across treatment arms with excellent tolerance of the intervention. The level of prostaglandin E2 in the colorectal mucosa was significantly decreased after treatment with naproxen when compared with placebo. Naproxen activated different resident immune cell types without any increase in lymphoid cellularity, and changed the expression patterns of the intestinal crypt towards epithelial differentiation and stem cell regulation. Naproxen demonstrated robust chemopreventive activity in a mouse co-clinical trial and gene expression profiles induced by naproxen in humans showed perfect discrimination of mice specimens with LS and PDOs treated with naproxen and control.

Conclusions: Naproxen is a promising strategy for immune interception in LS. We have discovered naproxen-induced gene expression profiles for their potential use as predictive biomarkers of drug activity.

Trial registration number: gov Identifier: NCT02052908.

Keywords: HNPCC syndrome; cancer syndromes; chemoprevention; gene expression; non-steroidal anti-inflammatory drugs.

Conflict of interest statement

Competing interests: JS has a consulting role with Janssen Research and Development, and Cancer Prevention Pharmaceuticals. IW has an advisory role with Genentech/Roche, Bayer, Bristol-Myers Squibb, AstraZeneca/Medimmune, Pfizer, HTG Molecular, Asuragen, Merck, GlaxoSmithKline, Guardant Health and MSD, has received speaker fees from Medscape, MSD, Genentech/Roche, Pfizer and received research support from Genentech, Oncoplex, HTG Molecular, DepArray, Merck, Bristol-Myers Squibb, Medimmune, Adaptive, Adaptimmune, EMD Serono, Pfizer, Takeda, Amgen, Karus, Johnson & Johnson, Bayer, Iovance, 4D, Novartis, and Akoya. SL and EV are co-principal investigators in an NIH/NCI U01 award with co-investigators employed by Nouscom, s.r.l. EV has a consulting and advisory role with Janssen Research and Development and Recursion Pharma.

© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Flow diagram of participants in the ‘Phase Ib biomarker trial of naproxen in patients at risk for DNA mismatch repair-deficient colorectal cancer’.

Figure 2

(A) Overall design and dataset. Human PGs and naproxen levels, pathology analysis of colorectal biopsies and mRNA-seq data were assessed in the context of the ‘Naproxen trial’ (NCT02052908), where 80 patients were randomised to naproxen 440 mg once a day, 220 mg once a day or placebo for a total of 6 months. A cohort of 83 mice with LS, Msh2fl/fl;Villin-Cre, was treated to assess the chemopreventive activity of naproxen and aspirin compared with control at a dose of 166 ppm and 400 ppm, respectively, in terms of survival outcomes and tumour burden. Human mRNA-seq results were validated in an additional cohort of six mice with LS treated with naproxen orally once a day at a dose of 30 mg/kg/day or sesame oil as control, and in a set of six PDO models generated from colorectal mucosa of patients with LS who were treated for 48 hours with naproxen at 1 mM and DMSO (control). (B) Frequency of AEs. Number of AEs reported during the 6 months of intervention by treatment group, grade and attribution to study treatment. One patient could report more than one AE and all AEs were counted. (C) Change in PGE2 levels in response to naproxen. Comparison of the change in levels of PGE2 from baseline after treatment among placebo, HD and LD naproxen-treated patients using a Wilcoxon rank sum test. The graph displays the percentage of change calculated using (100×((Post) – (Baseline)/Baseline); (**p≤0.05, ***p≤0.001) and the median per group. (D) Change in PGE-M levels in response to naproxen. Change in levels of PGE-M from baseline after treatment with placebo, HD and LD naproxen using a Wilcoxon rank sum test. The graph displays the percentage of change calculated using (100×((Post) – (Baseline)/Baseline); (***p≤0.001) and the median per group. (E) Change in the levels of other PGs in response to naproxen. Changes in the levels of 9a11b-PGF2a, PGF2a, 6-Keto-PGF1a, PGD2, TxB2 after treatment with placebo, HD and LD naproxen using a Wilcoxon rank sum test. The graph displays the percentage of change after treatment calculated using (100×((Post) – (Baseline)/Baseline); (*p≤0.05, **p≤0.01, ***p≤0.001) and the median per group. AEs, adverse events; HD, high dose; LD, low dose; PDO, patient-derived organoid; PGs, prostaglandins; ppm, parts per million.

Figure 3

(A) Volcano plots of DEGs. Genes expression data obtained using whole transcriptome sequencing from participants allocated to PL, LD and HD treatment arms are displayed in volcano plots with log2(FoldChange) on the X-axis and -log10(BH-adjusted p-value) on the Y-axis. Significant upregulated and downregulated genes with BH-adjusted p-value≤0.05 and absolute value of log2(FoldChange)≥0.5 are highlighted and annotated by pathways of interest using different colours. The horizontal line represents BH-adjusted p-value=0.05. The left and right vertical lines represent log2(FoldChange)=±0.5, respectively. (B and C) LD and HD DEGs in the ‘Naproxen trial’ samples. Significant DEGs with BH-adjusted p≤0.05 and absolute value of log2-(FoldChange)≥0.5 in human LD and HD post-treatment versus pretreatment comparison are used to perform an unsupervised hierarchical clustering of LD and HD samples, respectively. Expression levels for all samples are row centred and displayed in the heatmap with gene symbols as row names and sample IDs as column names. The column covariate bar indicates pre-treatment (blue) and post-treatment (gold) expression. Dendrogram illustrates sample clustering based on distances. DEGs, differentially expressed genes; HD, high dose; LD, low dose; PL, placebo.

Figure 4

(A) Gene pathways modulated by both HD and LD naproxen levels with the same direction of effect. Both HD and LD consistently induced immune activation with additional specific effects by HD on cytokine and chemokine signalling. Bubble chart plot displaying the results of the GSEA including pathways that met the following criteria: 1. BH-adjusted p-value≤0.05 in both LD and HD (common, bottom), LD only (LD specific, top) or HD only (HD specific, middle); 2. Direction of NESs are consistent in both LD and HD (same trend). The sizes of bubbles were determined by BH-adjusted p-value increasing the size as the significance increases. The colours of the circles are determined by the direction and amplitude of NES, with positive NES (positively enriched in post-treatment group) in red and negative NES (negatively enriched in post-treatment group) in green. (B) In silico dissection of immune cell types. HD and LD naproxen consistently activated different types of T cells, B cells, DC and macrophages. Sizes of circles were determined by BH-adjusted p-value increasing the size as the significance increases. The colours of circles were determined by the sign and amplitude of t-statistic (t-stat), with positive t-stat (positively enriched in post-treatment group) in red and negative t-stat (negatively enriched in post-treatment group) in green. Note that some pathways are more extensively annotated that others based on the number of gene markers. (C) Histomorphometric analysis of IELs and MALT. Digital images of H&E slides of colorectal mucosa were analysed using HALO software (Indica Labs) using the tissue classification module, CytoNuclear algorithm and manual click counter tool. (D) IELs (n=46 specimens) and MALT (n=18 specimens) were counted and their respective epithelial area or lymphoid tissue area was quantified to calculate the cell density, cell ratio and area ratios. Glass area and tissue artefacts were excluded from the analysis. The IELs and MALTs before and after treatment with placebo, LD ad HD naproxen were assessed. Cell density is expressed by the number of cells per mm2. GSEA, gene set enrichment analysis; HD, high dose; IELs, intraepithelial lymphocytes; LD, low dose; MALT, mucosa-associated lymphoid tissue; NES, normalised enrichment score.

Figure 5

(A) Human specimens, PDOs and mouse correlation. Upper left, we use a patient-wise Pearson correlation matrix for unsupervised clustering of LD, HD, mice and organoid samples. Dataset and patient IDs are concatenated and shown as column and row names. Note that human and PDOs were analysed individually while mouse specimens were combined together. Column and row covariate bars indicate datasets to which samples belong to. The dendrogram illustrates sample clustering based on distances and showing that mouse samples clustered with HD human samples and PDOs with LD samples. (B) Overlap among human, mouse and PDO datasets. Venn diagrams showing the number of overlapping significant genes with BH-adjusted p-value≤0.1 among human LD post versus pre, HD post versus pre and mouse naproxen versus control comparisons (Upper), and among human LD post versus pre, HD post versus pre and PDOs naproxen versus control comparisons (bottom). (C) Differential gene expression of key genes identified in humans using PDOs treated with naproxen and control. Naproxen induced downregulation of stem cell markers and upregulation of epithelial differentiation markers. Data derived from PDOs treated with naproxen at 1 mM (red triangles) and dimethyl sulfoxide (DMSO)/control (grey circles) is presented. Graphs show 2−ΔCt, where ΔCt represent cycle threshold (Ct) of the gene of interest normalised by Ct of Cyclophilin-A (PPIA). The graphs display mean values. *p≤0.05, **p≤0.01. HD, high dose; LD, low dose; PDOs, patient-derived organoids.