Controlling hyperlipidemia has reduced but not eliminated atherosclerotic cardiovascular disease as a predominant cause of human mortality. Here, we report that loss of the immunoregulatory gene GTPase of immunity-associated protein 6 ( GIMAP6 ), causes an inflammatory vasculopathy and accelerated atherosclerosis in the absence of hyperlipidemia. These pathologic changes in turn result in progressive cardiac ischemia, myocardial infarction, and heart failure, culminating in early death. In humans, rare deleterious GIMAP6 variants are associated with premature severe cardiovascular disease. These findings reveal GIMAP6 to play an important protective role against atherosclerotic cardiovascular disease whose identification offers opportunities for improved risk management and a target for new therapies.

Chimeric antigen receptor T-cell (CAR-T) therapy, a groundbreaking advancement in cancer immunotherapy, has demonstrated remarkable efficacy in treating hematological malignancies and autoimmune diseases. However, conventional ex vivo CAR-T therapy medicinal products face multiple limitations, including complex manufacturing processes, high production costs, and challenges in quality control and risk management. In recent years, the emergence of in vivo CAR-T therapy medicinal products using viral vector or lipid nanoparticle platforms has provided a promising new direction by simplifying manufacturing, enhancing scalability, lowering costs, and increasing accessibility, though it may introduce elevated risks such as off-target effects and immunogenicity. From a regulatory perspective, this article reviews the progress, technological strengths, and regulatory landscape of in vivo CAR-T therapy medicinal products. We analyze their potential benefits in manufacturability, scalability, and discuss challenges including quality control, safety risks, and mitigation strategies in drug development. We propose adaptive regulatory strategies together with early regulatory engagement and international coordination to accelerate the clinical translation and standardized use of this emerging modality.

GPR45 is an orphan G protein-coupled receptor (GPCR) that is widely expressed in the central nervous system. In mice, Gpr45 disruption results in reduced pro-opiomelanocortin (POMC) signaling, decreased energy expenditure, and severe obesity. Meanwhile, GPR45 has been suggested to function downstream of POMC to regulate metabolism and body weight. To better understand the role of GPR45 in energy homeostasis, this study aimed to investigate its function directly in POMC neurons. A Gpr45-EYFP knock-in allele and a polyclonal antibody against GPR45 were created to follow endogenous GPR45 expression. Additionally, a Gpr45flox allele was crossed with tissue-specific Cre recombinase to delete Gpr45 in POMC neurons (Gpr45POMC KO). GPR45-immunoreactive cells accounted for approximately 57.1

Inflammatory bowel disease (IBD) causes chronic suffering from gastrointestinal inflammation and dysfunction that can progress to colon cancer1,2. The prevalence of the disease is increasing, and there is an urgent need to better understand its pathogenic mechanisms to improve treatment. We show that GPR15-a G-protein-coupled receptor expressed in immune cells and described previously as an entry co-factor for human and simian immunodeficiency viruses3-is a marker and homing receptor for a subset of intramucosal GPR15-guided regulatory CD8+ T lymphocytes (CD8+ TIGR cells). Deleterious GPR15 gene variants in humans cause defective homing of CD8+ TIGR cells and are associated with severe early-onset IBD. Moreover, CD8+ TIGR cells are reduced in the intestinal mucosa of individuals with sporadic IBD. In mice, GPR15 deficiency impairs colonic homing of CD8+ TIGR cells, leading to accumulation of inflammatory macrophages and increased susceptibility to colitis. CD8+ TIGR cells potently kill macrophages activated by intestinal damage or disease using Fas ligand and TNF-related weak inducer of apoptosis (TWEAK). The identification of CD8+ TIGR cells yields new insights into organ-specific immune regulation and potential therapeutics for IBD.

Inborn errors of immunity (IEIs) are rare genetic anomalies that cause defective immune function. Over 500 IEIs have been identified to date, affecting millions of patients globally. These IEIs reveal the complex interplay between genetics, the environment and microorganisms that determine immune disease phenotypes. Progress in understanding the molecular and cellular mechanisms of IEIs provides a genetic framework for a functional understanding of the human immune system, disease pathogenesis and successful therapeutic interventions. This Review describes how IEIs impact infectious diseases, particularly coronavirus disease 2019, inflammatory bowel disease and cancer. Cui et al. discuss new molecular and cellular insights underlying the pathogenesis of rare human diseases that arise from genetic inborn errors of immunity.

OBJECTIVE:Ephrin type-B receptor 1 (EphB1) is a receptor tyrosine kinase involved in axon guidance, synaptic plasticity, and tumorigenesis. However, the role of EphB1 in metabolic regulation and obesity remains poorly understood. This study aims to uncover the role of EphB1 in energy metabolism and provide insights into the underlying mechanisms by which EphB1 regulates obesity. METHODS:Two Ephb1 mutations identified from a forward genetic screen for obesity-related loci in mice were examined for their effects in gene expression, energy metabolism, and endocrine changes. The impacts of EphB1 on neuropeptide expression and signal transduction were evaluated in both hypothalamic tissues and primary cells. Potential downstream signals were modified in Ephb1 mutants to verify the interaction. RESULTS:Ephb1 mutants develop obesity in adolescence and develop impaired glucose tolerance during adulthood. EphB1 deficiency caused lower body temperature, blunted cold-induced thermogenesis, and decreased locomotor activity, but it did not alter food intake. EphB1 promotes cyclic AMP-responsive element-binding protein (CREB) phosphorylation via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling in a cell-autonomous manner. EphB1 deficiency leads to reduced expression of corticotropin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH) in the brain. Intraventricular administration of either TRH or a CRH fragment suppressed obesity in Ephb1 mutants. CONCLUSIONS:EphB1 regulates hypothalamic CRH and TRH expression and promotes energy expenditure in mice.

In the original publication [...].

Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100 - 300 kHz) and low intensity (1 - 3 V/cm) regime - termed "Tumor Treating Fields" (TTFields) - have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro , we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0 - 6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines - U87, U118, GSC827, and GSC923 - for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0 - 3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.

Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

Cardiovascular disease (CVD) is the most common cause of death, environmental factors, such as arsenic, playing an important role in the progress of CVD. Vascular endothelial dysfunction (VED) is a crucial early feature for CVD, inorganic arsenic (iAs) can induce autophagy in various cells. However, the role of endothelial autophagy has rarely been studied in VED triggered by arsenic. Total of one hundred and twenty healthy male C57BL/6J mice weighing 18-22 g were randomly divided into an arsenic-exposure group and a control group for 3, 6, 9, and 12 weeks. The results showed that, independent of the exposure period, autophagy markers of p-ATG16L1 levels and Beclin 1 contents in the aortic arch endothelium increased significantly compared with those of the corresponding control group. And different exposure duration decreased NO contents in the serum significantly. Combined with the histological changes that endothelial injury aggravated gradually with the increasing exposure period, suggesting that under exposure to iAs over 9 weeks, VED was remarkably induced, and consistant high levels of endothelial autophagy may play an important role. Additionally, levels of p-AMPK alpha/AMPK alpha increased significantly and p-mTORC1/mTORC1 levels decreased remarkably in the aortic arch endothelium. Then, a NaAsO2-induced-VED in vitro model was used to explore the mechanism of arsenic-induced endothelial autophagy. Similarly, p-AMPK alpha/AMPK alpha level significantly increased, and p-mTORC1/mTORC1 level remarkably decreased induced by 30 mu mol/L NaAsO2 in HUAECs. Further, an AMPK inhibitor (Compound C) pre-treatment prior to arsenic exposure reversed the increased autophagy level, and alleviated the endothelial dysfunction in HUVECs, as shown by the significant increase in the intracellular NO content and the cell vitality. Mechanistically, we revealed that AMPK alpha is active in autophagy of endothelial cells in arsenic-induced VED by regulating mTORC1/p70S6K/ULK1. The present study provide a new promising target for prevention and control arsenicassociated CVD.

G Protein-Coupled Receptor 15 (GPR15) is a chemokine receptor, primarily expressed in T cells based on human single-cell RNA sequencing data. Its natural ligand, GPR15L, is found in the gastrointestinal basal epithelial region and presumably guide GPR15+ T cells to that location. Previous studies showed that Gpr15−/− mice were more susceptible to C. rodentium-induced colitis but the function of GPR15 and whether its deficiency plays a role in inflammatory bowel disease (IBD) in humans are unclear. We recruited three pediatric patients who developed severe early onset IBD and found novel GPR15 mutations using whole-exome sequencing. A reduction in T cells was observed in patient colon biopsies. Surface staining of patient T cells after TCR stimulation further revealed low expression of the mutant GPR15 alleles compared to the wildtype. Characterizing the GPR15 mutants in transduced stable cell lines, we found that the mutations led to lower GPR15L-dependent signaling, causing decreased cell chemotaxis towards the ligand. Accordingly, Gpr15−/− mouse colon also exhibited reduced T cell phenotype observed in patients. Our results indicate that GPR15 is critical for T cell and migration to the human colon and its absence may lead to immunodeficiency in the region, predisposing individuals to IBD induced by specific pathogenic microbiota. This research was supported NIAID by the Intramural Research Program of the NIH. This research was supported NIAID by the Intramural Research Program of the NIH.

Background Compared to normal cells, cancer cells exhibit a higher level of oxidative stress, which primes key cellular and metabolic pathways and thereby increases their resilience under oxidative stress. This higher level of oxidative stress also can be exploited to kill tumor cells while leaving normal cells intact. In this study we have found that isovalerylspiramycin I (ISP I), a novel macrolide antibiotic, suppresses cancer cell growth and tumor metastases by targeting the nucleolar protein selenoprotein H (SELH), which plays critical roles in keeping redox homeostasis and genome stability in cancer cells. Methods We developed ISP I through genetic recombination and tested the antitumor effects using primary and metastatic cancer models. The drug target was identified using the drug affinity responsive target stability (DARTS) and mass spectrum assays. The effects of ISP I were assessed for reactive oxygen species (ROS) generation, DNA damage, R-loop formation and its impact on the JNK2/TIF-IA/RNA polymerase I (POLI) transcription pathway. Results ISP I suppresses cancer cell growth and tumor metastases by targeting SELH. Suppression of SELH induces accumulation of ROS and cancer cell-specific genomic instability. The accumulation of ROS in the nucleolus triggers nucleolar stress and blocks ribosomal RNA transcription via the JNK2/TIF-IA/POLI pathway, causing cell cycle arrest and apoptosis in cancer cells. Conclusions We demonstrated that ISP I links cancer cell vulnerability to oxidative stress and RNA biogenesis by targeting SELH. This suggests a potential new cancer treatment paradigm, in which the primary therapeutic agent has minimal side-effects and hence may be useful for long-term cancer chemoprevention.

Paclitaxel-based chemotherapy is a treatment option for advanced esophageal squamous cell carcinoma (ESCC). However, the development of chemoresistance leads to treatment failure, and the underlying mechanism remains elusive. We investigated the mechanisms of nanoparticle albumin-bound paclitaxel (nab-PTX) resistance by establishing three nab-PTX resistant ESCC cell lines. Proteomics analysis revealed higher oxidative phosphorylation (OXPHOS) in resistant cell line DR150 than in its parental cell line KYSE150, which is likely caused by stabilized anti-apoptotic protein MCL1. Additionally, we discovered the elevated activity of protein phosphatase 2A (PP2A), the phosphatase that dephosphorylates and stabilizes MCL1, in nab-PTX resistant cell lines. Pharmacological inhibition of PP2A with small molecule compound LB-100 decreased MCL1 protein level, caused more apoptosis in nab-PTX resistant ESCC cell lines than in the parental cells in vitro, and significantly inhibited the tumor growth of nab-PTX resistant xenografts in vivo. Moreover, LB-100 pretreatment partially restored nab-PTX sensitivity in the resistant cell lines and synergistically inhibited the tumor growth of nab-PTX resistant xenografts with nab-PTX. In summary, our study identifies a novel mechanism whereby elevated PP2A activity stabilizes MCL1 protein, increases OXPHOS, and confers nab-PTX resistance, suggesting that targeting PP2A is a potential strategy for reversing nab-PTX resistance in patients with advanced ESCC.

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

Emerging evidence is demonstrating the extent of T-cell infiltration within the tumor microenvironment has favorable prognostic and therapeutic implications. Hence, immunotherapeutic strategies that augment the T-cell signature of tumors hold promising therapeutic potential. Recently, immunotherapy based on intratumoral injection of mannan-BAM, toll-like receptor ligands and anti-CD40 antibody (MBTA) demonstrated promising potential to modulate the immune phenotype of injected tumors. The strategy promotes the phagocytosis of tumor cells to facilitate the recognition of tumor antigens and induce a tumor-specific adaptive immune response. Using a syngeneic colon carcinoma model, MBTA's potential to augment CD8(+) T-cell tumor infiltrate when administered intratumorally or subcutaneously is demonstrated as part of a whole tumor cell vaccine. Both immunotherapeutic strategies prove effective at controlling tumor growth, prolong survival, and induce immunological memory against the parental cell line. Collectively, the investigation demonstrates MBTA's potential to trigger a potent anti-tumor immune response.

Purpose Glioblastoma (GBM) carries a dismal prognosis despite standard multimodal treatment with surgery, chemotherapy and radiation. Immune checkpoint inhibitors, such as PD1 blockade, for treatment of GBM failed to show clinical benefit. Rational combination strategies to overcome resistance of GBM to checkpoint monotherapy are needed to extend the promise of immunotherapy to GBM management. Emerging evidence suggests that protein phosphatase 2A (PP2A) plays a critical role in the signal transduction pathways of both adaptive and innate immune cells and that inhibition of PP2A could enhance cancer immunity. We investigated the use of a PP2A inhibitor, LB-100, to enhance antitumor efficacy of PD1 blockade in a syngeneic glioma model. Methods C57BL/6 mice were implanted with murine glioma cell line GL261-luc or GL261-WT and randomized into 4 treatment arms: (i) control, (ii) LB-100, (iii) PD1 blockade and (iv) combination. Survival was assessed and detailed profiling of tumor infiltrating leukocytes was performed. Results Dual PP2A and PD1 blockade significantly improved survival compared with monotherapy alone. Combination therapy resulted in complete regression of tumors in about 25% of mice. This effect was dependent on CD4 and CD8 T cells and cured mice established antigen-specific secondary protective immunity. Analysis of tumor lymphocytes demonstrated enhanced CD8 infiltration and effector function. Conclusion This is the first preclinical investigation of the effect of combining PP2A inhibition with PD1 blockade for GBM. This novel combination provided effective tumor immunotherapy and long-term survival in our animal GBM model.

You have accessJournal of UrologyKidney Cancer: Advanced (including Drug Therapy) I (MP14)1 Apr 2020MP14-19 A NOVEL ANTI-EPOR CAR-T CELL FOR THE TREATMENT OF CLEAR CELL RENAL CELL CARCINOMA Jingcheng Zhou*, Haibiao Xie, Kaifang Ma, Lei Li, Kenan Zhang, Kan Gong, and Lin Cai Jingcheng Zhou*Jingcheng Zhou* More articles by this author , Haibiao XieHaibiao Xie More articles by this author , Kaifang MaKaifang Ma More articles by this author , Lei LiLei Li More articles by this author , Kenan ZhangKenan Zhang More articles by this author , Kan GongKan Gong More articles by this author , and Lin CaiLin Cai More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000839.019AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chimeric antigen receptor (CAR) T cell is a promising treatment approach for renal cell carcinoma (RCC) but has been limited by the specificity of antigens. Here, we demonstrate that erythropoietin receptor (EPOR) is a tumor-associated antigen highly expressed in RCC. We developed a novel anti-EPOR CAR-T cell consisting of scFv against EPOR which demonstrates good efficacy in vitro and in vivo. METHODS: First, we illustrated the expression of EPOR in patient derived RCC tumor specimens, as well as various renal cell cancer cell lines. Then, we constructed the EPOR antigen and immunize mice to obtain monoclonal antibody from hybridoma cells. Validated monoclonal cell line with the strongest RCC cell killing ability was performed 5’RACE sequencing to get the scFv sequence, which was subsequently combined with 3rd generation CAR vector. CAR-T cells were generated against EPOR and efficacy was evaluated in RCC cell lines under normoxia, hypoxia and EPO stimulated condition. Cytotoxicity was determined via LDH, IFN-γ, TNF-α, and IL-2 levels released from tumor cells killed by anti-EPOR CAR-T cells. Finally, we evaluated the in vivo therapeutic effect with intravenous injection on xenograft mouse model using 786-0 cell line. RESULTS: EPOR was highly expressed in RCC cell lines and patient tumor samples. We demonstrated that anti-EPOR monoclonal antibodies and CAR-T cells have the ability to kill RCC cells in vitro. While the xenograft tumor volume in anti-EPOR CAR-T group being significantly suppressed after intravenous injection 2 weeks in vivo, the mice shown side effects (e.g., pulmonary consolidation, congestive hepatomegaly) probably due to the EPOR expression in specific organs. CONCLUSIONS: We developed a type of novel CAR-T cells targeting EPOR which shown cytotoxicity against RCC cells in vitro and in vivo. This data indicates that anti-EPOR CAR-T is promising to be a therapeutic target. However, considering the side effects, new CAR-T strategies should be used to optimize it in the future. Source of Funding: This work was supported by the National Natural Science Foundation of China (grant 81572506 and grant 81872081), the Fundamental Research Funds for the Central Universities (grant BMU2018JI002) and the NCI Intramural Research Program. The first author acknowledges a scholarship (201806010388) funded by the China Scholarship Council. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e203-e203 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jingcheng Zhou* More articles by this author Haibiao Xie More articles by this author Kaifang Ma More articles by this author Lei Li More articles by this author Kenan Zhang More articles by this author Kan Gong More articles by this author Lin Cai More articles by this author Expand All Advertisement PDF downloadLoading ...

Importance:Patients with the EPAS1 gain-of-function mutation syndrome (or Pacak-Zhuang syndrome) present with multiple paragangliomas or pheochromocytomas, duodenal somatostatinoma, polycythemia, headaches, and sometimes diminished visual acuity at an early age. The characteristic phenotype and known genetic cause of the syndrome provide an opportunity to study the role of hypoxia-inducible factor 2α (HIF-2α) in oxygen sensing, development in regions of physiologic hypoxia, and other pathological processes.Objectives:To describe the ocular lesions in EPAS1 gain-of-function mutation syndrome and to establish whether early-onset diminished visual acuity is developmental or associated with long-term physiologic sequelae of the syndrome.Design, Setting, and Participants:This clinical case series with a transgenic murine model study was conducted from July 2013 to June 2019. Participants were 3 patients referred by their primary care physicians to the National Institutes of Health for evaluation of recurrent and metastatic paragangliomas or pheochromocytomas accompanied by polycythemia. The syndrome and somatic mosaicism in patients were confirmed by the identification of gain-of-function mutations in the EPAS1 gene in resected tumors and other tissues.Main Outcomes and Measures:Ocular findings in patients with EPAS1 gain-of-function mutation syndrome.Results:A total of 3 patients (mean [SD] age, 29 [6.2] years) with confirmed ocular abnormalities were included in the study. Increased contrast accumulation at the posterior aspect of the globe was seen bilaterally on magnetic resonance imaging scans in all patients. Ophthalmoscopy images demonstrated fibrosis overlying the optic disc, tortuous and dilated retinal vessels, and retinal pigment epithelium changes. Optic disc edema and retinal exudates were also seen. Fluorescein angiography images showed leakage of dye from postcapillary venules surrounding the optic disc and highlighted aberrant retinal vascular patterns. Enhanced-depth imaging optical coherence tomography images showed substantial thickening of the choroid and dilation of choroidal vessels. The ocular features of the syndrome were confirmed with a transgenic model of mice with gain-of-function Epas1A529V mutation.Conclusions and Relevance:In this case series, HIF-2α and hypoxia signaling was found to have a role in vessel development within the choroid and retina, indicating that the marked permanent choroidal thickening and tortuous and dilated veins seen in the choroid and retina in patients with EPAS1 gain-of-function mutation syndrome were suggestive of the persistence of venous elements within the developing mesenchyme. These findings may explain other eye and vascular abnormalities whose pathogenesis remains unclear.

We examined the efficacy of selective inhibition of cyclin-dependent kinase 5 (CDK5) in glioblastoma by TP5. We analyzed its impact in vitro on CDK5 expression and activity, cell survival, apoptosis and cell cycle. DNA damage was analyzed using the expression of γH2A.X and phosphorylated ATM. Its tolerance and efficacy were assessed on in vivo xenograft mouse models. We showed that TP5 decreased the activity but not the expression of CDK5 and p35. TP5 alone impaired cell viability and colony formation of glioblastoma cell lines and induced apoptosis. TP5 increased DNA damage by inhibiting the phosphorylation of ATM, leading to G1 arrest. Whereas CDK5 activity is increased by DNA-damaging agents such as temozolomide and irradiation, TP5 was synergistic with either temozolomide or irradiation due to an accumulation of DNA damage. Concomitant use of TP5 and either temozolomide or irradiation reduced the phosphorylation of ATM, increased DNA damage, and inhibited the G2/M arrest induced by temozolomide or irradiation. TP5 alone suppressed the tumor growth of orthotopic glioblastoma mouse model. The treatment was well tolerated. Finally, alone or in association with irradiation or temozolomide, TP5 prolonged mouse survival. TP5 alone or in association with temozolomide and radiotherapy is a promising therapeutic option for glioblastoma.