Randomized Controlled Trial Substudy of Cell-specific Mechanisms of Janus Kinase 1 Inhibition With Upadacitinib in the Crohn's Disease Intestinal Mucosa: Analysis From the CELEST Study

Daniel Aguilar, Lluís Revilla, Alba Garrido-Trigo, Julian Panés, Juan J Lozano, Núria Planell, Miriam Esteller, Ana P Lacerda, Heath Guay, James Butler, Justin Wade Davis, Azucena Salas, Daniel Aguilar, Lluís Revilla, Alba Garrido-Trigo, Julian Panés, Juan J Lozano, Núria Planell, Miriam Esteller, Ana P Lacerda, Heath Guay, James Butler, Justin Wade Davis, Azucena Salas

Abstract

Background: Janus kinase (JAK) inhibition shows promise for treatment of patients with moderate to severe Crohn's disease. We aimed to provide mechanistic insights into the JAK1-selective inhibitor upadacitinib through a transcriptomics substudy on biopsies from patients with Crohn's disease from CELEST.

Methods: Seventy-four patients consented to this optional substudy. Ileal and colonic biopsies were collected during endoscopy at screening and week 12 or 16. RNA isolated from 226 samples was analyzed by RNAseq, with additional qPCR analysis. Additional biopsies from patients with Crohn's disease receiving anti-tumor necrosis factor (anti-TNF; n = 34) and healthy controls (n = 10) were used for qPCR. Single-cell RNAseq public profiles were used to evaluate treatment effects on specific cellular subsets, associations with endoscopic improvement, and indirect comparisons with the anti-TNF-treated cohort.

Results: In involved areas of mucosa with endoscopic remission after upadacitinib treatment, 1156 and 76 protein-coding genes were significantly regulated (false discovery rate < 0.05) at week 12/16 in colonic and ileal biopsies, respectively (60 overlapped), compared with baseline. Upadacitinib did not significantly affect transcriptomes of noninvolved intestinal areas. CELEST patients (mostly anti-TNF-refractory) showed baseline differences in gene expression compared with a separate cohort of biologic-naïve patients. Notably, upadacitinib reversed overexpression of inflammatory fibroblast and interferon-γ effector signature markers.

Conclusions: Upadacitinib modulates inflammatory pathways in mucosal lesions of patients with anti-TNF-refractory Crohn's disease, including inflammatory fibroblast and interferon-γ-expressing cytotoxic T cell compartments. This substudy is the first to describe the molecular response to JAK1 inhibition in inflammatory bowel disease and differential effects relative to anti-TNF treatment. (Clinical trial identifier: NCT02365649).

Keywords: Crohn’s disease; JAK inhibitor; tumor necrosis factor; upadacitinib.

© 2021 Crohn’s & Colitis Foundation. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation.

Figures

Figure 1.
Figure 1.
Effect of upadacitinib treatment on colonic and ileal transcriptional signatures. Heat map representations of differentially expressed genes (FDR 

Figure 2.

Effect of upadacitinib on cell-specific…

Figure 2.

Effect of upadacitinib on cell-specific gene subsets. Transcriptional changes observed by RNA sequencing…

Figure 2.
Effect of upadacitinib on cell-specific gene subsets. Transcriptional changes observed by RNA sequencing analysis of colonic and ileal biopsies in response to upadacitinib were assigned to different cell subsets. Each dot represents the median normalized expression of all samples at week 0 or at week 12/16 in endoscopic remitter and nonremitter patients. Genes that were significantly regulated in endoscopic R at week 12/16 relative to week 0 are shown in black. Abbreviations: CD8, cluster of differentiation 8; NR, nonremitter; R, remitter.

Figure 3.

Transcriptional changes in cohorts of…

Figure 3.

Transcriptional changes in cohorts of patients with Crohn’s disease before starting upadacitinib or…

Figure 3.
Transcriptional changes in cohorts of patients with Crohn’s disease before starting upadacitinib or anti-TNF treatment. Cell-specific transcripts were determined at week 0 before starting upadacitinib (n = 19–24) or anti-TNF (n = 19–23) treatment in biopsies by real-time qPCR. Gene expression is also shown in intestinal biopsies from healthy controls (n = 9–10) for comparison. Box plots (whiskers represent 10th and 90th percentiles) representing baseline colonic (A) and ileal (B) gene expression are shown in AU relative to ACTB (β-actin) mRNA expression. *FDR < 0.05; **FDR < 0.01; ***FDR < 0.001 vs control (Mann–Whitney–Wilcoxon test corrected for FDR). #FDR < 0.05; ##FDR < 0.01; ###FDR < 0.001 anti-TNF vs UPA (Mann–Whitney–Wilcoxon test corrected for FDR). Abbreviations: aTNF, anti-TNF; AU, arbitrary unit; FDR, false discovery rate; qPCR, quantitative polymerase chain reaction; TNF, tumor necrosis factor; UPA, upadacitinib.

Figure 4.

Upadacitinib and anti-TNF transcriptional changes…

Figure 4.

Upadacitinib and anti-TNF transcriptional changes in colonic Crohn’s disease. Cell-specific transcripts were determined…

Figure 4.
Upadacitinib and anti-TNF transcriptional changes in colonic Crohn’s disease. Cell-specific transcripts were determined by real-time qPCR in colonic biopsies of patients with Crohn’s disease at week 0 and at follow-up in remitters and non-remitters. Patients starting upadacitinib (A) or anti-TNF (B) were analyzed. Box plots (whiskers represent 10th and 90th percentiles) show gene expression in AU relative to ACTB (β-actin) mRNA expression. Dotted lines show the SEM for each gene in colonic biopsies from healthy controls. *FDR < 0.05; **FDR < 0.01; ***FDR < 0.001 compared with week 0 (Mann–Whitney–Wilcoxon test corrected for FDR). Upadacitinib: week 0, n = 19; remitters, n = 21; non-remitters, n = 11. Anti-TNF: week 0, n = 20–21; remitters, n = 15; non-remitters, n = 10. Abbreviations: AU, arbitrary unit; FDR, false discovery rate; NR, nonremitter; qPCR, quantitative polymerase chain reaction; R, remitter; SEM, standard error of mean; TNF, tumor necrosis factor.
Figure 2.
Figure 2.
Effect of upadacitinib on cell-specific gene subsets. Transcriptional changes observed by RNA sequencing analysis of colonic and ileal biopsies in response to upadacitinib were assigned to different cell subsets. Each dot represents the median normalized expression of all samples at week 0 or at week 12/16 in endoscopic remitter and nonremitter patients. Genes that were significantly regulated in endoscopic R at week 12/16 relative to week 0 are shown in black. Abbreviations: CD8, cluster of differentiation 8; NR, nonremitter; R, remitter.
Figure 3.
Figure 3.
Transcriptional changes in cohorts of patients with Crohn’s disease before starting upadacitinib or anti-TNF treatment. Cell-specific transcripts were determined at week 0 before starting upadacitinib (n = 19–24) or anti-TNF (n = 19–23) treatment in biopsies by real-time qPCR. Gene expression is also shown in intestinal biopsies from healthy controls (n = 9–10) for comparison. Box plots (whiskers represent 10th and 90th percentiles) representing baseline colonic (A) and ileal (B) gene expression are shown in AU relative to ACTB (β-actin) mRNA expression. *FDR < 0.05; **FDR < 0.01; ***FDR < 0.001 vs control (Mann–Whitney–Wilcoxon test corrected for FDR). #FDR < 0.05; ##FDR < 0.01; ###FDR < 0.001 anti-TNF vs UPA (Mann–Whitney–Wilcoxon test corrected for FDR). Abbreviations: aTNF, anti-TNF; AU, arbitrary unit; FDR, false discovery rate; qPCR, quantitative polymerase chain reaction; TNF, tumor necrosis factor; UPA, upadacitinib.
Figure 4.
Figure 4.
Upadacitinib and anti-TNF transcriptional changes in colonic Crohn’s disease. Cell-specific transcripts were determined by real-time qPCR in colonic biopsies of patients with Crohn’s disease at week 0 and at follow-up in remitters and non-remitters. Patients starting upadacitinib (A) or anti-TNF (B) were analyzed. Box plots (whiskers represent 10th and 90th percentiles) show gene expression in AU relative to ACTB (β-actin) mRNA expression. Dotted lines show the SEM for each gene in colonic biopsies from healthy controls. *FDR < 0.05; **FDR < 0.01; ***FDR < 0.001 compared with week 0 (Mann–Whitney–Wilcoxon test corrected for FDR). Upadacitinib: week 0, n = 19; remitters, n = 21; non-remitters, n = 11. Anti-TNF: week 0, n = 20–21; remitters, n = 15; non-remitters, n = 10. Abbreviations: AU, arbitrary unit; FDR, false discovery rate; NR, nonremitter; qPCR, quantitative polymerase chain reaction; R, remitter; SEM, standard error of mean; TNF, tumor necrosis factor.

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