Short title:
CSU Transcriptomic signature and benralizumab treatment
Corresponding Author:
Jonathan A. Bernstein, M.D.
University of Cincinnati College of Medicine
Cincinnati, Ohio 45267-0563
Email: Bernstja@ucmail.uc.edu
Phone: 513-235-7971
Fax: 513-558-3799
To the Editor,
We previously reported the efficacy of benralizumab (anti–IL5Rα biologic) in controlling CSU refractory to second generation H1-antihistamines (SGHA).1,2 Herein, we sought to identify CSU-relevant genes and pathways and their regulation by benralizumab.
CSU patients (N=10; unresponsive to up to 4x FDA-approved doses of SGHA) were enrolled in a single-blind, uniform-dose, multi-treatment trial (benralizumab 30mg × 3 treatments; Figure s1). Pre- and post-benralizumab skin and blood samples were analyzed by RNAseq. We identified a CSU signature using machine learning (ML) on an available CSU dataset (GSE72540) which was validated using our samples.3 Pre-, post-Rx symptom scores, and corresponding blood eosinophil counts are summarized in Table-S1. Comparison between lesional vs non-lesional CSU skin indicated upregulation of multiple interleukins (IL-6, IL-20, IL-1β), chemokines (CCL2, CCL3, CCL8, CCL13, CCL18 and CCL21) and adhesion molecules (CD209, ICAM, CD69), while keratin variants (KRT25, KRT26, KRT27, KRT85) were downregulated. Multiple enriched networks were identified with IL1β, IL6, CCL2 and CCL3 as major hub genes (Figure-1A). Machine learning and hierarchical clustering showed that a minimum of 9 genes (OSM, PTGS2, UTP14A, ZFP36, STAT3, CCL2, RPF2, IL6 and LILRB3) were required to stratify lesional from non-lesional samples with > 90% predictive efficacy (Figure-1B). Compared to healthy control skin, lesional CSU skin showed upregulation of adhesion molecules (SELE, ICAM1, CD69), low-affinity IgE-receptor (FcER2) and CC-chemokines (CCL2, CCL3, CCL4). Methods and further results are summarized in the on-line supplement.
To determine the effect of benralizumab, the transcripts (Visit 2 pre-treatment vs Visit 5 end of study) were arranged by significance (p-values) and the top 500 genes (fold-change value > 2.0; ~ top 2% of all detected transcripts) were used for downstream analysis.4,5 A significantly enriched treatment-relevant network showed IL6, TNF and IL1A as major hub-genes. Other relevant transcripts in the network were CD44, mir-24, IFNg, IL4, S100A10 and S100A12. Relevant differentially expressed genes (DEGs) were related to chemokeines/cytokines (CCR1, CCL2, IL6; log2FC -2.04, -2.24, -2.94 respectively), inflammation (PLA2G2A, PTGS2; log2FC -3.19, -1.02), adhesion (SELE; log2FC = -3.7) and barrier function (AQP4; Log2FC 4.27). Focusing on the three complete treatment responders, the same set of hub genes (IL6, TNF and IL1A) in addition to IL22, IL13, collagen-related genes (COL1A1, COL4A2) and NFkB gene members were identified (Figure-1C, D). In addition, PLA2G2A (Phospholipase A2, Group IIA), THBS1 (Thrombospondin-1) and SOCS3 (Suppressor Of Cytokine Signaling 3) were identified as significant DEGs (FDR-adjusted p-values < 0.05; log­2FC values -3.3, -1.9 and -2.5 respectively). The functional significance of these genes in regulating mast cell function, inflammation and cytokine function have previusly been described.6-8 Moreover, benralizumab responders also demonstrated a significant downregulation of PTGDS2 (log2FC = -1.59), a major driver of cutaneous inflammation. Baseline transcriptomic differences between CSU responder and non-responder skin indicated that the major significantly upregulated genes (log2 FC in perentheses) in benralizumab-resistant CSU might be FOS family members of proto-oncogenes FOSB (6.3), Activating Transcription Factor 3 ATF3 (4.5), and nuclear factor subfamily 4 members A1 and A2 (4.4 and 3.92, respectively). Enrichment analysis identified platelet activities in addition to glucocorticoid and phosphatase activities in the non-responsive versus responsive CSU lesional skin. Finally, blood transcriptomes pre vs post-benralizumab treatment demonstrated downregulation of transcripts associated with IL5 signaling (e.g., IL5RA and SIGLEC8 log2FC -2.79 and SIGLEC8 -5.47 respectively; FDR-adjusted p-value < 0.01). When this analysis focused on complete responders to benralizumab, a similar group of transcripts (IL5RA, IL1RL1, RPL34, ALOX15, SIGLEC8 and S100A8) related to IL5 signaling and inflammatory pathways were downregulated. Collectively, benralizumab was associated with blood IL5 downregulation and reversal of the cutaneous CSU transcriptomic signature. Benralizumab-refractory CSU could represent a distinct endotype warranting further investigation.
Debajyoti Ghosh, MSc., PhD1
Rashid Karim PhD2
Xiang Zhang PhD3
Umesh Singh MD, PhD1
and
Jonathan A. Bernstein, M.D1
1 Division of Rheumatology, Allergy and Immunology, Department of Internal Medicine, University of Cincinnati College of Medicine
2Department of Electrical Engineering and Computer Science, University of Cincinnati College of Engineering and Applied Science
3Department of Environmental & Public Health Sciences, University of Cincinnati College of Medicine.
AUTHOR CONTRIBUTIONS
DG and JB designed the study. DG, RK, XZ, US conducted the study, JB supervised the work. DG and JB wrote the manuscript. All authors contributed to the manuscript.
ETHICS STATEMENT
The study protocol was approved by the University of Cincinnati Internal Review Board.
CONFLICTS OF INTEREST
Dr. Bernstein reports grants and consultant fees from AstraZeneca, Sanofi-Regeneron, Novartis, Genentech, Amgen, Celldex, Allakos during the conduct of the study. In addition, he has been a speaker for AstraZeneca, Sanofi-Regeneron, Novartis and Genentech. Outside this work he has received grants and consultant fees from Shire/Takeda, CSL Behring, Pharming, Biocryst, Kalvista, Ionis, Escient, ONO, Incyte, Cycle, Biomarin and Blueprint Medicine. He is a member of the Joint Task Force for the AAAAI and ACAAI, a member of the CU UCARE guideline committee and a UCARE and ACARE center of excellence, and a member of the MAB of the HAEA organization. All other authors have nothing to disclose.