Targeting leucine-rich repeat kinase 2 overcomes resistance to oncolytic herpes simplex virus-based therapies in glioblastoma

Domain: chemistry
Published: 2026-02-28
Editorial outcome: major_revision

Abstract
Glioblastoma (GBM) is an incurable brain tumor characterized by high heterogeneity. Oncolytic viruses (OVs) are an approved therapy, but their efficacy is often limited by poor cellular permissiveness to infection. Here we screen bioactive compounds to identify enhancers of oncolytic herpes simplex virus (oHSV) in GBM and discover a brain-penetrant leucine-rich repeat kinase 2 (LRRK2) inhibitor as the strongest sensitizer. Pharmacological LRRK2 inhibition potentiates oHSV infection and oncolysis across diverse GBM cell lines and improves therapeutic outcomes in patient-derived xenograft and orthotopic models. Mechanistically, LRRK2 interacts with and promotes STAT1 phosphorylation at Y701, which drives interferon-independent antiviral responses. Consequently, LRRK2 inhibition attenuates these host defenses, thereby enhancing tumor-selective viral replication. Elevated LRRK2 levels in GBM correlate with reduced oHSV sensitivity, suggesting LRRK2 as a predictive biomarker for tumors amenable to oHSV therapy. Our findings support the combination of LRRK2 inhibitors with oHSV as a promising therapeutic strategy for aggressive GBM.

Introduction
Glioblastoma (GBM) is an aggressive brain cancer characterized by high mortality, primarily due to its invasive growth and notorious resistance to therapies. Surgical resection, chemotherapy, and radiotherapy represent the standard of care for GBM patients. However, these conventional therapies yield poor clinical outcomes, with a median survival of less than 15 months1. Due to GBM's diffusely infiltrative and highly proliferative nature, complete surgical resection is considered unachievable. Despite high occurrence rates, maximal safe tumor resection remains the ideal goal in GBM treatment. The high heterogeneity of GBM and the low central nervous system (CNS) permeability of drugs across the blood-brain tumor barrier (BBTB) profoundly reduce the therapeutic efficacy of anticancer drugs2, 3. Although extensive multidisciplinary treatments have emerged recently, tumor response rates are still unsatisfactory4. Thus, there is an urgent need for new strategies to improve outcomes for GBM patients.

The use of oncolytic viruses (OVs) has been recognized as a potential therapeutic approach for GBM due to their unique mechanism of action, which combines tumor-specific cell lysis with immune stimulation, effectively serving as an in situ cancer vaccine to combat invasive tumors systemically5. Several OVs have recently been approved for GBM treatment. PVS-RIPO, a recombinant oncolytic poliovirus, has shown durable complete responses in about 20% of patients with recurrent GBM6. G47∆ (Delytact), a conditionally approved triple-mutated oncolytic herpes simplex virus (oHSV), has proven safe and shown promising outcomes in GBM patients, according to Japanese regulatory agencies7. Although these clinical advances demonstrate the promise of OVs in treating GBM, they are effective only in subpopulations of GBM patients and often lead to drug resistance8. OVs have been shown to be ideal for combination approaches with other therapeutic agents, such as immune checkpoint inhibitors9, 10, 11, interleukins12, 13, small-molecule compounds14, 15, 16, 17, and other systemic agents18, 19, leading to improved response rates without increasing immune-related adverse events. Therefore, identifying CNS-penetrant pharmacological agents that enhance the activity of oncolytic viruses (OVs) in tumors is crucial for maximizing the therapeutic impact of OVs in GBM treatment.

Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional serine-threonine kinase and GTPase encoded by the PARK8 gene, with variations associated with increased risks of Parkinson's disease (PD) and Crohn's disease (CD)20, 21. Mutations in the LRRK2 gene, such as G2019S, pathologically increase kinase function, affecting vesicle trafficking, cytoskeletal dynamics, autophagy, lysosomal degradation, neurotransmission, mitochondrial function, immunity, and microglial responses22. Currently, various drugs targeting the LRRK2 protein for PD treatment are in clinical trials23. DNL201 is a CNS-penetrant, small-molecule LRRK2 kinase inhibitor (LRRK2i). A phase I study recently reported that DNL201 can correct lysosomal dysfunction in PD patients at safe therapeutic doses and is well tolerated in healthy volunteers24. Furthermore, BIIB122, a once-daily CNS-penetrant, small-molecule LRRK2 inhibitor, is currently undergoing a phase III study for treating patients with LRRK2-mutated PD (NCT05348785)23. In our study, we discovered specific sensitizers of oHSV-mediated oncolysis in GBM by screening a bioactive small-molecule compound library. We found that LRRK2is act cooperatively with oHSV to kill GBM cells by suppressing cellular innate antiviral responses and promoting oHSV-induced apoptosis in vitro and in vivo. Our results suggest that LRRK2 expression may be a useful biomarker for identifying subsets of GBM that are sensitive to oHSV monotherapy or may benefit from combination treatment, providing a rationale for clinical studies using LRRK2is with oHSV-based therapies for GBM treatment.

Results
Compound screening identifies an LRRK2 inhibitor that potentiates the efficacy of oHSV in GBM cells
To identify compounds that enhance oHSV activity in GBM, we conducted drug screening assays on the U-251 MG cell line, which exhibits modest sensitivity to oHSV (Fig. 1a). We screened a collection of 1,593 bioactive compounds, including natural products, enzyme inhibitors, receptor ligands, and drugs (Fig. 1b and Supplementary Table 1). Cell viability was first assessed after treatment with each compound (2.5 μM) in the presence or absence of oHSV-GFP, derived from the HSV-1 ICP0-null oncolytic vector25. A relatively low multiplicity of infection (MOI) of 0.25 plaque-forming units (PFU) per cell ensured minimal cytotoxic effects on U-251 MG cells from the virus alone. The response of U-251 MG cells to the combination treatment was evaluated using the Combination Index (CI), which compares the efficacy of oHSV monotherapy with the combination treatment. Synergistic effects were identified when the CI exceeded 0, with higher CI values indicating more significant synergy. Initial screening identified numerous compounds targeting different pathways that cooperated with oHSV-GFP to varying degrees (Supplementary Data 1). Notably, 118 out of 1,593 compounds demonstrated a CI of ≥20%, from which the top 90 candidates were selected for subsequent retesting. Upon reevaluation, the LRRK2 inhibitor LRRK2-IN-1 displayed the most pronounced synergistic effects with oHSV, achieving a CI of 45.94% (Fig. 1c and Supplementary Data 2). For further validation, the top 20 candidates demonstrating synergistic effects were selected for dose-dependent sensitization analysis. Cell viability was examined after treatment with increasing doses of compounds in the presence or absence of oHSV-GFP. LRRK2-IN-1 emerged as the top sensitizer, enhancing the anticancer activity of oHSV-GFP in U-251 MG cells in a dose-dependent manner (Fig. 1d and Supplementary Table 2). It significantly enhanced virus-encoded GFP expression in oHSV-GFP-infected GBM cells (P < 0.0001) (Fig. 1e and Supplementary Fig. 1a-c). To test the dose-dependent effect of LRRK2-IN-1 on enhancing oHSV-GFP's cytotoxicity, we examined cell viability after treatment with increasing doses of LRRK2-IN-1 in the presence of a nontoxic dose of oHSV-GFP. LRRK2-IN-1 exhibited a strong synergistic inhibitory effect with oHSV-GFP across all six GBM cell lines, including the patient-derived GBM-1 cell line (Fig. 1f-k). These results suggest that LRRK2-IN-1 is a potential sensitizer for oHSV in vitro.

Inhibition of LRRK2 sensitizes GBM cells to viral oncolysis
To further evaluate the sensitization effect of LRRK2-IN-1, we measured cell viability after treating cells with increasing doses of oHSV-GFP, both with and without a nontoxic concentration of LRRK2-IN-1. The dose required to kill 50% of cells (EC50) was determined using nonlinear regression. We calculated the EC50 of oHSV-GFP with and without LRRK2-IN-1 in U-251 MG, U-87 MG, U-118 MG, and LN-18 cells, and analyzed the EC50 shift for each GBM cell line. The EC50 shift refers to a change in the EC50 of oHSV-GFP in the absence versus the presence of LRRK2-IN-1. Our results demonstrated that pretreatment with LRRK2-IN-1 sensitized human GBM cell lines to oHSV-GFP oncolysis, with EC50 shifts of 10.42-fold for U-251 MG, 7.99-fold for U-87 MG, 20.17-fold for U-118 MG, 11.77-fold for LN-18, and 4.33-fold for GBM-1 cells (Fig. 2a-c and Supplementary Fig. 2a). Additionally, LRRK2-IN-1 sensitized two commercially available patient-derived GBM cell lines (PDM-502 and PDM-499) to oHSV-GFP by at least 2-fold (Fig. 2d). Given the role of GBM stem cells (GSCs) in tumor initiation and therapeutic resistance26, we evaluated whether LRRK2-IN-1 could augment the cytotoxic effects of oHSV-GFP in two clinically isolated GSC lines (GSC030 and GSC109), both of which express neural stem cell markers NESTIN and SOX2 (Supplementary Fig. 2b). LRRK2-IN-1 significantly increased the cytotoxic effects of oHSV-GFP on GSC030 (P = 0.0068) and GSC109 (P = 0.0237) (Fig. 2e).

To determine if this synergistic effect extended to other LRRK2 inhibitors, we tested three additional LRRK2 inhibitors (LRRK2is)—PFE-360, DCLK-IN-1, and MLi-2—which have distinct structures and off-target activities27. All three inhibitors sensitized multiple GBM cell lines to oHSV-GFP infection in vitro (P < 0.0001) (Fig. 2f and Supplementary Fig. 2c, d). LRRK2is potentiated the cytotoxicity of oHSV-GFP in GBM cells in a dose-dependent manner (Supplementary Fig. 2e). Specifically, pretreatment with MLi-2 sensitized U-251 MG and U-87 MG cells to oHSV-GFP oncolysis, with EC50 shifts of 4.03-fold and 3.09-fold, respectively (Fig. 2g).

To further validate the role of LRRK2 in resistance to oHSV replication, we use short hairpin RNA (shRNA) to knock down LRRK2 expression in GBM cells. This knockdown increased GBM cell sensitivity to oHSV-GFP infection (P < 0.0001) (Fig. 2h and Supplementary Fig. 3a, b) and enhanced oHSV replication by 2 to 4-fold (Fig. 2i). In contrast, the overexpression of either wild-type LRRK2 or the PD-associated LRRK2G2019S mutant, which has increased kinase activity, reduced GBM cell sensitivity to oHSV-GFP infection (Supplementary Fig. 3c). Specifically, overexpression of wild-type LRRK2 significantly impeded viral infection (P = 0.0003), while the LRRK2G2019S mutant further decreased viral infection (P = 0.0379) compared to wild-type LRRK2 overexpression. Furthermore, overexpression of the LRRK2G2019S mutant further reduced cytotoxicity toward oHSV-GFP oncolysis compared to wild-type LRRK2 overexpression (P = 0.0003), while overexpression of the kinase-dead LRRK2 (LRRK2D1994A) abolished LRRK2’s ability to decrease cytotoxicity toward oHSV-GFP oncolysis (P = 0.0257) (Fig. 2j). Notably, the reduced sensitivity to oHSV-GFP oncolysis induced by LRRK2 or LRRK2G2019S overexpression was reversed by LRRK2-IN-1 treatment (Fig. 2j). Inhibition of wild-type LRRK2 or the LRRK2G2019S mutant restored cytotoxicity toward oHSV-GFP oncolysis to baseline levels. Collectively, these findings indicate that LRRK2 inhibition sensitizes GBM cells to viral oncolysis.

The combination of oHSV and LRRK2 inhibition augments viral spread, oncolysis, and virus-induced apoptosis in vitro
Little is known about the effect of LRRK2 inhibition on viral infection. To address this, we first evaluated the impact of LRRK2-IN-1 on both the early and late stages of oHSV-GFP infection. GBM cells were treated with increasing doses of LRRK2-IN-1 and infected with oHSV-GFP at specified MOIs. Viral infectivity was assessed at 8 hours post-infection (indicating virus entry) and 24 hours post-infection (indicating virus spread) using flow cytometry to quantify the GFP signal. While LRRK2-IN-1 did not enhance viral entry at 8 hours, it significantly increased viral spread at 24 hours, indicating that LRRK2-IN-1 mainly promotes viral infection through enhanced viral spread (Fig. 3a, b). To further explore the effect of LRRK2-IN-1 on virus spread, GBM cells were pretreated with LRRK2-IN-1, infected with oHSV-GFP, and overlaid with methylcellulose to restrict viral diffusion. Fluorescence microscopy revealed that LRRK2-IN-1 increased the average plaque diameter of oHSV-GFP by 2- to 3- fold across all five tested GBM cell lines (Fig. 3c and Supplementary Fig. 4a). To explore the oncolytic effect of oHSV-GFP in the presence of LRRK2-IN-1, GBM cells were infected with oHSV-GFP at specified MOIs and subjected to crystal violet staining 48 hours post-infection. The combination of LRRK2-IN-1 and oHSV-GFP significantly enhanced cell killing in three GBM cell lines (Fig. 3d and Supplementary Fig. 4b).

Furthermore, we analyzed the effects of LRRK2 inhibition on the propagation of oHSV-GFP by generating multistep growth curves. Five GBM cell lines were pretreated with LRRK2-IN-1 and infected at a low MOI of 0.01, with virus titers measured at indicated time points. LRRK2-IN-1 markedly increased viral replication (P < 0.0001) at 48 hours post-treatment, enhancing virus growth by 5.33-fold in U-251 MG, 8.97-fold in U-87 MG, 27.57-fold in U-118 MG, 16.29-fold in GBM-1, and 8.77-fold in LN-18 cells (Fig. 3e, f and Supplementary Fig. 4c). Additionally, increased viral protein expression was observed in U-87 MG and U-251 MG cells treated with LRRK2-IN-1 and oHSV-GFP (Fig. 3g). Overall, LRRK2-IN-1 enhanced virus production by over 100-fold, although the degree of enhancement varied. Numerous studies have shown that OVs can induce cancer cell death through apoptosis28. Thus, we evaluated the effect of LRRK2-IN-1 on oHSV-induced apoptosis in U-87 MG and U-251 MG cells. Activities of both caspase-3/7 and caspase-9 were significantly upregulated following LRRK2-IN-1 treatment and subsequent oHSV-GFP infection (P < 0.0001) (Fig. 3h). We also observed a significant increase in both early and late apoptotic cells after combination treatment (P < 0.0001) (Fig. 3i and Supplementary Fig. 4d, e). Together, these data suggest that LRRK2 inhibition accelerates oHSV-induced cancer cell apoptosis. Collectively, LRRK2-IN-1 robustly enhanced oHSV spread, oncolysis, and virus-induced apoptosis in GBM cells in vitro.

LRRK2i enhances the oncolytic activity of oHSV by suppressing antiviral responses
To gain insight into the potential mechanisms underlying the enhanced oncolytic effect of oHSV by LRRK2is, we performed RNA-Seq analysis of U-251 MG and U-87 MG cells infected with oHSV-GFP in the presence or absence of LRRK2-IN-1. U-251 MG cells treated with either oHSV alone or LRRK2-IN-1 alone exhibited distinct gene expression profiles compared to those co-treated with both agents (Fig. 4a and Supplementary Fig. 5a). Similar results were observed in U-87 MG cells (Supplementary Fig. 5b, c). Four hours post-infection, multiple antiviral genes were upregulated as expected; however, LRRK2-IN-1 inhibited many of these genes, including OAS3, GBP3, IFI44L, and MX2 (Fig. 4b and Supplementary Fig. 5d). Gene Ontology (GO) term analysis revealed that LRRK2 inhibition attenuated the STAT signaling pathway and the response to IFNβ, suggesting that LRRK2-IN-1 suppresses cellular innate antiviral responses (Fig. 4c and Supplementary Fig. 5e). To gain further mechanistic insight into the effect of LRRK2-IN-1 on antiviral responses, we examined whether LRRK2-IN-1 could affect antiviral signaling by repressing the production of type I IFN and its downstream signaling through the JAK–STAT pathway. We first investigated the role of IFNα in the sensitizing effect of LRRK2-IN-1 by pretreating GBM cells with IFNα or an IFNα blocker (anti-IFNR1). IFNα treatment significantly reduced oHSV oncolysis (P < 0.0001), however, LRRK2 inhibition counteracted the antiviral effects of IFNα on oHSV-GFP oncolysis (P < 0.0001) (Fig. 4d). Notably, pretreatment with anti-IFNR1 did not alter the sensitizing effect of LRRK2-IN-1 on oHSV-GFP oncolysis, suggesting that LRRK2-IN-1 may modulate antiviral signaling by suppressing IFNα downstream signaling via the JAK–STAT pathway, rather than by repressing the production of IFNα. Furthermore, no changes in IFN and IFN production were observed after infection in the presence of LRRK2-IN-1 (Supplementary Fig. 6a, b), excluding the possibility that LRRK2-IN-1 affects antiviral signaling by repressing type I IFN production in GBM cells, which typically exhibit dysregulated type I IFN production29. Additionally, we investigated LRRK2-IN-1 could enhance cytotoxicity in combination with oHSV through the bystander effect. No cytotoxicity was detected when conditioned medium from virus-infected GBM cells was transferred to fresh GBM cells pretreated with or without LRRK2-IN-1 (Supplementary Fig. 7).

We next examined whether LRRK2 inhibition could affect antiviral signaling by direct inhibition of the JAK–STAT pathway. The levels of phosphorylated STAT1 (p-STAT1 Tyr701) were potently repressed by LRRK2-IN-1, with a suppression of 2.15-fold in U-251 MG cells and 3.08-fold in U-87 MG cells, 48 hours post-infection (Fig. 4e, f). Consistent with the RNA-Seq data, LRRK2-IN-1 attenuated antiviral signaling by robustly reducing ISG RNA levels (P < 0.0001), such as IFIT2, IFIT3, IRF9, OASL, IFIH1, and OAS2 (Fig. 4g). Furthermore, LRRK2-IN-1 reduced the levels of IRF9 by 2.00-fold in U-251 MG cells and 1.93-fold in U-87 MG cells, as well as the antiviral protein IFI16 by 2.25-fold in U-251 MG cells and 1.46-fold in U-87 MG cells, 24 hours post-infection (Fig. 4h). Together, these data suggest that LRRK2-STAT1-ISG signaling may serve as a potential mechanism underlying the oncolytic activity of oHSV.

LRRK2 regulates antiviral responses through direct interaction and phosphorylation of STAT1 at tyrosine residue 701
We further investigated the mechanism behind LRRK2-dependent STAT1 activation. LRRK2 appears to regulate the expression of interferon-stimulated genes (ISGs) potentially through STAT1, suggesting an association between the two proteins. To test this hypothesis, 293T cells were cotransfected with myc-LRRK2 and STAT1 or empty vector as a control. The presence of STAT1 in anti-myc immunoprecipitates from cells coexpressing myc-LRRK2 and STAT1 indicated an association between the ectopically expressed LRRK2 and STAT1 (Fig. 5a). We also observed that endogenous LRRK2 interacts with STAT1 in two GBM cell lines using primary antibodies against LRRK2 (Fig. 5b and Supplementary Fig. 8a). To identify the STAT1-binding domain, 293T cells were cotransfected with Flag-STAT1 and myc-tagged full-length or truncated LRRK2 proteins (Fig. 5c). Co-IP analysis of STAT1-Flag immunoprecipitates revealed that amino acids 1327–1842 of LRRK2, encompassing the RocCOR domain, mediate the interaction with STAT1 (Fig. 5c and Supplementary Fig. 8b). Similarly, to identify the LRRK2-binding domain, analysis of the anti-myc immunoprecipitates by immunoblotting with an anti-HA antibody showed that amino acids 316–488 of STAT1, which comprise the DB domain, and amino acids 576–683, forming the SH2 domain, are responsible for LRRK2 interaction (Fig. 5d, e and Supplementary Fig. 8c, d). In agreement with these results, baculovirus-purified full-length Flag-tagged LRRK2 interacted with E. coli-purified His-tagged STAT1 (1–712) in vitro (Fig. 5f). Collectively, these findings demonstrate a direct association between LRRK2 and STAT1 both in vitro and in vivo.

To determine whether STAT1 is a substrate of LRRK2, purified STAT1 was incubated with LRRK2 in the presence of ATP for an in vitro kinase assay. Immunoblotting of the reaction product with an anti-phospho-STAT1 (Tyr701) antibody confirmed that STAT1 is directly phosphorylated by LRRK2 at tyrosine residue 701 in vitro (Supplementary Fig. 8e). Flag-STAT1 expressed in 293T cells was also phosphorylated on Tyr701 by LRRK2 (Supplementary Fig. 8f). Notably, no phosphorylation of STAT1 at serine residue 727 by LRRK2 was detected (Fig. 5g, h). Furthermore, LRRK2-mediated STAT1 phosphorylation was reduced by approximately 6.28-fold upon treatment with LRRK2-IN-1 compared to untreated controls (Fig. 5g, h), indicating dependence on LRRK2 kinase activity. oHSV-GFP infection had minimal impact on the phosphorylation of LRRK2 at Ser935 and Ser1292 and did not activate its downstream substrates, Rab10 and Rab12, two well-established LRRK2 substrates in LN-229 cells (Supplementary Fig. 8g, h). Notably, the LRRK2 inhibitors LRRK2-IN-1 and MLi-2 significantly reduced the phosphorylation of LRRK2 (at Ser935 and Ser1292), Rab10 (at Thr73), and Rab12 (at Ser106) in LN-229 cells, although the degree of inhibition varied across these sites. In contrast, the inhibition of LRRK2-dependent Rab phosphorylation was not pronounced in U-251 MG cells, whereas inhibition of STAT1 phosphorylation was robust (Supplementary Fig. 8i, j). This disparity may be attributed to the lower expression levels of LRRK2 in U-251 MG cells, as well as a substrate preference for STAT1 over Rab10/12 in this specific cell line. To further examine the effects of LRRK2 kinase activity on STAT1 phosphorylation, kinase-active LRRK2G2019S enhanced STAT1 phosphorylation at Tyr701 by approximately 1.57-fold compared to wild-type LRRK2, while kinase-dead LRRK2D1994A reduced LRRK2-mediated STAT1 phosphorylation by approximately 2.47-fold (Fig. 5i). Treatment with LRRK2-IN-1 reduced LRRK2-mediated STAT1 phosphorylation by approximately 1.74-fold compared to untreated controls, a result echoed by the LRRK2 inhibitor MLi-2 (Supplementary Fig. 8k). These observations demonstrate that LRRK2 can phosphorylate and activate STAT1 at Tyr701 in a manner dependent on LRRK2 kinase activity.

To investigate whether LRRK2 regulates antiviral effects through STAT1 signaling, LRRK2 knockdown nearly abolished STAT1 phosphorylation at Tyr701 and enhanced oHSV-GFP infection by more than 89-fold (Supplementary Fig. 9a). Treatment with the STAT1 inhibitor Fludarabine significantly increased oHSV-GFP infection and replication in GBM cells independent of LRRK2 kinase activity by more than two-fold (Fig. 5j and Supplementary Fig. 9b, c), indicating that LRRK2 functions upstream of STAT1. In contrast, LRRK2 overexpression led to more than a 3.60-fold increase in STAT1 phosphorylation and nearly completely suppressed oHSV-GFP infection (Fig. 5k). Overexpression of the LRRK2D1994A mutant, however, resulted in an approximately 2.20-fold reduction in STAT1 phosphorylation relative to wild-type LRRK2 and maintained GBM cells' sensitivity to oHSV-GFP infection (Fig. 5k). Importantly, the reduced sensitivity to oHSV-GFP infection caused by LRRK2 overexpression could be reversed using the LRRK2-IN-1 inhibitor.

Consistent with previous studies indicating that LRRK2 is a direct substrate of both TBK1 and IKKε30, we confirmed the interactions between LRRK2 and TBK1 in GBM cells by a Co-IP assay (Supplementary Fig. 9d). We found that inhibition of TBK1/IKKε with BX795 suppressed TBK1 phosphorylation by approximately 5.20-fold and decreased the total level of LRRK2. Consequently, TBK1/IKKε inhibition reduced LRRK2 phosphorylation by approximately 1.89-fold (Fig. 5l) and attenuated the phosphorylation of downstream LRRK2 effectors, including STAT1 and Rab10. Our results demonstrated that BX795 markedly decreased phosphorylated STAT1 by approximately 10.33-fold and phosphorylated Rab10 by approximately 3.50-fold. Additionally, we assessed the effect of BX795 on oHSV-GFP infection in the presence of LRRK2-IN-1. BX795 significantly counteracted the effect of LRRK2-IN-1 in enhancing viral infection in a dose-dependent manner (Supplementary Fig. 9e). Together, these data identify a previously unrecognized TBK1–LRRK2–STAT1–ISG signaling axis that suppresses innate immune responses in GBM cells.

LRRK2i improves the therapeutic efficacy of oHSV in a patient-derived xenograft (PDX) model of GBM
Because LRRK2is have entered clinical trials and have been shown to broadly and robustly enhance the oncolytic activity of oHSV in vitro, we next evaluated the potential therapeutic efficacy of combining LRRK2is with oncolytic oHSV-GFP in vivo. To this end, we used an orthotopic PDX mouse model of GBM-1 cells. Initially, we determined the EC50 of oHSV-GFP with or without LRRK2-IN-1 in GBM-1 cells and analyzed the EC50 shift, finding a 4.33-fold sensitization (Fig. 6a). We then investigated whether LRRK2-IN-1 could enhance oHSV replication in vivo. In mice with orthotopic GBM-1 tumors, treated with intratumoral oHSV-Luc and/or a single intraperitoneal dose of LRRK2-IN-1, the combination therapy significantly enhanced viral replication on days 2 (P = 0.0333) and 3 (P = 0.0069) post-treatment compared to virotherapy alone (Fig. 6b, c). To determine whether LRRK2-IN-1 could improve the therapeutic potential of oHSV in vivo, mice with orthotopic GBM-1-Luc tumors received a single intratumoral dose of oHSV-GFP and/or intraperitoneal LRRK2-IN-1 for four consecutive days (Fig. 6d). The combination treatment significantly inhibited tumor growth in GBM-1 xenografts compared to either monotherapy, starting from day 15 post-treatment (P < 0.01) (Fig. 6e, f). By day 30 post-treatment, the combined therapy substantially reduced tumor burden relative to virotherapy alone (P = 0.0003) or LRRK2-IN-1 alone (P = 0.0018). Histopathology examination indicated that the combination treatment effectively restricted tumor growth compared with monotherapy (Fig. 6g). On day 2 post-virus treatment, cleaved-caspase-3, HSV-1, and IRF9 levels were examined in orthotopic xenograft tumor sections via immunohistochemistry (IHC) staining. The combination treatment significantly enhanced viral replication (P = 0.0091) and increased tumor cell apoptosis (P < 0.0001) compared to virotherapy alone (Fig. 6h). Additionally, the proportion of HSV+ Casp3+ cells was significantly higher (P < 0.0001) in the combination treatment group than in the virotherapy-alone group. Furthermore, LRRK2-IN-1 significantly reduced (P < 0.0001) the IRF9 upregulation induced by oHSV-GFP, leading to enhanced viral replication (Fig. 6h).

LRRK2i and intracranial delivery of oHSV improve outcomes in an immunocompetent GBM model
Although the efficacy of combination therapy was promising in PDX models of GBM, demonstrating effectiveness in an immunocompetent, orthotopic GBM mouse model would more convincingly support the systemic delivery of LRRK2 inhibitors alongside intratumoral oHSV administration. Therefore, we established orthotopic GL261-hNectin1-Luc tumors in immunocompetent mice. Initially, we calculated the EC50 of oHSV-GFP with or without LRRK2-IN-1 in GL261-hNectin1-luc cells and analyzed the EC50 shift. Our data showed that LRRK2-IN-1 sensitized GL261 cells to oHSV-GFP oncolysis, resulting in a 5.35-fold EC50 shifts (Fig. 7a). Mice with GL261 tumors received a single intratumoral dose of oHSV-GFP and/or LRRK2-IN-1 administered intraperitoneally, once daily for four doses on specified days (Fig. 7b). The combination treatment delayed tumor progression (P < 0.0001) and significantly prolonged survival (P < 0.0001) compared to monotherapy alone (Fig. 7c-e and Supplementary Fig. 10a-d). Consistent with findings in the GBM-1 PDX model, the combination therapy effectively restricted tumor growth compared to monotherapy. Approximately 75% of the mice achieved complete remission. Five out of eight mice in the oHSV/LRRK2i group showed no detectable luciferase signal 15 days post-treatment, and six survived over 120 days without GBM symptoms (Fig. 7e and Supplementary Fig. 10d). The cured GL261-bearing mice that received the combination therapy developed immune protection against rechallenge with the same cancer cells (Fig. 7f). Histopathology examination showed that combination treatment effectively restricted tumor growth compared to monotherapy (Fig. 7g). On day 2 post-virus treatment, the combination therapy significantly enhanced viral replication (P = 0.0343) and increased tumor cell apoptosis (P < 0.0001) relative to virotherapy alone. Notably, colocalization signals of HSV and cleaved caspase-3 were significantly stronger in the combination treatment group (P < 0.0001) (Fig. 7h). Moreover, LRRK2-IN-1 substantially reduced (P < 0.0001) the upregulation of IRF9 induced by oHSV-GFP, enhancing viral replication (Fig. 7h). Considering OVs can induce potent and potentially durable antitumor immunity, we hypothesized that LRRK2-IN-1 enhanced replication of oHSV might stimulate a stronger antitumor immune response. Analyses of the immune response within tumors revealed a significant increase in CD8+ T cells (P = 0.0025) and granzyme B (GZMB) + CD8+ T cells (P < 0.0001) in tumors treated with combination therapy (Fig. 7i). These findings suggest that combination therapy can remodel the tumor microenvironment (TME) and significantly enhance both CD8+ T cell infiltration and function. In contrast, no significant difference (P > 0.9999) in Foxp3+ Treg cell infiltration between the virus-treated and combination therapy groups. Together, these results indicate that LRRK2-IN-1 robustly improves the therapeutic efficacy of oHSV in GBM treatment in vivo and facilitates the generation of long-lasting antitumor immunity.

LRRK2 abundance regulating sensitivity to oHSV is general in human GBM
To test whether increased expression of LRRK2 conferred resistance to oncolytic virotherapy, we assayed LRRK2 and p-Rab10 protein levels (Fig. 8a) and calculated the EC50 for oHSV in eight GBM cell lines (Fig. 8b and Supplementary Fig. 11a). We found that high LRRK2 expression in GBM correlates with reduced sensitivity to oHSV, as indicated by higher EC50 values (R² = 0.7066, P = 0.0090; left panel of Fig. 8c). Similarly, elevated p-Rab10 levels were associated with decreased oHSV sensitivity, reflected by higher EC50 values (R² = 0.7585, P = 0.0049; right panel of Fig. 8c). These data suggest that LRRK2-hyperexpressing GBM cells were generally more refractory to oHSV treatment than cells with relatively low LRRK2 expression. Given that GBM may arise from brain astrocytes due to the accumulation of somatic mutations31, we selected primary astrocytes to assess their responsiveness to oHSV. To determine whether LRRK2 inhibition selectively increases oHSV potency in primary astrocytes, we evaluated the oncolytic effect of oHSV-GFP in the presence or absence of LRRK2-IN-1. Unlike in GBM cells, no synergistic effect between oHSV and LRRK2 inhibition was observed in primary astrocytes, as only minimal EC50 shifts occurred (Fig. 8d and Supplementary Fig. 11b), indicating that the increased potency of oHSV due to LRRK2 inhibition is specific to tumor cells. Given that macrophages, neurons, and microglia are also important components of the brain environment, we examined the synergistic effect of oHSV and LRRK2 inhibition in these cells as well. LRRK2 inhibition did not enhance oHSV potency in these cell types, as there were no significant EC50 shifts (Fig. 8e-g).

To explore the impact of LRRK2 abundance on the therapeutic benefit of combination therapy, we employed two other PDX models of GBM with varying LRRK2 expression levels (Fig. 8h and Supplementary Table 3). LRRK2 protein expression was significantly lower in GBM-PDX-1 xenografts than GBM-PDX-2 xenografts. Consistently with the GBM-1 PDX model findings, oHSV-GFP alone exerted strong inhibitory effects on tumor growth (P = 0.0025), and combination therapy significantly reduced tumor burden (P = 0.0104) compared to oHSV monotherapy in GBM-PDX-1 models (Fig. 8i, j). Although oHSV-GFP alone did not inhibit tumor growth (P = 0.0846), combination therapy significantly reduced tumor burden (P = 0.0008) compared to oHSV monotherapy in GBM-PDX-2 models (Fig. 8k). In these PDX models, combination therapy considerably delayed tumor progression compared to either monotherapy, although mice bearing with GBM xenografts with higher LRRK2 expression were more refractory to oncolytic virotherapy and responded better to combination therapy (Fig. 8j, k).

To assess the potential toxicity of combination therapy, mice were administered a single orthotopic dose of oHSV-GFP and/or an intraperitoneal dose of LRRK2-IN-1 and monitored for eight days. After treatment, we evaluated changes in body weight and measured serum levels of key inflammatory cytokines (IL-6, TNF-α, and IFN-γ). Results indicated that combination therapy did not cause significant changes in body weight or induce an inflammatory response (Supplementary Fig. 12a, b).

Thus, LRRK2 upregulation in human GBM may serve as a biomarker for identifying which GBM subsets are sensitive to oHSV monotherapy or may benefit from combination treatment. Together, these data further support the potential of LRRK2 inhibitors to enhance oncolytic activity in GBM treatment.

Methods
Study design
The primary objective of this study was to identify enhancers for oHSV therapy in the treatment of GBM. We evaluated the therapeutic potential of LRRK2 inhibitors in combination with oHSV-based therapy using both human and murine GBM cell lines, patient-derived GBM cells, and GBM stem cell (GSC) cultures, as well as clinically relevant human patient-derived xenograft (PDX) models, in both in vitro and in vivo settings. Female C57BL/6J and NOD/SCID mice (5 or 6 weeks old) were obtained from GemPharmatech (China) and housed under specific pathogen-free conditions in a temperature-controlled environment at 22‒24 °C with a 12-h light/dark cycle at the animal core facility of Xiamen University. All animal procedures were conducted under protocols approved by the Institutional Animal Care and Use Committee of Xiamen University (XMULAC20230314). In all experiments, mice were earmarked before grouping and randomly assigned to different groups, with 5 to 10 mice per experimental group. Mice were euthanized when tumor volume reached 1200 mm3. No tumor exceeded these limits during the study. The human studies were approved by the Ethics Boards of the First Affiliated Hospital of Xiamen University (XMFH-2025-090), and written informed consent was obtained from each patient in accordance with the Declaration of Helsinki.

Cell lines and constructs
Cell lines U-2 OS, U-87 MG, U-118 MG, LN-18, LN-229, A-172, T98G, 293T, PDM-502, and PDM-499 were obtained from the American Type Culture Collection (ATCC), while U-251 MG cells were acquired from the European Collection of Authenticated Cell Cultures (ECACC). GBM-1 cells were established in our laboratory from surgically dissected GBM specimens50. BT325 cells were sourced from MeisenCTCC, and GL261 cells were provided by J. Qiao from the University of Texas, Southwestern Medical Center at Dallas. Human astrocytes were purchased from iXCell Biotechnologies and cultured in astrocyte medium according to the manufacturer’s instructions. U-87 MG, U-251 MG, T98G, and BT325 cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin‒streptomycin (100 IU/ml, Gibco), and 1 × GlutaMax (Gibco). U-2 OS, U-118 MG, LN-18, LN-229, A-172, 293T, GBM-1, and GL261 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with the same supplements. Primary GBM lines were cultured using the NeuroCult™ NS-A Proliferation Kit (StemCell Technologies, 05751) supplemented with penicillin‒streptomycin (100 IU/ml, Gibco), heparin (2 µg/mL, MedChemExpress), recombinant human EGF (20 ng/mL, R&D systems), and recombinant human bFGF (20 ng/mL, R&D systems). Human peripheral blood mononuclear cells (PBMCs) from Cellverse (HUM-iCell-i010) were cultured in RPMI 1640 medium, supplemented with 20% FBS and 100 U/ml penicillin-streptomycin. PBMC-derived M0 macrophages were generated using RPMI 1640 medium, supplemented with 10% FBS, 1 × GlutaMax, 100 U/ml penicillin-streptomycin, and 50 ng/ml recombinant human M-CSF (Sino Biological, 11792-HNAH). IPSC-induced neurons (Cellapy, CA2316106) were cultured using the Neuron Differentiation Kit (Cellapy, CA2307100), and human microglia cells (iCell, HUM-iCell-n007) were maintained in microglia-specific medium (iCell, iCell-n007-002h). All cell lines were maintained at 37 °C with 5% CO2. The cell lines used in this study were authenticated by short tandem repeat (STR) profiling and tested routinely for mycoplasma contamination using the LookOut® Mycoplasma PCR Detection Kit (Sigma‒Aldrich, MP0035). The gene encoding human NECTIN1 was synthesized by GENEWIZ and cloned into the LentiCRISPRv2GFP vector, which contains a puromycin selection cassette (Addgene, 82416). Constructs expressing human NECTIN1, full-length (FL) LRRK2, truncated LRRK2, FL STAT1, truncated STAT1, and TBK1 were synthesized by GENEWIZ. LRRK2 and STAT1 mutants were generated using the QuikChange II site-directed mutagenesis kit (Agilent, 200523). Transient transfections were performed with Lipofectamine 3000 (Thermo Fisher, L3000015).

GSC isolation and cell assay
Fresh surgical GBM tumor tissues were dissociated into single cells using Accutase (Sigma) for 15-20 minutes at 37 °C. The digested cells were cultured in Neurobasal medium (Gibco, 12349015) supplemented with 1× GlutaMax (Gibco), 1× sodium pyruvate (Gibco), EGF (20 ng/ml, R&D Systems), bFGF (20 ng/ml, R&D Systems), and 2% B27 supplement (Gibco, 12587010). GSCs were expanded in suspension as neurospheres in low-attachment wells, then cultured adherently on mouse laminin-coated plates (Corning, 354232). Low-passage GSCs (passages 2-4) were used in this study. For cell viability assays, GSC spheres were dissociated into single cells, and 10,000 cells were plated per well in T96-well plates. Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, G7572) at specified time points. For immunofluorescence staining, GSC spheres were fixed overnight at 4 °C in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 in PBS for 10 minutes. The slides were washed with PBS and blocked with 10% serum from the secondary antibody host species. Primary antibody incubation was performed overnight, followed by three 20-minute washes with 0.2% Triton X-100 in PBS, and a 1-hour incubation with the secondary antibody. The sections were washed three times and mounted with antifade mounting medium containing DAPI (Beyotime, P0131).

Chemicals and recombinant proteins
The following chemicals and recombinant proteins were used: Bioactive Compound Library (MedChemExpress, HY-L001), LRRK2-IN-1 (MedChemExpress, HY-10875), DCLK1-IN-1 (MedChemExpress, HY-135985), MLi-2 (MedChemExpress, HY-100411), Fludarabine (MedChemExpress, HY-B0069), PFE-360 (MedChemExpress, HY-120085), BX795 (TopScience, T1830), Phosphatase inhibitor cocktail III (TopScience, C0004), Protease inhibitor cocktail (MedChemExpress, HY-K0011), and RIPA lysis buffer (Beyotime, P0013C). Crystal violet (BBI, CB0331), Recombinant Human CD300a Protein (Sinobiological, 12449-H08H), and Universal Type I IFN Protein (R&D Systems, 11200-2) were used.

Oligonucleotides
Quantitative polymerase chain reaction (qPCR) primers were synthesized by Sangon Biotech, and the sequences were as follows: Human IFIT2 primers, Forward: 5'-GACACGGTTAAAGTGTGGAGG-3', Reverse: 5'-TCCAGACGGTAGCTTGCTATT-3'; Human IFIT3 primers, Forward: 5'-AAAAGCCCAACAACCCAGAAT-3', Reverse: 5'-CGTATTGGTTATCAGGACTCAGC-3'; Human IRF9 primers, Forward: 5'-GCCCTACAAGGTGTATCAGTTG-3', Reverse: 5'-TGCTGTCGCTTTGATGGTACT-3'; Human OASL primers, Forward: 5'-CCATTGTGCCCTGCCTACAGAG-3', Reverse: 5'-CTTCAGCTTAGTTGGCCGATG-3'; Human IFIH1 primers, Forward: 5'-TCGAATGGGTATTCCACAGACG-3', Reverse: 5'-GTGGCGACTGTCCTCTGAA-3'; Human OAS2 primers, Forward: 5'-ACCATCTCGTCGATCAGTGTC-3', Reverse: 5'-CTCAGAAGCTGGGTTGGTTTAT-3'. A short hairpin RNA (shRNA) sequence targeting a nonspecific target (5'-CCTAAGGTTTAAGTCGCCCTCG-3') was used as a negative control. ShRNA sequences targeting human LRRK2 were as follows: 1, 5'-CCCAGGATGTTGGAAATTGATT-3'; 2, 5'-GCCAGAGGAAATGTCATTTAT-3'. Small interfering RNA (siRNA) sequences targeting human LRRK2 were as follows: 1, 5'-GACCUGCCUAGAAAUAUUATT-3'; 2, 5'-GCUGGGCACUAAAUAAUCUTT-3'. An siRNA sequence targeting a nonspecific target (5'-UUCUCCGAACGUGUCACGUTT-3') was used as a negative control.

Generation of stably expressing cell lines
Stable knockdown of LRRK2 in human GBM cells was achieved using lentiviruses produced in 293T cells with the packaging plasmid psPAX2 (Addgene, 12260), the envelope plasmid pMD2.G (Addgene, 12259), and the pLKO.1 vector (Addgene, 8453) containing specific shRNA against LRRK2. The infected cells were then selected with puromycin (0.8 μg/ml for U-87 MG and 1 μg/ml for U-251 MG) for five days, and target protein expression was assessed by western blotting. Stable overexpression of human NECTIN1 and luciferase in mouse GL261 cells was achieved using lentiviruses produced in 293T cells with the packaging plasmids Lenti-NECTIN1-Puro and pLenti-CMV-LUC-Blast (Addgene, 21474). The infected cells were selected using puromycin (1 μg/ml) and blasticidin (2.5 μg/ml) for eight days to obtain GL261-NECTIN1-Luc cells, and target protein expression was confirmed by western blotting. To achieve stable overexpression of luciferase in GBM-1 cells, lentiviruses were produced in 293T cells with the packaging plasmid pLenti-CMV-LUC-Blast. The infected cells were then selected with blasticidin (2.5 μg/ml) for eight days to obtain GBM-1-Luc cells, and target protein expression was verified by western blotting.

Antibodies
The following antibodies were used: anti-LRRK2 (Abcam, ab133474, 1: 500, RRID: AB_2713963), anti-glycoprotein D (Santa Cruz Biotechnology, sc-21719, 1: 1000, RRID: AB_627720), anti-β-actin (Proteintech, 60008-1-Ig, 1: 3000, RRID: AB_2289225), anti-ICP27 (Santa Cruz Biotechnology, sc-17544, 1: 1000, RRID: AB_2289225), anti-IRF9 (Proteintech, 14167-1-AP, 1: 1000, RRID: AB_2296227), anti-IFI16 (Proteintech, 67790-1-Ig, 1: 1000, RRID: AB_2918554), anti-STAT1 (Cell Signaling Technologies, 9172, 1: 1000, RRID: AB_2198300), anti-Phospho-STAT1 (Tyr701) (Cell Signaling Technologies, 9167, 1: 500, RRID: AB_561284), anti-GFP (Abcam, ab290, 1: 2000, RRID: AB_303395), anti-Phospho-LRRK2 (S935) (ABclonal, AP1154, 1: 200, RRID: AB_2864018), anti-STAT1 (Santa Cruz Biotechnology, sc-464, 1: 1000, RRID: AB_675899), anti-Phospho-TBK1 (S172) (Abcam, ab109272, 1: 1000, RRID: AB_10862438), anti-Phospho-Rab10 (T73) (Abcam, ab230261, 1: 500, RRID: AB_2811274) anti-LRRK2 (Cell Signaling Technologies, 13046, 1: 200, RRID: AB_2798098), anti-GFAP (Sigma‒Aldrich, HPA056030, 1: 6000, RRID: AB_2683015), anti-Ki67 (Proteintech, 27309-1-AP, 1: 32000, RRID: AB_2756525), anti-HSV-1 (Abcam, ab236307, 1: 3000, RRID: N/A), anti-Cleaved Caspase-3 (Asp175) (Cell Signaling Technologies, 9661, 1: 1000, RRID: AB_2341188), anti-HA tag (Abcam, ab128131, 1: 2000, RRID: AB_11143947), anti-Flag tag (Proteintech, HRP-66008, 1: 2000, RRID: AB_2883835), anti-myc tag (Proteintech, 16286-1-AP, 1: 2000, RRID: AB_11182162), anti-IFNAR (Abcam, ab10739, 1: 150, RRID: AB_2122635), anti-Granzyme B (Abcam, ab4059, 1: 100, RRID: AB_304251), anti-CD8 (Abcam, ab209775, 1: 3000, RRID: AB_2860566), anti-FoxP3 (D6O8R) (Cell Signaling Technologies, 12653, 1: 200, RRID: AB_2797979), anti-Phospho-LRRK2 (S1292) (Abcam, ab203181, 1: 200, RRID: AB_2921223) and anti-Phospho-Rab12 (S106) (Abcam, ab256487, 1: 200, RRID: AB_2884880).

Commercial kits
The following commercial kits were used: Caspase-Glo® 9 Assay System (Promega, G8210), Caspase-Glo® 3/7 Assay System (Promega, G8090), Zombie Aqua™ Fixable Viability Kit (BioLegend, 423102), Human IFN-alpha ELISA Kit (R&D Systems, 41100-1), Human IFN-beta ELISA Kit (R&D Systems, 41410-1), Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme, A211-01), 5× All-In-One RT MasterMix (abm, G592), Duolink® In Situ Red Starter Kit (Sigma‒Aldrich, DUO92101), Mouse TNF-α ELISA Kit (FineTest, EM0183-HS), Mouse IFN-γ ELISA Kit (FineTest, E-HSEL-M0007), Mouse IL-6 ELISA Kit (FineTest, E-HSEL-M0003), SuperSignal West Atto substrate (Thermo Scientific, A38556), and Clarity Western ECL Substrate (Bio-Rad, 1705060).

Viruses and titration
The OV oHSV-GFP was previously constructed on the wild-type HSV-1 (KOS strain) backbone, where both ICP0 coding sequences were replaced by eGFP genes50. The oHSV-Luc was derived from oHSV-GFP by inserting the luciferase gene between the UL37 and UL38 regions51. oHSV-GFP was propagated in U-2 OS cells and collected for experiments as previously described25. Viral titers were determined using a standard plaque assay with U-2 OS cells, and the data were converted to plaque-forming units (PFU).

Multistep virus growth curves
For each GBM cell line, confluent cells were pretreated with or without LRRK2 inhibitors (1 μM) for four hours and subsequently inoculated with the virus at a multiplicity of infection (MOI) of 0.01 PFU per cell. The infected cultures were incubated at 37°C and harvested at 0, 8, 24, 36, and 48 hours post-infection. Following infection, cells underwent three freeze-thaw cycles, were centrifuged at low speed to remove cellular debris, and aliquots of the resulting supernatant were stored at −80°C. Viral titers were measured by plaque assay using U-2 OS cells.

RNA extraction and real-time qPCR (RT‒qPCR)
Total RNA was extracted using Tripure Isolation Reagent according to the manufacturer’s instructions (Roche). RNA quantity was measured with a NanoDrop spectrophotometer. Reverse transcription of total cellular RNA was performed using 5 × All-In-One RT MasterMix. RT‒qPCR was conducted in triplicate using an Applied Biosystems 7500 Real-Time PCR system.

Cell viability assay
Cells were seeded in 96-well plates in triplicate at a density of 8,000 cells per well and allowed to attach for 12 hours. Following a four-hour pretreatment with various compounds, the cells were either infected with oHSV-GFP at specified MOI or mock-infected. Three days post-infection, 10 µl of Cell Counting Kit-8 (CCK-8) reagent (MedChemExpress, HY-K0301) was added, and the cells were incubated at 37 °C for an additional two hours. Optical absorbance was then measured at 450 nm using a Thermo Scientific Multiskan Go microplate reader.

Cell-based compound screening
U-251 MG cells were dispensed into 96-well plates using Multidrop (Thermo) dispensers, with each well containing 100 µL of medium and 10,000 cells. A library of 1,593 bioactive compounds, including natural products, enzyme inhibitors, receptor ligands, and pharmaceuticals, was obtained from MedChemExpress (HY-L001). For the initial screening of small chemical compounds, cells were pretreated for four hours with either various compounds (2.5 µM) or an equivalent volume of DMSO (control), followed by infection with oHSV-GFP at an MOI of 0.25. Three days post-infection, cell viability was assessed using the CCK-8 reagent. Virus-treated and mock-infected cells on each plate served as positive and negative controls, respectively, to ensure data quality. Cell viability effects were normalized to the results of each compound alone or DMSO. The Combination Index (CI) for each compound was calculated using the formula: A - B, where A represents the ratio of cell viability after treatment with oHSV alone relative to untreated cells, while B represents the ratio of cell viability after treatment with oHSV in combination with each compound (Combo) relative to cell viability after treatment with the compound alone. A CI value exceeding 0 signifies synergy, with a higher CI value correlating to a more pronounced synergistic effect. For secondary screening, cells were pretreated for 4 hours with escalating doses of each compound or an equivalent volume of DMSO (control), then infected with oHSV-GFP at an MOI of 0.25. Cells treated with escalating titers of the virus or mock-infected cells on each plate served as positive and negative controls, respectively, to ensure data quality.

High-content screening (HCS) microscopy and flow cytometry
GBM cells were seeded in CellCarrier™ 96-well microplates (PerkinElmer) at a density of 8,000 cells per well, cultured for 12 hours, and then treated or mock-treated with LRRK2-IN-1 (2.5 µM) for 4 hours. The cells were subsequently infected with oHSV-GFP at the indicated MOI or mock-infected. Two days post-infection, the cells were observed using the Opera Phenix® Plus High-Content Screening System (PerkinElmer). Quantitative analysis of the fluorescence area fraction was performed using ImageJ. For quantitative analysis of oHSV infection, GFP-positive cells were analyzed using a BD LSRFortessa™ X-20 flow cytometer.

Supernatant transfer experiment
GBM cells were seeded in 6-well plates in triplicate at a density of 5 × 105 cells per well and allowed to attach for 24 hours. The cells were pretreated with LRRK2i or the vehicle for four hours and then infected with oHSV-GFP at an MOI of 1 or mock-infected. After 24 hours of incubation, supernatants from the infected cells were UV-inactivated using a UV Stratalinker 2400 instrument (Stratagene, 360 mJ/cm2) for five minutes52 and subsequently filtered through a 100-kDa filter (Millipore) before being transferred to fresh GBM cells. A CCK-8 assay was performed to determine cell viability.

Western blot analysis
Cell lysates were prepared using RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktails, followed by SDS-polyacrylamide gel electrophoresis (PAGE). After transferring to nitrocellulose membranes, proteins were probed with specific primary antibodies, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. The membranes were visualized using a Fusion FX SPECTRA system (VILBER) with Clarity Western ECL Substrate. Quantification was performed using ImageJ software.

Co-IP assay
Cells were lysed using Pierce IP Lysis Buffer (Thermo Fisher Scientific, 87787) supplemented with protease and phosphatase inhibitor cocktails. Flag- or myc-tagged proteins were precipitated using anti-Flag® M2 Affinity Gel (Sigma‒Aldrich, A2220) or anti-c-myc magnetic beads (MedChemExpress, HY-K0206A). Endogenous proteins were immunoprecipitated using Protein A/G magnetic beads (MedChemExpress, HY-K0202) with specified primary antibodies. The resulting coprecipitates were separated by SDS‒PAGE and subsequently immunoblotted with specific antibodies.

Kinase assays
Recombinant human STAT1 protein (ABclonal, RP01251) was incubated with active, baculovirus-expressed LRRK2 protein (Thermo Fisher Scientific, A15197) in 1 × kinase buffer (Cell Signaling Technologies, 9802S) containing 200 µM ATP (Cell Signaling Technologies, 9804S) for 30 minutes at 30 °C. The reaction products were analyzed by Western blotting.

RNA-seq analysis
GBM cells were seeded at a density of 1 × 106 in 6-well plates and allowed to attach for 24 hours. The next day, cells were pretreated for four hours with LRRK2i (2.5 M) or the vehicle. After pretreatment, the cells were infected with oHSV-GFP at an MOI of 0.5 or mock-infected. Four hours-post infection, biological triplicates for each group were collected using Tripure Isolation Reagent (Roche). RNA-seq analysis was performed by GENEWIZ as previously described10. The fold change in gene expression was calculated for each gene relative to an uninfected and untreated control. Heatmaps of normalized expression values of enriched genes were generated using the R package pheatmap. Gene ontology (GO) enrichment analysis was performed using ClusterProfiler.

Subcutaneous PDX models of GBM
Fresh surgical GBM tumor tissues were sectioned into 2-3 mm3 pieces and transplanted subcutaneously into the flanks of female NOD/SCID mice. After 2 to 4 months of tumor growth, the established PDX tumors were transferred subcutaneously to another generation of NOD/SCID mice. Once the tumor volume reached approximately 100–150 mm3, mice were grouped and subjected to various treatment regimens. For oHSV treatment, the virus (1 × 107 PFU) in 50 μl of saline or saline alone (control) was injected intratumorally every two days for a total of three injections. For LRRK2i treatment, LRRK2-IN-1 (25 mg/kg/day) in 200 μl of corn oil vehicle or vehicle alone (control) was injected intraperitoneally every two days for three times. Tumor sizes and weights were measured daily or every three days.

Orthotopic GBM model
Mice were anesthetized and stereotactically injected with GBM cells (5 µl of PBS) into the right frontal lobe of the brain. For NOD/SCID mice, the injection coordinates were 2 mm lateral and 1 mm anterior to bregma at a depth of 3 mm53, while for C57BL/6J mice, the coordinates were 2 mm lateral and 1 mm caudal to bregma, at a depth of 3 mm54. NOD/SCID mice received an orthotopic transplant of 1 × 105 GBM-1-Luc cells, whereas C57BL/6J mice were orthotopically transplanted with 1 × 105 GL261-hNectin1-Luc cells. Nine days post-tumor implantation, the NOD/SCID mice were randomly divided into groups receiving either LRRK2-IN-1 (25 mg/kg/day) in 200 μl of corn oil vehicle or vehicle alone (control) via intraperitoneal injection, administered daily for four consecutive days. One day following LRRK2-IN-1 administration, these mice received a single intracranial (i.c.) injection of either 2 × 106 PFU oHSV-GFP in 10 μl of saline or saline alone (control). For C57BL/6J mice, four days post-tumor implantation, they were similarly divided into groups receiving either LRRK2-IN-1 (25 mg/kg/day) in 200 μl of corn oil vehicle or vehicle alone (control) via intraperitoneal injection, administered daily for four consecutive days. One day after LRRK2-IN-1 administration, the C57BL/6J mice received a single i.c. injection of either 1 × 107 PFU oHSV-GFP in 10 μl of saline or saline alone (control). In vivo luciferase-based imaging was performed on specified days to assess GBM progression. Body weight and clinical signs were monitored daily. Mice were euthanized when moribund, and brain tissues were harvested on specified days.

Tumor re-challenge
After 120 days of tumor-free survival following combination treatment, the C57BL/6J survivor mice were re-challenged with 1 × 106 GL261 cells via intracranial injection at the original tumor implantation site.

Toxicity study
C57BL/6 mice received a single orthotopic dose of oHSV-GFP on day 1 and/or intraperitoneal LRRK2-IN-1 starting from day 0 for four consecutive days. Post-treatment, blood samples were collected on days 0, 2, 4, and 8 following initial treatment, and serum levels of IL-6, TNF-α, and IFN-γ were measured using ELISA kits.

Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)
H&E staining were performed on formalin-fixed, paraffin-embedded tumor specimens and brain tissues. Sections (4–6 μm) were deparaffinized, rehydrated, and antigen retrieval was conducted as previously described50. Multiplex staining was performed with the indicated antibodies, followed by detection using Opal Anti-Ms+Rb HRP reagents, 1 × Plus Amplification Diluent, and corresponding fluorophores, as well as spectral DAPI, according to the manufacturer’s instructions (Akoya Biosciences). The slides were scanned, and images were captured using the DM6 B (Leica) with LAS X software at 400× magnification or the Pannoramic 250 Flash III (3DHISTECH) with Pannoramic Scanner software at 20× magnification. The images were subsequently cropped and analyzed using either SlideViewer software with its QuantCenter feature or with LAS X and ImageJ software.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 7 software (GraphPad Software). Differences were considered significant when P-values less than 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant). The EC50, representing the dose required to kill 50% of cells, was calculated by nonlinear dose-response regression analysis using GraphPad Prism 7. Two-way ANOVA was employed for multiple comparisons in tumor growth experiments. Log-rank tests were used for survival analyses of animals. For other experiments, comparisons between two groups were conducted using unpaired two-sided Student’s t-tests, while comparisons involving three or more groups were performed using ordinary one-way ANOVA. Spearman’s correlation was used to analyze the relationship between LRRK2 levels and the EC50 of oHSV in different GBM cells.

Data availability
mRNA sequencing data presented in this paper have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE246606. Source Data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors run a small molecule screen using historical, immortilized glioblastoma cell lines to identify combinations of small molecule + OV to increase the effectiveness of the oncolytic-virotherapy oHSV-GFP. The manuscript identifies a small molecule inhibitor of the protein, LRRK2 which synergizes with the oHSV to specifically target cancer cells with enhance viral infection and oncolysis by attenuating the INF-mediated antiviral effects, specifically through the STAT1 pathway. These results were also demonstrated in mouse models.\n\nKey Results:\nLRRK2 inhibitors are well tolerated in human clinical trials to date, are blood-brain barrier penetrant, and reach good cerebrospinal fluid/unbound plasma concentration ratios. In combination with OV to target GBM, they are an attractive therapeutic combination. The MOA is interesting and appears to offer a unique mechanism for direct phosphorylation of the STAT1 which mediates the innate immune response. The results show convincing functional enhancement of OVs in GBM 2D models and in vivo mouse models.\n\nValidity\nDrug Screening/Screen Validation\nThe small molecule screen lacks considerable rigour as the screen reports no quality metrics that I can see, no raw data, and does not describe the normalization techniques. It also reports a “synergistic index” which is poorly defined and is more commonly called the “Combination Index” in cancer applications. LRRK2-IN-1 and other LRRK2 inhibitors s are poorly defined but all which are absolutely necessary to determine the quality of the compound/prob being used. Although the screen did validate in 6-cell lines, the statement “the results suggest that LRRK2-IN-1 is a broad sensitizer for oHSV in vitro” is premature. Patient derived GBM cells, including the treatment refractory glioma stem cells, are now readily available from repositories around the world at relatively low costs. These could/should also be tested to acquire nearer tumour results.\n\nViral Spread, oncolysis and apopotosis\nPlease describe Figure 3 E/F in more detail. Figure G is not quantified but does demonstrate a very large difference between the two cell types suggesting that there is broad range of virus production between cell lines. What is the virus spread and replication in the other cell lines previously test (i.e. – U-118, LN-18, etc)? The statement, “Collectively, LRRK2-IN-1 robustly enhances oHSV replication, spread…” is relatively poorly supported.\n\nLRRK2i enhances the oncolytic activity of oHSV by suppressing antiviral responses\nAlthough substantial supporting data is presented, it is probably more accurate to say STAT1-LRRK2 complexes are pathways are one potential mechanism for oncolytic activity.\n\nAdditionally, the mechanism of action is critically important in understanding oncolytic synergistic effects with LRRK2 inhibition. GBM has a strong immunosuppressive microenvironments established most likely from the innate immune cell component of the tumour, including >50% macrophages, microglia and astrocytes. Single cell interactions in the case of GBM, focussing on the cancer cell mechanism in this tumour microenvironment, would be very supportive of the proposed MOA. Here, scRNA transcriptomic of multi-culture organoids, either in vitro, PDX, or patient derived organoids, could support the results.\n\nImmunocompetent GBM mouse model\nThe points of observation that the conbination treatment in enhanced viral replication and increase tumour cell apopotosis (Figure 7 H and I) are not supported empirically. Please quantify these results. These are links to in vivo results so are crucial to the conclusions for this section.\n\nLRRK2 abundance in human GBM\nAlthough it is encouraging that LRRK2 inhibition does not appear to target healthy astrocytes, macrophages and microgia, as well a neurons (all could be iPSC derived) and tested to interrogate all cell types found in primary and recurrent GBM.\n\nSignificance\nThe use of cell lines for screening are often an economically valid step as it considerably reduces experimental costs however, more complex and predictive models are now widely available using multicell organoids and patient derived organoid techniques. The authors should consider executing, or collaborating with laboratories, experiments with co-culture models incorporating the innate immune components of GBM or patient derived organoids (techniques are available for both). This with demonstrate effectiveness of cotreatment in a more biologically predictive model vs. mouse PDX models.\n\nClarity and content\nSeveral graphs could benefit from reformatting\nFigure 1A - Consider drawing a vertical line screen MOI\nFigure 3B - What are the timepoints beyond 24 hours if figure 3E includes a 24 hour timepoint\nFigure 3G- Can you quantify the westerns on a fluorescent western reader\nFigure 7E & F - Please include figure legend\nFigure 7H - Please quantify with histograms", "reviewer_2": "In this report, Drs. Yu et al reported the targeting of LRRK2 to overcome resistance to oHSV in GBMs. Given the poor prognosis of GBM, the established safety profiles and noticeable activities of oHSV, this study addresses an important clinical problem by potentially offering a new approach to enhance the anti-GBM activities of oHSV. The identification of LRRK2 as a key mediator of oHSV resistance appears new to the fields although the impact of innate anti-viral system on oncolytic activities have long been recognized. The overall experimental design is sound, extensive and appears to be well executed. Multiple GBM cell lines were included together with orthotopic xenograft mouse models both in immune-deficient and immune-competent animals. The inclusion of a fairly large panel of patient tumors in a tissue microarray provided additional support for the potential use of LRRK2 as a diagnostic marker. While this paper provided some potentially important information, there are some concerns that need to be addressed:\n\nThe major concern is the lack of data or “results” in the RESULT section. Only summary or conclusive type statements were included. There was no sufficient information on the actual changes of biological activities and statistical analysis to convincingly support their statement. Matching the data presented in the figures (although nicely done) with descriptions in the figure legends is also a big challenge. Proper evaluation of this manuscript will need major revision on data presentation. Listed below are some (a small fraction) of the missing data and P values:\n• Furthermore, upon treatment 221 with the LRRK2 inhibitor LRRK2-IN-1, LRRK2-mediated STAT1 phosphorylation was significantly reduced (Fig. 5I): how much?\n• To 226 investigate whether LRRK2 regulates antiviral effects through STAT1 signaling, LRRK2 227 knockdown reduced the phosphorylation of STAT1 at Tyr701 and enhanced oHSV-GFP infection 228 (Fig. 5K). How much?\n• Our data showed that treatment 256 with LRRK2-IN-1 sensitized GBM-1 cells to oHSV-GFP oncolysis (Fig. 6A). How much?\n• The 264 combination treatment resulted in significantly stronger inhibition of tumor growth in GBM-1 265 xenografts compared with either monotherapy alone (Fig. 6D-F). How much?\n\nMinor concerns:\n1. Figure 1 and 2: “Three florescent fields were analyzed”. How were the fields selected to prevent bias? And why only three fields. These data are not sufficient for quantitative analysis.\n2. The true identity of U87 is controversial. Since it was involved in multiple assays, this limitation should be discussed.\n3. Fig 4B: Cells treated with LRRK2in alone should be included as reference.\n4. In vivo treatment lacks sufficient scientific details about the timing and dosing, making it difficult to determine the potential clinical relevancy.\n5. Figure 8 A and B: The protein expression of LRRK2 was low/no in multiple cell lines that exhibited strong responses (low EC50). How were the relative expression determined? When these data combined with their findings in Fig 1, Fig 2 (e.g., U-251 of which the LRRK2 expression was nearly zero), it is difficult to conclude that the LRRK2 is critically important for oHSV. For example, U-251 does not express LRRK2 protein (Fig 8 A), but KRRK2 inhibitor was very active in sensitizing it to oHSV infection (Fig 2 A)\n6. A systematic lack of statistic analysis in the result section.", "reviewer_3": "In the manuscript by Yu and colleagues, titled “Targeting Leucine-Rich Repeat Kinase 2 Overcomes Resistance to Oncolytic Herpes Simplex Virus-Based Therapies in Glioblastoma”, the authors conduct a bioactive drug screen to identify compounds that enhance the activity of oncolytic herpes simplex virus (oHSV) in glioblastoma (GBM) cells. They identify LRRK2-IN1 as a potent enhancer of oHSV activity in these cells. Mechanistically, the authors propose that LRRK2 phosphorylates STAT1, thereby promoting antiviral responses. Although I am not an expert in cancer biology, I have extensive experience studying LRRK2. Accordingly, my comments will focus primarily on the biology of LRRK2. Please find my feedback below.\n1. The authors use student’s t-test in analysis of more than 2 groups, which is wrong. The authors must use the proper statistical tests (one or two-way ANOVA) when analyzing three or more groups.\n2. The authors should demonstrate LRRK2 kinase inhibition by analyzing pRab10, pRab12 and pS1292 levels. They should also see if oHSV increases LRRK2 kinase activity.\n3. LRRK2-IN-1 is not the most reliable LRRK2 inhibitor. The authors should repeat some of their cell viability and mechanistical experiments with MLi2.\n4. The authors should also repeat some of the viability assays from Fig.3 comparing WT vs kinase dead mutant vs G2019S.\n5. Even though no clear information can be found in the methods section, it seems that LRRK2 is tagged C terminally. LRRK2 needs to be tagged N terminally, as we and others in the field have seen that C terminal tagging can be artificial. If that would be the case, this reviewer finds hard to trust the results using the C terminally tagged vector.\n6. Western blots in Fig 4E-G, and 5I,J,M,L,O should be quantified.\n7. To convincingly claim that LRRK2 and STAT1 interact. The authors should only overexpress one the proteins, and blot for the other at the endogenous level. Co overexpression of two proteins in 293 cells is known to yield artificial results.\n8. The authors claim that STAT1’s DB domain binds to LRRK2, however, the CCDB construct shows minimal binding to LRRK2, suggesting that the binding with LRRK2 requires both the DB and SH2 domains.\n9. The idea that STAT1 can bind independently to three different LRRK2 domains (LRR, Kin and ANK) is hardly convincing and makes little sense.\n10. Authors need to include a WT vs G2019S comparison (with LRRK2i), analyzing pSTAT1-701 levels (quantified with multiple replicates).\n11. In my experience, LRRK2 antibodies do not work for staining. The authors should either validate their antibody for immunostaining or remove all the data involving LRRK2 staining.\n12. Typo in Fig. 5J (D2019S).", "reviewer_4": "This manuscript submitted by Mengqin and colleagues reported their findings that LRRK2i may sensitize oHSV replication, spreading and oncolysis in GBM through suppressing a noncanonical LRRK-and STAT1-pathways. Moreover, they found that the combination of LRRK2i and oHSV improved therapeutic efficacy as compared to monotherapy in murine or human GBM-PDXs established in immunocompromised mice, as well as in syngeneic mouse GMB model. Overall, this study is interesting, the identification of LRRK2 as a potential biomarker for oHSV therapy and the combination of LRRK2i with oHSV for GBM treatment are attractive approaches for further investigations.\nSpecific comments:\n1. Page 6, line 158-160, OVs induces cancer death, which could be immunogenic cell death, but cancer cell apoptosis may inhibit antitumor immune responses, please reconcile the sentence.\n2. In Fig. 4D, if add anti-IFNa in the treatment to oHSV and LRRK2-IN-1 group, would the cell viability be changed?\n3. In Fig. 7I, how about the immune suppressive regulatory T cells (Tregs), any changes in this population in the tumors?\n4. Did the toxicity study show any changes of body weight or levels of inflammatory cytokines in serum?"}, "author_response": "Response to Reviewers\n\nReviewer #1 (Remarks to the Author): expert in drug screens\nIn this manuscript, the authors run a small molecule screen using historical, immortilized glioblastoma cell lines to identify combinations of small molecule + OV to increase the effectiveness of the oncolytic-virotherapy oHSV-GFP. The manuscript identifies a small molecule inhibitor of the protein, LRRK2 which synergizes with the oHSV to specifically target cancer cells with enhance viral infection and oncolysis by attenuating the INF-mediated antiviral effects, specifically through the STAT1 pathway. These results were also demonstrated in mouse models.\nResponse: Thank you for reviewing our manuscript and providing valuable feedback.\nKey Results:\nLRRK2 inhibitors are well tolerated in human clinical trials to date, are blood-brain barrier penetrant, and reach good cerebrospinal fluid/unbound plasma concentration ratios. In combination with OV to target GBM, they are an attractive therapeutic combination. The MOA is interesting and appears to offer a unique mechanism for direct phosphorylation of the STAT1 which mediates the innate immune response. The results show convincing functional enhancement of OVs in GBM 2D models and in vivo mouse models.\nResponse: Thank you for your positive comments and valuable suggestions to improve the quality of our manuscript.\nValidity\nDrug Screening/Screen Validation\nThe small molecule screen lacks considerable rigour as the screen reports no quality metrics that I can see, no raw data, and does not describe the normalization techniques. It also reports a “synergistic index” which is poorly defined and is more commonly called the “Combination Index” in cancer applications. LRRK2-IN-1 and other LRRK2 inhibitors s are poorly defined but all which are absolutely necessary to determine the quality of the compound/prob being used. Although the screen did validate in 6-cell lines, the statement “the results suggest that LRRK2-IN-1 is a broad sensitizer for oHSV in vitro” is premature. Patient derived GBM cells, including the treatment refractory glioma stem cells, are now readily available from repositories around the world at relatively low costs. These could/should also be tested to acquire nearer tumour results.\nResponse: Thank you for your valuable suggestions. The screening methods, calculation techniques, and results are detailed in the Methods and Results sections (Tables S1, Lines 684-702). Supplementary tables (Tables S1–S4) provide compound information, the normalization method, and raw screening data. The term \"Combination Index\" is defined and used to evaluate the synergistic effects of small molecule inhibitors (SMIs) on oHSV cytotoxicity (Fig. 1B, C and D; Lines 102-105).\nIn addition to a patient-derived GBM cell line we isolated, we used two commercially available patient-derived GBM cell lines (PDM-502 and PDM-499) and two GBM stem cell lines (GSC030 and GSC109) to determine whether LRRK2-IN-1 serves as a potential sensitizer for oHSV. We revised the statement “these results suggest that LRRK2-IN-1 is a broad sensitizer for oHSV in vitro” by replacing “broad” with “potential.” The results are presented in the revised manuscript (Fig. 2D, E and S2A, B; Line 121, Lines 132-138).\nViral Spread, oncolysis and apopotosis\nPlease describe Figure 3 E/F in more detail. Figure G is not quantified but does demonstrate a very large difference between the two cell types suggesting that there is broad range of virus production between cell lines. What is the virus spread and replication in the other cell lines previously test (i.e. – U-118, LN-18, etc)? The statement, “Collectively, LRRK2-IN-1 robustly enhances oHSV replication, spread…” is relatively poorly supported.\nResponse: We sincerely appreciate your valuable suggestions. In response to Fig. 3E and 3F, we have provided more detailed methods for multistep virus growth curves in both the Methods and figure legend sections of the revised manuscript (Lines 665-671 and 1112-1115). Additionally, we quantified the Western blot results, which demonstrated that LRRK2-IN-1 enhances virus production by over 100-fold, although the degree of enhancement varies (Fig. 3G; Lines 184-187).\nTo further substantiate our conclusion that LRRK2-IN-1 enhances oHSV infection, replication, and spread, we evaluated virus spread in U-118MG, GBM-1, and LN-18 cells using a plaque-forming assay. We also examined virus replication in these cell lines by comparing viable cell staining results and multistep virus growth curves. The results are presented in the revised manuscript (Fig. S4A-C).\nLRRK2i enhances the oncolytic activity of oHSV by suppressing antiviral responses\nAlthough substantial supporting data is presented, it is probably more accurate to say STAT1-LRRK2 complexes are pathways are one potential mechanism for oncolytic activity.\nResponse: Thank you for your valuable suggestions. We agree that it is more accurate to describe LRRK2-STAT1 signaling as one potential mechanism underlying the oncolytic activity of oHSV. We have revised the relevant descriptions accordingly in the updated manuscript (Lines 229-231).\nAdditionally, the mechanism of action is critically important in understanding oncolytic synergistic effects with LRRK2 inhibition. GBM has a strong immunosuppressive microenvironments established most likely from the innate immune cell component of the tumour, including >50% macrophages, microglia and astrocytes. Single cell interactions in the case of GBM, focussing on the cancer cell mechanism in this tumour microenvironment, would be very supportive of the proposed MOA. Here, scRNA transcriptomic of multi-culture organoids, either in vitro, PDX, or patient derived organoids, could support the results.\nResponse: Thank you for your valuable suggestions and comments. We fully agree with your perspective and believe that exploring cell interactions within the TME will further support our proposed MOA. However, due to insufficient funding and the lack of available research methodologies, we were unable to establish a single-cell RNA transcriptomic method to dissect the potential mechanism, despite our best efforts to follow your suggestions. In this study, we focused primarily on the role and underlying MOA of LRRK2 in regulating oHSV activity in GBM tumor cells. The role of the GBM TME in the oncolytic synergistic effects with LRRK2 inhibition remains to be investigated in future studies. We have included these discussions and limitations in the revised manuscript (Lines 471-476, Lines 496-503).\nImmunocompetent GBM mouse model\nThe points of observation that the conbination treatment in enhanced viral replication and increase tumour cell apopotosis (Figure 7 H and I) are not supported empirically. Please quantify these results. These are links to in vivo results so are crucial to the conclusions for this section.\nResponse: Thank you for your valuable suggestions and comments. The percentages of HSV+, IRF9+, Casp3+, and HSV+Casp3+ cells in each immunohistochemical (IHC) staining were quantified. The combination therapy significantly enhanced viral replication (P = 0.0343) and increased tumor cell apoptosis (P < 0.0001) relative to virotherapy alone. Notably, colocalization signals of HSV and cleaved caspase-3 were significantly stronger in the combination treatment group (P < 0.0001). Moreover, LRRK2-IN-1 substantially reduced (P < 0.0001) the upregulation of IRF9 induced by oHSV-GFP, enhancing viral replication. These results have been included in the revised manuscript (Fig. 7H; Lines 344-349, Lines 817-821).\nLRRK2 abundance in human GBM\nAlthough it is encouraging that LRRK2 inhibition does not appear to target healthy astrocytes, macrophages and microgia, as well a neurons (all could be iPSC derived) and tested to interrogate all cell types found in primary and recurrent GBM.\nResponse: Thank you for your valuable suggestions. We conducted additional experiments to investigate the effects of LRRK2 inhibition on the cytotoxicity of oHSV in macrophages, microglial cells, and iPSC-derived neurons, which are the predominant cell types in primary and recurrent GBM. The results indicated that LRRK2 inhibition does not enhance oHSV activity in these cells. These findings have been incorporated into the revised manuscript (Fig. 8E-G; Lines 373-377).\nSignificance\nThe use of cell lines for screening are often an economically valid step as it considerably reduces experimental costs however, more complex and predictive models are now widely available using multicell organoids and patient derived organoid techniques. The authors should consider executing, or collaborating with laboratories, experiments with co-culture models incorporating the innate immune components of GBM or patient derived organoids (techniques are available for both). This with demonstrate effectiveness of cotreatment in a more biologically predictive model vs. mouse PDX models.\nResponse: Thank you for your valuable suggestions and comments. We fully agree with your perspective and believe that, in addition to the mouse PDX models used in our study, more complex and predictive models, such as multicellular organoids and patient-derived organoid techniques, could further support our findings. Despite our best efforts, it was challenging for our lab to establish organoid techniques or collaborate with other labs. We have emphasized the importance of organoid models in studying the oncolytic synergistic effects with LRRK2 inhibition in GBM in the Discussion section of our revised manuscript (Lines 474-476).\nClarity and content\nSeveral graphs could benefit from reformatting\nFigure 1A - Consider drawing a vertical line screen MOI\nResponse: Thank you for your suggestions. We have drawn a vertical line to screen the MOI (Fig. 1A).\nFigure 3B - What are the timepoints beyond 24 hours if figure 3E includes a 24 hour timepoint\nResponse: Thank you for your suggestions. In Figure 3B, we focused on two time points, 8 hours to examine the role of LRRK2 inhibition in virus entry, and 24 hours for viral spread, thus excluding time points beyond 24 hours. In Figure 3E, we examined the entire viral life cycle to assess the role of LRRK2 inhibition in viral growth, including the time points 0, 8, 24, 26, and 48 hours in the multistep virus growth curves. These details are included in our revised manuscript (Lines 167-168, Lines 178-184).\nFigure 3G- Can you quantify the westerns on a fluorescent western reader\nResponse: Thank you for your suggestions. We have quantified all Western blot results and incorporated these data into the revised manuscript.\nFigure 7E & F - Please include figure legend\nResponse: Thank you for your suggestions. We have incorporated figure legends in the revised Figures 7E and 7F.\nFigure 7H - Please quantify with histograms\nResponse: Thank you for your suggestions. We have quantified these results in the revised manuscript (Fig. 7H, Lines 817-821).\nReviewer #2 (Remarks to the Author): expert in glioblastoma\nIn this report, Drs. Yu et al reported the targeting of LRRK2 to overcome resistance to oHSV in GBMs. Given the poor prognosis of GBM, the established safety profiles and noticeable activities of oHSV, this study addresses an important clinical problem by potentially offering a new approach to enhance the anti-GBM activities of oHSV. The identification of LRRK2 as a key mediator of oHSV resistance appears new to the fields although the impact of innate anti-viral system on oncolytic activities have long been recognized. The overall experimental design is sound, extensive and appears to be well executed. Multiple GBM cell lines were included together with orthotopic xenograft mouse models both in immune-deficient and immune-competent animals. The inclusion of a fairly large panel of patient tumors in a tissue microarray provided additional support for the potential use of LRRK2 as a diagnostic marker. While this paper provided some potentially important information, there are some concerns that need to be addressed:\nResponse: Thank you for reviewing our manuscript and providing valuable feedback to improve the quality of our manuscript.\nThe major concern is the lack of data or “results” in the RESULT section. Only summary or conclusive type statements were included. There was no sufficient information on the actual changes of biological activities and statistical analysis to convincingly support their statement. Matching the data presented in the figures (although nicely done) with descriptions in the figure legends is also a big challenge. Proper evaluation of this manuscript will need major revision on data presentation.\nResponse: Thank you for your valuable suggestions and comments. We have revised the descriptions and expanded the analysis in the Results section and figure legends to provide comprehensive information on the observed changes in biological activities. For Western blot analyses, we quantified the band intensity using ImageJ and expressed the results as fold changes relative to the control group to illustrate the differences among the experimental groups. Additionally, we included the fold change compared to the control below each blot to improve data presentation. For animal experiments, we conducted a detailed comparison and performed statistical analyses across different groups. For cell-killing curve analyses, we calculated the EC values for each treatment 50 and used EC shifts to illustrate the actual changes in biological activity. All P-values 50 depicted in the graphs are clearly described in the Results section. Accordingly, we meticulously revised the Results section and figure legends of our manuscript to ensure improved data presentation.\nListed below are some (a small fraction) of the missing data and P values:\nResponse: Thank you for your valuable suggestions and comments. We meticulously revised the Results section of our manuscript to present the data and P-values as clearly as possible.\n• Furthermore, upon treatment 221 with the LRRK2 inhibitor LRRK2-IN-1, LRRK2-mediated STAT1 phosphorylation was significantly reduced (Fig. 5I): how much?\nResponse: Thank you for your valuable suggestions and comments. Upon treatment with LRRK2-IN-1, LRRK2-mediated STAT1 phosphorylation was significantly reduced by approximately 6-fold compared to untreated controls. These details are included in our revised manuscript (Lines 260-262).\n• To 226 investigate whether LRRK2 regulates antiviral effects through STAT1 signaling, LRRK2 227 knockdown reduced the phosphorylation of STAT1 at Tyr701 and enhanced oHSV-GFP infection 228 (Fig. 5K). How much?\nResponse: Thank you for your valuable suggestions and comments. LRRK2 knockdown nearly abolished STAT1 phosphorylation at Tyr701 and enhanced oHSV-GFP infection by at least 8.2-fold. These details are included in our revised manuscript (Lines 276-277).\n• Our data showed that treatment 256 with LRRK2-IN-1 sensitized GBM-1 cells to oHSV-GFP oncolysis (Fig. 6A). How much?\nResponse: Thank you for your valuable suggestions and comments. Our data showed that treatment with LRRK2-IN-1 sensitized GBM-1 cells to oHSV-GFP oncolysis, with EC shifts of 4.33-fold. These details are included in our revised manuscript (Lines 50 304-305).\n• The 264 combination treatment resulted in significantly stronger inhibition of tumor growth in GBM-1 265 xenografts compared with either monotherapy alone (Fig. 6D-F). How much?\nResponse: Thank you for your valuable suggestions and comments. The combination treatment demonstrated significantly greater inhibition (P < 0.01) of tumor growth in GBM-1 xenografts compared to either monotherapy alone, starting from day 15 post-treatment (Fig. 6D-F). By day 30 post-treatment, the combined therapy substantially reduced tumor burden in GBM-1 xenografts relative to virotherapy alone (P = 0.0003) or LRRK2-IN-1 alone (P = 0.0018). These details are included in our revised manuscript (Lines 311-315).\nMinor concerns:\n1. Figure 1 and 2: “Three florescent fields were analyzed”. How were the fields selected to prevent bias? And why only three fields. These data are not sufficient for quantitative analysis.\nResponse: Thank you for your valuable suggestions and comments. Our previous description was incorrect. The data were derived from the average fluorescence intensity of three independent experiments. For each experiment, three replicates per experimental group were analyzed, with three random fluorescence fields captured per replicate. Consequently, the data for each experimental group were calculated from the average fluorescence intensity of nine fluorescence fields. We have clearly described the quantitative analysis method in the corresponding figure legends (Lines 1062-1063, Lines 1092-1094).\n2. The true identity of U87 is controversial. Since it was involved in multiple assays, this limitation should be discussed.\nResponse: Thank you for your valuable suggestions. We acknowledge the controversy surrounding the identity of U-87 MG cells purchased from ATCC. Although our study utilized a substantial number of GBM cell lines and primary GBM cells to support our conclusions, the data derived from U-87 MG should be interpreted cautiously. This is because the commercially available U-87 MG cells differ from the original cells and likely represent a human GBM cell line of unknown patient origin. This limitation has been explicitly addressed in the Discussion section of our manuscript (Lines 503-506).\n3. Fig 4B: Cells treated with LRRK2in alone should be included as reference.\nResponse: Thank you for your valuable suggestions. In response, we re-analyzed the RNA-Seq data from U-251 MG and U-87 MG cells infected with oHSV-GFP or mock-infected, in the presence or absence of LRRK2-IN-1. Cells treated with LRRK2i alone were included as a reference. The results indicated that LRRK2-IN-1 inhibited antiviral genes, such as OAS3, GBP3, IFI44L, and MX2, regardless of viral infection status. These findings have been incorporated into the revised manuscript (Figure 4B, Figure S5A-D; Lines 198-201).\n4. In vivo treatment lacks sufficient scientific details about the timing and dosing, making it difficult to determine the potential clinical relevancy.\nResponse: Thank you for your valuable suggestions and comments. We have provided the experimental scheme for each in vivo experiment (Fig. 6D and Fig. 7B). The details regarding timing and dosing were clearly described in the corresponding figure legends and the Methods section of our manuscript (Lines 768-772, Lines 777-788; Lines 1190-1192, Lines 1196-1199, Lines 1216-1219).\n5. Figure 8 A and B: The protein expression of LRRK2 was low/no in multiple cell lines that exhibited strong responses (low EC50). How were the relative expression determined? When these data combined with their findings in Fig 1, Fig 2 (e.g., U-251 of which the LRRK2 expression was nearly zero), it is difficult to conclude that the LRRK2 is critically important for oHSV. For example, U-251 does not express LRRK2 protein (Fig 8 A), but KRRK2 inhibitor was very active in sensitizing it to oHSV infection (Fig 2 A)\nResponse: Thank you for your valuable suggestions and comments. The relative expression of LRRK2 was determined by analyzing the band density of western blot using Image J for relative quantification. Since the expression of LRRK2 is relatively lower in U-251 MG cells compared to other GBM cell lines, it was challenging to detect LRRK2 using standard western blot techniques. To address this issue, we conducted additional experiments employing an ultra-sensitive enhanced chemiluminescent (ECL) substrate for high attogram protein detection, enabling more accurate quantification of LRRK2 in U-251 MG cells. LRRK2 protein expression is lowest in U-251 MG cells compared to other tested GBM cell lines. Our data revealed a positive correlation between LRRK2 protein expression and EC across multiple GBM cell lines, 50 suggesting that LRRK2-hyperexpressing GBM cells were generally more refractory to oHSV treatment than cells with relatively low LRRK2 expression. On the other hand, results from LRRK2 inhibition, knockdown, and overexpression studies collectively support our conclusion that LRRK2 plays a critical role in oHSV cytotoxicity. Although LRRK2 inhibitors sensitize multiple GBM cell lines to oHSV infection, the degree of sensitization varies, suggesting that additional factors may influence oHSV cytotoxicity in some GBM cell lines. These results have been incorporated into the revised manuscript (Fig. 8A; Line 361-365; Lines 489-491).\n6. A systematic lack of statistic analysis in the result section.\nResponse: Thank you for your comments, we meticulously revised the Results section and included the statistical analyses in the Results section of our manuscript. Additionally, the specific statistical methods used for each figure are detailed in the corresponding figure legends and Methods section of the manuscript.\nReviewer #3 (Remarks to the Author): expert in LRRK2\nIn the manuscript by Yu and colleagues, titled “Targeting Leucine-Rich Repeat Kinase 2 Overcomes Resistance to Oncolytic Herpes Simplex Virus-Based Therapies in Glioblastoma”, the authors conduct a bioactive drug screen to identify compounds that enhance the activity of oncolytic herpes simplex virus (oHSV) in glioblastoma (GBM) cells. They identify LRRK2-IN1 as a potent enhancer of oHSV activity in these cells. Mechanistically, the authors propose that LRRK2 phosphorylates STAT1, thereby promoting antiviral responses. Although I am not an expert in cancer biology, I have extensive experience studying LRRK2. Accordingly, my comments will focus primarily on the biology of LRRK2. Please find my feedback below.\nResponse: Thank you for reviewing our manuscript and providing valuable feedback to improve the quality of our manuscript.\n1. The authors use student’s t-test in analysis of more than 2 groups, which is wrong. The authors must use the proper statistical tests (one or two-way ANOVA) when analyzing three or more groups.\nResponse: We sincerely appreciate your constructive suggestions. In response, we have revised the statistical analyses in the manuscript. For other experiments, comparisons between two groups were conducted using unpaired two-sided Student’s t-tests, while comparisons involving three or more groups were performed using ordinary one-way ANOVA (Lines 828-830).\n2. The authors should demonstrate LRRK2 kinase inhibition by analyzing pRab10, pRab12 and pS1292 levels. They should also see if oHSV increases LRRK2 kinase activity.\nResponse: We have conducted additional experiments to evaluate LRRK2 kinase activity following LRRK2 inhibition and/or oHSV infection. Notably, phosphorylation of LRRK2 at Ser935 and Ser1292, as well as phosphorylation of Rab10 at Thr73 and Ser106, were significantly reduced by two LRRK2 inhibitors (LRRK2-IN-1 and MLi-2), irrespective of viral infection status. These results have been incorporated into the revised manuscript (Figures S9H-K; Lines 263-267).\n3. LRRK2-IN-1 is not the most reliable LRRK2 inhibitor. The authors should repeat some of their cell viability and mechanistical experiments with MLi2.\nResponse: We have conducted additional cell viability and mechanistic experiments using MLi2. Both LRRK2-IN-1 and MLi-2 exhibited similar effects and mechanisms of action in enhancing the efficacy of oHSV in GBM cells. Together with the results from LRRK2 knockdown and overexpression studies, these findings collectively support our conclusions. These results have been incorporated into the revised manuscript (Figures 2F, 2G, S2C, S2D, S2E, S9H, S9I, and S9L).\n4. The authors should also repeat some of the viability assays from Fig.3 comparing WT vs kinase dead mutant vs G2019S.\nResponse: We have conducted additional viability assays comparing wild-type (WT), kinase-dead mutant, and G2019S LRRK2 (with LRRK2 inhibitor). Overexpression of the LRRK2G2019S mutant further reduced cytotoxicity toward oHSV-GFP oncolysis compared to wild-type LRRK2 overexpression, while overexpression of the kinase-dead LRRK2 (LRRK2D1994A) abolished LRRK2’s ability to decrease cytotoxicity toward oHSV-GFP oncolysis. Notably, the reduced sensitivity to oHSV-GFP oncolysis induced by LRRK2 or LRRK2G2019S overexpression was reversed by LRRK2-IN-1 treatment. Inhibition of wild-type LRRK2 or the LRRK2G2019S mutant restored cytotoxicity toward oHSV-GFP oncolysis to baseline levels. These results have been incorporated into the revised manuscript (Figure 2J; Lines 154-160).\n5. Even though no clear information can be found in the methods section, it seems that LRRK2 is tagged C terminally. LRRK2 needs to be tagged N terminally, as we and others in the field have seen that C terminal tagging can be artificial. If that would be the case, this reviewer finds hard to trust the results using the C terminally tagged vector.\nResponse: We sincerely appreciate your constructive suggestions. In this study, some LRRK2 constructs were C-terminally tagged, while others remained untagged. Following the reviewer’s recommendation, we constructed an additional N-terminally tagged LRRK2 and repeated all experiments previously conducted with the C-terminally tagged constructs. The results obtained from the N-terminally tagged LRRK2 constructs were largely consistent with those from the C-terminally tagged constructs, except for the findings that identified the key LRRK2 domain interacting with STAT1. All plasmids, whether tagged or not, are clearly indicated in the figures and the corresponding legends. These new data have been incorporated into the revised manuscript (Figure 5A, 5E-G and S9B-D).\n6. Western blots in Fig 4E-G, and 5I,J,M,L,O should be quantified.\nResponse: We sincerely appreciate your constructive suggestions. The quantification results of all Western blot analyses are now included in the revised manuscript.\n7. To convincingly claim that LRRK2 and STAT1 interact. The authors should only overexpress one the proteins, and blot for the other at the endogenous level. Co overexpression of two proteins in 293 cells is known to yield artificial results.\nResponse: We sincerely appreciate your constructive suggestions. We have confirmed the interaction between endogenous LRRK2 and endogenous STAT1 in two GBM cell lines. These findings have been incorporated into the revised manuscript (Figure 5D and S9A; Lines 242-244).\n8. The authors claim that STAT1’s DB domain binds to LRRK2, however, the CCDB construct shows minimal binding to LRRK2, suggesting that the binding with LRRK2 requires both the DB and SH2 domains.\nResponse: We sincerely appreciate your suggestions. Following the reviewer’s recommendation, we constructed an additional STAT1 DBSH2 truncation and evaluated the binding activity of the DB and SH2 domains to STAT1. Our results revealed that LRRK2 binds to both the DB and SH2 domains of STAT1, with a stronger affinity observed for the DBSH2 truncation, which simultaneously contains both domains. These findings have been incorporated into the revised manuscript (Figure 5F and S9C; Lines 246-249).\n9. The idea that STAT1 can bind independently to three different LRRK2 domains (LRR, Kin and ANK) is hardly convincing and makes little sense.\nResponse: We sincerely appreciate your comments. Our previous results indicated that STAT1 could independently bind to three distinct LRRK2 domains (LRR, Kin, and ANK) when the tag was positioned at the C-terminus of LRRK2. Following the reviewer’s suggestion, we adopted a more reasonable approach by using LRRK2 truncations with tags at their N-terminus. Through co-IP assays, we evaluated the binding activity of myc-LRRK2 truncations and STAT1, revealing that STAT1 specifically binds to the Roc domain of LRRK2. We hypothesize that C-terminal tagging of LRRK2 may induce artificial structures, enabling binding to multiple domains. Consequently, the new results obtained from co-IP assays using N-terminal tagged LRRK2 have replaced the previous data. These findings have been incorporated into the revised manuscript (Figure 5E-5G and S9B-9D; Lines 249-251).\n10. Authors need to include a WT vs G2019S comparison (with LRRK2i), analyzing pSTAT1-701 levels (quantified with multiple replicates).\nResponse: We sincerely appreciate your valuable suggestions. To assess the impact of LRRK2 activity on the phosphorylation of STAT1, we conducted three independent experiments, including conditions with wild-type (WT), D1994A, and G2019S LRRK2 (with LRRK2 inhibitor). Kinase-active LRRK2G2019S enhanced STAT1 phosphorylation at Tyr701 compared to wild-type LRRK2, while kinase-dead LRRK2D1994A reduced LRRK2-mediated STAT1 phosphorylation. Treatment with LRRK2-IN-1 reduced LRRK2-mediated STAT1 phosphorylation compared to untreated controls. The quantification results are presented in Fig. 5J and Fig. S9G (Lines 267-273).\n11. In my experience, LRRK2 antibodies do not work for staining. The authors should either validate their antibody for immunostaining or remove all the data involving LRRK2 staining.\nResponse: We sincerely appreciate your suggestions. To validate the feasibility of commercially available anti-LRRK2 antibodies for immunostaining, we conducted additional experiments. Our results confirmed that the anti-LRRK2 antibodies from Abcam (cat. # ab133474) are suitable for LRRK2 immunostaining. These findings have been incorporated into the revised manuscript (Figure S8A and S8B).\n12. Typo in Fig. 5J (D2019S).\nResponse: We sincerely appreciate your valuable suggestions. The typo has been corrected in Figure 5J of the revised manuscript.\nReviewer #4 (Remarks to the Author): expert in oncolytic viruses and antiviral response\nThis manuscript submitted by Mengqin and colleagues reported their findings that LRRK2i may sensitize oHSV replication, spreading and oncolysis in GBM through suppressing a noncanonical LRRK-and STAT1-pathways. Moreover, they found that the combination of LRRK2i and oHSV improved therapeutic efficacy as compared to monotherapy in murine or human GBM-PDXs established in immunocompromised mice, as well as in syngeneic mouse GMB model. Overall, this study is interesting, the identification of LRRK2 as a potential biomarker for oHSV therapy and the combination of LRRK2i with oHSV for GBM treatment are attractive approaches for further investigations.\nResponse: Thank you for reviewing our manuscript and providing valuable feedback to improve the quality of our manuscript.\nSpecific comments:\n1. Page 6, line 158-160, OVs induces cancer death, which could be immunogenic cell death, but cancer cell apoptosis may inhibit antitumor immune responses, please reconcile the sentence.\nResponse: We sincerely appreciate your valuable suggestions. The sentence \"OVs can induce cancer cell death through apoptosis\" is more appropriate in this context and has been adopted accordingly. We have revised the relevant sentence in our manuscript (Line 187).\n2. In Fig. 4D, if add anti-IFNa in the treatment to oHSV and LRRK2-IN-1 group, would the cell viability be changed?\nResponse: We sincerely appreciate your valuable suggestions. To investigate the impact of IFNα neutralization (anti-IFNa) on the combined effects of LRRK2-IN-1 and oHSV, we conducted additional experiments. Our findings demonstrated that the introduction of anti-IFNAR1 in the treatment group combining oHSV and LRRK2-IN-1 did not affect cell viability. These results have been incorporated into the revised manuscript (Figure 4D; Lines 208-215).\n3. In Fig. 7I, how about the immune suppressive regulatory T cells (Tregs), any changes in this population in the tumors?\nResponse: We sincerely appreciate your valuable suggestions. To investigate changes in Tregs within tumors subjected to various treatments, we conducted further experiments and analyses. Our findings revealed that the combination treatment of oHSV and LRRK2-IN-1 did not alter the abundance of Tregs in the TME. These results have been included in the revised manuscript (Figure 7I; Line 355-356).\n4. Did the toxicity study show any changes of body weight or levels of inflammatory cytokines in serum?\nResponse: We sincerely appreciate your valuable suggestions. To evaluate the potential toxicity of the combination therapy, we conducted additional animal experiments. Following orthotopic administration of various agents, we monitored the mice for changes in body weight and measured key inflammatory cytokines (IL-6, TNF-α, and IFN-γ) in serum. The results demonstrated that the combination therapy did not induce any alterations in body weight or trigger an inflammatory response. These findings have been incorporated into the revised manuscript (Figure S13A and B; Line 392-396, Lines 796-800)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Following a thorough review of the revised manuscript, focussing specifically on the authors responses to my comments, the authors have satisfactorily answered my comments to improve the manuscript. I support publication of this new manuscript.", "reviewer_3": "The authors have significantly improved the manuscript; however, key concerns remain regarding the LRRK2 staining and the quantification of western blot data, particularly in relation to experimental replicates. Please see my detailed comments below:\nFigure S9 was not included in the submission. Please ensure that all supplemental figures referenced in the text are provided.\nThe authors use the Abcam antibody (cat. ab133474) for LRRK2 staining and argue that it is suitable because it recognizes overexpressed LRRK2. However, the YCharOS dataset (https://zenodo.org/records/7971965) clearly demonstrates that this antibody fails to detect endogenous LRRK2 in immunocytochemistry applications. Consistent with this, we have also been unable to stain for endogenous LRRK2 in our own lab using this antibody.\nGiven this lack of validation for endogenous conditions, the reliability of the PLA results shown in Figure S8 is questionable. Without robust antibody validation in the relevant cellular context, the conclusions drawn from these experiments remain unconvincing.\nThe authors need to specify whether the error bars in the western blot quantifications represent standard deviation (SD) or standard error of the mean (SEM). Additionally, the manuscript lacks details on the number of biological replicates and the statistical tests used to support the quantification. As it stands, it appears that some conclusions may be based on data from a single experiment (n=1), which is not statistically robust. These concerns were previously raised with respect to Figures 4 and 5 and remain unaddressed.", "reviewer_4": "The authors have addressed my comments and concerns.", "reviewer_5": "The authors have thoroughly responded to the reviewers comments."}, "author_response": "Response: Thank you for your positive comments and valuable suggestions to improve the quality of our manuscript.\n\nFigure S9 was not included in the submission. Please ensure that all supplemental figures referenced in the text are provided.\nResponse: We appreciate the valuable feedback and valuable suggestions. The supplementary figures (including Figure S8, previously labeled Figure S9) and their corresponding legends are included in the supplementary data file and are appropriately referenced in the main text.\n\nThe authors use the Abcam antibody (cat. ab133474) for LRRK2 staining and argue that it is suitable because it recognizes overexpressed LRRK2. However, the YCharOS dataset (https://zenodo.org/records/7971965) clearly demonstrates that this antibody fails to detect endogenous LRRK2 in immunocytochemistry applications. Consistent with this, we have also been unable to stain for endogenous LRRK2 in our own lab using this antibody.\nGiven this lack of validation for endogenous conditions, the reliability of the PLA results shown in Figure S8 is questionable. Without robust antibody validation in the relevant cellular context, the conclusions drawn from these experiments remain unconvincing.\nResponse: Thank you for your valuable suggestions. After re-evaluating the YCharOS dataset using the LRRK2 knockout cell line, we concur with your assessment that no commercially available LRRK2 antibodies can reliably detect endogenous LRRK2 in immunostaining assays. Consequently, we have removed the PLA results from our revised manuscript.\n\nThe authors need to specify whether the error bars in the western blot quantifications represent standard deviation (SD) or standard error of the mean (SEM). Additionally, the manuscript lacks details on the number of biological replicates and the statistical tests used to support the quantification. As it stands, it appears that some conclusions may be based on data from a single experiment (n=1), which is not statistically robust. These concerns were previously raised with respect to Figures 4 and 5 and remain unaddressed.\nResponse: We sincerely appreciate your valuable suggestions. Western blot analyses were routinely performed in triplicate (or in duplicate where indicated), with consistent results across experimental replicates. Given space constraints, representative blots are presented in the main and supplementary figures, while complete replicate datasets are available in the resource data file. In accordance with reviewer recommendations, we have summarized the replicate data in bar charts, which are provided alongside the corresponding figures (Fig. 4, Fig. 5 and Figure S8).\nThe data are presented as the mean ± SD from three independent experiments, and western blot analyses were repeated multiple times independently with similar results. These details are included in the corresponding figure legends."}, {"round": 3, "reviewer_comments": {"reviewer_3": "While the manuscript has improved, there are still inaccurate statements in the manuscript regarding LRRK2. You can find my detailed comments below.\n1) The authors claim that “Interestingly, oHSV-GFP infection increased LRRK2 kinase activity and activated its downstream substrate Rab10 and Rab12, two well-established LRRK2 substrates (Supplementary Fig. 8g–j).” This statement is false. Supplementary Fig. 8g–j does not show an increase in pRab10 or pRab12 following oHSV-GFP infection and therefore does not support the conclusion of increased LRRK2 kinase activity by this readout.\n2) The subsequent statement is also inaccurate: “Notably, phosphorylation of LRRK2 at Ser935 and Ser1292, and phosphorylation of Rab10 at Thr73 and Rab12 at Ser106, were significantly reduced by the LRRK2 inhibitors LRRK2-IN-1 and MLi-2, irrespective of viral infection status.” In Supplementary Fig. 8h, pRab12 levels are not reduced by MLi-2 treatment alone, and in Supplementary Fig. 8i–j, phosphorylation of both Rab10 and Rab12 remains largely unchanged following exposure to the LRRK2 kinase inhibitor. These data do not demonstrate effective inhibition of LRRK2-dependent Rab phosphorylation.\nTaken together, although the data obtained using the LN-229 cell line appear largely reliable, these results raise serious concerns regarding the use of the U-251 MG cell line. Specifically, the positive controls for LRRK2 kinase activity fail to respond appropriately to kinase inhibition, indicating a major lack of experimental oversight. More importantly, the authors’ claims are clearly incorrect, which shows a worrying lack of rigor. Additionally, the fact that oHSV-GFP infection does not increase pRab levels while increasing the auto-phosphorylation site S1292, should be addressed in the discussion.\n3) “We found that inhibiting TBK1/IKKε with BX795 can suppress TBK1 phosphorylation by approximately 5.20-fold and consequently reduce the phosphorylation of its substrate, LRRK2 by approximately 1.89-fold (Fig. 5l). We further examined the effects of TBK1/IKKε inhibition on the downstream mediators of LRRK2, specifically STAT1 and Rab10. Our results demonstrated that BX795 significantly reduced phosphorylated STAT1 levels by approximately 10.33-fold. Similarly, phosphorylated Rab10 levels were markedly decreased by approximately 3.50-fold following BX795 treatment”. BX795 reduces total LRRK2 expression, which likely explains the reduction in pRab10 and pSTAT1. Please fix the text accordingly.\n4) As the authors could not validate the LRRK2 antibody for immunostaining, the should remove LRRK2 from Figure 8g, and Figure 8k-m.\n5) Lastly, I asked the authors to include the SD in the WB quantification and they have not done so in Figure S8."}, "author_response": "Response: Thank you for reviewing our manuscript and providing valuable feedback. Your comments are significantly important for improving the quality of our manuscript. We have made every effort to address these issues and strengthen the revised manuscript. If you have any comments on my feedback or suggestions regarding my responses, please feel free to let me know. Thank you.\n\n1) The authors claim that “Interestingly, oHSV-GFP infection increased LRRK2 kinase activity and activated its downstream substrate Rab10 and Rab12, two well-established LRRK2 substrates (Supplementary Fig. 8g–j).” This statement is false. Supplementary Fig. 8g–j does not show an increase in pRab10 or pRab12 following oHSV-GFP infection and therefore does not support the conclusion of increased LRRK2 kinase activity by this readout.\nResponse: We sincerely appreciate your valuable suggestions. Based on Western blot quantification (reported as mean ± SD), we observed that while oHSV-GFP infection led to a marginal increase in LRRK2 phosphorylation at Ser935 and Ser1292 in LN-229 cells, these changes were not statistically significant. This suggests that oHSV-GFP infection does not substantially enhance LRRK2 kinase activity. In the previous version of our manuscript, we mistakenly described that oHSV-GFP infection activated its downstream substrates Rab10 and Rab12. We have rephrased these texts into “oHSV-GFP infection had minimal impact on the phosphorylation of LRRK2 at Ser935 and Ser1292 and did not activate its downstream substrates, Rab10 and Rab12, two well-established LRRK2 substrates in LN-229 cells (Supplementary Fig. 8g, h).”。\n\n2) The subsequent statement is also inaccurate: “Notably, phosphorylation of LRRK2 at Ser935 and Ser1292, and phosphorylation of Rab10 at Thr73 and Rab12 at Ser106, were significantly reduced by the LRRK2 inhibitors LRRK2-IN-1 and MLi-2, irrespective of viral infection status.” In Supplementary Fig. 8h, pRab12 levels are not reduced by MLi-2 treatment alone, and in Supplementary Fig. 8i–j, phosphorylation of both Rab10 and Rab12 remains largely unchanged following exposure to the LRRK2 kinase inhibitor. These data do not demonstrate effective inhibition of LRRK2-dependent Rab phosphorylation.\nResponse: We sincerely appreciate your valuable suggestions. In LN-229 cells, Rab12 phosphorylation was significantly reduced by MLi-2 treatment alone (P = 0.007), although the magnitude of this reduction was smaller than that observed for Rab10 phosphorylation. Because LRRK2 expression in U-251 MG cells was relatively low compared with the other glioblastoma cell lines we tested, LRRK2 phosphorylation—particularly at Ser935—was difficult to detect reliably and was associated with high background noise. Although we observed a slight decrease in Rab12 phosphorylation following exposure to the LRRK2 kinase inhibitor, the effect was not robust. Notably, STAT1 phosphorylation was clearly inhibited after inhibitor treatment (Fig. S8n), suggesting that an as-yet-unknown pathway may regulate the LRRK2–Rab axis in U-251 MG cells. Therefore, we have revised the corresponding text and discussed these observations in the Discussion section.\nWe have rephrased these texts into “Notably, the LRRK2 inhibitors LRRK2-IN-1 and MLi-2 significantly reduced the phosphorylation of LRRK2 (at Ser935 and Ser1292), Rab10 (at Thr73), and Rab12 (at Ser106) in LN-229 cells, although the degree of inhibition varied across these sites. In contrast, the inhibition of LRRK2-dependent Rab phosphorylation was not pronounced in U-251 MG cells, whereas inhibition of STAT1 phosphorylation was robust. This disparity may be attributed to the lower expression levels of LRRK2 in U-251 MG cells, as well as a substrate preference for STAT1 over Rab10/12 in this specific cell line.”.\n\nTaken together, although the data obtained using the LN-229 cell line appear largely reliable, these results raise serious concerns regarding the use of the U-251 MG cell line. Specifically, the positive controls for LRRK2 kinase activity fail to respond appropriately to kinase inhibition, indicating a major lack of experimental oversight. More importantly, the authors’ claims are clearly incorrect, which shows a worrying lack of rigor. Additionally, the fact that oHSV-GFP infection does not increase pRab levels while increasing the auto-phosphorylation site S1292, should be addressed in the discussion.\nResponse: Thank you for pointing out our mistake and offering valuable suggestions. In addition to revising the corresponding text as described above, we have incorporated the following points into the Discussion section:\n“Interestingly, we observed that oHSV-GFP infection led to a marginal increase in LRRK2 phosphorylation at Ser935 and Ser1292 in LN-229 cells, suggesting that viral infection may partially influence LRRK2 signaling. Although Rab protein phosphorylation is widely used as a readout of LRRK2 kinase activity1, 2, we also observed differential regulation of the downstream signaling substrates Rab10 and Rab12 by LRRK2 inhibitors in different GBM cell lines. This suggests that cell-type-specific intracellular factors may influence LRRK2 substrate preference. These findings warrant further investigation in future studies.”\n\n3) “We found that inhibiting TBK1/IKKε with BX795 can suppress TBK1 phosphorylation by approximately 5.20-fold and consequently reduce the phosphorylation of its substrate, LRRK2 by approximately 1.89-fold (Fig. 5l). We further examined the effects of TBK1/IKKε inhibition on the downstream mediators of LRRK2, specifically STAT1 and Rab10. Our results demonstrated that BX795 significantly reduced phosphorylated STAT1 levels by approximately 10.33-fold. Similarly, phosphorylated Rab10 levels were markedly decreased by approximately 3.50-fold following BX795 treatment”. BX795 reduces total LRRK2 expression, which likely explains the reduction in pRab10 and pSTAT1. Please fix the text accordingly.\nResponse: We sincerely appreciate your valuable suggestions. We fully agree that it is more appropriate to conclude that BX795 reduces total LRRK2 expression. We have revised this statement into “We found that inhibition of TBK1/IKKε with BX795 suppressed TBK1 phosphorylation by approximately 5.20-fold and decreased the total level of its substrate, LRRK2. Consequently, TBK1/IKKε inhibition reduced LRRK2 phosphorylation by approximately 1.89-fold (Fig. 5l) and attenuated the phosphorylation of downstream LRRK2 effectors, including STAT1 and Rab10. Our results demonstrated that BX795 markedly decreased phosphorylated STAT1 by approximately 10.33-fold and phosphorylated Rab10 by approximately 3.50-fold.”.\n\n4) As the authors could not validate the LRRK2 antibody for immunostaining, the should remove LRRK2 from Figure 8g, and Figure 8k-m.\nResponse: We sincerely appreciate your valuable suggestions. We have removed the IHC results from the revised manuscript.\n\n5) Lastly, I asked the authors to include the SD in the WB quantification and they have not done so in Figure S8.\nResponse: We sincerely appreciate your valuable suggestions. Accordingly, we have provided a full set of WB quantifications, including SD, in the revised Figure S8 (see the revised Figure S8 attached below).\n\nReference\n1. Kluss JH, et al. Detection of endogenous S1292 LRRK2 autophosphorylation in mouse tissue as a readout for kinase activity. NPJ Parkinsons Dis 4, 13 (2018).\n2. Dhekne HS, et al. Genome-wide screen reveals Rab12 GTPase as a critical activator of Parkinson's disease-linked LRRK2 kinase. Elife 12, (2023).\n\nReviewer #3 Response to tentative rebuttal letter:\nMy only issue is the third point; the want to modify the text to : \"We found that inhibition of TBK1/IKKε with BX795 suppressed TBK1 phosphorylation by approximately 5.20-fold and decreased the total level of its substrate, LRRK2. Consequently, TBK1/IKKε inhibition reduced LRRK2 phosphorylation by approximately 1.89-fold (Fig. 5l) and attenuated the phosphorylation of downstream LRRK2 effectors, including STAT1 and Rab10. Our results demonstrated that BX795 markedly decreased phosphorylated STAT1 by approximately 10.33-fold and phosphorylated Rab10 by approximately 3.50-fold.\".\nThe idea that TBK1 phosphorylates LRRK2 is far from established. I do not think that the evidence is strong is that direction, so I would modify the statement removing “its substrate”, so the sentence reads “ We found that inhibition of TBK1/IKKε with BX795 suppressed TBK1 phosphorylation by approximately 5.20-fold and decreased the total level of LRRK2.”. The authors knowledge on LRRK2 biology is very poor, which may lead to misleading conclusions and statements. They should avoid calling TBK1 a LRRK2 kinase.\nResponse: We sincerely appreciate your valuable suggestions. Accordingly, we have revised this sentence into “We found that inhibition of TBK1/IKKε with BX795 suppressed TBK1 phosphorylation by approximately 5.20-fold and decreased the total level of LRRK2.” in our revised manuscript."}]

Decision letter