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Gilbert Aaron Lee,1,2 Wan-Li Lin,1 Duen-Pang Kuo,3 Yi-Tien Li,4,5 Yu-Wei Chang,1 Yung-Chieh Chen,4,6 Shiu-Wen Huang,1,7,8 Justin Bo-Kai Hsu,1 Cheng-Yu Chen3,4,6,9 1Department of Medical Research, Taipei Medical University Hospital, Taipei, Taiwan; 2Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 3Department of Medical Imaging, Taipei Medical University Hospital, Taipei, Taiwan; 4Translational Imaging Research Center, Taipei Medical University Hospital, Taipei, Taiwan; 5Neuroscience Research Center, Taipei Medical University, Taipei, Taiwan; 6Research Center of Translational Imaging, College of Medicine, Taipei Medical University, Taipei, Taiwan; 7Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 8Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan; 9Department of Radiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, TaiwanCorrespondence: Cheng-Yu ChenDepartment of Radiology, School of Medicine, College of Medicine, Taipei Medical University, No. 251 Wu Hsing Street, Taipei City 110, Taipei, TaiwanEmail sandy0932@gmail.comPurpose: Targeted superparamagnetic iron oxide (SPIO) nanoparticles are a promising tool for molecular magnetic resonance imaging (MRI) diagnosis. Lipid-coated SPIO nanoparticles have a nonfouling property that can reduce nonspecific binding to off-target cells and prevent agglomeration, making them suitable contrast agents for molecular MRI diagnosis. PD-L1 is a poor prognostic factor for patients with glioblastoma. Most recurrent glioblastomas are temozolomide resistant. Diagnostic probes targeting PD-L1 could facilitate early diagnosis and be used to predict responses to targeted PD-L1 immunotherapy in patients with primary or recurrent glioblastoma. We conjugated lipid-coated SPIO nanoparticles with PD-L1 antibodies to identify PD-L1 expression in glioblastoma or temozolomide-resistant glioblastoma by using MRI.Methods: The synthesized PD-L1 antibody-conjugated SPIO (PDL1-SPIO) nanoparticles were characterized using dynamic light scattering, zeta potential assays, transmission electron microscopy images, Prussian blue assay, in vitro cell affinity assay, and animal MRI analysis.Results: PDL1-SPIO exhibited a specific binding capacity to PD-L1 of the mouse glioblastoma cell line (GL261). The presence and quantity of PDL1-SPIO in temozolomide-resistant glioblastoma cells and tumor tissue were confirmed through Prussian blue staining and in vivo T2* map MRI, respectively.Conclusion: This is the first study to demonstrate that PDL1-SPIO can specifically target temozolomide-resistant glioblastoma with PD-L1 expression in the brain and can be quantified through MRI analysis, thus making it suitable for the diagnosis of PD-L1 expression in temozolomide-resistant glioblastoma in vivo.Keywords: PD-L1, superparamagnetic iron oxide, SPIO, magnetic resonance imaging, MRI, lipid-coated nanoparticle, glioblastoma

RIPK1/RIPK3-MLKL signaling molecules are fundamental in initiating necroptotic cell death, but their roles in the development of colon cancer are unclear. This study reports that RIPK3 interacted with SPOP, a component of the E3 ligase within the Cul3 complex. This interaction leads to K48-linked ubiquitination and subsequent proteasomal degradation of RIPK3. Two distinct degron motifs, PETST and SPTST, were identified within the linker domain of RIPK3 for SPOP. RIPK3 phosphorylations at Thr403 by PIM2 and at Thr412/Ser413 by ERK2 are essential to facilitate its interaction with SPOP. Computational docking studies and immunoprecipitation analyses showed that these PIM2 and ERK2 phosphorylations bolster the stability of the RIPK3-SPOP interaction. In particular, mutations of RIPK3 at the degron motifs extended the half-life of RIPK3 by preventing its phosphorylation and subsequent ubiquitination. The deletion of SPOP, which led to increased stability of the RIPK3 protein, intensified LPS/sMAC/zVAD-induced necroptotic cell death in colon cancer cells. These findings underscore the critical role of the SPOP-mediated RIPK3 stability regulation pathway in controlling necroptotic cell death.

Cyclic guanosine-adenosine monophosphate synthase (cGAS) is a key cytosolic DNA sensor that plays a pivotal role in the innate immune response. Although a decade of research on the cGAS has advanced our understanding of inflammasome formation, cytokine production, and signaling pathways, the role of cGAS in the nucleus remains unclear. In this study, we found that the nuclear localization of endogenous and stably expressed cGAS differed from transiently expressed cGAS, which mainly localized in the cytosol. In the nucleus, cGAS is tightly bound to chromatin DNA. The chromatin DNA binding of cGAS was dependent on RSK2. Our molecular mechanism study indicated that the N-lobe of RSK2 harboring 1-323 interacted with the NTase domain of cGAS harboring residues 213-330. This interaction increased RSK2-induced cGAS phosphorylation at Ser120 and Thr130, resulting in the tightly binding of cGAS to chromatin. Importantly, epidermal growth factor (EGF)-induced cell transformation and anchorage-independent colony growth showed an increase in growth factors, such as EGF or bFGF, in cGAS stable expression compared to mock expression. Notably, the cGAS-S120A/T130A mutant abolished the increasing effect of cell transformation of JB6 Cl41 cells and colony growth of SK-MEL-2 malignant melanoma cells. The results suggested that cGASs chromatin DNA binding, which is indispensable to RSK2-dependent phosphorylation of cGAS at Ser120/Thr130, provides the first clue to how cGAS may participate in chromatin remodeling in the nucleus.

The AKT signaling pathway plays critical roles in the resolution of inflammation. However, the underlying mechanisms of anti-inflammatory regulation and signal coordination remain unclear. Here, we report that anti-inflammatory AKT signaling is coordinated by glutamyl-prolyl-tRNA synthetase 1 (EPRS1). Upon inflammatory activation, AKT specifically phosphorylates Ser999 of EPRS1 in the cytoplasmic multi-tRNA synthetase complex, inducing release of EPRS1. EPRS1 compartmentalizes AKT to early endosomes via selective binding to the endosomal membrane lipid phosphatidylinositol 3-phosphate and assembles an AKT signaling complex specific for anti-inflammatory activity. These events promote AKT activation-mediated GSK3β phosphorylation, which increase anti-inflammatory cytokine production. EPRS1-deficient macrophages do not assemble the early endosomal complex and consequently exacerbate inflammation, decreasing the survival of EPRS1-deficient mice undergoing septic shock and ulcerative colitis. Collectively, our findings show that the housekeeping protein EPRS1 acts as a mediator of inflammatory homeostasis by coordinating compartment-specific AKT signaling.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.