Enhanced proteasome activity in perifascicular
myofibres is a hallmark of dermatomyositis and its
inhibition is efficient in preclinical models

Léa Debrut, Margherita Giannini, Giulia Quiring, Simone Perniola, Daniela

Rovito, Céline Keime, Béatrice Lannes, Aleksandra Nadaj-Pakleza,
Jean-Baptiste Chanson, Anne-Laure Charles, et al.

► To cite this version:

Léa Debrut, Margherita Giannini, Giulia Quiring, Simone Perniola, Daniela Rovito, et al.. Enhanced proteasome activity in perifascicular myofibres is a hallmark of dermatomyositis and its inhibition is efficient in preclinical models. Annals of the Rheumatic Diseases, 2025, 10.1016/j.ard.2025.08.031. hal-05375014

HAL Id: hal-05375014

https://univoak.hal.science/hal-05375014v1

Submitted on 20 Nov 2025

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

ELSEVIER

Contents lists available at ScienceDirect

Annals of the Rheumatic Diseases

journal homepage: https://www.sciencedirect.com/journal/annals-of-the-rheumatic-diseases

<figure><img src="image_3.png" /></figure>

Myositis

Enhanced proteasome activity in perifascicular
myofibres is a hallmark of dermatomyositis and its
inhibition is efficient in preclinical models

Léa Debrut<sup>1,2,#</sup>, Margherita Giannini<sup>1,3,#,*</sup>, Giulia Quiring<sup>1</sup>,
Simone Perniola<sup>4</sup>, Daniela Rovito<sup>2</sup>, Céline Keime<sup>2</sup>, Béatrice Lannes<sup>5</sup>,
Aleksandra Nadaj-Pkleza<sup>6</sup>, Jean-Baptiste Chanson<sup>6</sup>, Anne-Laure Charles<sup>1</sup>,
Bernard Geny<sup>1,7</sup>, Daniel Metzger<sup>2</sup>, Gilles Laverny<sup>2,#</sup>, Alain Meyer<sup>1,3,#</sup>

ARTICLE INFO

Article history:

Received 30 April 2025

Received in revised form 29 August 2025

Accepted 30 August 2025

Keywords:

Dermatomyositis, DM

Polymyositis, PM

Inclusion body myositis, IBM

antisynthetase syndrome, ASyS

scleromyositis

immune-mediated necrotising myopathy, IMNM

necrotising autoimmune myopathy

overlap myositis

inflammatory myopathies, IIM

proteasome

immunoproteasome

PSMB8

ixazomib

interferon

IFN

KZR

experimental myositis

A B S T R A C T

**Objectives:** This study aims to characterise the molecular pathways underpinning perifascicular muscle fibre alterations to identify repositionable molecules for dermatomyositis (DM) treatment, and validate potential candidates in preclinical models.

**Methods:** RNA-sequencing was performed to determine the transcriptome of microdissected peri- and endofascicular myofibres from 7 patients with untreated and early DM (symptoms <6 months), 6 with other inflammatory myopathies and 5 without neuromuscular disease. The molecular pathways characterising DM perifascicular fibres were determined and used to explore databases of repositionable drugs. The top candidate molecule was validated in cellular and animal preclinical models.

**Results:** Microdissected myofibre transcriptomic analysis revealed that upregulation of the proteasome pathway, prevailing in perifascicular myofibres, is a hallmark of DM. Immunostaining experiments conducted in a validation cohort showed that in patients with DM, β5i (encoded by PSMB8) was predominantly expressed in the cytoplasm of muscle fibres within the perifascicular region, which also predominantly expressed MxA and HLA-I. Experiments in human myotubes showed that interferon (IFN)-β enhances the expression of proteasome catalytic subunit β5i and chymotrypsin-like activity in myofibres. Computational drug repurposing analysis based on the DM perifascicular myofibre transcriptomic signature identified ixazomib (a proteasome inhibitor) as the drug with the highest therapeutic potential. In human myotubes, ixazomib prevented IFN-β-induced DM-like lesions (atrophy, major histocompatibility complex class I [MHC-I] expression and mitochondrial dysfunctions) without impacting the expression of interferon-

https://doi.org/10.1016/j.ard.2025.08.031

0003-4967/© 2025 The Authors. Published by Elsevier B.V. on behalf of European Alliance of Associations for Rheumatology (EULAR). This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

<footnote n="1">¹ UR3072 Centre de Recherche en Biomédecine, Université de Strasbourg, Strasbourg, France</footnote>

<footnote n="2">² Université de Strasbourg, CNRS, Inserm, IGBMC UMR 7104-UMR-S 1258, Illkirch, France</footnote>

<footnote n="3">³ Explorations Fonctionnelles Musculaires, Service de Physiologie, Centre de Référence des Maladies Autoimmunes Rares, Hôpitaux Universitaires de Strasbourg, Strasbourg, France</footnote>

<footnote n="4">⁴ Clinical Immunology Unit, Fondazione Policlinico Universitario A. Gemelli-IRCCS, Rome, Italy</footnote>

<footnote n="5">⁵ Département d'Anatomopathologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France</footnote>

<footnote n="6">⁶ Service de Neurologie, Centre de Référence des Maladies Neuromusculaires NEIdF, ERN EURO-NMD, Hôpitaux Universitaire de Strasbourg, Strasbourg, France</footnote>

<footnote n="7">⁷ Service de Physiologie et Explorations fonctionnelles, Hôpitaux Universitaires de Strasbourg, Strasbourg, France</footnote>

<footnote>* Correspondence to Dr Margherita Giannini, Pr Alain Meyer.</footnote>

<footnote>E-mail addresses: gianninim@unistra.fr (M. Giannini), alain.meyer7@gmail.com (A. Meyer).</footnote>

<footnote>Handling editor Josef S. Smolen.</footnote>

<footnote># These authors are equal contributed.</footnote>
stimulated genes (ISGs). Moreover, experimental myositis (EM) mice treated with ixazomib recovered muscle strength and normalised serum creatine kinase levels. While ISG expression was unchanged in muscles of ixazomib-treated EM mice, sarcolemmal MHC-I expression was abolished, myofibre size variability was normalised, and inflammatory infiltrate was decreased.

*Conclusions:* Enhanced proteasome activity in perifascicular myofibres is a hallmark of DM; its inhibition, deemed effective in preclinical models, may represent a new therapeutic strategy for this disease.

### WHAT IS ALREADY KNOWN ON THIS TOPIC

* Dermatomyositis (DM) is defined by a specific alteration pattern of the perifascicular myofibres. However, the underlying mechanisms are unknown, and current treatments are empirical and only partially effective.

### WHAT THIS STUDY ADDS

* Perifascicular muscle fibres in early DM are characterised by interferon-β-induced upregulation of the proteasome pathway that contributes to the other key characteristics of the disease (ie, major histocompatibility complex class I expression, mitochondrial dysfunctions, and atrophy of perifascicular fibres).

* Proteasome inhibitors, predicted *in silico* as candidate repurposed drugs based on this new insight, are effective in preclinical models of DM.

### HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

* These findings encourage future trials testing proteasome inhibition in patients with DM.

## INTRODUCTION

Inflammatory myopathies (IMs) are a group of rare autoimmune diseases leading to muscle weakness, although the underlying mechanisms are not fully known. Dermatomyositis (DM) is the most frequent IM subset and induces the highest mortality rate [1]. At the myopathological level, DM is defined by specific alterations of perifascicular fibres, that include atrophy, expression of type I interferon (IFN-I)-stimulated genes (ISGs) and of the major histocompatibility complex class I (MHC-I), as well as mitochondrial dysfunctions [2]. Inflammatory infiltrates and capillary injuries are also present in the muscles of patients with DM. The prevalence and severity of these histological features vary according to the autoantibody status, indicating several DM subsets [3].

Current treatments, based on the empirical use of corticosteroids and immunomodulators, allow only a partial recovery and expose to numerous side effects and frequent relapses [4]. Hence, to identify new DM treatment strategies, the underlying mechanisms need to be unravelled.

There is evidence that muscle fibres play a direct role in DM pathophysiology. Indeed, DM muscle cells deliver an IFN-I autocrine effect [5] while mitochondrial dysfunctions in DM muscle fibres sustain inflammation [6]. Myofibre sarcolemmal MHC-I expression may contribute to the activation, expansion, and survival of CD8+ T cells [7]. Moreover, we have recently shown that the selective inactivation of the glucocorticoid receptor in muscle fibres suppresses the therapeutic response of glucocorticoids in experimental myositis (EM) [8]. Thus, targeting muscle fibres may represent a relevant therapeutic strategy for DM.

Bulk transcriptomic analysis has revealed that IFN-I signaling, pattern recognition receptors, dendritic cell maturation, and T-cell exhaustion signalling are higher in DM muscle

compared to other IM subsets [9,10]. However, these analyses did not identify molecular pathways specifically deregulated in myofibres (as opposed to the other components of the muscle cells), nor the mechanisms underpinning the distinctive changes in perifascicular muscle fibres in DM.

Only 2 studies have analysed microdissected DM muscle fibres with 1 highlighting a reduction in titin protein in DM perifascicular muscle fibres [11], while the second revealed increased expression of several interferonogenic pattern recognition receptors in MHC-I expressing DM muscle fibres [12]. Neither study used RNA-sequencing for their analysis.

To further unveil the deregulated pathways in DM perifascicular fibres, using RNA-sequencing, we established the transcriptome of laser capture microdissected peri- and endofascicular myofibres from early untreated patients with DM, patients with other IMs as well as individuals without neuromuscular disease (NMD). The resulting findings revealed molecular pathways that characterise DM perifascicular fibres and allowed the identification of repositionable molecules for DM treatment. The efficacy of the candidate drug with the highest therapeutic potential was validated in both cellular and animal preclinical models.

## METHODS

Methods are detailed in the Supplementary material.

## RESULTS

### Proteasome pathway upregulation prevailing in perifascicular muscle fibres is a hallmark of DM

To unravel the molecular pathways underpinning perifascicular muscle fibre alterations in DM, we compared the transcriptome of microdissected deltoid perifascicular (n > 300) and endofascicular (n > 300) myofibres (Supplementary Fig S1) from 13 early (symptoms for less than 6 months) untreated patients with IM (DM: n = 7; IM other than DM: n = 6) and 5 patients without NMD, the characteristics of which are shown in Table 1.

Next-generation sequencing analysis revealed that 865 genes were differentially expressed among the perifascicular fibres of the 3 groups of patients (Fig 1A, left panel). Among the latter, 479 genes were selectively deregulated in patients with DM (Fig 1B, left panel). The levels of the top 15 deregulated genes in DM perifascicular fibres compared to no NMD perifascicular fibres are presented in Figure 1C (left panel) and D. The most differentially expressed transcript in the perifascicular fibres of patients with DM was PSMB8 (encoding the proteasome subunit beta type-8, which is responsible for the chymotrypsin-like activity of the immunoproteasome), whose expression was 47-fold higher than in the patients without NMD (P = 1.31E-21) and 3-fold higher than in the patients without DM IM (P = .008) (Fig 1C left panel, D). Genes coding for previously reported hallmark biomarkers of DM perifascicular fibres (ie, IFI6, ISG15, MHC-I subunits) were also found in this top list (Fig 1C left panel, D). The most deregulated pathway in DM
<table><caption>**Table 1**

Characteristics of the 18 patients included for transcriptomic analysis</caption><thead><tr><th>Patient</th><th>Age/sex</th><th>Muscle symptoms (wk)</th><th>CK (IU/L)</th><th>Autoantibodies</th><th>Extramuscular involvement</th></tr></thead><tbody><tr><td>DM 1</td><td>57/M</td><td>8</td><td>696</td><td>None</td><td>DM skin rash</td></tr><tr><td>DM 2</td><td>51/F</td><td>24</td><td>1700</td><td>Anti-Mi2β</td><td>Dysphagia</td></tr><tr><td>DM 3</td><td>42/F</td><td>18</td><td>4700</td><td>Anti-Mi2β</td><td>DM skin rash + polyarthritis</td></tr><tr><td>DM 4</td><td>54/F</td><td>3</td><td>16,616</td><td>Anti-Mi2β</td><td>None</td></tr><tr><td>DM 5</td><td>65/F</td><td>12</td><td>1420</td><td>None</td><td>DM skin rash</td></tr><tr><td>DM 6</td><td>76/F</td><td>17</td><td>154</td><td>Anti-TIF1γ</td><td>DM skin rash + ILD</td></tr><tr><td>DM 7</td><td>26/F</td><td>12</td><td>NA</td><td>Anti-TIF1γ</td><td>DM skin rash</td></tr><tr><td>Other IM 1 (IMNM)</td><td>61/F</td><td>13</td><td>1490</td><td>Anti-SRP</td><td>None</td></tr><tr><td>Other IM 2 (IBM)</td><td>80/M</td><td>24</td><td>292</td><td>None</td><td>None</td></tr><tr><td>Other IM 3 (IMNM)</td><td>75/F</td><td>24</td><td>1135</td><td>None</td><td>Dysphagia</td></tr><tr><td>Other IM 4 (IMNM)</td><td>27/F</td><td>4</td><td>11,139</td><td>None</td><td>Dysphagia</td></tr><tr><td>Other IM 5 (IMNM/ scleromyositis)</td><td>60/F</td><td>24</td><td>391</td><td>None</td><td>Dysphagia, myocarditis, Raynaud's phenomenon, scleroderma pattern on capillaroscopy</td></tr><tr><td>Other IM 6 (IMNM)</td><td>52/M</td><td>13</td><td>4843</td><td>Anti-SRP</td><td>None</td></tr><tr><td>No NMD 1</td><td>56/F</td><td>None</td><td>112</td><td>None</td><td>None</td></tr><tr><td>No NMD 2</td><td>47/M</td><td>None</td><td>77</td><td>None</td><td>None</td></tr><tr><td>No NMD 3</td><td>51/F</td><td>None</td><td>66</td><td>None</td><td>None</td></tr><tr><td>No NMD 4</td><td>42/F</td><td>None</td><td>125</td><td>None</td><td>None</td></tr><tr><td>No NMD 5</td><td>43/F</td><td>None</td><td>63</td><td>None</td><td>None</td></tr></tbody></table>

CK, creatine kinase blood level at the time of muscle biopsy; DM, dermatomyositis; F, female; IBM, inclusion body myositis; ILD, interstitial lung disease; IM, inflammatory myopathy; IMNM, immune-mediated necrotising myopathy; IU, international unit; M, male; NA, not applicable; NMD, neuromuscular disorder.

No patient was treated with glucocorticoids or immunosuppressants. Anti-SSA, -SSB, -U1-RNP, -Scl70, -centromere, -Mi2β, -TIF1γ, -SAE1, -SAE2, -MDA5, -NXP2, -Jo-1, PL-7, -PL-12, -EJ, -OJ, -Zo, -SRP, and -HMGCR antibodies were investigated using D-Tek line immunoassay; anti-cN1A were investigated using Euroimmun line immunoassay.

perifascicular muscle fibres (compared to individuals without NMD and other patients with IM) was proteasome (P = 9.23E-6), followed by antiviral response (human papillomavirus infection pathway) (P = 1.87E-4) (Fig 1E left panel).

A fewer number of genes (n = 566) were differentially expressed between the endofascicular fibres of the 3 groups of patients (Fig 1A right panel). Of the latter, 218 genes were selectively deregulated in patients with DM (Fig 1B right panel), 113 (51%) of which were also selectively deregulated in the perifascicular fibres of this group (Supplementary Fig S2A). The levels of the top 15 deregulated genes in DM endofascicular fibres are shown in Figure 1C right panel. Among these 15 genes, 6 (40%) were shared with the top 15 deregulated genes in the perifascicular fibres of this group (Supplementary Fig S2B). However, the extent of the deregulation of these genes was lower in the endofascicular fibres compared to the perifascicular fibres (Fig 1D). The most differentially expressed transcript in the endofascicular fibres of patients with DM compared to patients without NMD was PSMB8, although the overexpression of this transcript was approximately 3-fold lower than in the perifascicular fibres (Fig 1C right panel, D). Similarly to the perifascicular fibres, the most deregulated pathway in DM endofascicular muscle fibres (compared to individuals without NMD and other patients with IM) was proteasome, followed by antiviral response (hepatitis C pathway), although the weight of these 2 pathways (P value) was lower in the endofascicular vs the perifascicular fibres (6.31E-4 and 0.0058, respectively, Fig 1E right panel).

To validate transcriptomic findings from microdissected fibres in the discovery cohort, we performed immunostaining experiments in an independent validation cohort (Table 2). In patients with DM, β5i (encoded by PSMB8) was predominantly expressed in the cytoplasm of perifascicular muscle fibres, colocalising with MxA and MHC-I. In contrast, β5i expression was: i) confined to a few MxA-positive fibres in antisynthetase syndrome (ASyS) and scleromyositis (SM), ii) limited to inflammatory cells and rare fibres in inclusion body myositis (IBM), iii)

absent in immune-mediated necrotising myopathy (IMNM), and non-NMD subjects (Fig 1F).

Taken together, these data highlight that the enhanced proteasome pathway, prevailing in perifascicular muscle fibres, is a hallmark of DM.

*In early untreated patients with DM, the physiological transcriptomic signature of perifascicular muscle fibres is replaced by a pathological signature characterised by proteasome dysregulation*

There are very few data regarding the physiological differences between muscle fibres according to their spatial location within the muscle fascicules. In addition to previously reported biomarkers of DM, PSMB8 expression prevailed in DM perifascicular fibres, but was also observed in DM endofascicular fibres. Thus, to further determine the molecular differences between endo- and perifascicular fibres in physiological conditions and in IM, the transcriptome of paired perifascicular and endofascicular fibres was compared in each of the 3 groups (Fig 2A). In patients without NMD, 83 genes were upregulated and 54 down-regulated in perifascicular fibres compared to the endofascicular fibres (Supplementary Fig S3A). Pathway analyses revealed that these genes were mainly related to muscle tissue development and the generation of precursor metabolites as well as energy signalling pathways (Supplementary Fig S3B). The levels of the top 15 differentially expressed genes are presented in Supplementary Figure S3C. Of note, physiological PSMB8 expression was 2.1-fold lower in perifascicular fibres compared to the endofascicular fibres (P = .0001) (Supplementary Fig S4). In patients with IM, this transcriptomic signature of the perifascicular fibres was lost, and replaced by a specific perifascicular signature consisting of 18 overexpressed and 10 underexpressed genes (Fig 2A,B), mainly involved proteasome, mitophagy, mitochondrial functioning, and antiviral response pathways (Fig 2B).
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Deregulation of the proteasome pathway in muscle fibres is a hallmark of early untreated dermatomyositis (DM). (A) Heatmaps depicting the mean-centred normalised expression of differentially expressed genes (adj. *P* < .05) in perifascicular (n = 865) and endofascicular (n = 566) fibres of the deltoid muscles from patients with DM (n = 7), inflammatory myopathies (IMs) other than DM (n = 6), and without neuromuscular disease (NMD) (n = 5). (B) Venn diagram representation of the number of genes deregulated (*P* value <0.05 and |log₂(fold-change)| > log₂(1.5)) in perifascicular and endofascicular fibres of patients with DM (compared to other patients with IM and patients without NMD). (C) Volcano plot of the genes deregulated in DM perifascicular and endofascicular fibres compared to patients without NMD. The 15 most deregulated genes (according to *P* value; *P* value <0.05 and |log₂(fold-change)| > log₂(1.5)) in DM perifascicular and endofascicular fibres are indicated in red. (D) Expression of the 15 most deregulated genes in DM perifascicular fibres (according to *P* value; *P* value <0.05 and |log₂(fold-change)| > log₂(1.5)). The expression of each gene (normalised reads) is represented as the ratio between patients with IM and patients without NMD. Data are expressed as the mean + SEM. (E) Specifically deregulated pathways (upregulated [red] and downregulated [green]) in perifascicular and endofascicular fibres of patients with DM (compared to other patients with IM and patients without NMD). (F) Representative figures of immunostaining for β5i, MxA, MHC-I, and MHC-II in muscle from DM, antisynthetase syndrome (ASyS), scleromyositis (SM), immune-mediated necrotising myopathy (IMNM), inclusion body myositis (IBM), and patients without NMD. Black arrows indicate PSMB8 and MxA-positive fibres. Black arrowheads indicate PSMB8 and MxA-positive inflammatory cells. Scale bar: 100 µm. MHC-I, major histocompatibility complex class I.</figcaption></figure>

### IFN-β enhances myofibre expression of the proteasome catalytic subunit β5i and chymotrypsin-like activity

The IFN-I signature is an established hallmark of DM muscle fibres [9]. The present analysis revealed that proteasome upregulation represents an additional characteristic of DM perifascicular muscle fibres and a potential therapeutic target. The proteasome harbours 6 catalytic subunits in its catalytic core (20S), 3 of which are constitutive (β5, β1, and β2, respectively encoded by PSMB5, PSMB6, and PSMB7 and carrying the chymotrypsin-like, caspase-like, and trypsin-like activity, respectively) and 3 are inducible (β5i, β1i, and β2i, respectively encoded by PSMB8, PSMB9, and PSMB10 and carrying the same activities as the constitutive catalytic subunits) in inflammatory conditions (immunoproteasome) [13,14] (Fig 3A).

The proteasome also involves regulatory subunits PSMF1 (encoded by PI31) and the proteasome maturation protein (encoded by POMP) [13,14]. The expression and activity of each catalytic subunit of the 20S proteasome and their respective association with type I IFN signalling were hence studied in DM muscle fibres. PSMB8 was the most highly differentially expressed proteasome subunit in DM fibres (Fig 3A). Expression of the 2 other inducible subunits (β1i and β2i) and 1 of the constitutive subunits (β2) was increased, albeit to a much lesser extent (Fig 3A, Table 3). Transcript levels of genes encoding these subunits positively correlated with the expression of ISG15 in DM muscle fibres (R² from 0.36 to 0.51, *P* < .05) (Fig 3B). In contrast, the transcript levels of PSMF1, encoding the endogenous proteasome inhibitor PI31, were decreased in DM muscle fibres (1.2-fold vs no NMD in perifascicular fibres, *P* = .042 and 1.03-fold
<figure><img src="image_5.png" /><figcaption>Figure 1. Continued.</figcaption></figure>

in endofascicular fibres, *P* = .792) and negatively correlated with type I ISGs (R² from 0.36 to 0.51, *P* < .05) (Table 3).

IFN-β levels are known to be increased in the blood of patients with DM compared to patients without NMD and to patients with IM other than DM [15,16], and IFN-β-treated myotubes recapitulate *in vitro* the canonical characteristics of DM perifascicular muscle fibres (ie, IFN-I signature, MHC-I expression, mitochondrial dysfunctions, and atrophy) [6,10,17,18].

In a series of experiments designed to determine whether IFN-β stimulates the expression of proteasome subunits that is increased in DM muscle fibres, 5-day treatment of human myotubes with IFN-β (500 U/mL) was found to increase β5i subunit (PSMB8) protein levels by 5.6-fold (Fig 3C,D). In addition, IFN-β also induced proteasome chymotrypsin-like activity in human myotubes in a dose-dependent manner (Fig 3E).

Altogether, these results indicate that IFN-β, whose level is selectively increased in DM, contributes to the increased expression of catalytic proteasome subunits (mainly β5i) and chymotrypsin-like activity in DM muscle fibres.

### *In DM perifascicular muscle fibres, PSMB8 expression is mediated by type I IFN*

Type I and type II IFNs exhibit partially overlapping transcriptional signatures [19]. While PSMB8 has been reported to be induced by both IFN types [20–25], MxA is primarily induced by type I IFNs, whereas major histocompatibility complex class II (MHC-II) is upregulated by type II IFNs [9,26]. Both IFN-I and IFN-II are elevated in the blood of patients with DM [16], but only type I ISGs and proteins (MxA)—and not type II

(MHC-II)—are detected in perifascicular fibres (Fig 1F, Supplementary Fig S5). To further investigate the relative contribution of each IFN type to PSMB8 expression in DM perifascicular fibres (Fig 4A), we assessed the ability of IFN-β (type I) and IFN-γ (type II) to induce PSMB8, in parallel with canonical IFNs response markers, MxA and MHC-II.

We queried the Interferome database, which compiles transcriptomic data from various cell types exposed to IFNs [27]. In agreement with previous reports [28,29], IFN-β preferentially upregulated MxA, whereas IFN-γ induced MHC-II levels. In addition, both IFN-β and IFN-γ had a similar potency to induce PSMB8 expression (Fig 4B). These results support that while PSMB8 is inducible by both IFN types, MxA and MHC-II are biomarkers of IFN-β and IFN-γ, respectively. Given that Interferome does not include transcriptomic datasets from skeletal muscle, we next assessed the expression of PSMB8, MxA, and MHC-II in human myotubes treated with IFN-β (500 U/mL) or IFN-γ (500 U/mL). In line with the *in silico* results, IFN-β and IFN-γ both induced PSMB8 transcript levels (Fig 4C) and chymotrypsin-like activity of the proteasome (Supplementary Fig S6A,B). In contrast, MxA, a hallmark of DM perifascicular fibres, was preferentially induced by IFN-β, while MHC-II expression, a hallmark of non-DM IM, was preferentially induced by IFN-γ (Fig 4C). These results indicate that PSMB8 expression in perifascicular fibres of DM is mediated by IFN-β rather than IFN-γ.

### Proteasome inhibitors are repurposed candidate drugs for DM

To identify currently available candidate drugs targeting the molecular pathways underpinning perifascicular muscle fibres
<table><caption>**Table 2**

Individual characteristics of PSMB8 and MxA immunostainings in myofibres and immune cells of patients with inflammatory myopathies and without neuromuscular diseases</caption><thead><tr><th rowspan="2">N</th><th rowspan="2">Sex/age (y)</th><th rowspan="2">IM-specific autoantibody</th><th rowspan="2">Diagnosis</th><th colspan="2">PSMB8 positivity</th></tr><tr><th>Fibres</th><th>Immune cells</th></tr></thead><tbody><tr><td>1</td><td>M, 39</td><td>Anti-NXP2</td><td>DM</td><td>Perif > endof</td><td>+</td></tr><tr><td>2<sup>a</sup></td><td>F, 20</td><td>None</td><td>DM</td><td>Perif > endof</td><td>+</td></tr><tr><td>3</td><td>M, 71</td><td>Anti-TIF1γ</td><td>DM</td><td>Perif</td><td>—</td></tr><tr><td>4</td><td>F, 60</td><td>Anti-MDA5</td><td>DM</td><td>Perif > endof</td><td>+</td></tr><tr><td>5</td><td>F, 39</td><td>Anti-SAE1/2</td><td>DM</td><td>Perif > endof</td><td>+</td></tr><tr><td>6</td><td>F, 54</td><td>Anti-MI2β</td><td>DM</td><td>Perif > endof</td><td>+</td></tr><tr><td>7<sup>a</sup></td><td>F, 44</td><td>Anti-Jo-1</td><td>ASyS</td><td>Patchy</td><td>+</td></tr><tr><td>8</td><td>F, 73</td><td>Anti-PL-7</td><td>ASyS</td><td>Patchy</td><td>+</td></tr><tr><td>9</td><td>F, 46</td><td>Anti-Jo-1</td><td>ASyS</td><td>Patchy</td><td>+</td></tr><tr><td>10</td><td>F, 64</td><td>Anti-U1-RNP</td><td>SM</td><td>Patchy</td><td>+</td></tr><tr><td>11</td><td>F, 49</td><td>Anti-U1-RNP</td><td>SM</td><td>Patchy</td><td>+</td></tr><tr><td>12<sup>a</sup></td><td>F, 27</td><td>Anti-U1-RNP</td><td>SM</td><td>Patchy</td><td>+</td></tr><tr><td>13<sup>a</sup></td><td>M, 60</td><td>None</td><td>IMNM</td><td>—</td><td>+</td></tr><tr><td>14</td><td>F, 47</td><td>Anti-SRP</td><td>IMNM</td><td>—</td><td>+</td></tr><tr><td>15</td><td>M, 48</td><td>None</td><td>IMNM</td><td>—</td><td>+</td></tr><tr><td>16</td><td>M, 73</td><td>None</td><td>IBM</td><td>—</td><td>+</td></tr><tr><td>17</td><td>F, 49</td><td>Anti-cN1A</td><td>IBM</td><td>—</td><td>+</td></tr><tr><td>18<sup>a</sup></td><td>M, 64</td><td>Anti-cN1A</td><td>IBM</td><td>Rare</td><td>+</td></tr><tr><td>19</td><td>M, 57</td><td>None</td><td>no NMD</td><td>—</td><td>—</td></tr><tr><td>20</td><td>M, 59</td><td>None</td><td>no NMD</td><td>—</td><td>—</td></tr><tr><td>21</td><td>F, 45</td><td>None</td><td>no NMD</td><td>—</td><td>—</td></tr></tbody></table>

ASyS, antisynthetase syndrome; DM, dermatomyositis; Endof, endofascicular fibres; IBM, inclusion body myositis; IM, inflammatory myopathy; IMNM, immune-mediated necrotising myopathy; NMD, neuromuscular disorder; Perif, perifascicular fibres; SM, scleromyositis.

No patient was treated with glucocorticoids or immunosuppressants. Anti-SSA, -SSB, -U1-RNP, -Scl70, -centromere, -Mi2β, -TIF1γ, -SAE1, -SAE2, -MDA5, -NXP2, -Jo-1, PL-7, -PL-12, -EJ, -OJ, -Zo, -SRP, and -HMGCR antibodies were investigated using D-Tek line immunoassay; anti-cN1A were investigated using Euroimmun line immunoassay.

alterations in DM, the transcriptomic signature of DM perifascicular fibres (28 genes involved in proteasome, mitophagy, mitochondrial functioning, and antiviral response pathways, Fig 2B) was used for computational drug repurposing analysis, as described by Karatzas et al [30]. Nine chemical compounds were identified by at least 2 tools, with a predictive score <-.50, thus possibly reversing the transcriptomic signature observed in DM perifascicular muscle fibres (Fig 5A,B). Among these, prednisolone (the current standard of care in DM treatment [4]), and a Janus kinase (JAK) inhibitor (a drug class for which case reports and series indicate efficacy in patients with DM) were highlighted, supporting the validity of this analysis. However, the drug with the highest predicted therapeutic potential was ixazomib, an inhibitor of the chymotrypsin-like activity of the constitutive catalytic subunits and the immunoproteasomes. An additional proteasome inhibitor, MG-132, was also identified with a high therapeutic score, which further reinforced the probability that targeting the chymotrypsin-like activity of the proteasome may constitute an effective therapeutic strategy for patients with DM.

These results indicate that targeting proteasome hyperactivity in DM perifascicular fibres is a potential innovative therapeutic strategy.

## Proteasome inhibition is effective in an in vitro model of DM

To validate the *in silico* drug repurposing prediction gained from patients’ muscle biopsies, and to investigate the link between high blood IFN-β, canonical DM perifascicular fibre lesions (atrophy, mitochondrial dysfunction, IFN-I signature, and MHC-I expression) and proteasome pathway upregulation,

IFN-β (500 U/mL)-treated human myotubes were cultured in the presence of ixazomib (40 nM added from day 2 to day 5 of IFN-β treatment) (Fig 6A).

As expected, ixazomib normalised chymotrypsin-like activity in IFN-β-treated myotubes (Fig 6B). Ixazomib prevented the IFN-β-mediated 40% reduction in myotube diameter (Fig 6C) as well as the 54% reduction in maximal mitochondrial respiration (Vmax) (Fig 6D).

Ixazomib treatment also reduced the IFN-β-induced expression of the MHC-I-protein (Fig 6E). By contrast, the 12.5-fold increase in IFN-β-induced ISG15 transcript levels was only partially mitigated by ixazomib (9-fold increase vs control) (Fig 6F). A similar partial effect of ixazomib was observed on other ISG (ie, IFIT3, IFI44, RSAD2, and IFIT 27) (Fig 6F).

Hence, proteasome inhibition prevents IFN-β-induced atrophy, mitochondrial dysfunction, and MHC-I without significantly impacting ISG expression in human myotubes indicating that proteasome mediates IFN-β-induced myotoxicity, downstream of the ISG activation.

We observed that, in DM perifascicular fibres, enhanced PSMB8 expression is mediated by IFN-β, rather than by IFN-γ. Accordingly, IFN-γ-treated myotubes have previously been used as an *in vitro* model of non-DM [29]. In human myotubes treated with IFN-γ (500 U/mL), other key features of DM perifascicular muscle fibres, such as atrophy, mitochondrial dysfunction, MHC-I expression, and the expression of ISGs other than MxA, were less prominent than in IFN-β-treated myotubes (Supplementary Fig S6A-F). To test the specificity of the therapeutic effect of proteasome inhibition on the myopathic actions of IFN-β, we repeated the *in vitro* experiment described above

<footnote><sup>a</sup> Patients whom representative pictures have been provided in Figure 1.</footnote>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2.** The transcriptomic signature of perifascicular muscle fibres is lost in early untreated patients with inflammatory myopathies (IMs) and replaced by a pathological signature in patients with dermatomyositis (DM). (A) Venn diagram of differently expressed genes (adj. *P* < .05 and |log₂(fold-change)| > log₂(1.5)) between peri- and endofascicular fibres of the deltoid muscles from DM (n = 7), IM other than DM (n = 6), and patients without neuromuscular disease (NMD) (n = 5). (B) List of the 28 differentially expressed genes (adj. *P* < .05 and |log₂(fold-change)| > log₂(1.5)) between peri- and endofascicular fibres of the deltoid muscles from patients with DM. Bar charts indicate fold change for DM (red), other IM (yellow), and no NMD (blue).</figcaption></figure>

using human myotubes treated with IFN-γ (500 U/mL). Ixa-zomib normalised chymotrypsin-like activity in IFN-γ-treated myotubes, but did not reverse atrophy and mitochondrial dysfunction (Supplementary Fig S6B-D). Thus, unlike IFN-β, the myopathic effects of IFN-γ are independent of protea-some activation.

*Proteasome inhibition is effective in an in vivo model of myositis*

There is currently no animal model fully mimicking DM. However, we previously generated an EM model [31], repre-senting an optimised version of C-protein-induced myositis [32] and shown to recapitulate key characteristics of DM (Fig 7A),
<figure><img src="image_8.png" /><figcaption>**Figure 3.** Interferon (IFN)-β enhances expression of proteasome catalytic subunit β5i and chymotrypsin-like activity in myofibres. (A) Heatmap depicting the expression of genes encoding the 20S proteasome and immunoproteasome subunit in perifascicular and endofascicular fibres of dermatomyositis (DM) compared to patients without neuromuscular disease (NMD). (B) Correlation between the expression of ISG15 and genes coding for proteasome catalytic subunits of the constitutive (PSMB5, PSMB6, PSMB7) and the immunoproteasome (PSMB8, PSMB9, PSMB10) in perifascicular (red circle) and endofascicular (pink triangle) DM fibres. (C) Representative immunoblot and quantification of proteasome β5i subunits (encoded by PSMB8) protein expression in human myotubes treated with IFN-β (500 U/mL) or vehicle (control, CTRL) for 5 days. n ≥ 4 for each condition. \*: P < .05. (D) Representative immunostaining of PSMB8 expression in human myotubes treated with IFN-β (500 U/mL) or vehicle (control, CTRL) for 5 days. Scale bar: 100 µm. (E) Chymotrypsin-like activity in human myotubes treated with IFN-β (100 U/mL, 500 U/mL or 1000 U/mL) or vehicle (control, CTRL) for 5 days. Data are expressed as mean + SEM. \*:P < .05, \*:*:P < .001. ISG, interferon-stimulated gene; POMP, proteasome maturation protein; PSMF1, proteasome inhibitor PI31 subunit.</figcaption></figure>

including muscle weakness, elevated serum creatine kinase (CK) levels, muscle fibres atrophy, sarcolemmal MHC-I expression, elevated expression of several ISG and inflammatory infiltrate in the muscle [31] (Fig 7B-H, Supplementary Fig S7). Importantly, PSMB8 transcript levels (Fig 7I) and chymotrypsin-like activity (Fig 7J) were also increased in muscles of EM mice. Thus, the EM model enables further exploration in vivo of the potential effect of proteasome inhibition in DM at a preclinical stage.

Given that EM muscle defects are maximal from day 14 (D14) to day 21 (D21) after immunisation, we subsequently explored the therapeutic potential of proteasome inhibition using a single dose of ixazomib (5 mg/kg) [33,34] vs vehicle

only administered by gavage at D14 (Fig 7A). Ixazomib reduced chymotrypsin-like activity at D21 in gastrocnemius muscles of EM mice (Fig 7J). Moreover, ixazomib-treated EM mice normalised muscle strength and CK serum levels at D21 (Fig 7B,C). In agreement with the in vitro data, myofibre sarcolemmal MHC-I expression in ixazomib-treated EM mice was abolished (Fig 7F), fibre size variability was normalised (Fig 7D), and inflammatory infiltrates decreased (Fig 7H), while muscle IFIT3 expression remained high (Fig 7G). Taken together, these results show that proteasome inhibition is an effective therapeutic strategy in an in vivo preclinical model of DM.
<caption>Table 3</caption>

<table><caption>Genes encoding the proteasome subunits in dermatomyositis perifascicular and endofascicular fibres compared to patients without neuromuscular disease and their correlation with ISG15 expression in DM</caption><thead><tr><th rowspan="2">Gene name</th><th rowspan="2">Protein</th><th colspan="3">DM perif. vs no NMD perif.</th><th colspan="3">DM endo. vs no NMD endo.</th><th rowspan="2">Correlation with ISG15 expression</th></tr><tr><th>Fold change</th><th>P value</th><th>Adj. P value</th><th>Fold change</th><th>P value</th><th>Adj. P value</th></tr></thead><tbody><tr><td>PSMD3</td><td>26S proteasome regulatory subunit RPN3</td><td>-1.14</td><td>.461</td><td>.744</td><td>-1.01</td><td>.909</td><td>.974</td><td>R<sup>2</sup>: 0.00028<br>P val: .9549</td></tr><tr><td>PSMD9</td><td>26S proteasome regulatory subunit p27</td><td>1.18</td><td>.324</td><td>.631</td><td>-1.10</td><td>.612</td><td>.849</td><td>R<sup>2</sup>: 0.4501<br>P val: .0086</td></tr><tr><td>PSMD12</td><td>26S proteasome regulatory subunit RPN5</td><td>1.23</td><td>.227</td><td>.532</td><td>1.16</td><td>.229</td><td>.581</td><td>R<sup>2</sup>: 0.04923<br>P val: .4459</td></tr><tr><td>PSMD11</td><td>26S proteasome regulatory subunit RPN6</td><td>1.17</td><td>.382</td><td>.683</td><td>1.10</td><td>.528</td><td>.803</td><td>R<sup>2</sup>: 0.5294<br>P val: .0032</td></tr><tr><td>PSMD6</td><td>26S proteasome regulatory subunit RPN7</td><td>1.18</td><td>.191</td><td>.488</td><td>1.22</td><td>.046</td><td>.273</td><td>R<sup>2</sup>: 0.0002<br>P val: .9605</td></tr><tr><td>PSMD7</td><td>26S proteasome regulatory subunit RPN8</td><td>1.31</td><td>.081</td><td>.318</td><td>1.23</td><td>.255</td><td>.606</td><td>R<sup>2</sup>: 0.5513<br>P val: .0024</td></tr><tr><td>PSMD13</td><td>26S proteasome regulatory subunit RPN9</td><td>-1.11</td><td>.561</td><td>.807</td><td>1.07</td><td>.559</td><td>.823</td><td>R<sup>2</sup>: 0.01682<br>P val: .6586</td></tr><tr><td>PSMD14</td><td>26S proteasome regulatory subunit RPN11</td><td>-1.13</td><td>.660</td><td>.861</td><td>1.27</td><td>.118</td><td>.433</td><td>R<sup>2</sup>: 0.0053<br>P val: .804</td></tr><tr><td>PSMD8</td><td>26S proteasome regulatory subunit RPN12</td><td>1.62</td><td>.014</td><td>.112</td><td>1.37</td><td>.069</td><td>.332</td><td>R<sup>2</sup>: 0.0016<br>P val: .893</td></tr><tr><td>SHFM1</td><td>26S proteasome complex subunit SEM1</td><td>1.86</td><td>.014</td><td>.113</td><td>1.35</td><td>.111</td><td>.422</td><td>R<sup>2</sup>: 0.0437<br>P val: .476</td></tr><tr><td>PSMD4</td><td>26S proteasome regulatory subunit RPN10</td><td>1.09</td><td>.536</td><td>.791</td><td>1.00</td><td>.947</td><td>.986</td><td>R<sup>2</sup>: 0.1984<br>P val: .1105</td></tr><tr><td>PSMD2</td><td>26S proteasome regulatory subunit RPN1</td><td>-1.30</td><td>.073</td><td>.301</td><td>-1.29</td><td>.122</td><td>.441</td><td>R<sup>2</sup>: 0.2218<br>P val: .0891</td></tr><tr><td>PSMD1</td><td>26S proteasome regulatory subunit RPN2</td><td>-1.01</td><td>.937</td><td>.981</td><td>-1.05</td><td>.774</td><td>.925</td><td>R<sup>2</sup>: 0.1248<br>P val: .2153</td></tr><tr><td>ADRM1</td><td>Proteasome regulatory particle non-ATPase 13 (RPN13)</td><td>-1.02</td><td>.868</td><td>.955</td><td>-1.01</td><td>.930</td><td>.979</td><td>R<sup>2</sup>: 0.0014<br>P val: .8959</td></tr><tr><td>PSMC2</td><td>26S proteasome AAA-ATPase sub-unit RPT1</td><td>1.04</td><td>.812</td><td>.930</td><td>1.23</td><td>.075</td><td>.344</td><td>R<sup>2</sup>: 0.1389<br>P val: .1895</td></tr><tr><td>PSMC1</td><td>26S proteasome AAA-ATPase sub-unit RPT2</td><td>1.06</td><td>.755</td><td>.906</td><td>-1.01</td><td>.940</td><td>.984</td><td>R<sup>2</sup>: 0.2092<br>P val: .1001</td></tr><tr><td>PSMC5</td><td>26S proteasome AAA-ATPase sub-unit RPT6</td><td>-1.00</td><td>.982</td><td>.996</td><td>1.00</td><td>.943</td><td>.984</td><td>R<sup>2</sup>: 0.1279<br>P val: .2093</td></tr><tr><td>PSMC6</td><td>26S proteasome AAA-ATPase sub-unit RPT4</td><td>1.04</td><td>.826</td><td>.935</td><td>1.34</td><td>.012</td><td>.126</td><td>R<sup>2</sup>: 0.0014<br>P val: .8994</td></tr><tr><td>PSMC3</td><td>26S proteasome AAA-ATPase sub-unit RPT5</td><td>-1.05</td><td>.576</td><td>.816</td><td>-1.08</td><td>.364</td><td>.698</td><td>R<sup>2</sup>: 0.0265<br>P val: .5784</td></tr><tr><td>PSMC4</td><td>26S proteasome AAA-ATPase sub-unit RPT3</td><td>1.38</td><td>.054</td><td>.254</td><td>1.38</td><td>.035</td><td>.235</td><td>R<sup>2</sup>: 0.6509<br>P val: .0005</td></tr><tr><td>PSME1</td><td>PA28α<br>(Proteasome activator complex subunit 1)</td><td>5.02</td><td>4.60E-11</td><td>3.01E-08</td><td>4.13</td><td>4.17E-08</td><td>1.55E-05</td><td>R<sup>2</sup>: 0.2128<br>P val: .0969</td></tr><tr><td>PSME2</td><td>PA28β<br>(Proteasome activator complex subunit 2)</td><td>10.16</td><td>2.92E-11</td><td>1.99E-08</td><td>8.49</td><td>2.76E-08</td><td>1.17E-05</td><td>R<sup>2</sup>: 0.3579<br>P val: .0238</td></tr><tr><td>PSME3</td><td>PA28γ<br>(Proteasome activator complex subunit 3)</td><td>-1.09</td><td>.688</td><td>.874</td><td>1.44</td><td>.007</td><td>.093</td><td>R<sup>2</sup>: 0.128<br>P val: .2091</td></tr><tr><td>PSME4</td><td>PA200<br>(Proteasome activator complex subunit 4)</td><td>1.18</td><td>.566</td><td>.809</td><td>1.14</td><td>.584</td><td>.835</td><td>R<sup>2</sup>: 0.0028<br>P val: .8558</td></tr><tr><td>PSMA6</td><td>Proteasome subunit α1</td><td>1.38</td><td>.147</td><td>.428</td><td>1.24</td><td>.193</td><td>.542</td><td>R<sup>2</sup>: 0.6617<br>P val: .0004</td></tr><tr><td>PSMA2</td><td>Proteasome subunit α2</td><td>1.01</td><td>.938</td><td>.981</td><td>1.09</td><td>.561</td><td>.824</td><td>R<sup>2</sup>: 0.1182<br>P val: .2288</td></tr><tr><td>PSMA4</td><td>Proteasome subunit α3</td><td>1.22</td><td>.174</td><td>.463</td><td>1.48</td><td>.010</td><td>.115</td><td>R<sup>2</sup>: 0.4151<br>P val: .0129</td></tr><tr><td>PSMA5</td><td>Proteasome subunit α5</td><td>2.27</td><td>8.06E-05</td><td>.003</td><td>1.92</td><td>.001</td><td>.034</td><td>R<sup>2</sup>: 0.4742<br>P val: .065</td></tr><tr><td>PSMA1</td><td>Proteasome subunit α6</td><td>1.20</td><td>.329</td><td>.635</td><td>1.53</td><td>.006</td><td>.085</td><td>R<sup>2</sup>: 0.097<br>P val: .2784</td></tr><tr><td>PSMA3</td><td>Proteasome subunit α7</td><td>1.52</td><td>1.61E-04</td><td>.005</td><td>1.62</td><td>4.40E-07</td><td>1.11E-04</td><td>R<sup>2</sup>: 0.1721<br>P val: .1402</td></tr><tr><td>PSMB6</td><td>Proteasome subunit β1</td><td>1.09</td><td>.538</td><td>.792</td><td>-1.06</td><td>.598</td><td>.842</td><td>R<sup>2</sup>: 0.0437<br>P val: .473</td></tr><tr><td>PSMB7</td><td>Proteasome subunit β2</td><td>1.48</td><td>5.85E-04</td><td>.013</td><td>1.37</td><td>.006</td><td>.080</td><td>R<sup>2</sup>: 0.3619<br>P val: .0229</td></tr></tbody></table>

(continued)
<table><caption>Table 3 (Continued)</caption><thead><tr><th rowspan="2">Gene name</th><th rowspan="2">Protein</th><th colspan="3">DM perf. vs no NMD perf.</th><th colspan="3">DM endo. vs no NMD endo.</th><th rowspan="2">Correlation with ISG15 expression</th></tr><tr><th>Fold change</th><th>P value</th><th>Adj. P value</th><th>Fold change</th><th>P value</th><th>Adj. P value</th></tr></thead><tbody><tr><td>PSMB3</td><td>Proteasome subunit β3</td><td>1.47</td><td>.006</td><td>.068</td><td>1.19</td><td>.130</td><td>.456</td><td>R<sup>2</sup>: 0.0848<br>P val: .3123</td></tr><tr><td>PSMB2</td><td>Proteasome subunit β4</td><td>1.49</td><td>.013</td><td>.107</td><td>1.56</td><td>.007</td><td>.092</td><td>R<sup>2</sup>: 0.4825<br>P val: .0058</td></tr><tr><td>PSMB5</td><td>Proteasome subunit β5</td><td>1.07</td><td>.520</td><td>.781</td><td>1.07</td><td>.520</td><td>.799</td><td>R<sup>2</sup>: 0.0162<br>P val: .6644</td></tr><tr><td>PSMB1</td><td>Proteasome subunit β6</td><td>1.23</td><td>.278</td><td>.588</td><td>1.04</td><td>.695</td><td>.889</td><td>R<sup>2</sup>: 0.1423<br>P val: .1836</td></tr><tr><td>PSMB4</td><td>Proteasome subunit β7</td><td>1.74</td><td>.005</td><td>.056</td><td>1.22</td><td>.130</td><td>.456</td><td>R<sup>2</sup>: 0.4243<br>P val: .0116</td></tr><tr><td>PSMB9</td><td>Proteasome subunit β1i</td><td>17.16</td><td>3.82E-10</td><td>1.66E-07</td><td>9.29</td><td>3.62E-06</td><td>5.12E-04</td><td>R<sup>2</sup>: 0.5061<br>P val: .0043</td></tr><tr><td>PSMB10</td><td>Proteasome subunit β2i</td><td>3.00</td><td>2.46E-04</td><td>.007</td><td>2.15</td><td>.015</td><td>.146</td><td>R<sup>2</sup>: 0.3629<br>P val: .0226</td></tr><tr><td>PSMB8</td><td>Proteasome subunit β5i</td><td>47.14</td><td>1.31E-21</td><td>2.22E-17</td><td>16.64</td><td>7.75E-14</td><td>5.92E-10</td><td>R<sup>2</sup>: 0.4121<br>P val: .0133</td></tr><tr><td>PSMF1</td><td>Proteasome inhibitor PI31 subunit</td><td>-1.20</td><td>.003</td><td>.042</td><td>-1.03</td><td>.792</td><td>.932</td><td>R<sup>2</sup>: 0.0588<br>P val: .4037</td></tr><tr><td>POMP</td><td>Proteasome maturation protein</td><td>1.49</td><td>.006</td><td>.065</td><td>1.12</td><td>.485</td><td>.779</td><td>R<sup>2</sup>: 0.1984<br>P val: .1105</td></tr></tbody></table>

DM, dermatomyositis; endo., endofascicular; NMD, neuromuscular disorder; perfif., perifascicular.

R² value: coefficient of determination.

DISCUSSION

The lack of knowledge regarding the molecular mechanisms underpinning perifascicular fibre abnormalities in DM limits the development of treatments targeting the pathomechanisms of this disease.

We show herein that IFN-β-induced upregulation of the proteasome pathway in perifascicular muscle fibres is an early hallmark of DM that contributes to certain other key characteristics of the disease, including MHC-I expression, mitochondrial dysfunctions and perifascicular fibre atrophy. In keeping with both the role of IFN-I in DM [11,15] and the regulation of the proteasome expression [24], we found that PSMB8 is the most upregulated proteasome subunit in the perifascicular fibres of patients with DM. In addition, proteasome inhibitors, predicted in silico as candidate repurposed drugs based on this new insight, were observed to be effective in preclinical models of DM.

Bulk analysis has previously shown an increased expression of PSMB8 and chymotrypsin-like activity in the muscle of patients with DM as well as in other IM subtypes, mainly in IBM and ASyS, and to a lesser extent in IMNM [35,36]. Muscle β5i immunostaining has suggested that, in contrast to non-DM IM, enhanced proteasome expression and chymotrypsin-like activity in DM are prevalent in perifascicular fibres, although such activity has not been systematically assessed [35]. Our findings, based on an unbiased RNA-sequencing analysis of microdissected fibres, validated at the protein level in an independent cohort spanning all IM subtypes, confirm and expand these previous reports. The analyses were performed in untreated, early patients with DM (symptom duration <6 months). In muscle fibres, proteasome subunits overexpression colocalised with, and positively correlated to, the type I IFN signature. Moreover, IFN-β enhanced proteasome activity in human myotubes. Altogether, these findings indicate that increased proteasome activation in perifascicular fibres of patients with DM is associated with disease activity. These results pave the way for future studies aimed at assessing the sensitivity and specificity of PSMB8 immunostaining as a potential diagnostic and prognostic biomarker for DM in larger cohorts.

IFN-β is increased in the blood of patients with DM [15,16] and recapitulates the canonical characteristics of DM perifascicular muscle fibres in myotubes (ie, IFN-I signature, MHC-I expression, mitochondrial dysfunctions, and atrophy) [6,10,17,18]. However, the mechanism underlying the IFN-β effect on myofibres has remained largely unknown. In particular, while ISG15 was previously found to be highly upregulated in DM perifascicular fibres and in IFN-β-treated cells, targeting ISG15 did not prevent the effects of IFN-β on muscle cells [17]. The data presented herein shed new light on DM pathophysiology by revealing that IFN-β enhances proteasome activity in muscle fibres that subsequently underpin myofibre i) atrophy, ii) MHC-I expression, and iii) mitochondrial dysfunction, downstream of ISG upregulation. This finding is in line with previous independent observations, including i) the seminal pharmaceuticals development of proteasome inhibitors for muscle wasting [37], ii) the reversion of IFN-induced MHC-I expression in myotubes [35], iii) the improvement of muscle function with bortezomib in a mouse model of myositis [38], and iv) the rescue of mitochondrial complex IV deficiency by proteasome inhibitors in fibroblasts derived from patients with mitochondrial monogenic diseases [39,40].

In contrast to DM, IFN-γ has been reported to play a prominent role in the pathogenesis of non-DM myopathies such as ASyS, SM, and IBM [36,29]. Consistent with these data, we observed β5i expression in muscle tissue from these patients, with a distribution distinct from the perifascicular pattern seen in DM. Additionally, in vitro, IFN-γ exerted a distinct myopathic effect compared to IFN-β, more closely resembling the features of non-DM muscle fibres [29]. Finally, in further contrast with IFN-β, the myopathic effects of IFN-γ occurred independently of proteasome activation. Comparative data on IFN-β- and IFN-γ-mediated proteasome regulation have so far been scarce [20,24], and, to our knowledge, absent in muscle cells. Here, we show that, in human myotubes, both IFN-β and IFN-γ similarly enhanced β5i proteasome activity, while IFN-β induced higher PSMB8 expression, suggesting differential regulation of proteasome components by the 2 cytokines. However, given that the in vivo biological activity of IFN-β and IFN-γ in the muscle tissue of
**Figure 4.** Interferons (IFNs) regulation of PSMB8 gene expression in an *in vitro* model of dermatomyositis (DM). (A) IFNs activation of JAK-STAT pathway. (B) Heatmap depicting the expression of PSMB8, MxA, and major histocompatibility complex (MHC)-II in fibroblasts treated with IFN-β (500 U/mL) or IFN-γ (500 U/mL), according to the datasets available on Interferome database. (C) PSMB8, MxA, and MHC-II gene expression in human myotubes treated with IFN-β (500 U/mL) or IFN-γ (500 U/mL). Data are expressed as the mean ± SEM. \*P < .05, \*\*P < .01, \*\*\*P < .001, \*\*\*\*P < .0001 vs the comparator indicated by the line. ISG, interferon-stimulated gene; JAK, Janus kinase.

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

patients with IM remains undefined, the clinical significance of this difference is uncertain. Myofibre mitochondrial dysfunctions [6] and MHC-I expression [41] participate in the development and maintenance of muscle inflammation in DM. In keeping with these observations, we found that inflammatory infiltrate was decreased in the muscle of EM mice treated with ixazomib further supporting a crosstalk between muscle fibres and immune cells, and therefore positioning muscle fibres as a therapeutic target in DM. However, in this model, a direct effect of ixazomib on immune cells.

Several strategies aiming at blocking the IFN-β pathway in DM are currently under investigation, which include direct targeting IFN-β itself (NCT05895786), its receptors

(NCT06455449), and downstream JAK (NCT04972760) and/or Tyk2 (NCT05437263, NCT05335447) activation. Our data reveal that proteasome blockade is an additional putative strategy to block IFN-β pathway in DM. An 'on-target' therapeutic effect of proteasome inhibitors—consistent with their known pharmacodynamics—occurring downstream of IFN-β-driven transcriptomic stimulation, was supported by their ability to inhibit IFN-β-induced proteasome activation and IFN-β-induced myopathic effects without altering ISG expression. Comparative studies assessing the efficacy and tolerability of these candidate therapies warrant further investigation. Proteasome inhibition with bortezomib (included solely in the L1000CDS² database and identified as a candidate drug
<figure><img src="image_13.png" /></figure>

<table><caption>**Figure 5.** Drug repurposing analysis. (A) Venn diagram of molecules identified by 3 drug repurposing tools. (B) Candidate repurposed drugs identified by at least 2 tools with a predictive score (see Methods section) <-50. HSP, heat shock protein; IKK, IκB kinase; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; PKC, protein kinase C.</caption><thead><tr><th>Drug</th><th>Predictive score</th><th>Target</th></tr></thead><tbody><tr><td>Ixazomib</td><td>-84.50</td><td>Proteasome inhibitor</td></tr><tr><td>Fedratinib</td><td>-81.70</td><td>JAK2 inhibitor</td></tr><tr><td>TPCA-1</td><td>-81.68</td><td>IKK inhibitor</td></tr><tr><td>Rottlerin</td><td>-79.69</td><td>MAPK inhibitor, PKC inhibitor</td></tr><tr><td>MG-132</td><td>-77.63</td><td>Proteasome inhibitor</td></tr><tr><td>Geldanamycin</td><td>-62.11</td><td>HSP inhibitor</td></tr><tr><td>Prednisolone</td><td>-62.5</td><td>Glucocorticoid receptor agonist</td></tr><tr><td>Lobelanidine</td><td>-61.5</td><td>Acetylcholine receptor antagonist</td></tr><tr><td>Alvespimycin</td><td>-54.49</td><td>HSP inhibitor</td></tr></tbody></table>

repurposed for DM by this tool) has been reported as an effective strategy in real-life treatment of systemic lupus erythematosus (SLE) [42,43], an autoimmune disease sharing clinical, pathophysiological, and genetic features with DM [6,44]. In this setting, the efficacy of proteasome inhibition has been hypothesised to be driven by the induction of apoptosis in plasma cells [42,43]. Although this mechanism might also participate in the therapeutic response observed herein in our EM mice model, our results derived from microdissected fibres and myotube experiments additionally support immunomodulation through the targeting of β5i hyperactivation in resident cells (ie, myofibres). Notably, selective inhibition of β5i has been shown to block the presentation of MHC-I-restricted antigens during the antiviral response [45], 2 pathways found to be specifically increased in DM perifascicular fibres in the present study. In both IFN-β-treated myotubes and EM myofibres, proteasome inhibition abolished MHC-I expression without affecting ISG expression. As a result, inflammatory infiltrate (mainly T cells in EM) [31] was decreased in ixazomib-treated mice muscle tissue.

Zetomipzomib (KZR-616), an inhibitor of the β5i and β1i immunoproteasome subunits not included in the databases of repositionable drugs, is currently in development for several autoimmune diseases. While it has shown positive effects in SLE in an open-label phase 1b/2 study [46], a 16-week intake of the treatment in 25 patients with active IM (including 13 DM and 12 non-DM IM), did not lead to a higher improvement vs placebo (the improvement was clinically meaningful in both groups).

Notwithstanding, improvement was nonetheless numerically greater with zetomipzomib with baseline correction to W16 (cross over) continuing throughout the open-label extension period up to 96 weeks [47]. Whether the improvement was greater in patients with DM vs patients without DM IM has not been provided to date. Our data further support the pursuit of the development of this drug in DM rather than in other IM subtypes.

Adverse events in patients with SLE treated with bortezomib have been frequently reported (ranging from 50% to 90%), although they were generally mild [42,43]. To improve the safety profile of this class of drugs, next-generation proteasome inhibitors have been developed. Ixazomib (used in our study) has demonstrated a lower incidence of serious adverse events in patients with multiple myeloma compared to bortezomib [48]. Although no serious adverse events related to zetomipzomib were reported in patients with IM [47] or in a Phase IIa trial in autoimmune hepatitis (NCT05569759), four patients died (including one in the placebo arm) in a Phase IIb trial evaluating this drug in active lupus nephritis (NCT05781750). Evidence suggestive of co-morbidities immediately prior to zetomipzomib exposure (e.g., possible systemic infection) and/or underlying disease characteristics (e.g., cardiovascular conditions) was present in these fatal cases [49]. Nevertheless, this study was stopped following a Food and Drug Administration hold. A partial hold imposed on the Phase IIa trial in autoimmune hepatitis (NCT05569759) was lifted in July 2025, and this study is currently ongoing.
<figure><img src="image_14.png" /><figcaption>**Figure 6.** Proteasome inhibition is effective in an *in vitro* model of dermatomyositis (DM). (A) Experimental design: human myotubes treated with IFN-β (500 U/mL for 5 days) and ixazomib (Ixa, 40 nM) or vehicle added at day 2. (B) Chymotrypsin-like activity in human myotubes expressed as pmol/min/well (n ≥ 4). (C) Assessment of human myotube diameter (n ≥ 49 per group). (D) Mitochondrial respiration (n ≥ 5 per group). (E) Representative figure of major histocompatibility complex-I (MHC-I) immunostaining (green). Nuclei were stained with DAPI (blue). Scale bar: 100 µm. (F) Expression of interferon-stimulated genes MHC-I immunostaining (green). Nuclei were stained with DAPI (blue). Scale bar: 100 µm. (F) Expression of interferon-stimulated genes (n ≥ 9 per group). Data are expressed as the mean ± SEM. \*P < .05, \*\*P < .01, \*\*\*P < .001, \*\*\*\*P < .0001. CTRL, control; IFN, interferon; V0: basal oxygen consumption with glutamate and malate; Vmax: ADP-stimulated respiration; Vsucc: succinate-stimulated respiration. ADP: adenosine diphosphate; DAPI: 4',6-diamidino-2-phenylindole.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 7.** Proteasome inhibition is effective in experimental myositis (EM). (A) Experimental design: EM was induced at day 0 (D0), ixazomib (Ixa) or vehicle (V) was administered at D14, and mice were sacrificed at day 21 (D21). (B) Grip strength measurements (n ≥ 5) in mice at D14 and D21. (C) Serum creatine kinase (CK) levels at D21 in EM mice treated with Ixa or V, and in unimmunised mice (control, CTRL). (D) Coefficient of fibre size variability in gastrocnemius muscle of EM mice treated with Ixa or V, n ≥ 3. (E) Representative hematoxylin & eosin (H&E) staining of gastrocnemius cryo-sections at × 200 magnification. Scale bar: 100 µm. (F) Representative major histocompatibility complex-I (MHC-I) immunostaining (green) in the gastrocnemius muscle of EM mice treated with Ixa or V. Nuclei were stained with DAPI (blue). IgG was used as negative (Neg) control. Scale bar: 100 µm. (G) IFIT3 transcript levels normalised for housekeeping gene expression (18S), n ≥ 9 for each condition. (H) Histological inflammatory infiltrate score in H&E-stained slides from gastrocnemius muscle of EM mice treated with Ixa or V and in CTRL mice, at D21. Each dot represents the average score of 3 sections for each mouse (n ≥ 3 mice for each condition; 3 sections from each mouse were analysed). (I) PSMB8 transcript level normalised for housekeeping gene expression (18S), n ≥ 7 for each condition. (J) Chymotrypsin-like activity in gastrocnemius muscle of EM mice treated with Ixa or V and in CTRL mice, at D21 (n ≥ 3 for each condition). Data are expressed as the mean ± SEM. ns: not significant (*P* ≥ 0.05), \*P\* < .05, \*\*P\* < .01, \*\*\*P\* < .001, \*\*\*\*P\* < .0001, \*\*\*\*\*P\* < .0001 vs D14. DAPI: 4',6-diamidino-2-phenylindole.</figcaption></figure>
In conclusion, enhanced proteasome activity in perifascicular myofibres is a hallmark of DM and a likely therapeutic target.

## Competing interests

None declared.

## Acknowledgements

We thank the IGBMC animal facility, cell culture, and histology services. We acknowledge the support of the Light Microscopy Facility at the IGBMC imaging center, a member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04), as well as that of GenomEast, a member of the 'France Génomique' consortium (ANR-10-INBS-0009). We thank the CRBS imaging facility members (PIC-STRA, Pascal Kessler).

We acknowledge the support of the Anatomopathological Department of the Strasbourg University Hospital for histology services (Angélique Werck, Fabienne Moegling). We thank Prof. Hermen Overkleeft and Prof. Bobby Florea for fruitful discussions. We thank Mr Pierre Pothier for proofreading.

## Contributors

LD, MG, GL, AM: substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. LD, MG: are joint junior authors. GL, AM: are joint senior authors. GQ, SP, DR, CK, BL, A-LC, BG, DM: substantial contributions to the acquisition of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published. LD, MG, GL, AM: accept full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish. LD and MG are guarantors.

## Funding

This study was supported by a post-doctoral fellowship from the AFM-Telethon attributed to Léa Debrut (24236) and by a grant from the AFM-Telethon (22425), from the French Society of Rheumatology (Bourse mobilité SFR 2018), and from CSL Behring (Bourse en immunologie 2019).

## Patient consent for publication

Written informed consent was obtained from all participants. Patients were not involved in the design and conduct of this research, but patient associations will participate in disseminating the results of this research.

## Ethics approval

This study was approved by the local Ethics Committee (No 6431). Written informed consent was obtained from all participants. The animal study was approved by the IGBMC Ethical Committee and the French Ministry (#35432-2022021417533158).

## Provenance and peer review

Not commissioned; externally peer reviewed.

## Data availability statement

Data will be shared on request from any qualified investigator.

## Supplementary materials

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.ard.2025.08.031.

## Orcid

Léa Debrut: http://orcid.org/0000-0003-1534-8088

Margherita Giannini: http://orcid.org/0000-0002-4834-5804

## REFERENCES

[1] Debrut L, Giannini M, Klein D, Spielmann L, Mertz P, Martin T, et al. Refining incidence and characteristics of inflammatory myopathies: a quadruple-source capture-recapture survey using the 2017 European League Against Rheumatism/American College of Rheumatology Classification Criteria. Arthritis Rheumatol. 2023 Oct;75(10):1850-5.

[2] Mammen AL, Allenbach Y, Stenzel W, Benveniste O. ENMC 239th Workshop Study Group. 239th ENMC International Workshop: classification of dermatomyositis, Amsterdam, the Netherlands, 14-16 December 2018. Neuromuscul Disord 2020 Jan;30(1):70-92.

[3] Pinal-Fernandez I, Casciola-Rosen LA, Christopher-Stine L, Corse AM, Mammen AL. The prevalence of individual histopathologic features varies according to autoantibody status in muscle biopsies from patients with dermatomyositis. J Rheumatol 2015 Aug;42(8):1448-54.

[4] Meyer A, Scirè CA, Talarico R, Alexander T, Amoura Z, Avcin T, et al. Idiopathic inflammatory myopathies: state of the art on clinical practice guidelines [corrected]. RMD Open 2018;4(suppl 1):e000784.

[5] Gallay L, Fermon C, Lessard L, Weiss-Gayet M, Viel S, Streichenberger N, et al. Involvement of type I interferon signaling in muscle stem cell proliferation during dermatomyositis. Neurology 2022 May 24;98(21):e2108-19.

[6] Meyer A, Laverny G, Allenbach Y, Grelet E, Ueberschlag V, Echaniz-Laguna A, et al. IFN-β-induced reactive oxygen species and mitochondrial damage contribute to muscle impairment and inflammation maintenance in dermatomyositis. Acta Neuropathol 2017 Oct;134(4):655-66.

[7] Fasth AER, Dastmalchi M, Rahbar A, Salomonsson S, Pandya JM, Lindroos E, et al. T cell infiltrates in the muscles of patients with dermatomyositis and polymyositis are dominated by CD28null T cells. J Immunol 2009 Oct 1;183(7):4792-9.

[8] Giannini M, Rovito D, Debrut L, Keime C, Lannes B, Charles A, et al. Muscle fibres play a critical role in therapeutic response of myositis to glucocorticoids through polarisation of the inflammatory infiltrate by a paracrine mechanism [abstract]. Arthritis Rheumatol 2022;74(suppl 9).

[9] Greenberg SA, Pinkus JL, Pinkus GS, Burleson T, Sanoudou D, Tawil R, et al. Interferon-alpha/beta-mediated innate immune mechanisms in dermatomyositis. Ann Neurol 2005 May;57(5):664-78.

[10] Pinal-Fernandez I, Casal-Dominguez M, Derfoul A, Pak K, Miller FW, Milienda JC, et al. Machine learning algorithms reveal unique gene expression profiles in muscle biopsies from patients with different types of myositis. Ann Rheum Dis 2020 Sep;79(9):1234-42.

[11] Salajegheh M, Kong SW, Pinkus JL, Walsh RJ, Liao A, Nazareno R, et al. Interferon-stimulated gene 15 (ISG15) conjugates proteins in dermatomyositis muscle with perifascicular atrophy. Ann Neurol 2010 Jan;67(1):53-63.

[12] Suárez-Calvet X, Gallardo E, Pinal-Fernandez I, De Luna N, Lleixà C, Díaz-Manera J, et al. RIG-I expression in perifascicular myofibers is a reliable biomarker of dermatomyositis. Arthritis Res Ther 2017 Dec;19(1):174.

[13] Kloetzel PM. Antigen processing by the proteasome. Nat Rev Mol Cell Biol 2001 Mar;2(3):179-87.

[14] Collins GA, Goldberg AL. The logic of the 26S proteasome. Cell 2017 May 18;169(5):792-806.
[15] Liao AP, Salajegheh M, Nazareno R, Kagan JC, Jubin RG, Greenberg SA. Interferon β is associated with type 1 interferon-inducible gene expression in dermatomyositis. Ann Rheum Dis 2011 May;70(5):831–6.

[16] Bolko L, Anquetil C, Llibre A, Maillard S, Amelin D, Dorgham K, et al. Ultra-sensitive interferons quantification reveals different cytokine profile secretion in inflammatory myopathies and can serve as biomarkers of activity in dermatomyositis. Front Immunol 2025 Feb 12;16:1529582.

[17] Franzi S, Salajegheh M, Nazareno R, Greenberg SA. Type 1 interferons inhibit myotube formation independently of upregulation of interferon-stimulated gene 15. PLoS One 2013 Jun 4;8(6):e65362.

[18] Ladislau L, Suárez-Calvet X, Toquet S, Landon-Cardinal O, Amelin D, Depp M, et al. JAK inhibitor improves type I interferon induced damage: proof of concept in dermatomyositis. Brain 2018 Jun 1;141(6):1609–21.

[19] Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signaling. Nat Rev Immunol 2005 May;5(5):375–86.

[20] Hisamatsu H, Shimbara N, Saito Y, Kristensen P, Hendil KB, Fujiwara T, et al. Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma. J Exp Med 1996 Apr 1;183(4):1807–16.

[21] Niewerth D, Kaspers GJ, Assaraf YG, van Meerloo J, Kirk CJ, Anderl J, et al. Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines. J Hematol Oncol 2014 Jan 13;7:7.

[22] Jamaluddin M, Wang S, Garofalo RP, Elliott T, Casola A, Baron S, et al. IFN-beta mediates coordinate expression of antigen-processing genes in RSV-infected pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 2001 Feb;280(2):L248–57.

[23] Shin EC, Seifert U, Kato T, Rice CM, Feinstone SM, Kloetzel PM, et al. Virus-induced type I IFN stimulates generation of immunoproteasomes at the site of infection. J Clin Invest 2006 Nov;116(11):3006–14.

[24] Seifert U, Bialy LP, Ebstein F, Bech-Otschir D, Voigt A, Schröter F, et al. Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress. Cell 2010 Aug 20;142(4):613–24.

[25] Duncan CJ, Mohamad SM, Young DF, Skelton AJ, Leahy TR, Munday DC, et al. Human IFNAR2 deficiency: lessons for antiviral immunity. Sci Transl Med 2015 Sep 30;7(307) 307ra154.

[26] Lessard LER, Robert M, Fenouil T, Mounier R, Landel V, Carlesimo M, et al. Contribution of major histocompatibility complex class II immunostaining in distinguishing idiopathic inflammatory myopathy subgroups: a histopathological cohort study. J Neuropathol Exp Neurol 2024 Dec 1;83(12):1060–75.

[27] Rusinova I, Forster S, Yu S, Kannan A, Masse M, Cumming H, et al. Interferome v2.0: an updated database of annotated interferon-regulated genes. Nucleic Acids Res 2013 Jan;41 part 1:000000–000000.

[28] Der SD, Zhou A, Williams BR, Silverman RH. Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc Natl Acad Sci USA 1998 Dec 22;95(26):15623–8.

[29] Hou C, Periou B, Gervais M, Martin L, Berthier J, Baba-Amer Y, et al. Interferon-γ causes myogenic cell dysfunction and senescence in immune myopathies. Brain 2025 Apr 29;148(8):2883–98.

[30] Karatzas E, Kolios G, Spyrou GM. An application of computational drug repurposing based on transcriptomic signatures. Methods Mol Biol 2019;1903:149–77.

[31] Giannini M, Rovito D, Oulad-Abdelghani M, Messaddeq N, Debrut L, Quiring G, et al. Experimental myositis: an optimised version of C-protein-induced myositis. RMD Open 2025 Mar 5;11(1):e004558.

[32] Sugihara T, Sekine C, Nakae T, Kohyama K, Harigai M, Iwakura Y, et al. A new murine model to define the critical pathologic and therapeutic mediators of polymyositis. Arthritis Rheum 2007 Apr;56(4):1304–14.

[33] Micheletto MLJ, Hermes T de A, Bertassoli BM, Petri G, Perez MM, Fonseca FLA, et al. Ixazomib, an oral proteasome inhibitor, exhibits potential effect in dystrophin-deficient mdx mice. Int J Exp Pathol 2021 Feb;102(1):11–21.

[34] Millennium Pharmaceuticals Inc.. NINLARO (ixazomib) capsules, for oral use. Prescribing information. U.S. Food and Drug Administration; 2015 Available from: https://www.accessdata.fda.gov/drugsatfda_docs/label/2015/208462lbl.pdf.

[35] Bhattarai S, Ghannam K, Krause S, Benveniste O, Marg A, de Bruin G, et al. The immunoproteasomes are key to regulate myokines and MHC class I expression in idiopathic inflammatory myopathies. J Autoimmun 2016 Dec;75:118–29.

[36] Pinal-Fernandez I, Casal-Dominguez M, Derfoul A, Pak K, Plotz P, Miller FW, et al. Identification of distinctive interferon gene signatures in different types of myositis. Neurology 2019 Sep 17;93(12):e1193–204.

[37] Sánchez-Serrano I. Success in translational research: lessons from the development of bortezomib. Nat Rev Drug Discov 2006 Feb;5(2):107–14.

[38] Rayavarapu S, Coley W, Van der Meulen JH, Cakir E, Tappeta K, Kinder TB, et al. Activation of the ubiquitin proteasome pathway in a mouse model of inflammatory myopathy: a potential therapeutic target. Arthritis Rheum 2013 Dec;65(12):3248–58.

[39] Mohanraj K, Wasilewski M, Benincá C, Cysewski D, Poznanski J, Sakowska P, et al. Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7. EMBO Mol Med 2019 May;11 (5):e9561.

[40] Habich M, Riemer J. Stop wasting protein-proteasome inhibition to target diseases linked to mitochondrial import. EMBO Mol Med 2019 May;11(5):e10441.

[41] Okiyama N, Ichimura Y, Shobo M, Tanaka R, Kubota N, Saito A, et al. Immune response to dermatomyositis-specific autoantigen, transcriptional intermediary factor 1γ can result in experimental myositis. Ann Rheum Dis 2021 Sep;80(9):1201–8.

[42] Walhelm T, Gunnarsson I, Heijke R, Leonard D, Tryssberg E, Eriksson P, et al. Clinical experience of proteasome inhibitor bortezomib regarding efficacy and safety in severe systemic lupus erythematosus: a nationwide study. Front Immunol 2021;12:756941.

[43] Alexander T, Sarfert R, Klotsche J, Kühl AA, Rubbert-Roth A, Lorenz HM, et al. The proteasome inhibitor bortezomib depletes plasma cells and ameliorates clinical manifestations of refractory systemic lupus erythematosus. Ann Rheum Dis 2015 Jul;74(7):1474–8.

[44] Bianchi M, Kozyrev SV, Notarnicola A, Sandling JK, Pettersson M, Leonard D, et al. Unraveling the genetics of shared clinical and serological manifestations in patients with systemic inflammatory autoimmune diseases. Arthritis Rheumatol 2025 Feb;77(2):212–25.

[45] Muchamuel T, Basler M, Aujay MA, Suzuki E, Kalim KW, Lauer C, et al. A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis. Nat Med 2009 Jul;15(7):781–7.

[46] Rovin B, Saxena A, Leff R, Li E, Park E, Henig N. Zetomipzomib (KZR-616) demonstrates anti-inflammatory and immunomodulatory potential in patients with active lupus with or without lupus nephritis: results from the openlabel phase 1B/2 mission study. Lupus Sci Med 2023;10(suppl 1):A1-171.

[47] Aggarwal R, Goyal N, Lam D, Ng A, Leff R, Ray K, et al. Zetomipzomib demonstrates favorable long-term safety and tolerability profile without signs of immunosuppression: results from the PRESIDIO study and its open-label extension study in patients with dermatomyositis and polymyositis [abstract]. Arthritis Rheumatol 2023;75(suppl 9).

[48] Huang J, Zhang M, Lin J, Yang X, Huang P, Zheng X. Safety assessment of proteasome inhibitors real world adverse event analysis from the FAERS database. Sci Rep 2025 Apr 4;15(1):11628.

[49] Furie R, Anand N, Desai S, Lowe E, Muchamuel T, Palaniswamy K, et al. Efficacy and Safety Results of Zetomipzomib from the PALIZADE Phase 2b Clinical Trial in Patients with Lupus Nephritis [abstract]. Arthritis Rheumatol 2025;77(suppl 9).