[EN] Benralizumab, a monoclonal antibody targeting IL5RA, significantly improves asthma symptoms and quality of life in severe asthmatic patients. However, understanding the factors influencing treatment response remains a challenge. In this prospective observational study, we analyzed transcriptomic changes in peripheral whole blood samples from 15 severe asthmatic patients treated with benralizumab for 6 months. The study included patients with two frequent comorbidities: nasal polyposis (CRSwNP) and NSAID-exacerbated respiratory disease (N-ERD)Instituto de Salud Carlos III (ISCIII); European Union -NextGeneration EU; Junta de Castilla y Leó

Dávila González, Ignacio Jesús

Estravís Sastre, Miguel

García Sánchez, María Asunción

Gil Melcón, María

Gómez‐García, Manuel

Isidoro García, María

Martín García, Cristina

Moreno‐Jimenez, Emma

Morgado, Natalia

Muñoz Bellido, Francisco Javier

Pérez Pazos, Jacqueline

Ramos González, Jacinto

Sanz Lozano, Catalina Sofía

Triviño, Juan Carlos

Allergy

English

Gestion del Repositorio Documental de la Universidad de Salamanca

Allergy. 2023;00:1–4.   | 1wileyonlinelibrary.com/journal/allReceived: 8 June 2023  | Revised: 27 July 2023  | Accepted: 12 August 2023DOI: 10.1111/all.15869  L E T T E RTranscriptomics reveals new regulatory mechanisms involved in benralizumab responseTo the Editor,Benralizumab, a monoclonal antibody targeting IL5RA, signifi-cantly improves asthma symptoms and quality of life in severe asthmatic patients. However, understanding the factors influenc-ing treatment response remains a challenge. In this prospective observational study, we analyzed transcriptomic changes in pe-ripheral whole blood samples from 15 severe asthmatic patients treated with benralizumab for 6 months. The study included pa-tients with two frequent comorbidities: nasal polyposis (CRSwNP) and NSAID- exacerbated respiratory disease (N- ERD) (Appen-dix S1 and Table S1).We analyzed differential gene expression on paired samples, comparing gene expression levels before and after treatment, and identified 216 differentially expressed genes (DEGs) (Figure S1A,B; Appendix S1). Previous studies have shown a reduction in gene ex-pression inherent to the depletion of eosinophil counts upon ben-ralizumab treatment.1,2 While gene expression generally decreased after treatment, we observed that many differentially expressed genes were not correlated with eosinophil counts (Figure 1A, Fig-ure S1B). In a previous transcriptome analysis of asthmatic patients (Asthma DEG study), we identified 109 DEGs.3 By comparing both studies, we identified 16 common genes (Table 1†, Figure S2A). Among them, we validated CYSLTR2, IL5RA, CLC, PTGDR2, and SMPD3 as potential asthma biomarkers in our study population (Figure S3).Furthermore, we identified three DEG clusters after benrali-zumab treatment (Figure 1A, File S1). Cluster 1 was comprised of downregulated eosinophil- correlated genes. Cluster 2 comprised predominantly upregulated genes, and Cluster 3 consisted mainly of downregulated genes, both uncorrelated to eosinophils. The presence of non- protein coding genes in Clusters 2 and 3 suggested the involvement of additional layers of regulatory processes. Thus, we examined the trans- acting regulatory target RNAs for six dif-ferentially expressed long non- coding RNAs (lncRNAs), consider-ing their base pairing with complementary sequences (Table S2, Appendix S1). Such interactions between lncRNAs and RNAs can regulate gene expression, including mRNA stability, localization, or translation.4 Our findings suggest that specific lncRNAs may contribute to the observed pathway changes during benralizumab treatment, and interestingly, these non- eosinophil- correlated lncRNAs share biological processes with the DEGs such as “Im-mune system process” and “Regulation of signaling” (File S2). That implies that expression changes in other blood cell types might be masked by eosinophil counts, highlighting the regulatory role of non- coding RNAs and other DEGs and their potential in uncov-ering the broader transcriptomic effects of benralizumab beyond eosinophil reduction.CRSwNP and N- ERD are frequent comorbidities in severe asthma, worsening symptoms, and decreasing quality of life.5 We investigated DEG patterns in asthma patients with and without CRSwNP and N- ERD, identifying 20 shared downregulated genes (Table 1‡, Figure 1B). When comparing these genes with those up-regulated in asthma (Asthma DEG Study), we identified a common subset of 10 genes that could define asthma- related inflammation (Figure 1C, Table 1†‡).A random forest model was used to identify gene expression changes that could predict benralizumab treatment response in se-vere asthma patients. First, we applied a holistic approach, catego-rizing the response to benralizumab by considering relevant clinical variables and quality of life measures (Table S3, Appendix S1). Out of the three candidate genes (FBN1, CCR3, and SRGAP3) identified in the transcriptomic study as potential predictors of benralizumab response, FBN1 could be validated as a potential biomarker for super response (Figure 1D,E, Figure S4). Interestingly, mutations in FBN1 have been linked to impaired lung function.6 Further research is needed to explore the clinical implications of FBN1 in asthma and its potential role in benralizumab response.These findings provide novel insights into possible regulatory mechanisms underlying treatment response with benralizumab, thus contributing to the development of personalized precision medicine for severe asthma management. Further studies are needed to vali-date these findings in larger patient cohorts to explore the underly-ing molecular mechanisms.FUNDING INFORMATIONThis study has been funded by the projects PI20/00268, funded by Instituto de Salud Carlos III (ISCIII) and co- funded by the European Union; RD21/0002/0054 and PMP22/00124, funded by Instituto de Salud Carlos III (ISCIII) and European Union - NextGeneration EU, Mecanismo para la Recuperación y la Resiliencia (MRR); IMP/00009, This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.© 2023 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.2  |    LETTERfunded by Instituto de Salud Carlos III (ISCIII) and European Regional Development Fund “A way to make Europe;” by the Grant PID2021- 125117OB- I00 funded by MCIN/AEI/https://doi.org/10.13039/ 50110 0011033 and by “ERDF A way of making Europe,” by the “Eu-ropean Union;” and by the Grant IES161P20, funded by Consejería de Educación de la Junta de Castilla y León co- funded by FEDER funds.AUTHOR CONTRIBUTIONSID, MI- G, CS, and ME designed the study. ID, MG- M, JR- G, CM- G, and FJM- B recruited patients and collected blood samples and clini-cal data. JCT performed the bioinformatics analyses for sequenc-ing data. ME, JP- P, JCT, AG- S, and CS performed analyses for data. ME, JP- P, AG- S, and ID performed the analyses for clinical data. ME, JP- P, EM- J, AG- S, MG- G, and NM performed the experimental methodology and validation experiments. ME, CS, ID, MI- G, and JP- P were the major contributors in writing the manuscript. All au-thors read and approved the final manuscript.ACKNOWLEDG MENTSThe authors would like to express their gratitude to the staff of the Allergy Department at Salamanca University Hospital, particularly Milagros González Prieto, Cristina Regalado González, and Rosa Aguadero Martín, for their valuable collaboration in providing sam-ples and retrieving clinical information. The authors also thank the Genomic Unit of the Salamanca Cancer Research Center (CSIC- Universidad de Salamanca) for their technical guidance and support, also to Fundación del Instituto de Estudios de Ciencias de la Salud de Castilla y León (IECSCYL) and the Institute of Biomedical Research of Salamanca (IBSAL).F I G U R E  1  (A) Hierarchical clusters of DEGs based on their normalized fold changes from baseline to Month 6 of treatment. The right color bars indicate the three clusters. Brown bars highlight genes correlated with eosinophils. (B) DEGs associated with Asthma (n = 5), Asthma with CRSwNP (n = 7), and N- ERD (n = 3). (C) DEGs common to the three phenotypes and DEGs in asthmatic patients. (D) FBN1 normalized expression in Responder (R) and Super Responder patients (SR). (E) ROC curve of the predictive super response value of FBN1 expression based on the probabilities of classifying patients as SR or R according to FBN1 normalized expression. **p < 0.01. 13989995, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/all.15869 by Universidad De Salamanca, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License    | 3LETTERCONFLIC T OF INTERE S T S TATEMENTIn the last 3 years, Miguel Estravís has received payment for lectures from SANOFI. Asunción García- Sánchez has received payment for lectures from Leti. Jacinto Ramos- González has received payments for lectures from Astra- Zeneca, Chiesi, GSK, Novartis, Sanofi, Me-narini, and Boehringer; for a consultancy from Astra- Zeneca, GSK, Novartis, and Sanofi. María Gil- Melcón has received payment for lectures from Astra- Zeneca, GSK, Novartis, and Sanofi. Igna-cio Dávila has received payment for lectures, including service on speaker's bureaus from Allergy Therapeutics, Astra- Zeneca, Chiesi, Diater, GSK, Leti, Novartis, and Sanofi; for a consultancy from Al-lergy Therapeutics, ALK- Abello, Astra- Zeneca, GSK, Merck, MSD, Novartis, and Sanofi; and grants for Thermofisher Diagnostics. The rest of the authors declare no conflict of interest. The funders had no role in the study's design, in the collection, analyses, or interpre-tation of data, in the writing of the manuscript, or in the decision to publish the results.DATA AVAIL ABILIT Y S TATEMENTThe data that support the findings of this study are openly available in Sequence Read Archive (NCBI) at https://www.ncbi.nlm.nih.gov/sra, reference number PRJNA997234.Miguel Estravís1,2Jacqueline Pérez- Pazos1,3Emma Moreno- Jimenez1,4Juan Carlos Triviño5Asunción García- Sánchez1,2,6Manuel Gómez- García1,3Natalia Morgado1,6Jacinto Ramos- González7María Gil- Melcón8Cristina Martín- García1,9Francisco Muñoz- Bellido1,2,9Catalina Sanz1,2,4TA B L E  1  Differentially expressed genes shared among study groups.Ensembl ID Gene nameAsthma DEGs Benralizumab DEGsFold change pAdj Fold change pAdj† ‡ ENSG00000161905 ALOX15 +2.45 3.91E- 05 −334.53 3.20E- 37‡ ENSG00000103355 PRSS33 −154.76 1.87E- 28† ‡ ENSG00000250696 AC111000.4 +2.01 3.31E- 05 −50.42 4.37E- 27‡ ENSG00000205927 OLIG2 −32.06 1.68E- 31‡ ENSG00000213759 UGT2B11 −29.51 4.83E- 43† ‡ ENSG00000091181 IL5RA +2.22 8.63E- 08 −25.74 2.52E- 41‡ ENSG00000115602 IL1RL1 −14.89 1.62E- 29† ‡ ENSG00000105205 CLC +2.05 4.63E- 06 −14.22 7.44E- 31‡ ENSG00000131203 IDO1 −8.95 1.53E- 21† ENSG00000268758 ADGRE4P +1.94 2.47E- 04 −8.93 8.25E- 17† ENSG00000134489 HRH4 +1.93 7.21E- 07 −7.90 8.29E- 21† ‡ ENSG00000183134 PTGDR2 +1.99 2.64E- 06 −6.44 1.74E- 17† ‡ ENSG00000103056 SMPD3 +2.17 1.44E- 07 −4.14 1.67E- 28‡ ENSG00000171657 GPR82 −3.91 2.08E- 13† ‡ ENSG00000255587 RAB44 +1.74 8.10E- 06 −3.83 8.29E- 26‡ ENSG00000163751 CPA3 −3.82 6.97E- 20† ‡ ENSG00000276231 PIK3R6 +1.67 9.12E- 06 −3.50 1.92E- 24† ENSG00000152207 CYSLTR2 +1.84 1.15E- 09 −3.14 2.84E- 09‡ ENSG00000186204 CYP4F12 −2.97 7.14E- 20† ENSG00000133317 LGALS12 +1.58 8.83E- 05 −2.89 9.70E- 06† ‡ ENSG00000188822 CNR2 +1.53 3.41E- 02 −2.82 2.53E- 13‡ ENSG00000140263 SORD −2.73 2.75E- 09† ‡ ENSG00000171659 GPR34 +1.83 1.27E- 08 −2.13 1.59E- 11† ENSG00000174837 ADGRE1 +1.65 2.08E- 06 −2.11 5.69E- 06‡ ENSG00000131018 SYNE1 −2.09 1.06E- 16† ENSG00000050438 SLC4A8 +1.60 1.23E- 04 −1.60 2.58E- 04Note: (†) Genes upregulated in asthma (Asthma DEG study) and downregulated after benralizumab treatment; (‡) DEGs common in the three phenotypes (asthma, asthma with CRSwNP, and NERD) upon benralizumab treatment (benralizumab DEG study). 13989995, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/all.15869 by Universidad De Salamanca, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License4  |    LETTERMaría Isidoro- García1,2,10,11Ignacio Dávila1,2,5,91Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain2Red de Enfermedades Inflamatorias – Instituto de Salud Carlos III, Madrid, Spain3Clinical Biochemistry Department Salamanca, Pharmacogenetics and Precision Medicine Unit, Hospital Universitario de Salamanca, Salamanca, Spain4Microbiology and Genetics Department Salamanca, Universidad de Salamanca, Salamanca, Spain5Synlab Group, Sistemas Genómicos SL, Valencia, Spain6Biomedical and Diagnostics Sciences Department, Universidad de Salamanca, Salamanca, Spain7Pneumology Department, Hospital Universitario de Salamanca, Salamanca, Spain8Otorhinolaryngology and Head and Neck Surgery Department, Hospital Universitario de Salamanca, Salamanca, Spain9Allergy Department, Hospital Universitario de Salamanca, Salamanca, Spain10Medicine Department, Universidad de Salamanca, Salamanca, Spain11Clinical Biochemistry Department, Hospital Universitario de Salamanca, Salamanca, SpainCorrespondenceCatalina Sanz, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.Email: catsof@usal.esMaría Isidoro- García and Ignacio Dávila should be considered joint senior author.ORCIDMiguel Estravís  https://orcid.org/0000-0001-9788-4540 Jacqueline Pérez- Pazos  https://orcid.org/0000-0002-2023-781X Emma Moreno- Jimenez  https://orcid.org/0000-0003-1819-3077 Asunción García- Sánchez  https://orcid.org/0000-0002-9655-4918 Manuel Gómez- García  https://orcid.org/0000-0001-7766-8736 Natalia Morgado  https://orcid.org/0000-0002-2779-4512 Cristina Martín- García  https://orcid.org/0000-0003-3458-3593 Francisco Muñoz- Bellido  https://orcid.org/0000-0003-4071-9928 Catalina Sanz  https://orcid.org/0000-0003-2974-0873 María Isidoro- García  https://orcid.org/0000-0002-9013-9422 Ignacio Dávila  https://orcid.org/0000-0001-8485-5513 R E FE R E N C E S 1. Nakajima M, Matsuyama M, Arai N, et al. Identification of whole blood gene expressions correlated with responsiveness to benrali-zumab. J Allergy Clin Immunol. 2021;147:772- 775. 2. Sridhar S, Liu H, Pham T- H, Damera G, Newbold P. Modulation of blood inflammatory markers by benralizumab in patients with eo-sinophilic airway diseases. Respir Res. 2019;20:14. 3. García- Sánchez A, Estravís M, Martin MJ, et al. PTGDR2 expression in peripheral blood as a potential biomarker in adult patients with asthma. J Pers Med. 2021;11:827. 4. Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non- coding RNAs and its biological functions. Nat Rev Mol Cell Biol. 2021;22:96- 118. 5. Stevens WW, Peters AT, Hirsch AG, et al. Clinical characteristics of patients with chronic rhinosinusitis with nasal polyps, asthma, and aspirin- exacerbated respiratory disease. J Allergy Clin Immunol Pract. 2017;5:1061- 1070.e3. 6. Singh D, Bassi M, Balzano D, et al. COPD patients with chronic bronchitis and higher sputum eosinophil counts show increased type- 2 and PDE4 gene expression in sputum. J Cell Mol Med. 2021;25:905- 918.SUPPORTING INFORMATIONAdditional supporting information can be found online in the Supporting Information section at the end of this article. 13989995, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/all.15869 by Universidad De Salamanca, Wiley Online Library on [07/09/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

Transcriptomics reveals new regulatory mechanisms involved in benralizumab response

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Transcriptomics reveals new regulatory mechanisms involved in benralizumab response

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