Statistical significance was assessed by Students em t /em \test. S1 MOL2-14-2701-s002.pdf Vidofludimus (4SC-101) (2.1M) GUID:?EDAA5CCA-17E7-41E4-B4C4-4A8D1DCB2B0D Appendix S2 MOL2-14-2701-s003.xlsx (72K) GUID:?A7353C6B-DFBE-4C91-978C-C3378508B489 Appendix S3 MOL2-14-2701-s004.xls (65K) GUID:?D2F589D2-CBC4-4568-A549-2DB39CC96844 Appendix S4 Vidofludimus (4SC-101) MOL2-14-2701-s005.xlsx (22K) GUID:?CE080D30-F2E9-4829-AF76-4F6A2CE60162 Data Availability StatementAll data generated or analyzed during this study are included in this published article and its supplementary information files. Gene correlation assay in TCGA dataset was performed by online tool Ulcan (http://ualcan.path.uab.edu/analysis.html). Abstract Here, we demonstrate that moesin (MSN) phosphorylation by Rho\associated protein kinase (ROCK) stabilizes PD\L1 protein levels by blocking SPOP mediating PD\L1 degradation. Administration of ROCK inhibitor Y\27632 into immunocompetent mice bearing breast tumors suppressed tumor progression and triggers antitumor T\cell activity. and em in?vivo /em . Administration of Y\27632 into immunocompetent Balb/c mice bearing breast tumors suppressed tumor progression and enhanced CD4+ and CD8+ T\cell infiltration. RNA\seq analysis of Y\27632\treated mouse tumors revealed that ROCK inhibition upregulated several immune response genes. However, the combination of Y\27632 and an anti\PD\1 antibody did not show additive or synergistic effects due to reduced PD\L1 in the presence of Y\27632. Our study unravels a previously unappreciated mechanism of PD\L1 regulation through the ROCK\MSN pathway. Moreover, we found that ROCK inhibitors could be combined with breast cancer immunotherapy. AbbreviationsCMTM4/6CKLF\like MARVEL transmembrane domain\containing 4/6ERMezrin, radixin, and moesinGFPgreen fluorescent proteinGOGene OntologyIHCimmunohistochemistryMSNmoesinPARPpoly\ADP\ribose polymerasePD\1programmed cell death protein 1PD\L1programmed cell death ligand 1PHAphytohemagglutininPMAphorbol myristate acetateROCKrho\associated protein kinaseSPOPspeckle\type BTB/POZ protein 1.?Introduction Immune checkpoint inhibitors, such as programmed cell death ligand 1 (PD\L1)/programmed cell death protein 1 (PD\1) antibodies, have achieved significant clinical outcome in several cancer types NOTCH1 [1, 2]. The application of anti\PD\L1 therapy is effective in the treatment of triple\negative breast cancer [3]. The main challenges of Vidofludimus (4SC-101) applying anti\PD(L)\1 therapies in cancer treatment are low response rates and immune resistance [4]. Combination therapy of PD\L1 and antitumor drugs is a reasonable strategy to improve anti\PD\L1 therapy efficiency. For example, a CDK4/6 inhibitor and anti\PD\1 immunotherapy enhanced tumor regression and overall survival rates of tumor\borne mice [5], and blockade of PD\L1 potentiated poly\ADP\ribose polymerase (PARP) inhibitor anticancer therapies [6]. Understanding the regulation of PD\L1 expression will provide insights into new targets for combination therapy in breast cancer. The PD\L1 binds to PD\1 in T cells, resulting in T\cell anergy and apoptosis [7], while the efficiency of immunotherapy is closely associated with the number and activity of intratumoral CD8+ T cells [8]. PD\L1 is induced Vidofludimus (4SC-101) by several cytokines, such as type I and type II interferons, tumor necrosis factor , and vascular endothelial growth factor. The 3\UTR of PD\L1 mRNA is usually disrupted in multiple cancers, leading to downregulation of the PD\L1 transcriptional level [9]. As a transmembrane protein, PD\L1 expression is extensively regulated by post\translational modifications, such as phosphorylation, glycosylation, acetylation, palmitoylation, and ubiquitination [10, 11]. Additionally, membrane\associated proteins CMTM6 and CMTM4 were recently identified to stabilize the membrane PD\L1 level by preventing PD\L1 from lysosome\mediated degradation [12, 13]. Moesin (MSN) belongs to the ezrin, radixin, and moesin (ERM) family. ERM plays a pivotal role in regulating cell surface structures, such as microvilli and membrane ruffles, and specialized membrane domains [14]. With regard to biochemical characteristics, MSN consists of an FERM (4.1 protein, ezrin, radixin, and moesin) domain in the amino terminus and an ERM\association domain (C\ERMAD) in the C terminus, which is capable of binding to the FERM domain or F\actin. In inactive conformation, the N\terminal FERM domain binds to the C\terminal region, while in active conformation, phosphorylation mediates the release of the FERM domain to interact with membrane proteins, such as CD44 and CD43 [15, 16]. In the disease setting, interaction of MSN and CD44 promotes glioma progression [16]. Activation of MSN is regulated by several kinases, including rho\associated protein kinase (ROCK), which phosphorylates MSN on threonine 558 (Thr558) to inhibit intermolecular head\to\tail association [17]. Clinical trials have found that inhibition of the ROCK pathway contributes to cardiovascular benefits of statin therapy. Furthermore, ROCK inhibitors may also inhibit tumor metastasis [18]. More recently, they have been studied for the treatment of glaucoma [19]. In view of the limited understanding of regulation of PD\L1 in breast cancer, we utilized mass spectrometry to screen for PD\L1 interaction protein and found that MSN interacts with and regulates PD\L1 expression. Moreover, we elucidated that ROCK\mediated MSN Thr558 phosphorylation was required for PD\L1 stabilization. In vivo studies proved that ROCK inhibitors achieved an antitumor activity similar to that of the anti\PD\1 antibody by stimulating T\cell activity. 2.?Materials and methods.
