Prasugrel inhibits TLR7-driven autoimmunity in systemic lupus erythematosus by acetylating cGAS

Domain: biology
Published: 2026-03-18
Editorial outcome: minor_revision

Abstract
Systemic lupus erythematosus (SLE) has a complex, multifactorial etiology, which contributes to a lack of definitive cure and limited treatment efficacy. Here, we report that cyclic GMP-AMP synthase (cGAS) is significantly activated in SLE patients. We further demonstrate that cGAS deletion protects mice from lupus-like symptoms induced by the TLR7 agonist imiquimod (IMQ). In a screen of 3,159 FDA-approved drugs, we identify the antiplatelet agent prasugrel as a potent cGAS inhibitor. Mechanistically, prasugrel disrupts the DNA-triggered liquid phase condensation and activation of cGAS via direct acetylation. Strikingly, we find that prasugrel exhibits remarkable efficacy in treating SLE in both mouse models and patient cells. Importantly, we report elevated plasma cyclic GMP-AMP (cGAMP) in SLE patients and identify it as a potential biomarker for predicting prasugrel response. Thus, our work elucidates the essential role of cGAS in SLE pathogenesis and presents prasugrel as a promising therapeutic option with immediate translational potential.

Introduction
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of autoantibodies and the activation of immune cells that can attack a variety of tissues and organs throughout the body1. The pathogenesis of SLE is complicated and its clinical manifestations are heterogeneous, posing a formidable challenge in finding a definitive cure for SLE2,3. The existing treatment approaches typically focus on addressing individual pathological conditions and aim to alleviate symptoms2,3. However, these treatments lack precise targets, leading to various side effects and limited efficacy3. Studies revealed that the dysregulation of nucleic acid tolerance can lead to lupus-like diseases4,5. It is also reported that the transcriptional features closely associated with type I interferon (IFN) and the production of various autoantibodies, especially anti-double-stranded DNA antibodies and anti-nuclear antibodies, are prominent hallmarks of SLE2,3. Therefore, the involvement of the key DNA sensor, cyclic GMP-AMP synthase (cGAS), in SLE is of great interest6.

By sensing cytosolic DNA, cGAS plays a critical role in immune responses against infections7. The emergence of DNA in the cytoplasm is normally an important danger signal for pathogen invasion8. cGAS detects cytoplasmic DNA and catalyzes the synthesis of 2’3’-cyclic GMP-AMP (cGAMP), a second messenger molecule9,10. 2’3’-cGAMP then binds to the stimulator of interferon genes (STING, also known as MITA, MPYS or ERIS), an endoplasmic reticulum-localized adaptor protein9-14, mediating the production of type I IFNs and a spectrum of proinflammatory cytokines to elicit anti-infection immunity.

Besides the pathogen infection-introduced DNA, self-DNA can also stimulate cGAS activation4,15-19. For example, the release of mtDNA triggers immune responses by activating cGAS, and this process is implicated in multiple diseases4,17,18. In addition, the three-prime repair exonuclease 1 (TREX1) is responsible for metabolizing self-DNA fragments20-22. The deficiency of TREX1 has been identified in patients with SLE23 and Aicardi-Goutières syndrome (AGS) 24-26, and the Trex1–/– mice exhibit similar symptoms seen in human AGS patients26. The deletion of Cgas in Trex1–/– mice effectively ameliorates the disease manifestations, demonstrating that TREX1 deficiency leads to cytosolic DNA accumulation that stimulates autoimmune responses via cGAS16,19,27. Similarly, it is postulated that cGAS may be a key driver for SLE pathogenesis because of the type I IFN-associated transcriptional profiles and the aberrant nucleic acid-metabolizing processes found in SLE1,5. Despite this association, a recent study reported that cGAS did not promote autoimmunity in both pristane-induced murine SLE model and the MRL/lpr model28, suggesting that the specific mechanisms by which cGAS influences SLE, and its potential as a therapeutic target, remain obscure. Therefore, investigating the role of cGAS in SLE using clinical samples is of significant importance to elucidate the mechanisms and assess the therapeutic potential of cGAS.

In the current study, we find that cGAS is significantly activated in SLE patients and cGAS deletion protects mice from lupus-like symptoms induced by TLR7 agonist, imiquimod (IMQ). We further identify prasugrel, a widely used antiplatelet agent for blood clot prevention, as a potent cGAS inhibitor. Prasugrel directly acetylates cGAS and disrupts DNA-triggered liquid phase condensation, thereby blocking cGAS activation. Importantly, prasugrel exhibits remarkable efficacy in treating SLE in both IMQ-induced mouse models and patient cells. Our study elucidates the critical role of cGAS activation in SLE etiology and uncovers a novel therapeutic application for prasugrel.

Results
cGAS mediates the autoimmunity in TLR7-driven systemic lupus erythematosus

To investigate the involvement of cGAS in the pathogenesis of SLE, we measured the plasma concentrations of cGAMP, the product synthesized by the activated cGAS9,10, in samples obtained from healthy donors and SLE patients. Consistent with a previous study29, our data showed significantly higher cGAMP levels in plasma of SLE patients, compared to those of healthy donors (Fig.1a). Notably, no such elevation was observed in peripheral blood samples from patients with rheumatoid arthritis (RA) or dermatomyositis (DM) (Fig.1a). This finding was further confirmed in an independent validation cohort comprising 44 healthy donors and 44 SLE patients (Supplementary Fig.1a, b). These observations imply that cGAS activation is involved in SLE and may contribute to the pathogenic processes of this disease.

Although SLE is recognized as a complex autoimmune disorder with a multifactorial etiology, a growing number of evidence suggest that TLR7 is a driving factor for human SLE30-32. For example, a functional polymorphism of TLR7 was identified predisposing to SLE in humans30. The unrestricted TLR7 signaling is associated with human lupus31. Moreover, a gain-of-function mutation in TLR7 (Y264H) has been reported to cause human SLE32. Therefore, we sought to investigate whether cGAS plays a role in TLR7-mediated SLE. We first analyzed the correlation between TLR7 expression and cGAMP levels in samples from the above cohort (Supplementary Fig.1a) and found that the samples with higher TLR7 expression levels exhibited higher abundance of plasma cGAMP (Fig.1b). We further found that the TLR7 agonist, R837 or R84833, stimulated cGAS activation in cells (Fig.1c and Supplementary Fig.1c). These findings indicate the potential interplay between cGAS and TLR7 signaling.

In mice, the exclusive topical application of the TLR7 agonist, imiquimod (IMQ) 33, has been shown to induce lupus-like symptoms34. Employing this model, we administrated IMQ to wild-type (WT) mice three times a week for a duration of four weeks. We first measured the plasma cGAMP levels in mice and found that IMQ treatment led to a rapid increase in plasma cGAMP levels (Fig.1d, e), suggesting that cGAS activation is an early event upon TLR7 engagement. During this procedure, we monitored the representative phenotypes of SLE. Our results indicated that while the body weights remained stable (Supplementary Fig.1d), there was a notable enlargement of the spleen (Supplementary Fig.1e), and a progressive increase in serum levels of antinuclear antibody (ANA), anti-double-stranded DNA (dsDNA) antibody and IFN- was observed (Supplementary Fig.1f-h). The IMQ-induced SLE mice also exhibited renal pathological manifestations, indicated by the IgG and IgM depositions within the glomeruli (Supplementary Fig.1i) and the pronounced inflammatory cell infiltration (Supplementary Fig.1j). Strikingly, when Cgas–/– mice were treated with IMQ, the splenomegaly (Fig.1f, g and Supplementary Fig.1k) and other SLE-associated indicators were significantly reduced compared to that of WT mice (Fig.1h-k). Cgas deletion also alleviated the inflammatory cell infiltration induced by IMQ treatment (Fig.1l). The full kidney histological images from independent mice showed consistent results (Supplementary Fig.1l). According to a previous report28, cGAS deletion did not alleviate the autoimmunity in pristane-induced SLE or MRL/lpr mice. In our study we obtained similar results (Supplementary Fig.2a-i). These data indicates that cGAS plays an important role in TLR7-mediated SLE pathogenesis.

We then explored how cGAS is activated in TLR7 signaling. We found that the TLR7-stimulated IFN- production is markedly dampened in the absence of cGAS in both bone marrow cells and plasmacytoid dendritic cells (pDC) (Fig.1m, n and Supplementary Fig.2j-l). We further used ruxolitinib, a Janus Kinase (JAK) inhibitor35, to block the IFN cascade downstream of TLR7. Under such condition, TLR7 agonist stimuli still effectively triggered the synthesis of cGAMP (Fig.1o), suggesting that the involvement of cGAS in TLR7 signaling is independent of the feedback of IFN axis. In contrast, we observed that R837/R848 stimuli failed to induce cGAMP production in Tlr7 deletion cells (Supplementary Fig.2m, n). We also used RU.521, a cGAS inhibitor36, and M5049, a TLR7 inhibitor37, and found that the inhibition of either cGAS or TLR7 attenuated cGAMP production stimulated by R837 or R848 (Supplementary Fig.2o). We next treated Tlr7–/– bone marrow cells with RU.521 or treated Cgas–/– bone marrow cells with M5049 and found that the blockade of both proteins with each strategy dampened the R837/R848-induced cGAMP production (Supplementary Fig.2p). Together, TLR7 engagement triggers cGAS activation.

Considering that cGAS is a DNA sensor, and mtDNA is known to be released into cytosol under several pathological conditions to activate cGAS4,17,18, we reasoned that cGAS may also be activated by mtDNA downstream TLR7 engagement. To test this, we quantified the mtDNA abundance in cytosolic fractions and found that TLR7 activation led to a significantly increased level of cytoplasmic mtDNA (Supplementary Fig.3a, b). A previous study reported that alterations in mitochondrial permeability significantly influence the release of mtDNA17. As indicated by the openness of the inner membrane molecular machinery, mitochondrial permeability transition pore (mPTP) 17, R837 or R848 treatment induced an increased mitochondrial permeability (Supplementary Fig.3c-e).

The BCL2 antagonist/killer 1 (BAK) and BCL2 associated X, apoptosis regulator (BAX) macropores on the outer mitochondrial membrane are key channels for the releasement of mtDNA38. Using the BAK/BAX oligomerization inhibitor, MSN-12539, we found that BAK/BAX inhibition robustly reduced the mtDNA release (Supplementary Fig.3f, g) and the IFN- production (Supplementary Fig.3h) that are induced by TLR7 activation. Thus, TLR7 activation facilitated the release of mtDNA into the cytosol, thereby triggering cGAS activation and the subsequent IFN- production. Collectively, these data indicated the important role of cGAS in mediating the IFN production downstream of TLR7.

Prasugrel blocks cGAS activation by acetylation

Given that our findings imply cGAS inhibition as a potential therapeutic strategy for SLE, we explored approaches to inhibit cGAS. We focused on seeking small molecules capable of inducing cGAS acetylation, since our previous work reported the acetylation-mediated cGAS inhibition27. In a compound library comprising 3,159 FDA-approved small molecule drugs, we first selected 167 molecules that contained an acetyl group, and then performed in silico docking simulations with cGAS (PDB: 4O67) using Discovery Studio 2022 (Fig.2a). Among them, 106 molecules with potential cGAS-binding capabilities were ranked according to their CDOCKER interaction energy (Supplementary table 1). The top 15 candidates from this screening were then experimentally verified for their ability to acetylate cGAS. Using aspirin as a positive control27, we found that prasugrel was the only agent that induced cGAS acetylation, specifically at the key regulatory sites, Lys384, Lys394 and Lys414, which are known to inactivate cGAS upon acetylation (Fig.2b). The docking model showed the special accessibility of prasugrel to the key regulatory lysine sites of cGAS (Fig.2c-f).

We further examined the prasugrel-mediated cGAS acetylation and found that compared to aspirin, prasugrel can achieve a similar cGAS acetylation effect at one-thousandth of the concentration of aspirin (Fig.2g and Supplementary Fig.4a). With a human monocytic cell line, U937, we demonstrated that prasugrel (100 M) robustly acetylated endogenous cGAS in cells (Fig.2h). As expected, prasugrel treatment significantly suppressed the DNA-stimulated cGAS activation (Fig.2i), IRF3 phosphorylation (Fig.2j) and IFN production (Fig.2k, l). At this concentration, prasugrel had no cytotoxicity effect (Supplementary Fig.4b), and did not inhibit cGAMP-triggered downstream events (Fig.2m and Supplementary Fig.4c). These data suggested that prasugrel specifically inhibits the DNA-induced IFN production by acetylating cGAS. We then challenged U937 cells with intracellular RNA mimics, poly(I:C), a synthetic analogue of dsRNA that activates intracellular RNA-sensing receptors40-42. Our data indicated that prasugrel did not inhibit poly(I:C)-induced IFN production signaling (Fig.2n and Supplementary Fig.4d, e). These findings were further confirmed in murine bone marrow-derived macrophages (BMDM) (Fig.2o, p and Supplementary Fig.4f). We also treated BMDMs with TLR1/2 agonist, Pam3CSK4, and TLR4 agonist, LPS. Our data showed that prasugrel did not suppress the expression of Il6 downstream of these TLRs (Supplementary Fig.4g, h). Additionally, we examined the effect of prasugrel in cGAS-deficient cells and found that prasugrel had no inhibitory effect on the residual IFNB1 expression stimulated by DNA (Supplementary Fig.4i). These data collectively suggest that prasugrel inhibits DNA-induced cGAS activation with certain specificity.

Acetylation disrupts DNA-induced phase condensation of cGAS

Recent studies highlighted phase condensation as a crucial step in cGAS activation43,44. To understand how acetylation inhibits cGAS activation, we examined the effect of acetylation on cGAS phase condensation. By incubating the purified recombinant protein cGASNon-Ac and the site-specific acetylated variants, cGASK384Ac, cGASK394Ac and cGASK414Ac (Fig.3a) with FAM-labeled double-stranded DNA (dsDNA), we observed a robust and rapid condensate formation of DNA and cGASNon-Ac. In contrast, the acetylated cGAS exhibited a significant reduction in liquid droplet formation (Fig.3b-e and Supplementary Fig.5a). Because we previously reported that acetylation of cGAS at single-site (K384, K394 and K414) had only a limited effect on DNA-binding27, we next used PEG-8000 to promote condensate formation of cGAS proteins. We found that the unmodified cGAS proteins formed more robust condensates than those of acetylated variants (Supplementary Fig. 5b), indicating that acetylation attenuates the phase separation capacity of cGAS. We further assessed whether prasugrel could inhibit the DNA-triggered phase condensation of cGAS. Recombinant cGASNon-Ac proteins were pretreated with prasugrel, G140, an inhibitor that occupies the ATP/GTP-binding site of cGAS45 or XQ2B, an inhibitor targeting cGAS–DNA binding46, before the incubation with DNA. Our data showed that while all three chemicals strongly suppressed cGAS activation (Fig.3f), only prasugrel and XQ2B restrained the formation of the cGAS condensates (Fig.3g, h). Consistently, using cell lines stably expressing mEGFP-tagged cGAS (cGAS-mEGFP), we show that prasugrel or XQ2B significantly reduced the formation of DNA-stimulated cGAS droplets in cells (Fig.3i, j and Supplementary Fig.5c).

These results demonstrate that acetylation disturbs the DNA-induced phase condensation of cGAS, thereby blocking cGAS activation.

Prasugrel suppresses cGAS-dependent autoimmune responses

We next examined whether prasugrel could be used to treat cGAS-mediated autoimmune responses. To do so, we used the Trex1-deficient model. We first deleted Trex1 in L929 cells to induce the expression of interferon-stimulated genes (ISG), indicators of autoimmune responses in cells16,19. Prasugrel effectively inhibited the expression of ISGs (Fig.4a, b). We further evaluated the effect of prasugrel using primary bone marrow cells from Trex1-deficient mice and obtained consistent results (Fig.4c, d). The half-maximal inhibitory concentration (IC50) of prasugrel for inhibiting ISG expression was calculated (Fig.4d). A daily administration of prasugrel (100 mg/kg) for 14 days showed that this compound was well tolerated by mice (Fig.4e). According to previous publications16,19,27, we used the ISG expression levels in the heart tissues of the Trex1–/– mice as the indicator for disease severity16, 19,27, we showed that the daily intramuscular injection of prasugrel at 20 mg/kg for 7 days significantly reduced ISG expression in the hearts (Fig.4f). Furthermore, prasugrel treatment prolonged the survival of Trex1–/– mice (Fig.4g). Thus, prasugrel exhibits promising effect in alleviating the autoimmunity in mice by inhibiting cGAS.

Prasugrel ameliorates autoimmunity in SLE mice

We then examined the effectiveness of prasugrel using the IMQ-induced mouse model. We gave daily intramuscular injections of prasugrel to IMQ-induced mice for 4 weeks and collected the sera and tissues for analysis (Fig.5a). To rule out the possibility that the inhibitory effect of prasugrel could also be mediated through its known target, the P2Y12 receptor47, we utilized clopidogrel, another P2Y12-targeting medication used for treating clotting47, as comparative control. Our data showed that prasugrel significantly reduced the splenomegaly (Fig.5b, c), serum levels of autoantibodies (Fig.5d, e) and serum IFN- levels (Fig.5f), compared to that of clopidogrel treated group. In addition, the renal pathological manifestations were also relieved by prasugrel (Fig.5g-i). This trend was also confirmed by full kidney histological images from independent mice (Supplementary Fig.6a). Because cGAS deletion did not alleviate the autoimmunity in MRL/lpr mice (Supplementary Fig.2a-d), prasugrel treatment showed consistent results in these mice (Supplementary Fig.6b-d). Collectively, these data suggest that prasugrel ameliorates the autoimmunity in SLE mice, likely independent of its known target, P2Y12.

Prasugrel exhibits potential therapeutic effects in SLE patient cells

To further demonstrate the potential therapeutic effect of prasugrel, we obtained peripheral blood mononuclear cells (PBMC) from a cohort with 10 SLE patients (Fig.6a). A concentration of 50 M of prasugrel, which was determined as a non-toxic dosage (Fig.6b), was used to treat PBMCs before an RNA-seq analysis. Because the systemic disease characteristics of SLE were unable to evaluate in patient cells, we used several published IFN signatures to reflect the SLE-associated immune responses in these cells32,48-50. Significantly, prasugrel ameliorated the SLE-associated immune responses in patient cells, as indicated by the expression change of IFN signatures. As a positive control, the JAK1/2 inhibitor ruxolitinib showed a comparable effect (Fig.6c).

To determine whether cGAS activation correlates with the inhibitory effect of prasugrel, we further collected PBMCs and plasma from an additional cohort of 26 SLE patients (Fig.6d). In addition to examine the IFN signature change in patient PBMCs that treated with or without prasugrel, we also measured the plasma cGAMP levels. Importantly, our analysis revealed that the expression of IFN signature genes was substantially more suppressed in SLE patients exhibiting elevated plasma cGAMP levels (Fig.6e, f). This result was further validated using three additional IFN signatures (Supplementary Fig. 7a- f), according to previous reports32,49,50. These findings suggest that plasma cGAMP levels could serve as a predictive biomarker to identify SLE patients who are likely to respond favorably to prasugrel treatment. Collectively, our work presents prasugrel as a therapeutic candidate for SLE. Given its status as an FDA-approved medication, our findings also underscore its immediate clinical translation.

Methods
Mice
Trex1+/– of C57BL/6 background were from D. Barnes and T. Lindahl (Cancer Research UK). Wild-type and Trex1–/– mice (3-week-old) were given daily (i.m.) injection of prasugrel (20 mg/kg). Cgas+/– mice were generated in our laboratory. Knockout of the Cgas gene in mice was achieved using CRISPR-Cas9 technology. The following sgRNA sequences were used: sgRNA1: 5’-GTGAGGTCTTGCCAGTAGAG-3’, sgRNA2: 5’- TGCTCTTCGGAGAGTAGGCC-3’). MRL (000486) and MRL/lpr (000485) mice were purchased from The Jackson Laboratory. Cgas+/– MRL/lpr were generated from Cyagen Biosciences (sgRNA1: 5’-TTCGAAGAAAGGCCGCGAAA-3’, sgRNA2: 5’-GAAGTGTGTCACCGCCATAG-3’, sgRNA3: 5’-ATATATGGCGGGAACGTAGC-3’, sgRNA4: 5’- AAGCAGGTTTCGTGTACCTC-3’). 8-16-week-old females of MRL/MpJ and MRL/lpr were used in the study. Tlr7+/– mice (S-KO-02979) were purchased from Cyagen Biosciences. All mice were maintained under controlled temperature (22 ± 1°C) and humidity (50%-60%) with a 12h light-dark cycle, and housed in a non-specific pathogen-free facility with ad libitum access to food and water. Throughout the study, experimental and control animals were bred and housed separately. Euthanasia was performed by carbon dioxide inhalation. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee (No. IACUC-DWZX-2022-720).

Imiquimod-induced lupus model
All BALB/c mice used in the imiquimod-induced lupus model were 8-week-old females. The skin on the ears of the mice was treated with 5% imiquimod cream, 3 times weekly. Mice serum was collected and measured for autoantibodies and IFN-. Kidneys were used to evaluate the deposition of IgG and IgM for tissue immunofluorescence and inflammatory infiltration for HE staining.

Pristane-induced lupus model
12-16-week-old females C57BL/6N mice received a single intraperitoneal injection of 0.5 mL of pristane and serum samples were collected for analyzing autoantibodies and IFN- level 6 months after pristane injection.

Cell culture
U937 (CRL-1593.2, ATCC), L929 (CCL-1, ATCC), H1299 (CRL-5803, ATCC) and pDCs were cultured in RPMI 1640 (CM10040, MACGENE) supplemented with 10% Fetal Bovine Serum (FBS, FSD500, EXCELL), 1% penicillin-streptomycin (CC004, MACGENE). BMDMs and bone marrow cells were cultured in DMEM (CM10013, MACGENE) supplemented with 10% FBS, 2 mM glutamine (CC009, MACGENE), 1% streptomycin-penicillin. Cells were cultured in a 5% CO2 incubator at 37 °C. U937 cells were differentiated with PMA (0.1 M) (524400, Sigma-Aldrich) for 36-48 h before transfection or other treatment. pDCs were isolated from spleen of mice using plasmacytoid dendritic cell isolation kit (130-107-093, Miltenyi Biotec). Bone marrow cells were isolated from the femurs of mice and RBCs were removed using RBC Lysis Buffer (A1049201, Thermo Fisher Scientific) before stimulating. BMDMs were differentiated from bone marrow cells with recombinant mouse M-CSF (25 ng/mL, 416-ML-050, R&D systems) for 7 days. All cell lines were tested to be mycoplasma free by PCR.

Human PBMCs were isolated from whole blood of consenting donors with Histopaque-1077 (10771, Sigma-Aldrich) according to the manufacturer’s instructions. Informed consent was obtained from the blood donors by the institutions. The study was approved by the ethics committee of Jiaxing Central Blood Station (No. 2024-005), the ethics committee of Peking University People’s Hospital (No. 2019PHB089-01) and the Renji Hospital Ethics Committee of Shanghai Jiao tong University School of Medicine (No.2013-126). It was conducted in compliance with all relevant ethical regulations.

Antibodies
For western blotting, antibodies against acetylated-Lysine (9441s, RRID: AB_331805), cGAS (mouse, 31659s, RRID: AB_2799008) and TLR7 (82E658s, RRID: AB_3662102) were obtained from Cell Signaling Technology. Antibodies against IRF3 (Lab68481, RRID: AB_11155653) and p-IRF3 (ab76493, RRID: AB_1523836) were from Abcam. Antibodies against cGAS, Ac-cGAS (K384+K394) and Ac-cGAS (K414) were generated from our laboratory and validated in our previous work27. Antibodies against -actin (66009-1-Ig, RRID: AB_2687938), GAPDH (10494-1-AP, RRID: AB_2263076) and -Tubulin (66031-1-Ig, RRID: AB_11042766) were purchased from Proteintech Groups. Antibody against TREX1 (611986, RRID: AB_399407) was from BD Biosciences. For Immunofluorescence, Goat Anti-Mouse IgM mu chain (Alexa Fluor 488) (ab150121, RRID: AB_2801490) was from Abcam. Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (Alexa Fluor 647) (A21236, RRID: AB_2535805) was from Invitrogen. Antibody against G3BP1 (13057-2-AP, RRID: AB_2232034) was from Proteintech Groups. For fluorescence-activated cell sorting (FACS) analysis, PE anti-mouse CD11c antibody (117307, RRID: AB_313776) and APC anti-mouse PDCA-1 antibody (127016, RRID: AB_1967127) were from Biolegend. The antibody dilution rates are provided in Supplementary table 2.

Reagents
Laninamivir octanoate (T9124), cefathiamidine (T8134), cefotaxime (T7733), bisacodyl (T6414), cephapirin sodium (T5038), racecadotril (T1176), acebutolol hydrochloride (T1012), cephalothin sodium (T1122), idarubicin hydrochloride (T6010), diltiazem hydrochloride (T0112), apremilast (T2923), famciclovir (T1646) and roxatidine acetate hydrochloride (T0157) were purchased from TargetMOI Chemicals Inc. Prasugrel (HY-15284), clopidogrel (HY-15283), R837 (HY-B0180), R848 (HY-13740), PEG-8000 (HY-Y0873J), G140 (HY-133916), XQ2B (HY-P5997), ruxolitinib (HY-50856), MSN-125 (HY-120079) and PEG-8000 (HY-Y0873J) were from MedChemExpress. Aspirin (A2093), pristane (P2870) and HT-DNA (D6898) were purchased from Sigma-Aldrich. RU.521 (S6841) and M5049 (S9931) were from Selleck. Imiquimod was from Mingxin Pharmaceutical Tech. Poly(I:C) (tlrl-pic) and cGAMP (tlrl-nacga23) were from InvivoGen.

Cell viability assay
U937, BMDMs or PBMCs were seeded into 96-well plates and incubated with prasugrel at indicated concentrations for 24 h. Cell viability was analyzed by CellTiter One Solution Cell Proliferation Assay (MTS) (G3580, Promega) according to the manufacturer’s instruction.

Immunoblotting and Immunoprecipitation
PMA-differentiated U937 cells treated with prasugrel or aspirin were lysed in M2 buffer (20 mM Tris-HCl pH 7.5, 0.5% Nonidet P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM dithiothreitol) supplemented with complete protease inhibitor cocktail (HY-K0010, MedChemExpress), 1 M trichostatin A (TSA, HY-15144, MedChemExpress) and 10 mM nicotinamide (NAMN, N0636, Sigma-Aldrich), followed by sonication and centrifugation at 15,000 g for 10 min at 4°C. The supernatants were immunoprecipitated with cGAS antibody for 8 h at 4°C. The immunoprecipitants were washed six times with M2 buffer and boiled in SDS-loading buffer for immunoblot analysis to detect cGAS acetylation.

RNA extraction and quantitative PCR
For siRNA-mediated knockdown in L929 cells, siRNAs were transfected with Lipofectamine RNAiMAX (13778150, Invitrogen) at a final concentration of 50 nM. After 24 h siRNA transfection, cells were treated with prasugrel for 24 h. Mouse Trex1 (MSS238570, 5’-ACCGACAGACUCACAUACUGCUGAA-3’) siRNA was from Invitrogen. Total RNAs were isolated from cells or tissues using TRI reagent. Total RNAs (500 ng) were reversed-transcribed to cDNA using the PrimeScript RT Reagent Kit (RR037A, Takara). mRNA expression was analyzed by qPCR with PowerUp SYBR Green Master Mix (A25742, Thermo Fisher Scientific) on Applied Biosystems Step OnePlus system. Primers are listed in Supplementary table 3.

Quantification of cGAMP
Serum of clinical samples or murine were mixed with 800 L of cold extraction solvent (40:40:20 (v/v/v) methanol-acetonitrile-water) and then stored at -30°C for 30 min and centrifuged at 15,000 g for 15 min. The supernatants were evaporated at room temperature. For LC-MS/MS quantitation, the pellets were resuspended in 200 L of ammonium acetate buffer (0.05% acetate in water) and then were submitted to a triple-quadrupole mass spectrometer (Xevo TQ-S, Waters Corp.) equipped with an electrospray ionization source. The nebulizer gas was 99.95% nitrogen, and the collision gas was 99.99% argon with a pressure of 3×10−3 mbar in the T-Wave cell. The gas flows of the cone and desolvation were set as 150 and 800 l/h, respectively. The cGAMP measurements were performed in the positive mode with a 3.5 kV capillary voltage, 120°C source temperature and 450°C desolvation temperature. The optimized ion transitions were: cGAMP m/z 675→524; m/z 675→136. Levels of cGAMP in cell lysates and serum were also measured by 2'3'-cGAMP ELISA Kit (501700, Cayman Chemical) according to the manufacturer’s instruction.

Enzyme-linked immunosorbent assay
Mouse serum was collected and measured for different readouts, such as anti-nuclear antibody (ANA), anti-dsDNA antibody and IFN-. These above readouts were analyzed with mouse ELISA kits (JL17316, Mouse ANA ELISA kit; JL37836, Mouse anti-dsDNA ELISA kit; EMC035a.96, Mouse IFN- ELISA kit) according to the manufacturer’s instructions. The supernatants of U937 cell with HT-DNA or poly(I:C) stimulation were collected and measured for IFN- concentration using a human IFN- ELISA kit (EHC026b.96, Neobioscience) according to the manufacturer’s instructions.

Tissue staining and immunofluorescence
Kidneys were fixed in 4% PFA (P1110, MACGENE) for 24 h and then dehydrated overnight. The dehydrated samples were embedded in paraffin and sectioned at a thickness of 4 m. For HE staining, the sections were stained with hematoxylin and eosin. After drying, the sections were mounted and scanned on NDP.view2. For immunofluorescence staining, the sections were deparaffinized in xylene, serial solutions of alcohol and water, and then were performed antigen retrieval by steaming in citric acid. After cooling to room temperature, the sections were washed with PBS twice and blocked in PBS containing 5% normal goat serum (NGS, ZLI-9056, ZSGB-BIO) and 0.3% Triton X-100 (V900502, Sigma-Aldrich). Next, the tissue regions were incubated with AF488-conjunted IgM antibody or AF647-conjunted IgG antibody at 4°C for overnight. On the second day, the sections were washed with PBS twice and then were stained with Hoechst 33342 (H3570, Thermo Fisher Scientific) for 15 min. Finally, the sections were mounted and visualized on the SLIDEVIEW VS200 slide scanner (Olympus).

Detection of mtDNA in cytosolic extracts
Bone marrow cells were treated with 5 g/mL R837 or R848 for indicated times. Cytosolic fractions were obtained by bone marrow cells with 10 g/mL digitonin (D141, Sigma-Aldrich) in isolation buffer (AAPR406, PythonBio) for 10 min on ice, then centrifuged at 1,000 g for 5 min at 4°C. The nuclear DNA extracted from the pellet cells served as the normalization control. The cytosolic supernatants were transferred to fresh tubes and spun at 15,000 g for 10 min at 4°C. MtDNA of supernatants was measured by qPCR.

Calcein-quenching assay
Bone marrow cells were treated with 5 g/mL R837 or R848 for 6 h, followed by the cells collection and stained with Fixable Viability Dye eFluo 780 (65-0865-14, Thermo Fisher Scientific) in media with 1% serum for 15 min. Then, the cells were stained with Mitochondrial Permeability Transition Pore Assay Kit (40756ES60, YEASEN) according to the manufacturer's instruction. Cells were sorted and analyzed on a Beckman CytoFlex using CytExpert (Beckman Coulter).

Purification of site-specific acetylated cGAS recombinant proteins
The site-specific acetylated cGAS recombinant proteins were purified as previous described. Briefly, the pCDF-cGASK384Ac, pCDF-cGASK394Ac and pCDF-cGASK414Ac were individually transformed with pAcKRS-3 into BL21 (DE3) cells. The cells were cultured overnight in LB medium containing 50 g/mL kanamycin and 50 g/mL spectinomycin. 2 mL cultured bacteria were transferred into 200 mL fresh LB medium containing 50 g/mL kanamycin, 50 g/mL spectinomycin and 2 mM acetyl-lysine for further culturing at 37 °C, 220 rpm. When the OD reached 0.6, 30 mM NAM and 20 mM acetyl-lysine were added and incubated with the bacteria for 20-30 min at 37 °C, 220 rpm. 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) (VA20321, GenStar) was then added and the bacteria were cultured for 16-20 h to express the cGAS proteins at 16 °C, 180 rpm. The cells were harvested and resuspended in lysis buffer (20 mM Na3PO4, 1.5 M NaCl, 20 mM imidazole, pH7.5). Following the sonication and centrifugation, the cleared supernatants were purified with His-tag Purification Resin (P2218, Beyotime) according to the manufacturer's protocol.

In vitro phase separation assay
All the assays were performed in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.4). Recombinant cGAS protein was diluted to a final concentration of 10 M in PBS. Subsequently, 4 M FAM-labeled dsDNAs or 5% PEG-8000 were added to the protein solutions. The reaction mixture was transferred to a glass-bottom culture dish after gently mixed, and the droplet formation was visualized by microscopy at the indicated time points. For assays involving inhibitors pre-treatment, cGAS was incubated with prasugrel (5 M), XQ2B (10 M), or G140 (10 M) for 1 h. The reaction buffer was exchanged for PBS prior to performing the LLPS assay. FAM-labeled dsDNAs were prepared from equimolar amounts of the sense and antisense DNA oligonucleotide (sense strand sequence: 5'-ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCATCATATGCATTTTTTGTG-3')

Time-lapse and Immunofluorescence of cells
H1299-cGAS-mEGFP cells were seeded in a glass bottom cell culture dish and cultured overnight, followed by the treatment of prasugrel (100 M) for 24 h or XQ2B (10 M) for 3 h. For time-lapse, the cells were then transfected with 2 g/mL HT-DNA and the images were acquired by DeltaVision deconvolution microscope at 5-minute intervals. For immunofluorescence, the cells were transfected with 4 g/mL Cy3-DNA and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 10 min and blocked in 5% NGS for 1 h. Cells were incubated with anti-G3BP1 antibody for 1 h at room temperature. Images were acquired using ZEISS LSM 900 Confocal Microscopy. Cy3-DNA was prepared from equimolar amounts of the sense and antisense DNA oligonucleotide (sense strand sequence: 5'-ATGGGCAAAGGAGATCCTAAGAAGCCGAGAGGCAAAATGTCATCATATGCATTTTTTGTG-3')

RNA sequencing
Total RNA samples were assessed for purity and integrity using a NanoDrop 2000 spectrophotometer and a LabChip GX Touch system, respectively. Eukaryotic mRNA was enriched from total RNA using Oligo (dT) magnetic beads, and sequencing libraries were prepared using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (NR604, Vazyme) according to the manufacturer's instructions. Sequencing of 2 × 150 bp paired-end reads was performed on Illumina NovaSeq X Plus platform.

RNA-seq data analysis
Quality control and pre-processing of the RNA-seq data were performed using fastp (v0.23.2). Clean reads were mapped to human reference genome GRCh38 using HISAT2 (v2.2.1). Gene expression levels were determined using featureCounts (v2.0.1), and were normalized using DESeq2 (v1.38.3). To facilitate comparison between prasugrel-treated and DMSO-treated PBMC samples from each SLE patient, we employed a gene-wise normalization strategy on paired samples from each individual. For each gene within each patient, the expression level in the prasugrel-treated and corresponding DMSO-treated samples were scaled so that the mean expression of that gene across the pair was set to 1. This was achieved by dividing each gene's expression value by the average expression of that gene across the two samples. This approach aims to adjusts for gene-specific expression variability within each patient and accounts for inter-individual heterogeneity, allowing for direct comparison of expression changes induced by prasugrel treatment. For heatmap visualization, genes were first clustered into functional similarity groups using DAVID gene functional classification and were ordered based on these groups58. Z-score normalization was performed on the expression levels of each gene across all samples. The z-scores were calculated by subtracting the mean expression of each gene and dividing by the standard deviation, resulting in standardized expression values with a mean of 0 and a standard deviation of 1. For comparison of DMSO-treated, prasugrel-treated and JAK inhibitor-treated PBMC samples from each SLE patient, the similar gene-wise normalization strategy was performed. Specifically, for each gene within each patient, the expression levels in the DMSO-treated, prasugrel-treated and JAK inhibitor-treated samples were scaled so that the mean expression of that gene across the three samples was set to 1. Z-score normalization was then performed on the expression levels of each gene across all samples.

Statistical analysis
GraphPad Prism (version 10.1) was used for statistical analysis. A standard two-tailed unpaired Student’s t-test was used for statistical analysis of two groups. Statistical analyses from experiments with multiple groups were performed with one-way or two-way ANOVA with Turkey comparisons test. Survival curve comparison was performed with Log-rank (Mantel-Cox) test. A two-tailed paired student’s t-test was used for statistical analysis of IFN signature expression differences between DMSO- and prasugrel-treated PBMC samples and of summed IFN signature expression differences between DMSO-, prasugrel- and JAK inhibitor-treated PBMC samples. Association between cGAMP levels and mean log2-transformed fold change of expression levels of IFN signature genes quantified using Pearson’s correlation coefficient. The error bands represent the 95% confidence intervals. P values < 0.05 were considered as statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Guo et al presents a novel cGAS inhibitor, prasugel, which they identified by in silico screening of 3,159 FD approved drugs. They initially present data to justify the targeting of cGAS – looking at CGAMP levels in SLE plasma and showing a positive correlation between TLR7 (an important driver of SLE) and cGAS. They next show that in the TLR7 imiquimod model of inducible lupus that Imiquimod induces an early and sustainsble level of cGAMP in mice (measured in plasma) and that loss of cGAS ameliorates lupus in this model, thus providing a strong rationale for targeting cGAS in SLE. Having ideintified prasugel, they show that it acetylates cGAS on lysines 384, 394 and 414 and in doing so prevents its phase separation and mediates its activity. They use Trex1-/- cells and mice to show prasugel rescues pathology and the imiquimod model to show it protects in an SLE model of disease. Using SLE patients cells they also demonstrate that it reduces ISG expression. Importantly their data shows that prasugel has a 40 fold higher potency than aspirin, another known drug that acetylates cGAS. This is a well presented and nicely controlled study. Although the role of acetylation of cGAS in regulating its activity is not particularly novel, the identification and characterization of prasugrel as a novel cGAS inhibitor is an important observation. The data presented initially showing that TLR7 can drive cGAMP induction in vivo is intriguing and very interesting. However, the correlations described in figure 1 and extended data showing an association with TLR7 and cGAS cannot be interpreted as showing association as cGAS is IFN inducible and this association most likely reflects this. The experiments showing that acetylation of cGAS at the specific lysines (K384/394/414) disrupts DNA-induced liquid phase condensates of cGAS suggest this is the mechanism behind prasugrel’s activity.\n\nIn summary, this is a well conceived study delivering important new insights into crosstalk between TLR7 and cytosolic nucleic acid sensing albeit without the details. It also demonstrates that acetylation of cGAS on these 3 residues reduces the formation of DNA:cGAS LLS condensates, presumably resulting in decreased function. A number of minor points have been raised above. Additional points are:\nThe inclusion of polyI:C as a control in figure 2 is appropriate and a good control, however, the language used to describe its use in the main body of the text needs to be clearer.\nAlso minor typographical errors were spotted – one including re. 32.", "reviewer_2": "The paper by Guo et al. reports that the antiplatelet drug prasugrel inhibits cGAS activity by preventing DNA-induced cGAS condensation. They also demonstrate that cGAS is significantly activated in SLE patients, as evidenced by elevated cGAMP levels in serum, and that cGAS deletion safeguards mice against lupus induced by TLR7 activation. They also demonstrate that the in vivo administration of prasugrel in TREX1-deficient mice alleviates lupus symptoms and that the treatment of whole blood from SLE patients reduces the expression of interferon-stimulated genes. The paper is well written, the findings are interesting, and they are potentially of important biomedical relevance. However, several experimental studies are needed to support the conclusions.\nSpecific comments\n1. Line 54: The deficiency of TREX1 has been identified in patients with SLE 22 and 54 Aicardi-Goutières syndrome (AGS) 23-25. Instead of reference 22, the paper by Lee-Kirsch et al. (Nat Genet. 2007 Sep;39(9):1065-7. doi: 10.1038/ng2091) should be cited.\n2. cGAMP levels were found to be increased in SLE patients, but not in RA or DM patients. Like SLE patients, DN patients exhibit exquisitely strong type I IFN activation, yet apparently no elevated cGAMP levels. Were the DM patients studied in Figure 1 tested for ISG expression? How do the authors reconcile this discrepancy?\n3. cGAMP levels and cGAS expression were found to correlate with TLR7 gene expression. The authors argue that TLR7 hyperactivity drives lupus. However, this correlation was weak (R = 0.46). As both cGAS and TLR7 are ISGs, this correlation is not surprising and does not imply a causal relationship. Similarly, reduced R837-induced IFN-alpha production in cGAS-deficient mice or increased cGAS activity in cells stimulated with R837 likely results from the general negative or positive effects of alerting the IFN axis, which has an effect on ISGs such as TLR7 and cGAS.\n4. The authors found that prasugrel specifically acetylates cGAS at K384, K394 and K414. Previous studies had shown that this process inactivates cGAS. They also show that prasugrel specifically inhibits DNA-induced IFN production in BMDMs. To demonstrate that this is dependent on cGAS acetylation, the authors should test DNA-induced IFN production in BMDMs or mice, in which the cGAS acetylation sites have been mutated.\n5. The authors do not provide a detailed description of the experimental set-up for in vitro condensation of cGAS (buffer, pH, concentrations). This makes it difficult to assess the validity of the experiments. The authors need to include positive and negative controls.\n6. cGAS-mEGFP-expressing cells showed reduced foci formation upon prasugrel treatment. The authors should stain these foci for G3BP1 and DNA to confirm condensate formation. The authors should use known inhibitors of phase separation as control.\n7. In addition to the trex1-deficient mouse, the authors should validate their findings in pristane-induced SLE mice and MRL/lpr mice, which are bona fide mouse models of complex SLE, by assessing cGAS activation (cGAMP levels) and by testing the therapeutic effect of prasugrel. What was the effect of prasugrel treatment of trex1-deficient mice on ISG expression and on B cells?\n8. For comparison of the IFN-inhibitory potential of prasugrel, the authors should also treat whole blood from SLE patients with other inhibitors of the IFN axis, such as JAK inhibitors, anifrolumab or TLR7 antagonists. Presumably, cGAS hyperactivity will only be the primary disease-causing factor in a fraction of patients. It is surprising to observe such a profound effect of prasugrel in all patients. What were the clinical characteristics of these patients? Did the authors identify any variables that could be used for patient stratification?", "reviewer_3": "This manuscript presents interesting results that support the thesis that an anti-platelet drug, prasugrel, already used in the clinic as anti-clotting treatment, can also inhibit cGAS activation by acetylation of specific lysines. The same group published on Cell 5 years ago that cGAS activation can be inhibited through lysine acetylation by aspirin. Here the authors show that prasugrel can do what aspirin can do at a lower dose. Since the high doses of aspirin required to inhibit cGAS would have many side effects in patients, having found a different drug that carry less side effects is very significant.\nThe paper also shows results supporting the thesis that prasugrel can treat lupus autoimmunity, shown in two models. One model is cGAS dependent, the Trex-/- mice, and one is TLR7 dependent, Balb/c mice painted on the skin with imiquimod. The inhibition by prasugrel of the autoimmunity in Trex1-/- mice is clear and convincing, suggesting a novel treatment in the cGAS dependent interferonopaties, like Aicardi-Gutierriez. The results of the effects of prasugrel on Imiquimod autoimmunity are also clear for the part of the splenomegaly and the expression of I-IFNs and autoantibodies, but the data on the glomerulonephritis are not strong (see below).\nMoreover, the manuscript proposes that cGAS is downstream of and contributes to TLR7 activation and TLR7-dependent autoimmunity by imiquimod.\nThe lack of some important controls weakens the rigor of the contribution of cGAS to TLR7 activation and autoimmunity.\nIn details:\nThe authors start the manuscript with the hypothesis that cGAMP, product of cGAS activation, is higher in the plasma of SLE patients because is induced by TLR7 activation, which is high because the TLR7 expression is higher in SLE patients than healthy controls. These results could be explained in many other ways. For example, cGAMP and TLR7 RNA expression could correlate because higher cGAMP could be induced by some form of DNA through the activation of cGAS and STING and the production of type I-IFNs, and the latter has been shown to increases TLR7 expression. Therefore, TLR7 expression could be the effect rather than the cause of the high levels of cGAMP in SLE.\nFig.1c-d are important to support a direct induction of cGAS activation by TLR7 stimuli, but the legend does not clarify whether the three dots per each bar are technical replicates from one culture or from three individual cultures from three mice. More repeats of the experiments are required to support the rigor of these figures.\nMoreover, there is no justification for using a mix population of bone marrow precursors instead of BMDMs or BMDCs to test TLR7 responses. Furthermore, IFNalpha is mostly produced by plasmacytoid dendritic cells (pDCs) and therefore testing the production of IFNalpha in cGAS-/- pDCs would provide stronger data to support the dependence of the IFNalpha production on cGAS. Alternatively, the authors could measure IFN beta produced by BMDMs and BMDCs.\nThe conclusion that R837/imiquimod induces cGAS activation and production of cGAMP is a very novel one that requires stronger results. It would be important to provide the control of stimulating with R837 and measuring cGAMP production in cells that are both TLR7-/- and TLR7-/-/cGAS-/-, to exclude the possibility that R837 induces cGAMP in a TLR7 independent manner. Moreover, would other TLR7 ligands induce the same cGAMP production or is it something specific of imiquimod? Does it require TLR7?\nThe role of cGAS in the Imiquimod-induced lupus is still unclear. Fig. 1m shows the staining for IgM in murine glomeruli as sign of lupus disease, but it is standard procedure to show the staining for IgG in the glomeruli, since IgG are the sign of a memory response against the kidney, as it is regularly observed in autoimmune mice.\nThe histology of the Imiquimod kidney presented in Fig. 1n does not show the standard features of glomerulonephritis that are usually present in mice developing lupus, including the imiquimod type (PMID 35297032, 35260898). It is recommended that the H&E slides of the kidney are read by an expert in murine lupus nephritis. Same comments for Figure 5g and h.\nThe potential of prasugrel as novel therapeutic strategy in human SLE is overstated. The results in Fig. 6 indicate that prasugrel decreases the IFN Signature in cells from SLE patients. These effects are not direct indicators that prasugrel will be therapeutic in SLE since anifrolumab, which decreases the IFN Signature, has only mild effects on lupus disease. Moreover, Figure 6F reports the meta-analyses of public data that indicate that the expression of the IFN Signature inversely correlates with the cGAMP levels in the plasma, but it is not clear what is the source of the measurements of the cGAMP levels (the cited papers?) and how could serve as a predictive biomarker to identify SLE patients who are likely to respond favorably to prasugrel treatment.\nMethods\nStatistical significance was assessed using two-tailed student’s unpaired t-test even for data in which 3 or more conditions were analyzed, which would require ANOVA and post-hoc analyses.\nThe authors state that antibodies against cGAS, Acetylated-cGAS (K384+K394) and Ac-cGAS (K414) were generated in their laboratory, but it is not mentioned whether those Antibodies had been validated before and how. This validation is crucial. If it was done in a previous publication, please say so and cite the paper.\nMinor points\nPlease spell out what AcK means.\nThe following sentence infers a translational potential in the results of Figure 6d-e and Extended Data Fig.3c,d that goes beyond the effective potential of those data. “Because the systemic disease characteristics of SLE were unable to evaluate in patient cells, we used several published IFN signatures30,37-39 to reflect the autoimmune status in these cells. Significantly, prasugrel ameliorated the autoimmune status in patient cells, as indicated by the expression change of IFN signatures (Fig.6d,e and Extended Data Fig.3c,d). The mentioned figures instead show an inverse correlation between the levels of cGAMP and those of the IFN Signature. Any comment on the “autoimmune status” is unjustified and not necessary to support the relevance of the paper. Please change the wording of these sentences."}, "author_response": "Point-by-Point Response:\nReviewer #1:\nThe manuscript by Guo et al presents a novel cGAS inhibitor, prasugrel, which they\nidentified by in silico screening of 3,159 FDA approved drugs. They initially present\ndata to justify the targeting of cGAS – looking at CGAMP levels in SLE plasma and\nshowing a positive correlation between TLR7 (an important driver of SLE) and cGAS.\nThey next show that in the TLR7 imiquimod model of inducible lupus that Imiquimod\ninduces an early and sustainable level of cGAMP in mice (measured in plasma) and\nthat loss of cGAS ameliorates lupus in this model, thus providing a strong rationale for\ntargeting cGAS in SLE. Having identified prasugrel, they show that it acetylates cGAS\non lysines 384, 394 and 414 and in doing so prevents its phase separation and mediates\nits activity. They use Trex1-/- cells and mice to show prasugrel rescues pathology and\nthe imiquimod model to show it protects in an SLE model of disease. Using SLE patients\ncells they also demonstrate that it reduces ISG expression. Importantly their data shows\nthat prasugrel has a 40-fold higher potency than aspirin, another known drug that\nacetylates cGAS. This is a well presented and nicely controlled study. Although the role\nof acetylation of cGAS in regulating its activity is not particularly novel, the\nidentification and characterization of prasugrel as a novel cGAS inhibitor is an\nimportant observation. The data presented initially showing that TLR7 can drive\ncGAMP induction in vivo is intriguing and very interesting. However, the correlations\ndescribed in figure 1 and extended data showing an association with TLR7 and cGAS\ncannot be interpreted as showing association as cGAS is IFN inducible and this\nassociation most likely reflects this. The experiments showing that acetylation of cGAS\nat the specific lysines (K384/394/414) disrupts DNA-induced liquid phase condensates\nof cGAS suggest this is the mechanism behind prasugrel’s activity.\nIn summary, this is a well conceived study delivering important new insights into\ncrosstalk between TLR7 and cytosolic nucleic acid sensing albeit without the details. It\nalso demonstrates that acetylation of cGAS on these 3 residues reduces the formation\nof DNA:cGAS LLS condensates, presumably resulting in decreased function. A number\nof minor points have been raised above.\nResponse: The reviewer indicated that our work is a well presented and nicely\ncontrolled study. We greatly appreciate the reviewer’s encouraging comments.\nFollowing the reviewer’s suggestions, we revised our manuscript accordingly.\n1. Regarding the reviewer mentioned points: “Correlations described in figure 1 and\nextended data showing an association with TLR7 and cGAS cannot be interpreted\nas showing association, as cGAS is IFN inducible and this association most likely\nreflects this” and “this is a well conceived study delivering important new insights\ninto crosstalk between TLR7 and cytosolic nucleic acid sensing albeit without the\ndetails”.\nResponse: We thank the reviewers for the insightful comments. We agree with\nthe reviewer that both TLR7 and cGAS are interferon-stimulated genes (ISGs),\nand thus the observed coordination in their expression is expected. Reviewer#2\nand #3 also raised similar points. We apologize for our unclear descriptions.\nAccording to previous publications, TLR7 is proposed as a key driver of\nlupus in humans. For example, a functional polymorphism of TLR7 was\nidentified predisposing to SLE in humans (PNAS, 2010, PMID: 20733074). The\nunrestricted TLR7 signaling is associated with human lupus (Science Immunology,\n2024, PMID:38207015). Moreover, a gain-of-function mutation in TLR7 (Y264H) was\nreported to cause human SLE (Nature, 2022, PMID:35477763). In our study, we sought\nto investigate whether cGAS plays a role in TLR7-driven SLE. Therefore, we\nfirst determined whether cGAS is activated in SLE patients and then assessed\nthe correlation between TLR7 expression and cGAMP levels (indicator of cGAS\nactivation) (Fig.1b in our revised manuscript). These results may reflect the potential\ninterplay between cGAS and TLR7 signaling.\nUsing mouse models, we further showed that in the TLR7-driven SLE mice,\ncGAS was indeed activated (Fig.1d, 1e in our revised manuscript) and required\n(Fig.1f-l in our revised manuscript). We also confirmed that TLR7-stimulated IFN-\nproduction was markedly dampened in the absence of cGAS (Fig.1m, 1n and\nSupplementary Fig.1l, 1n in our revised manuscript). As suggested by Reviewer#2, we\nused ruxolitinib, a JAK1/2 inhibitor (The New England journal of medicine, 2012,\nPMID:22375970), to block the IFN cascade downstream of TLR7. Under such\ncondition, TLR7 agonist stimuli still effectively triggered the synthesis of cGAMP\n(Fig.1o in our revised manuscript), suggesting that the involvement of cGAS in TLR7\nsignaling is independent of the feedback of IFN axis. Thus, cGAS may regulate\nthe activation of TLR7 signaling.\nWe further studied the role of cGAS in TLR7 signaling. We found that TLR7\nactivation led to the increased mitochondrial permeability and the cytosol\nmtDNA releasement (Supplementary Fig.2a-c in our revised manuscript). The\nreleased mtDNAs stimulated the activation of cGAS, which is important for the\neffective production of IFN- downstream TLR7 (Fig. 1c, 1m, 1n and Supplementary\nFig.1c, 1l, 1n in our revised manuscript). We further show that the mtDNAs are\nreleased via BAX and BAK macropores, as the BAK/BAX oligomerization\ninhibitor MSN-125 (Cell Chemical Biology 2017, PMID: 28392146) robustly suppressed the\nmtDNA release induced by TLR7 activation (Supplementary Fig.2d-f in our revised\nmanuscript).\nCollectively, our new data demonstrate that cGAS is essential for the TLR7-\ndriven IFN- production. With these new data, we uncovered the mechanism\nunderlying the role of cGAS in TLR7-signaling and provided important new\ninsights into crosstalk between TLR7 and cytosolic DNA sensing. With the\nadding of these new data, our manuscript is significantly improved. In the\nrevised manuscript, we rephrased our rationale for studying TLR7-driven SLE\nand removed data regarding the correlation between cGAS mRNA levels and\nTLR7 expression.\nAdditional Points:\n2. The inclusion of polyI:C as a control in figure 2 is appropriate and a good control,\nhowever, the language used to describe its use in the main body of the text needs to\nbe clearer.\nResponse: We thank the reviewer for this suggestion and revised our\nmanuscript to add detailed descriptions of poly(I:C). We also explained why we\nused poly(I:C) as control (Line.143 – Line.146 in our revised manuscript).\n3. Also minor typographical errors were spotted – one including re. 32.\nResponse: We thank the reviewer for the suggestion. Accordingly, we\ncorrected the reference citation style and checked our revised manuscript\ncarefully.\nReviewer #2:\nThe paper by Guo et al. reports that the antiplatelet drug prasugrel inhibits cGAS\nactivity by preventing DNA-induced cGAS condensation. They also demonstrate that\ncGAS is significantly activated in SLE patients, as evidenced by elevated cGAMP levels\nin serum, and that cGAS deletion safeguards mice against lupus induced by TLR7\nactivation. They also demonstrate that the in vivo administration of prasugrel in\nTREX1-deficient mice alleviates lupus symptoms and that the treatment of whole blood\nfrom SLE patients reduces the expression of interferon-stimulated genes. The paper is\nwell written, the findings are interesting, and they are potentially of important\nbiomedical relevance. However, several experimental studies are needed to support the\nconclusions.\nResponse: The reviewer indicated that our study presents interesting findings.\nWe greatly appreciate the reviewer’s encouraging comments. We also thank\nthe reviewer for his/her important suggestions to further improve our work.\nFollowing the reviewer’s suggestions, we conducted substantial experiments\nand revised our manuscript extensively. As detailed below, point-by-point, we\ncan address all concerns with new data and discussions.\nSpecific Comments:\n1. Line 54: The deficiency of TREX1 has been identified in patients with SLE (22 and 54)\nAicardi-Goutières syndrome (AGS) (23-25). Instead of reference 22, the paper by Lee-\nKirsch et al. (Nat Genet. 2007 Sep;39(9):1065-7. doi: 10.1038/ng2091) should be cited.\nResponse: Following the reviewer's suggestion, we replaced the original\nreference 22 with the paper by Lee-Kirsch et al. (Nat Genet. 2007 Sep;39(9):1065-7.\ndoi: 10.1038/ng2091) in our revised manuscript (Ref # 23).\n2. cGAMP levels were found to be increased in SLE patients, but not in RA or DM patients.\nLike SLE patients, DM patients exhibit exquisitely strong type I IFN activation, yet\napparently no elevated cGAMP levels. Were the DM patients studied in Figure 1 tested for\nISG expression? How do the authors reconcile this discrepancy?\nResponse: We thank the reviewer for this important point. To address this point,\nwe newly collected peripheral blood samples from 6 DM patients and 10 SLE\npatients, and measured the plasma cGAMP levels, in line with our data in the\noriginal manuscript, the cGAMP levels in DM patients were not significantly\nelevated compared to that of SLE patients (Figure R_1).\nWe also determined the expression of ISGs in PBMCs of DM patients and\nheathy controls using RNA-seq. Our new data showed that the DM patients\nexhibit exquisitely strong type I IFN activation (Figure R_2). This is also consistent\nwith previous studies (Current Rheumatology Reports, 2011, PMID: 20425524; Arthritis &\nRheumatology, 2023, PMID: 37096447).\nFigure R_1. Plasma cGAMP concentrations of healthy donors (n = 6), SLE patients (n = 10) and DM\npatients (n = 6). Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test.\nFigure R_2. The expression of ISGs in healthy donors (n = 6) and DM patients (n = 6). Data were\nanalyzed by unpaired Student’s t-test.\nAccording to previous publications, the type I IFN expression in DM is likely\ntriggered by the RIG-I-like receptors (RLRs), such as RIG-I and MDA-5, which\nare activated upon the detection of intracellular RNAs (Journal of Pathology, 2014,\nPMID: 24604766; Nature Reviews Rheumatology, 2024, PMID: 38057474; Arthritis Research & Therapy,\n2019, PMD: 31142372; Arthritis Research & Therapy, 2017, PMID: 28738907). As shown in Figure\nR_3 below, the RNA mimics poly(I:C) triggers dramatic transcription of type I\nIFN, but not the synthesis of cGAMP. This may explain why the cGAMP levels,\nindicator of the activation of intracellular DNA sensor cGAS, were not elevated\nin DM patients.\nFigure R_3. Analysis of IFNB mRNA and cGAMP levels in U937 cells with indicated stimulation.\nData were analyzed by one-way ANOVA with Tukey’s multiple comparisons test.\n3. cGAMP levels and cGAS expression were found to correlate with TLR7 gene\nexpression. The authors argue that TLR7 hyperactivity drives lupus. However, this\ncorrelation was weak (R = 0.46). As both cGAS and TLR7 are ISGs, this\ncorrelation is not surprising and does not imply a causal relationship. Similarly,\nreduced R837-induced IFN-alpha production in cGAS-deficient mice or increased\ncGAS activity in cells stimulated with R837 likely results from the general negative\nor positive effects of alerting the IFN axis, which has an effect on ISGs such as\nTLR7 and cGAS.\nResponse: We thank the reviewers for the insightful comments. We agree with\nthe reviewer that both TLR7 and cGAS are interferon-stimulated genes (ISGs),\nand thus the observed coordination in their expression is expected. Reviewer#1\nand #3 also raised similar points. We apologize for our unclear descriptions.\nAccording to previous publications, TLR7 is proposed as a key driver of lupus\nin humans. For example, a functional polymorphism of TLR7 was identified\npredisposing to SLE in humans (PNAS, 2010, PMID: 20733074). The unrestricted\nTLR7 signaling is associated with human lupus (Science Immunology, 2024, PMID:\n38207015). Moreover, a gain-of-function mutation in TLR7 (Y264H) was reported\nto cause human SLE (Nature, 2022, PMID: 35477763). In light of these important\nfindings, we sought to investigate whether cGAS plays a role in TLR7-driven\nSLE. Therefore, we first determined whether cGAS is activated in SLE patients\nand then assessed the correlation between TLR7 expression and cGAMP\nlevels (indicator of cGAS activation) (Fig.1b in our revised manuscript). These results\nmay reflect the potential interplay between cGAS and TLR7 signaling.\nUsing mouse models, we further showed that in the TLR7-driven SLE mice,\ncGAS was indeed activated (Fig.1d, 1e in our revised manuscript) and required\n(Fig.1f-l in our revised manuscript). We also confirmed that TLR7-stimulated IFN-\nproduction was markedly dampened in the absence of cGAS (Fig.1m, 1n and\nSupplementary Fig.1l, 1n in our revised manuscript). Thus, cGAS may regulate the\nactivation of TLR7 signaling.\nFollowing the reviewer’s suggestion, we used ruxolitinib, a JAK1/2 inhibitor\n(The New England journal of medicine, 2012, PMID:22375970), to block the IFN cascade\ndownstream of TLR7. Under such condition, TLR7 agonist stimuli still effectively\ntriggered the synthesis of cGAMP (Fig.1o in our revised manuscript), suggesting that\nthe involvement of cGAS in TLR7 signaling is independent of the feedback of\nIFN axis.\nWe further studied the role of cGAS in TLR7 signaling and found that TLR7\nactivation led to the increased mitochondrial permeability and the cytosol\nmtDNA releasement (Supplementary Fig.2a-c in our revised manuscript). The\nreleased mtDNAs stimulated the activation of cGAS, which is important for the\neffective production of IFN- downstream TLR7 (Fig.1c, 1m, 1n and Supplementary\nFig.1c, 1l, 1n in our revised manuscript). We further show that the mtDNAs are\nreleased via BAX and BAK macropores, as the BAK/BAX oligomerization\ninhibitor MSN-125 (Cell Chemical Biology, 2017, PMID: 28392146) robustly suppressed\nthe mtDNA release induced by TLR7 activation (Fig.2d-f in our revised manuscript).\nCollectively, our new data demonstrate that cGAS is required for the TLR-\n7-driven IFN- production. With these new data, we uncovered the mechanism\nunderlying the role of cGAS in TLR7-signaling and provided important new\ninsights into the crosstalk between TLR7 and cytosolic DNA sensing. In the\nrevised manuscript, we rephrased our rationale for studying TLR7-driven SLE\nand removed data regarding the correlation between cGAS mRNA levels and\nTLR7 expression.\n4. The authors found that prasugrel specifically acetylates cGAS at K384, K394 and\nK414. Previous studies had shown that this process inactivates cGAS. They also\nshow that prasugrel specifically inhibits DNA-induced IFN production in BMDMs.\nTo demonstrate that this is dependent on cGAS acetylation, the authors should test\nDNA-induced IFN production in BMDMs or mice, in which the cGAS acetylation\nsites have been mutated.\nResponse: We thank the reviewer for this important suggestion. We agree with\nthe reviewer that using an acetylation-site-mutant cGAS-expressing cell will\nprovide the most compelling evidence.To do so, we will need to mutant all three\nsites (K384/K394/K414) that can be acetylated by prasugrel. Unfortunately,\nbased on our data, some of the lysine sites are critical for cGAS function and\ncannot be mutated. For example, we found that K414 is a critical residue for\ncGAS activity, the mutation of K414 to other amino acids abolished cGAS\nactivity (Figure R_4). Therefore, it is impossible to create a prasugrel-resistant but\nfunctional cGAS mutant.\nFigure R_4. The effect of lysine mutations on cGAS activity.\nAs discussed in our response to Point #7 of this reviewer, prasugrel\ntreatment showed no effect in treating MRL/lpr mice (Figure R_5). This is\nconsistent with the observation that deletion of Cgas in this model did not\nameliorate autoimmunity (Figure R_6). These data indicate that prasugrel likely\nsuppresses the autoimmune response in mice through inhibiting cGAS.\n5. The authors do not provide a detailed description of the experimental set-up for in\nvitro condensation of cGAS (buffer, pH, concentrations). This makes it difficult to\nassess the validity of the experiments. The authors need to include positive and\nnegative controls.\nResponse: We thank the reviewer for this point. Following the reviewer’s\nsuggestions, we included the following controls.\nFor liquid-liquid phase separation (LLPS):\n Negative control: cGAS proteins were not incubated with dsDNAs.\n Positive control: we used PEG-8000, a known LLPS inducer (Nature\nCommunications, 2023, PMID: 37794023).\nOur new results showed that cGAS proteins failed to form condensates in\nthe absence of dsDNAs (Supplementary Fig.4a in our revised manuscript). We also\nused PEG-8000 to promote the condensate formation of wild-type and the site-\nspecifically acetylated cGAS proteins (cGASK384Ac, cGASK394Ac and cGASK414Ac).\nWe find that the unmodified cGAS proteins formed more robust condensates\nthan those of acetylated variants (Supplementary Fig.4b in our revised manuscript),\nindicating that acetylation attenuates the phase separation capacity of cGAS.\nWe also introduced controls in our study:\n Negative control: G140, an inhibitor that occupies the ATP/GTP-binding site\nof cGAS (Nature Communications, 2019, PMID: 31113940), which has no effect on\ncGAS LLPS (Nature Communications, 2025, PMID: 40461475).\n Positive control: XQ2B, an inhibitor targeting cGAS–DNA binding and\nblocks cGAS LLPS (Nature Communications, 2023, PMID: 37783727).\nOur results showed that both prasugrel and XQ2B, but not G140, restrained\nthe formation of cGAS-DNA condensates, while they strongly suppressed\ncGAS activation (Fig.3f-h in our revised manuscript). Together, these results further\nsupport the conclusion that acetylation disturbs the formation of DNA-cGAS\ncondensates.\nRegarding the experimental design, we apologize for our unclear\ndescriptions in the original manuscript. As suggested by the reviewer, we\nrevised the Methods section by adding detailed descriptions of the\nexperimental set-up as follows:\nAll the assays were performed in 1× PBS (137 mM NaCl, 2.7 mM KCl, 10\nmM Na HPO , 1.8 mM KH PO , pH7.4).Recombinant cGAS protein was diluted\n2 4 2 4\nto a final concentration of 10 M in PBS. Subsequently, 4 M FAM-labeled\ndsDNAs or 5% PEG-8000 were added to the protein solutions. The reaction\nmixture was transferred to a glass-bottom culture dish after gently mixed, and\nthe droplet formation was visualized by microscopy at the indicated time points.\nFor assays involving inhibitors pre-treatment, cGAS was incubated with\nprasugrel (5 M), XQ2B (10 M), or G140 (10 M) for 1 hour. The reaction buffer\nwas exchanged for PBS prior to performing the LLPS assay.\n6. cGAS-mEGFP-expressing cells showed reduced foci formation upon prasugrel\ntreatment. The authors should stain these foci for G3BP1 and DNA to confirm\ncondensate formation. The authors should use known inhibitors of phase\nseparation as control.\nResponse: Following the reviewer’s suggestion, we performed\nimmunofluorescence (IF) staining for G3BP1 and DNA in H1299-cGAS-mEGFP\ncells to further validate the condensate formation.\nH1299-cGAS-mEGFP (green) cells were treated with DMSO or prasugrel\nfor 24 hours, followed by transfection with Cy3-labeled dsDNA (red) to induce\ncGAS condensation. XQ2B was used as a positive control.\nOur results showed a clear colocalization of cGAS with DNA (Supplementary\nFig.4c in our revised manuscript), which is consistent with our previous findings\n(Nature immunology, 2018, PMID: 30510222; The EMBO reports, 2022, PMID: 34779554).\nImportantly, treatment with prasugrel or XQ2B significantly reduced the\nformation of these cGAS-DNA droplets (Supplementary Fig.4c in our revised\nmanuscript). These findings support a specific role for prasugrel in inhibiting\ncGAS condensate formation.\n7. In addition to the trex1-deficient mouse, the authors should validate their findings\nin pristane-induced SLE mice and MRL/lpr mice, which are bona fide mouse\nmodels of complex SLE, by assessing cGAS activation (cGAMP levels) and by\ntesting the therapeutic effect of prasugrel. What was the effect of prasugrel\ntreatment of trex1-deficient mice on ISG expression and on B cells?\nResponse: We thank the reviewers for the important comments. We agree with\nthe reviewer that pristane-induced mice and MRL/lpr mice are widely used\nmodels for SLE study.\nAccording to a previous report (Frontiers in Immunology, 2021, PMID:33854495), cGAS\ndeletion did not alleviate the autoimmunity in these models. In our study we\nobtained similar results (Figure R_6). We also show that prasugrel treatment\nfailed to ameliorate autoimmunity in MRL/lpr mice (Figure R_5). Following the\nreviewer’s suggestion, we measured the serum cGAMP levels in different SLE\nmouse models and found that the elevated serum cGAMP could be detected in\nIMQ-induced SLE mice, but not in the pristane-induced SLE or MRL/lpr mice\n(Figure R_7). These data indicates that cGAS likely promotes the pathogenesis\nof SLE in IMQ-induced mouse model.\nFigure R_5. ELISA detection of serum IFN- concentrations, anti-nuclear antibodies and anti-dsDNA\nantibodies of MRL/lpr mice (n = 6) with DMSO or prasugrel treatment. Data were statistically\nanalyzed by two-tailed Student’s t-test.\nFigure R_6. Immunoblot analysis of WT and Cgas–/– bone marrow cells from MRL/lpr or\nC57BL/6N mice with indicated antibodies. ELISA detection of serum IFN- concentrations, anti-\nnuclear antibodies and anti-dsDNA antibodies of WT (n = 8 to 10) and Cgas–/– mice (n = 8 to 10).\nData were statistically analyzed by two-tailed Student’s t-test.\nFigure R_7. For IMQ-induced mice and Pristane-induced mice, we examined serum cGAMP levels\none week after the initial treatments. 8-10-week-old MRL/lpr mice were used to measure the serum\ncGAMP levels. Data were statistically analyzed by two-tailed Student’s t-test.\nIn addition, as suggested by the reviewer, we used the ISG expression\nlevels in the heart tissues of the Trex1–/– mice as the indicator for disease\nseverity. Prasugrel significantly reduced ISG expression in the heart tissues\n(Fig.4f in our revised manuscript).\nWe further evaluated the effect of prasugrel on B cells of Trex1–/– mice.\nTREX1 deficiency is known to cause aberrant B cell activation and autoantibody\nproduction (Cell 2008, PMID: 18724932; European Journal of Immunology 2022, PMID: 35112355).\nWe thus examined the abundancies of GC B (GL7 CXCR5 B220 ) and\nplasma cells (PCs, CD138 B220 ) in Trex1–/– mice that treated with or without\n⁺ ⁺ ⁺\nprasugrel. We found a significant reduction of both cells in the prasugrel-treated\n⁺ ⁻\nmice (Figure R_8).\nFigure R_8.The percentage of GC B (GL7+ CXCR5+ B220+) and plasma cells (PCs; CD138+ B220−)\nin splenic cells from WT mice (n = 5) and Trex1–/– mice (n = 5) were analyzed by FACS. Statistical\nanalysis was performed using two-way ANOVA.\n8. For comparison of the IFN-inhibitory potential of prasugrel, the authors should\nalso treat whole blood from SLE patients with other inhibitors of the IFN axis, such\nas JAK inhibitors, anifrolumab or TLR7 antagonists. Presumably, cGAS\nhyperactivity will only be the primary disease-causing factor in a fraction of\npatients. It is surprising to observe such a profound effect of prasugrel in all\npatients. What were the clinical characteristics of these patients? Did the authors\nidentify any variables that could be used for patient stratification?\nResponse: We thank the reviewer for these valuable points and suggestions.\nFollowing these suggestions, we isolated PBMCs from peripheral blood of 10\nSLE patients and treated the cells with prasugrel or JAK1/2 inhibitor ruxolitinib.\nOur results showed a comparable inhibiting effect of both treatments (Fig.6c in\nour revised manuscript).\nRegarding the effect of prasugrel on patient cells, we re-analyzed our data\nin original Figure 6D and labeled the cGAMP levels for each sample. Our results\nshowed that patients with higher cGAMP levels exhibited greater sensitivity to\nprasugrel treatment (Fig.6e, f in our revised manuscript), suggesting that plasma\ncGAMP levels could serve as a predictive biomarker to identify SLE patients\nwho are likely to respond favorably to prasugrel treatment.\nGiven the complexity of SLE as a systemic disease, the activation of cGAS\nmay vary across different stages of the disease. While a subset of patients\ntested positive for plasma cGAMP at certain timepoint, it is possible that cGAS\nactivation may be relevant to a larger patient population. Our study suggests\nthat SLE patients with higher cGAMP could be benefit from prasugrel treatment.\nReviewer #3:\nThis manuscript presents interesting results that support the thesis that an anti-\nplatelet drug, prasugrel, already used in the clinic as anti-clotting treatment, can also\ninhibit cGAS activation by acetylation of specific lysines. The same group published on\nCell 5 years ago that cGAS activation can be inhibited through lysine acetylation by\naspirin. Here the authors show that prasugrel can do what aspirin can do at a lower\ndose. Since the high doses of aspirin required to inhibit cGAS would have many side\neffects in patients, having found a different drug that carry less side effects is very\nsignificant.\nThe paper also shows results supporting the thesis that prasugrel can treat lupus\nautoimmunity, shown in two models. One model is cGAS dependent, the Trex-/- mice,\nand one is TLR7 dependent, Balb/c mice painted on the skin with imiquimod. The\ninhibition by prasugrel of the autoimmunity in Trex1-/- mice is clear and convincing,\nsuggesting a novel treatment in the cGAS dependent interferonopaties, like Aicardi-\nGutierriez. The results of the effects of prasugrel on Imiquimod autoimmunity are also\nclear for the part of the splenomegaly and the expression of I-IFNs and autoantibodies,\nbut the data on the glomerulonephritis are not strong (see below).\nMoreover, the manuscript proposes that cGAS is downstream of and contributes to\nTLR7 activation and TLR7-dependent autoimmunity by imiquimod. The lack of some\nimportant controls weakens the rigor of the contribution of cGAS to TLR7 activation\nand autoimmunity.\nResponse: The reviewer indicated that “this manuscript is interesting, and the\ninhibition by prasugrel of the autoimmunity in Trex1–/– mice is clear and\nconvincing”. We appreciate the reviewer’s encouraging comments and thank\nthe reviewer for the constructive suggestions to improve our work. Following\nthese suggestions, we conducted substantial experiments and revised our\nmanuscript extensively.\nIn details:\n1. The authors start the manuscript with the hypothesis that cGAMP, product of cGAS\nactivation, is higher in the plasma of SLE patients because is induced by TLR7\nactivation, which is high because the TLR7 expression is higher in SLE patients\nthan healthy controls. These results could be explained in many other ways. For\nexample, cGAMP and TLR7 RNA expression could correlate because higher\ncGAMP could be induced by some form of DNA through the activation of cGAS and\nSTING and the production of type I-IFNs, and the latter has been shown to increases\nTLR7 expression. Therefore, TLR7 expression could be the effect rather than the\ncause of the high levels of cGAMP in SLE.\nResponse: We thank the reviewers for the important comments. Reviewer#1\nand #2 also raised similar points. We apologize for our unclear descriptions.\nAccording to previous publications, TLR7 is proposed as a key driver of lupus\nin humans. For example, a functional polymorphism of TLR7 was identified\npredisposing to SLE in humans (PNAS, 2010, PMID: 20733074). The unrestricted\nTLR7 signaling is associated with human lupus (Science Immunology, 2024, PMID:\n38207015). Moreover, a gain-of-function mutation in TLR7 (Y264H) was reported\nto cause human SLE (Nature, 2022, PMID: 35477763). In light of these important\nfindings, we sought to investigate whether cGAS plays a role in TLR7-driven\nSLE. Therefore, we first determined whether cGAS is activated in SLE patients\nand then assessed the correlation between TLR7 expression and cGAMP\nlevels (indicator of cGAS activation) (Fig.1b in our revised manuscript). These results\nmay reflect the potential interplay between cGAS and TLR7 signaling.\nUsing mouse models, we further showed that in the TLR7-driven SLE mice,\ncGAS was indeed activated (Fig.1d, 1e in our revised manuscript) and required\n(Fig.1f-l in our revised manuscript). We also confirmed that TLR7-stimulated IFN-\nproduction was markedly dampened in the absence of cGAS (Fig.1m, 1n and\nSupplementary Fig.1l, 1n in our revised manuscript). As suggested by Reviewer#2, we\nused ruxolitinib, a JAK1/2 inhibitor (The New England journal of medicine, 2012,\nPMID:22375970), to block the IFN cascade downstream of TLR7. Under such\ncondition, TLR7 agonist stimuli still effectively triggered the synthesis of cGAMP\n(Fig.1o in our revised manuscript), suggesting that the involvement of cGAS in TLR7\nsignaling is independent of the feedback of IFN axis. Thus, cGAS may regulate\nthe activation of TLR7 signaling.\nWe further studied the role of cGAS in TLR7 signaling and found that TLR7\nactivation led to the increased mitochondrial permeability and the cytosol\nmtDNA releasement (Supplementary Fig.2a-c in our revised manuscript). The\nreleased mtDNAs stimulated the activation of cGAS, which is important for the\neffective production of IFN- downstream TLR7 (Fig.1c, 1m, 1n and Supplementary\nFig.1c, 1l, 1n in our revised manuscript). We further show that the mtDNAs are\nreleased via BAX and BAK macropores, as the BAK/BAX oligomerization\ninhibitor MSN-125 (Cell Chemical Biology, 2017, PMID: 28392146) robustly suppressed\nthe mtDNA release induced by TLR7 activation (Supplementary Fig.2d-f in our revised\nmanuscript).\nThus, our new data demonstrate that cGAS is essential for the TLR7-driven\nIFN- production. With these new data, we uncovered the mechanism\nunderlying the role of cGAS in TLR7-signaling and provided important new\ninsights into crosstalk between TLR7 and cytosolic nucleic acid sensing.\nWe agree with the reviewer that both TLR7 and cGAS are interferon-\nstimulated genes (ISGs), thus the observed coordination in their expression is\nexpected. In the revised manuscript, we rephrased our rationale for studying\nTLR7-driven SLE and removed data regarding the correlation between cGAS\nmRNA levels and TLR7 expression.\n2. Fig.1c-d are important to support a direct induction of cGAS activation by TLR7\nstimuli, but the legend does not clarify whether the three dots per each bar are\ntechnical replicates from one culture or from three individual cultures from three\nmice. More repeats of the experiments are required to support the rigor of these\nfigures. Moreover, there is no justification for using a mix population of bone\nmarrow precursors instead of BMDMs or BMDCs to test TLR7 responses.\nFurthermore, IFNalpha is mostly produced by plasmacytoid dendritic cells (pDCs)\nand therefore testing the production of IFNalpha in cGAS-/- pDCs would provide\nstronger data to support the dependence of the IFNalpha production on cGAS.\nAlternatively, the authors could measure IFN beta produced by BMDMs and\nBMDCs.\nResponse: We thank the reviewer for the important suggestions to improve our\nwork. We apologize for our unclear description. In our original manuscript, the\nthree dots per each bar of Figure 1c-d represent the results from three\nindependent experiments (each dot represents an individual mouse). Following\nthe reviewer’s suggestions, we repeated the experiments using six mice in each\ngroup and obtained consistent results (Fig.1c, 1m and Supplementary Fig. 1c, 1l in\nour revised manuscript). In the revised manuscript, we added the descriptions of\nexperimental design in each figure legend.\nIn our study, we tried to examine TLR7 activation in BMDMs. The results\nshowed that R837 or R848 treatment failed to induce Ifna1, Ifna4 and Ifnb\nexpressions in BMDMs (Figure R_9). As control, these TLR7 agonists markedly\ninduced the expression of Il6 and Tnfa in BMDMs (Figure R_10), which are\nconsistent with previous reports (Nature, 2022, PMID:35477763; The Journal of Immunology,\n2018, PMID: 30355788). Because we found that R837 stimulated IFN-α production\nin bone marrow cells, we used bone marrow cells in our study for TLR7\nactivation.\nFollowing the reviewer’s suggestion, we employed plasmacytoid dendritic\ncells (pDCs) to validate the role of cGAS in TLR7-driven IFN-α production. We\nisolated pDCs from spleen of mice using plasmacytoid dendritic cell isolation\nkit of Miltenyi Biotec (130-107-093). The purity of the isolated pDCs was\nconfirmed by flow cytometry, with mouse pDC surface markers, PDCA-1 and\nCD11c (The Journal of experimental medicine, 2017, PMID: 28356390) (Supplementary\nFig.1m in our revised manuscript). We then challenged pDCs from WT and Cgas–/–\nmice with TLR7 agonists, R837 or R848, and measured IFN-productionIn\nline with our data in the manuscript, these new results demonstrated that TLR7-\nstimulated IFN- production is markedly dampened in the absence of cGAS\n(Fig.1n and Supplementary Fig.1n in our revised manuscript).\nFigure R_9.qPCR analysis of Ifna1, Ifna4 and Ifnb expression with indicated stimuli.\nFigure R_10.qPCR analysis of Il6 and Tnfa expression with indicated stimuli.\n3. The conclusion that R837/imiquimod induces cGAS activation and production of\ncGAMP is a very novel one that requires stronger results. It would be important to\nprovide the control of stimulating with R837 and measuring cGAMP production in\ncells that are both TLR7-/- and TLR7-/-/cGAS-/-, to exclude the possibility that\nR837 induces cGAMP in a TLR7 independent manner. Moreover, would other TLR7\nligands induce the same cGAMP production or is it something specific of imiquimod?\nDoes it require TLR7?\nResponse: We appreciate the reviewer’s insightful comments. Following the\nreviewer’s suggestion, we first isolated wild-type and Tlr7–/– bone marrow cells\nand challenged the cells with R837 or R848, another known agonist for TLR7\n(Nature Immunology, 2002, PMID: 11812998). We observed that R837/R848 stimuli\nfailed to induce cGAMP production in Tlr7 deletion cells (Supplementary Fig.1o, p). As\nthe reviewer suggested, the use of Tlr7–/–Cgas–/–mice would be an ideal control\nto demonstrate the conclusion. However, given the challenge of acquiring the\nTlr7–/–Cgas–/– mice, we adopted an alternative experimental approach. We\nused RU.521, a cGAS inhibitor, and M5049, a TLR7 inhibitor (The Journal of\npharmacology and experimental therapeutics 2021, PMID: 33328334), and found that the\ninhibition of either cGAS or TLR7 attenuated cGAMP production stimulated by\nR837 or R848 (Figure R_11). We next treated Tlr7–/–cells with RU.521 (cGAS\ninhibitor) or treated Cgas–/–cells with M5049 (TLR7 inhibitor) and found that the\nblockade of both proteins with each strategy dampened the R837/R848-\ninduced cGAMP production. (Figure R_12).\nTogether, besides R837 (Imiquimod), R848 can also stimulate cGAMP\nproduction, and this induction is TLR7-dependent.\nFigure R_11. Analysis of cGAMP levels with indicated treatments in cells. Data were statistically\nanalyzed by two-way ANOVA with Turkey’s multiple comparisons test.\nFigure R_12. Analysis of cGAMP levels with indicated treatments in cells. Data were statistically\nanalyzed by two-way ANOVA with Turkey’s multiple comparisons test.\n4. The role of cGAS in the Imiquimod-induced lupus is still unclear. Fig. 1m shows the\nstaining for IgM in murine glomeruli as sign of lupus disease, but it is standard\nprocedure to show the staining for IgG in the glomeruli, since IgG are the sign of a\nmemory response against the kidney, as it is regularly observed in autoimmune mice.\nResponse: We appreciate the reviewer’s important suggestions. Accordingly,\nwe performed immunofluorescence staining of IgG on kidney sections from\nIMQ-induced lupus mice. Consistent with previous study (Arthritis & Rheumatology,\n2014, PMID:24574230), we observed the deposition of IgG in the glomeruli of IMQ-\ninduced wild-type mice at 4 weeks. Importantly, the IgG deposition was\nmarkedly attenuated by either genetic deletion of Cgas or by treatment with\nprasugrel (Fig.1k and Fig.5g in our revised manuscript).\nRegarding the role of cGAS in the TLR7 signaling, we performed additional\nexperiments and found that TLR7 activation led to the increased mitochondrial\npermeability and the cytosol mtDNA releasement (Supplementary Fig.2a-c in our\nrevised manuscript). The released mtDNAs stimulated the activation of cGAS,\nwhich is important for the effective production of IFN- downstream TLR7\n(Fig.1c, 1m, 1n and Supplementary Fig.1c, 1l, 1n in our revised manuscript). We further\nshow that the mtDNAs are released via BAX and BAK macropores, as the\nBAK/BAX oligomerization inhibitor MSN-125 (Cell Chemical Biology, 2017, PMID:\n28392146) robustly suppressed the mtDNA release induced by TLR7 activation\n(Supplementary Fig.2d-f in our revised manuscript). Thus, our new data demonstrate\nthat cGAS is essential for the TLR7-driven IFN- production. With these new\ndata, we uncovered the mechanism underlying the role of cGAS in TLR7\nsignaling and provided important new insights into the role of cGAS in the\npathogenesis of TLR7-driven SLE.\n5. The histology of the Imiquimod kidney presented in Fig. 1n does not show the\nstandard features of glomerulonephritis that are usually present in mice developing\nlupus, including the imiquimod type (PMID 35297032, 35260898). It is\nrecommended that the H&E slides of the kidney are read by an expert in murine\nlupus nephritis. Same comments for Figure 5g and h.\nResponse: We thank the reviewer for this valuable advice. As the reviewer\nindicated that our H&E slides of the kidney did not show the standard features\nof glomerulonephritis, we carefully read the papers mentioned by the reviewer\n(PMID: 35297032, 35260898). In these publications, the authors either treated\nNZB/NZW F1 mice with IMQ or used IMQ to treat FVB/NJcl mice. In contrast,\nwe treated BALB/c mice with IMQ, and the standard features of\nglomerulonephritis are not obvious, which can be attributed to the difference of\nadministration time and the different genetic backgrounds of mice. In the whole-\nkidney slides shown below, obvious inflammatory cell infiltration can be found\nin certain renal areas of the IMQ-induced lupus mice, which were significantly\nalleviated by either Cgas deletion or prasugrel treatment (Figure R_13).\nAdditionally, as suggested by the reviewer, we performed\nimmunofluorescence staining of IgG on kidney sections from IMQ-induced\nlupus mice. We observed the deposition of IgG in the glomeruli of IMQ-induced\nlupus mice at 4 weeks. Importantly, the IgG deposition was markedly attenuated\nby either genetic deletion of Cgas or by treatment with prasugrel (Fig.1k and\nFig.5g in our revised manuscript).\nFollowing the reviewer’s suggestions, all H&E-stained kidney slides (Fig.1l\nand Fig.5i in our revised manuscript) were reevaluated by a renal pathologist, Dr.\nChunmei Wu (Annals of the Rheumatic Disease, 2024, PMID: 39419536), who is one of\nour authors in this manuscript.\nA\nB\nFigure R_13. A. HE-stained of kidney from WT and Cgas–/– mice treated with IMQ for 4 weeks. B.\nHE-stained of kidneys from the mice treated with IMQ for 4 weeks in indicate treatment group.\n6. The potential of prasugrel as novel therapeutic strategy in human SLE is overstated.\nThe results in Fig. 6 indicate that prasugrel decreases the IFN Signature in cells\nfrom SLE patients. These effects are not direct indicators that prasugrel will be\ntherapeutic in SLE since anifrolumab, which decreases the IFN Signature, has only\nmild effects on lupus disease. Moreover, Figure 6F reports the meta-analyses of\npublic data that indicate that the expression of the IFN Signature inversely\ncorrelates with the cGAMP levels in the plasma, but it is not clear what is the source\nof the measurements of the cGAMP levels (the cited papers?) and how could serve\nas a predictive biomarker to identify SLE patients who are likely to respond\nfavorably to prasugrel treatment.\nResponse: The reviewer indicated that “The potential of prasugrel as novel\ntherapeutic strategy in human SLE is overstated”, we agree with the reviewer\nthat IFN signatures only serve as cellular readouts that may reflect the disease\nstate but are not direct indicators of SLE severity. Therefore, the effects of\nprasugrel, as assessed by IFN signature change, require further validation in\nclinical trials involving SLE patients. Following this suggestion, we toned down\nour descriptions of conclusion statements in our manuscript. For example, in\nthe revised manuscript, we added “potential” in the subheading of the\nparagraph (Line.200: Prasugrel exhibits potential therapeutic effects in SLE patient cells).\nWe also deleted “promising” in the conclusion sentence (Line.217:Collectively, our\nwork presents prasugrel as a promising therapeutic candidate for SLE).\nRegarding the correlation between cGAMP levels and the inhibition of IFN\nsignatures. In our manuscript, we showed a positive correlation between the\nplasma cGAMP levels and the inhibition of IFN signatures. The expression of\nIFN signature genes was substantially more suppressed in SLE patients\nexhibiting elevated plasma cGAMP levels.\nWe apologize for our unclear descriptions. The original Figure 6F was not a\nmeta-analysis of public data. We analyzed our data from the cohort including\nUniversity School of Medicine. We measured the plasma cGAMP levels and\nexamined the IFN signatures using PBMCs that treated with or without\nprasugrel. In the original Figure 6F, we show the trend that the expression of\nIFN signature genes was substantially more suppressed in SLE patients\nexhibiting elevated plasma cGAMP levels. The labels, such as “IFN signature\n(Ref. PMID: 35477763)” under the panels, only indicated the reference for the\ngene list we used. We apologize again for our unclear description and confusing\nlabeling, and we improved the labeling and figure legends in our revised\nmanuscript (Fig.6c, e, f and Supplementary Fig.5a-f in our revised manuscript).\n7. Methods: Statistical significance was assessed using two-tailed student’s unpaired\nt-test even for data in which 3 or more conditions were analyzed, which would\nrequire ANOVA and post-hoc analyses.\nResponse: We thank the reviewer for this important instruction. Following the\nreviewer’s suggestion, we re-performed all relevant statistical analyses\nthroughout our manuscript. The study designs with 3 or more conditions are\nnow analyzed with ANOVA and post-hoc analyses (Fig.1a, 1c-e,1g-j, 1m-o, 2i, 2k-\np, 3c, 3e, 3f, 3h, 3j, 4b, 5c-h and Supplementary Fig.1c, 1e-h, 1l, 1n, 1p, 2a-f, 3d, 3g-iin our\nrevised manuscript).\n8. The authors state that antibodies against cGAS, Acetylated-cGAS (K384+K394)\nand Ac-cGAS (K414) were generated in their laboratory, but it is not mentioned\nwhether those Antibodies had been validated before and how. This validation is\ncrucial. If it was done in a previous publication, please say so and cite the paper.\nResponse: We apologize for our unclear description regarding the validation\nof cGAS antibodies. Antibodies against cGAS, Ac-cGAS (K384+K394) and Ac-\ncGAS (K414) used in this study were generated in our laboratory and were\nvalidated in our previous work (Cell, 2019, PMID: 30799039). We included this\ninformation in the Methods section of the revised manuscript (Line. 472- Line.\n474 in our revised manuscript).\nMinor points:\n9. Please spell out what AcK means.\nResponse: Following the reviewer’s suggestion, we added the full name of AcK\n(pan-acetyl-lysine antibody) in the revised manuscript (Line. 305).\n10.The following sentence infers a translational potential in the results of Figure 6d-e\nand Extended Data Fig.3c,d that goes beyond the effective potential of those data.\n“Because the systemic disease characteristics of SLE were unable to evaluate in\npatient cells, we used several published IFN signatures30,37-39 to reflect the\nautoimmune status in these cells. Significantly, prasugrel ameliorated the\nautoimmune status in patient cells, as indicated by the expression change of IFN\nsignatures (Fig.6d,e and Extended Data Fig.3c,d). The mentioned figures instead\nshow an inverse correlation between the levels of cGAMP and those of the IFN\nSignature. Any comment on the “autoimmune status” is unjustified and not\nnecessary to support the relevance of the paper. Please change the wording of these\nsentences.\nResponse: We apologize for our unclear descriptions. In our manuscript, we\nshowed a positive correlation between the plasma cGAMP levels and the\ninhibition of IFN signature. The expression of IFN signature genes was\nsubstantially more suppressed in SLE patients exhibiting elevated plasma\ncGAMP levels.\nFollowing the reviewer’s suggestions, we replaced \"autoimmune status\"\nwith “SLE-associated immune responses” (Line. 205 – Line. 206 in our revised\nmanuscript) according to the publications we referenced for IFN signatures\n(Nature Immunology, 2020, PMID: 32747814; Genome Research, 2021, PMID: 33674349;\nNature, 2022, PMID: 35477763; Science Immunology, 2024, PMID: 38207055)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns and the additional data requested by reviewers 2&3 have strengthened the findings. I recommend acceptance", "reviewer_3": "The authors of this paper have answered to the critiques with great effort. I especially appreciate the openness to show raw data, like the full kidney histology, and the large number of unpublished results for the reviewers’s eye only. These data have dissipated past concerns. Moreover, they discovered the mechanism that link TLR7 and cGAS, making this paper very novel indeed.\nWithout asking new experiments, I have few suggestions to improve the clarity of the manuscript.\n1. The results of the inhibitors of M5049 and Ru.521 showed in Figure R11 and R12 are very interesting and strongly support the paper. Can they be added to this manuscript?\n2. The results of R5-, R6, R7 are also very important (R7 above all) to put in perspective the conclusion of this paper with previous papers suggesting that cGAS is not important in lupus. It would be great to add them to this paper. If the authors are already planning to put them in another paper, maybe they can mention them in the discussion as data in a manuscript in preparation.\n3. Regarding the histology of the kidney in Balb/c mice treated with Imiquimod, Figure R13 shows that this model does not have the classis signs of lupus glomerulonephritis, but rather immune infiltrates in the lower tubular and medullary areas, and those infiltrates do not form with prasugrel treatment or in cgas-/- mice. Therefore, figure R13 strongly supports the conclusions of the paper and would be helpful to show it. Moreover, it would remind the reader that Balb/c or in general WT mice have mild forms of kidney disease that are not exactly lupus nephritis. Showing R13 in Supplemental would NOT diminish the strength of the conclusions and would be didactic for the readers, reminding them of the limitations of the murine models with work with.\n4. Figure 6 supports the conclusion that prasugrel inhibits the expression of genes linked to cGAS activation and inflammation in general, and it has more effect in SLE patients with high levels of cGAMP. In Fig. 6e the general effect of prasugrel is clear with a naked eye, but the link between the levels of cGAMP and the effects of prasugrel is not that evident. After spending some time on the Figure, I am convinced but it could help the readers if the genes, rather than in alphabetical order, were clustered in functional groups, like ISGs, vs. chemokines, vs. other groups and etc, since it seems that they behave similarly inside groups, facilitating the recognition of trends and behaviors with the eye."}, "author_response": "Point-by-Point Response:\nReviewer #3:\nThe authors of this paper have answered to the critiques with great effort. I\nespecially appreciate the openness to show raw data, like the full kidney histology, and\nthe large number of unpublished results for the reviewers’s eye only. These data have\ndissipated past concerns. Moreover, they discovered the mechanism that link TLR7 and\ncGAS, making this paper very novel indeed.\nResponse: We thank the reviewer for the supportive comments.\nWithout asking new experiments, I have few suggestions to improve the clarity of the\nmanuscript.\n1. The results of the inhibitors of M5049 and Ru.521 showed in Figure R11 and R12\nare very interesting and strongly support the paper. Can they be added to this\nmanuscript?\nResponse: Following the reviewer’s suggestion, we added these data to our\nrevised manuscript (Supplementary Fig. 2o, p).\n2. The results of R5-, R6, R7 are also very important (R7 above all) to put in perspective\nthe conclusion of this paper with previous papers suggesting that cGAS is not important\nin lupus. It would be great to add them to this paper. If the authors are already planning\nto put them in another paper, maybe they can mention them in the discussion as data in\na manuscript in preparation.\nResponse: We thank the reviewer for the valuable advice. Following the\nreviewer’s suggestion, we added these data to our revised manuscript\n(Supplementary Fig. 2a-i).\n3. Regarding the histology of the kidney in Balb/c mice treated with Imiquimod, Figure\nR13 shows that this model does not have the classis signs of lupus glomerulonephritis,\nbut rather immune infiltrates in the lower tubular and medullary areas, and those\ninfiltrates do not form with prasugrel treatment or in cgas-/- mice. Therefore, figure\nR13 strongly supports the conclusions of the paper and would be helpful to show it.\nMoreover, it would remind the reader that Balb/c or in general WT mice have mild\nforms of kidney disease that are not exactly lupus nephritis. Showing R13 in\nSupplemental would NOT diminish the strength of the conclusions and would be\ndidactic for the readers, reminding them of the limitations of the murine models with\nwork with.\nResponse: We thank the reviewer for the insightful comments. Following the\nreviewer’s suggestion, we added these data to our revised manuscript\n(Supplementary Fig.1l and Supplementary Fig.6a).\n4. Figure 6 supports the conclusion that prasugrel inhibits the expression of genes\nlinked to cGAS activation and inflammation in general, and it has more effect in SLE\npatients with high levels of cGAMP. In Fig. 6e the general effect of prasugrel is clear\nwith a naked eye, but the link between the levels of cGAMP and the effects of prasugrel\nis not that evident. After spending some time on the Figure, I am convinced but it could\nhelp the readers if the genes, rather than in alphabetical order, were clustered in\nfunctional groups, like ISGs, vs. chemokines, vs. other groups and etc, since it seems\nthat they behave similarly inside groups, facilitating the recognition of trends and\nbehaviors with the eye.\nResponse: We thank the reviewer for this important point. Accordingly, we\nupdated Figure.6e, in which genes were first clustered into functional similarity\ngroups using DAVID gene functional classification (tool) and were ordered\nbased on these group (https://davidbioinformatics.nih.gov/gene2gene_new.jsp)\n(2022, Nucleic Acids Research, PMID: 35325185)."}]

Decision letter