Effects Of Inhibitors Research Articles

Thrombospondin 1 Mediates Autophagy Upon Inhibition of the Rho-Associated Protein Kinase Inhibitor

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Abstract B022: Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer (BC) cases worldwide, yet due to its aggressive phenotype and lack of targeted therapies it has the worst prognosis amongst BC subtypes. Up to 40% of TNBC patients will relapse after treatment and develop distant metastasis. Metastasis is the complex process by which disseminated tumor cells (DTCs) leave the primary tumor and grow at distal sites. Since most metastases occur within 3 years of diagnosis, the rapid onset and lack of effective treatments in metastatic TNBC (mTNBC) means less than 20% of patients survive after 4 years. Even though it is the most “immune-activated” BC subtype, and immune checkpoint blockade (ICB) has shown clinical benefits in early TNBC, there is a clear need for novel immunotherapeutic approaches for patients with mTNBC. Natural killer (NK) cells are innate lymphocytes that function as the body’s first line defense against metastatic cancer cells. NK cells eliminate DTCs in the blood or at distal sites via NK cell cytotoxicity (NKCC) and this process is known to be impaired in successful metastasis. We hypothesized that other cells within the tumor microenvironment (TME) can inhibit NKCC at the earliest stages of metastatic outgrowth. We have used Cherry-niche, an in vivo labelling tool, to characterize how TME cells change in early and established lung micro-metastases by single-cell RNA sequencing (scRNA-seq) and flow cytometry. Our initial analyses showed that monocytes are highly enriched, not only in the entire metastatic lung, but specifically in the established niche surrounding DTCs. Although we detected a slight increase in total NK cells, there was a shift in maturation status with fewer mature NK cells (CD27-CD11b+) and more immature NK cells (CD27+/-CD11b-) in the metastatic lung. Furthermore, NK cells were largely absent from the metastatic niche and those that were present displayed a dysfunctional phenotype. CellChat and NicheNet analyses of our scRNA-seq data predicted that several receptor-ligand interactions, including the cytokine macrophage migration inhibitory factor (MIF), can mediate the crosstalk between NK cells and monocytes. Next, we developed an in vitro 3D co-culture system to study the effect of blood-derived monocytes on human NKCC against mTNBC cells. We found that monocytes can suppress NKCC against MDA-MB-231 cells and decrease the viability of mature NK cells. Furthermore, recombinant MIF was able to directly inhibit NKCC, whilst a selective MIF antagonist was able to rescue monocyte-induced impairment of NKCC. These data implicate MIF as a critical monocyte-derived factor in the suppression of NK cell function against mTNBC cells. Future work will aim to identify the exact mechanism by which MIF inhibits NKCC, whilst also evaluate the effect of monocyte depletion and MIF inhibition on metastatic outgrowth and NK cell activation/maturation in vivo. This study will reveal novel inter-cellular dynamics occurring in the metastatic lung, as well as immunotherapeutic targets that may restore NKCC in mTNBC. Citation Format: Christos Ermogenous, Luigi Ombrato, Gordon Beattie. Investigating how monocytes impair NK cell cytotoxicity in lung metastasis of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B022.

Inhibition of PCSK9 Protects against Cerebral Ischemia‒Reperfusion Injury via Attenuating Microcirculatory Dysfunction.

Proprotein convertase substilin/kexin type 9 (PCSK9), a pivotal protein regulating lipid metabolism, has been implicated in promoting microthrombotic formation and inflammatory cascades, thereby contributing to cardiovascular ischemia/reperfusion (I/R) injury. However, its involvement in cerebral I/R injury and its potential role in microcirculation protection remain unexplored. In this investigation, we utilized a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to simulate ischemic stroke. Different concentrations of evolocumab (1, 5, 10mg/kg, i.v.), a PCSK9 inhibitor, were administered to assess its impact. Immunofluorescence staining was employed to analyze changes in the expression of occludin, claudin-5, thrombocyte, ICAM-1, VCAM-1, and CD45, providing insights into blood-brain barrier integrity, platelet adhesion, and immune cell infiltration. Moreover, the Morris water maze and elevated plus maze were utilized to evaluate neurological and behavioral functions in MCAO/R mice, shedding light on the effects of PCSK9 inhibition. Our findings revealed a surge in plasma PCSK9 levels post-MCAO/R, peaking at 24h post-reperfusion. Evolocumab (10mg/kg) treatment significantly mitigated brain infarction and neurological deficits, evidenced by enhanced locomotor function and reduced post-stroke anxiety. However, it did not ameliorate cognitive impairment following MCAO/R. Additionally, evolocumab administration led to diminished leakage of evans blue solution and upregulated expression of occludin and claudin-5. Thrombocyte, ICAM-1, VCAM-1, and CD45 levels were notably reduced in the penumbral area post-evolocumab treatment. These protective effects are speculated to be mediated through the inhibition of the ERK/NF-κB pathway. The PCSK9 inhibitor evolocumab holds promise as a therapeutic agent during the acute phase of stroke, exerting its beneficial effects by modulating the ERK/NF-κB signaling pathway.

Clinical and Immune Effects of Peri-Transplantation JAK Inhibition for Myelofibrosis.

Despite the introduction of JAK inhibitors, allogeneic hematopoietic cell transplant remains the only potentially curative treatment for patients with myelofibrosis but has considerable treatment-related complications. Whether the incorporation of JAK inhibition into the transplant algorithm leads to improved outcomes is still unclear. Here, we analyzed different transplant platforms in myelofibrosis patients undergoing a first transplant, comparing immune profiles and outcomes of (1) 33 patients continuing JAK inhibition at start of conditioning until stable engraftment (PERI-group), (2) 38 patients receiving JAK inhibition prior to transplant until start of conditioning (PRE-group), and (3) 38 patients that had never received JAK inhibition (NON-group). Patients in the PERI-group showed significantly higher early B-cell recovery as well as significantly increased late recovery of gamma-delta T cells and NK cells. We observed excellent neutrophil and platelet engraftment (100% for both) in the PERI-group and no hematotoxic effects or increased rates of infections following peri-transplant JAK inhibition. Cumulative incidence of acute graft-versus-host disease (GvHD) grade II-IV at day 100 after transplant was 15% in the PERI-group versus 29% in the PRE-group versus 34% in the NON-group. Cumulative incidence of relapse at 1 year after transplant was 9% in the PERI-group compared with 16% in the PRE-group and 18% in the NON-group. In conclusion, peri-transplant JAK inhibition was feasible with promising engraftment and acute GvHD rates, though deserves further investigation.

GRK2 mediates cisplatin-induced acute liver injury via the modulation of NOX4.

The present study investigated the function of G protein-coupled receptor kinase 2 (GRK2) in acute liver injury (ALI) by cisplatin, and investigated the protective effect of pharmacological inhibition of GRK2. ALI models were generated in global adult hemizygous (ALI-Grk2±) mice and wild-type (WT) mice. Liver biochemistry parameters and histopathology were used to evaluate the severity of ALI and the protective effect of pharmacological inhibition of GRK2. GRK2-siRNA was used to knock down the expression of GRK2 in AML12 cells in vitro. ALI model mice exhibited increased blood levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and abnormal liver pathology accompanied by imbalanced L-glutathione (GSH) levels. Cisplatin administration upregulated GKR2, p-GRK2 and NADPH oxidase 4 (NOX4) expression in the liver tissues of ALI model mice. Compared to WT mice injected with cisplatin, Grk2± mice that received cisplatin showed significant improvements in liver function and pathological performance, decreased NOX4 levels, reduced endoplasmic reticulum (ER) stress, and diminished liver cell apoptosis. In vitro, the transfection of AML12 cells with siRNA significantly reduced NOX4 expression and inhibited cisplatin-induced reactive oxygen species production, ER stress (increased levels of GRP94, GRP78, p-elF2α and CHOP) and apoptotic death. Moreover, pharmacological treatment with drugs that inhibit GRK2 (CP-25 or paroxetine) significantly ameliorated cisplatin-induced ALI by improving liver pathological manifestations, inhibiting oxidative stress and ER stress, and reducing liver cell apoptosis. Similar results were observed in vitro. GRK2 mediates the development of cisplatin-induced ALI by modulating NOX4 and ER stress. Pharmacological inhibition of GRK2 with CP-25 or paroxetine effectively alleviated ALI. GRK2 can be used as a potential target for the prevention and treatment of liver injury.

Aedes aegypti control in breeding sites through an insecticidal coating with dual effect: Laboratory trials and safety assessment.

Ground water tanks are known to be preferred Aedes aegypti oviposition places providing opportunities for adult and larvae control. Therefore, a dual-effect insecticidal coating (IC) (alphacypermethrin/ pyriproxyfen) with a slow-release mechanism and safe for users could be applied within Aedes spp. breeding sites, representing a promising option. Bioassays were designed to determine the mortality and sterilizing effects on gravid mosquitoes exposed to IC. The effect of inhibition of emergence was evaluated in eggs, larvae and pupae exposed in different containers. For the water safety assessment concentrations of active ingredients were determined by reverse phase high performance liquid chromatography (RP-HPLC) and the health risk was calculated. The IC applied to the interior walls of water-holding containers showed efficacy against Ae. aegypti in terms of high gravid-female mortality (81% at 24 h, p < 0.01), sterilizing effect (inhibition of oviposition by 63%, p < 0.01) and emergence inhibition (100% in eggs, L3 and L4; 97% in pupae). The offspring rate was reduced [only 0.15 (38/250) new adults emerged per exposed gravid females as against 11.90 per unexposed female (2976/250) at baseline]. Emergence inhibition was recorded up to 12 months and adult mortality >80% up to 6 months. The use of water stored in treated containers, either for washing or drinking, is not expected to pose a health risk to users. IC applied to domestic water containers has dual and complementary action that reduces Ae. aegypti densities (immature and adult stages). This represents baseline information for a cluster randomized efficacy trial in Colombia.

Normothermic Machine Perfusion Reconstitutes Porcine Kidney Tissue Metabolism But Induces an Inflammatory Response, Which Is Reduced by Complement C5 Inhibition

Normothermic machine perfusion (NMP) is a clinical strategy to reduce renal ischemia-reperfusion injury (IRI). Optimal NMP should restore metabolism and minimize IRI induced inflammatory responses. Microdialysis was used to evaluate renal metabolism. This study aimed to assess the effect of complement inhibition on NMP induced inflammatory responses. Twenty-two pig kidneys underwent 18 h of static cold storage (SCS) followed by 4 h of NMP using a closed-circuit system. Kidneys were randomized to receive a C5-inhibitor or placebo during SCS and NMP. Perfusion resulted in rapidly stabilized renal flow, low renal resistance, and urine production. During SCS, tissue microdialysate levels of glucose and pyruvate decreased significantly, whereas glycerol increased (p &amp;lt; 0.001). In the first hour of NMP, glucose and pyruvate increased while glycerol decreased (p &amp;lt; 0.001). After 4 h, all metabolites had returned to baseline. Inflammatory markers C3a, soluble C5b-9, TNF, IL-6, IL-1β, IL-8, and IL-10 increased significantly during NMP in perfusate and kidney tissue. C5-inhibition significantly decreased perfusate and urine soluble C5b-9 (p &amp;lt; 0.001; p = 0.002, respectively), and tissue IL-1β (p = 0.049), but did not alter other inflammatory markers. Microdialysis can accurately monitor the effect of NMP on renal metabolism. Closed-circuit NMP induces inflammation, which appeared partly complement-mediated. Targeting additional immune inhibitors should be the next step.

Abstract 4138799: SGK1 inhibition shortens action potential duration in LQT3 mice and LQT2 rabbits via inhibition of late sodium current

Background: Serum and glucocorticoid regulated kinase 1 (SGK1) activation during pathological conditions has been shown to increase late sodium current, prolong action potential duration (APD), and induce ventricular arrhythmias, thus recapitulating a long QT syndrome (LQTS) phenotype. Hence, pharmacological SGK1 inhibition may be a novel therapeutic approach in inherited LQTS. Aim: We aimed to investigate potential effects of SGK1 inhibition in animal models of different LQTS subtypes, and the potential involvement of late I Na inhibition. Methods: Cardiomyocytes (CMs) isolated from wild-type (WT) and LQT3 mice ( Scn5a -1798insD) as well as from WT, LQT1 ( KCNQ1 -Y315S) and LQT2 ( KCNH2 -G628S) rabbits, were incubated for 3-5 hours with SGK1-inhibitor (SGK1-inh, 300 nM-3 μM) or DMSO (vehicle). Patch-clamp experiments were performed to assess AP properties, short-term variability (STV) of APD and late I Na . Result: SGK1-inh incubation significantly shortened the prolonged APD 90 in LQT3 CMs by 23% and in LQT2 CMs by 25% (restoring them towards the WT level), without affecting other AP properties. In contrast, in WT and LQT1 CMs, no effect of SGK1-inh on APD 90 was observed. Furthermore, short-term-variability (STV) of APD, a measure of pro-arrhythmia, was pathologically increased in LQT3 (5.6±0.2 ms vs WT 2.7±0.3 ms, p&lt;0.05) and LQT2 (14.7±2.8 ms vs WT 5.9 ±1.1 ms, p &lt; 0.0001), but not in LQT1. SGK1-Inh markedly reduced STV in LQT3 (to 2.9±0.1 ms) and LQT2 CMs (to 7.2± 1.2 ms), but had no effect in WT and LQT1 CMs. While enhanced late I Na was previously demonstrated in LQT3 CMs, we now also observed increased late I Na in LQT2 CMs as compared to WT (-0.14 ± 0.02 pA/pF vs WT -0.08 ± 0.02 pA/pF, p &lt; 0.005), but not in LQT1 CMs. SGK1-Inh significantly reduced late I Na in LQT3 (by 32%) and LQT2 CMs (by 43%), but not in LQT1 CMs, confirming that SGK1-inh directly targets late I Na . Conclusion: SGK1-inhibition exerts a beneficial APD-shortening and anti-arrhythmic effect in LQT3 and LQT2 by reducing the enhanced late I Na associated with these LQTS genetic subtypes. These findings furthermore underscore the contribution of increased late I Na to the LQT2 phenotype.

Abstract 4137996: Effects of Empagliflozin on diuresis in heart failure: Results from the CINNAMON-study and in-vivo experiments

Background and Purpose: Heart failure is associated with renal dysfunction suggesting a pathophysiological link between heart and kidney. Empagliflozin, a SGLT2 inhibitor, showed beneficial effects on both cardiovascular and renal endpoints. However, mechanistically, it is unclear if empagliflozin-dependent kidney protection is mediated via inhibition of tubular SGLT2 or more indirectly via improved cardiac function. We hypothesized that Empagliflozin treatment improves left ventricular ejection fraction (LVEF) independent of diuretic effect of renal SGLT2 inhibition. Methods: We evaluated NTproBNP, hematocrit, hemoglobin and bodyweight in patients with HF with reduced (n=32) and preserved ejection fraction (n=59) after 30 and 180 days (prospective, single-arm CINNAMON-study, DRKS00031101). Furthermore, we conducted transverse aortic constriction (TAC) or sham surgery in C57BL/6J (wildtype, WT) and SGLT2 deficient mice (SGLT2 -/- ). Animals received either Empagliflozin (10 mg/kg bodyweight) or vehicle by daily oral gavage. Water intake and output was evaluated by metabolic cages at 10 weeks and cardiac function by echocardiography. Results: Empagliflozin treatment reduced NT-proBNP levels and increased hematocrit and hemoglobin levels especially in patients with reduced ejection fraction (Fig. A-C). Surprisingly, there was no change in body weight (Fig. D), which would be expected if the diuretic effects were dominant. In mice after 10 weeks, echocardiography confirmed TAC induced pressure-overload, leading to reduced LVEF. Empagliflozin attenuated changes in LVEF (Fig. G) and improved diastolic dysfunction (isovolumetric relaxation time, data not shown). To test if direct inhibition of SGLT2 in the heart and kidney is mechanistically involved, TAC surgery was repeated in SGLT2-deficient mice (SGLT2-KO). In fact, exposure to TAC resulted in comparable reduction of LVEF in SGLT2-KO and Empagliflozin prevented this deterioration similar to WT mice (Fig. G vs. I). Surprisingly, Empagliflozin did not increase drinking and urine volume neither in wildtype nor in SGLT2 deficient mice, suggesting a mechanism of cardioprotection independent of diuretic effects. Conclusion and Outlook: This is the first study investigating the role of SGLT2 in Empagliflozin treated patients and mice with heart failure. Importantly, empagliflozin treatment prevented deterioration of LVEF independent of the presence of SGLT2 and diuresis.

CSIG-30. EXPLORING THE ROLE OF CAPICUA (CIC) IN RESISTANCE MECHANISMS OF MEK INHIBITORS IN GLIOBLASTOMA

Abstract Glioblastoma multiforme (GBM) is the most common and fatal primary brain tumor, resistant to conventional therapy. Despite its heterogeneous nature, a key feature of GBM is aberrant kinase signaling. Specifically, hyperactivation of receptor tyrosine kinases (RTK) and their downstream tumorigenic effectors Ras/MEK/Extracellular Signal Regulated Kinase (ERK) pathway are attributed to the aggressive nature of GBM. Capicua (CIC) is a High Mobility Group (HMG) box transcriptional repressor that directly binds to DNA and counteracts the transcription of genes typically expressed only in response to RTK activation such as oncogenic transcription factors ETV1/4/5. Hyperactive MEK/ERK signaling in GBM causes CIC degradation. The mechanism of CIC’s repressor function is not well understood, however, transcriptional repressors (like CIC) typically form co-repressor complexes to repress target genes. We discovered a novel interaction between CIC and the transcriptional regulator yin yang 1 (YY1), a context-dependent transcription factor that can repress or activate gene expression. We uncovered that 90-kDa ribosome S6 protein kinase (p90RSK), a downstream effector of ERK and a known CIC regulator, mediates the CIC/YY1 interaction. Interestingly, we found re-activation of p90RSK following MEK/ERK inhibition implicating p90RSK in the resistance mechanism of RTK/MEK/ERK inhibition in GBM. We hypothesize that p90RSK inhibition will stabilize the CIC/YY1 co-repressor complex and re-sensitize towards MEK/ERK inhibition. Patient-derived glioma stem-like cells (GSCs) were treated with increasing doses of BI-D1870 or LJI308 (p90RSK inhibitors) with and without selumetinib (MEK inhibitor) and various functional assays were performed to examine the effect of p90RSK inhibition on the CIC/YY1 complex and sensitization to MEK/ERK inhibition. Treatment with BI-D1870 and LJI308 markedly reduced viability and mRNA expression of ETV1/4/5 indicating that p90RSK inhibition may mediate CIC /YY1 DNA binding ability. Importantly, inhibiting p90RSK sensitized GSCs to MEK/ERK inhibition. These results highlight the importance of downstream p90RSK inhibition in stabilizing CIC/YY1 repressor complex function and sensitizing cells to RTK/MEK/ERK inhibition.

EXTH-83. CHARACTERIZATION OF SPLICING ABERRATIONS INDUCED BY PRMT5 INHIBITION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) are lethal tumors with limited treatment options, with a high unmet need for novel therapies. PRMT5, an arginine methyltransferase that regulates several histone and non-histone targets, is overexpressed in cancers including GBM and is a promising therapeutic target given its role in promoting oncogenic processes. Here, we characterized the effects of pharmacological inhibition of PRMT5 by the brain-penetrant, orally bioavailable inhibitors, PRT811 and PRT808 on treatment-induced splicing aberrations using a panel of patient-derived glioma stem cells (GSC) and organotypic human glioma slice cultures. Changes in protein expression upon PRMT5 inhibition were assessed using reverse phase protein array (RPPA) analysis. Additionally, splicing aberrations due to PRMT5 inhibition in were tested in several GSCs using RNA-seq. We observed a potent reduction of symmetrical dimethylation of arginine (SDMA protein marks by both PRMT5 inhibitors across various GSC lines and ex-vivo brain tumor tissue slices. Notably, GSCs with both mutant and wild-type p53 displayed similar sensitivity to PRMT5 inhibition, independent of MTAP status. Moreover, PRMT5 inhibition elicited reduced cell proliferation, altered cell survival pathways, and apoptosis. RNA-seq analysis revealed that splicing alterations affecting numerous oncogenes and regulatory factors, encompassing 42 cancer driver genes, 80 splicing factors, and 42 transcriptional factors across these GSC lines, with specific splicing events observed in nuclear and cytoplasmic subcellular compartments. Additional analysis is ongoing to determine which of the aberrant transcripts are translated to yield tumor specific neoantigens that can be potential targets for tumor specific immune strategies. These results underscore the efficacy of pharmacological PRMT5 inhibition in genetically and epigenetically diverse GSCs, and highlight its potential for developing novel immune and non-immune therapeutic strategies against GBM.

CNSC-31. INVESTIGATING THE THERAPEUTIC POTENTIAL OF STK17A INHIBITION IN GLIOBLASTOMA

Abstract For decades, the survival rate for glioblastoma (GBM) has remained nearly stagnant, with a 5-year survival rate of only 5%. There is an unmet medical need for therapeutic development, because while GBM is the most common primary brain tumor, only four FDA approved drugs have been developed for GBM over the last century. By taking advantage of RNA sequencing, a potential target for GBM has been identified: the kinase STK17A (Serine/Threonine Kinase17A). We confirmed that STK17A is overexpressed in gliomas by analyzing public databases and single-cell RNA sequencing from our own GBM patients, and further associated it with poor patient outcome. Little is known about STK17A, but it has been found to be involved in cell proliferation and tumorigenesis. The role of STK17A in GBM pathophysiology and its therapeutic potential as a drug target remains a critical knowledge gap. To better understand STK17A’s role in GBM pathophysiology, we first knocked down STK17A in GBM cell lines and determined that STK17A had functional roles in proliferation and morphology. Similar conclusions were drawn after removing STK17A in GBM xenograft mouse models. These findings support STK17A as a viable therapeutic target. We then designed and optimized novel STK17A inhibitors for evaluations through kinase enzymatic and biological assays, in vitro proliferation and toxicity assays, and in vivo Drug Metabolism and Pharmacokinetics (DMPK) assays. We then used these potent, selective, and highly brain penetrant STK17A inhibitors to investigate the anti-tumor effects of STK17A inhibition in GBM and demonstrate the efficacy of our novel STK17A inhibitors in vivo. In conclusion, this work seeks to contribute to the field of targeted therapies for GBM by optimizing and examining a novel brain penetrant STK17A inhibitor, initiating studies for the treatment of GBM in vitro and in vivo, and providing proof of efficacy for further studies in GBM patients.

CNSC-25. CONCURRENT IMMUNE CHECKPOINT BLOCKADE ENHANCES THE SURVIVAL BENEFIT OF CLINICALLY RELEVANT MAPK PATHWAY INHIBITORS IN A T CELL-DEPENDENT MANNER

Abstract BACKGROUND Despite the availability of targeted therapies, gliomas carrying the BRAFV600E mutation remain challenging to treat. Combined pharmacologic BRAFV600E and MEK inhibition is clinically used in patients with BRAFV600E mutant gliomas, but primary and acquired resistance is reported in ~70% of high-grade glioma patients. OBJECTIVES To gain mechanistic insights into therapy adaptation and escape for developing combination therapies that enhance clinical effects of combined BRAFV600E inhibition with Dabrafenib and MEK inhibition with Trametinib (BRAFi+MEKi) in high-grade gliomas. APPROACH Patients’ pre- and post-treatment glioma tissues, patient-derived and mouse cell lines, and two novel immunocompetent mouse models for BRAFV600E mutant high-grade gliomas were analyzed for the effect of BRAFi+MEKi using a range of techniques including bulk and single-cell RNA sequencing, immunofluorescence staining, flow cytometry, and mass cytometry. RESULTS We present new data showing that the BRAFi+MEKi combination significantly reduced glioma cell viability while inducing glial differentiation programs, potentially associated with programmed death receptor (PD-1) signaling. This suggests a profound shift in the glioma cell differentiation state that is linked to an inclination toward T cell inhibition. This prompted us to combine BRAFi+MEKi with immune checkpoint inhibitors targeting PD-L1 and CTLA-4, which significantly decreased T cell inhibition. Furthermore, from mouse studies, this combination therapy conferred a survival benefit over single BRAFi+MEKi combination therapy alone. Additionally, BRAFi+MEKi treatment triggered an interferon-gamma response, indicative of cytotoxicity, and enhanced the expression of MHC molecules associated with antigen presentation, only in drug-sensitive but not drug-resistant gliomas. The lack of improved therapeutic efficacy of immune checkpoint blockade in athymic mice, which lack T cells, emphasized the critical role of T cell activity in driving the success of the combination treatment. CLINICAL IMPACT Our preclinical findings highlight the potential of concurrent BRAFi+MEKi treatment and immune checkpoint blockade for overcoming therapy resistance in BRAF mutant high-grade glioma patients.

DDDR-48. EZH2 INHIBITION AS A POTENTIAL THERAPEUTIC AVENUE IN THE TREATMENT OF MENINGIOMA

Abstract INTRODUCTION High grade meningiomas provide challenges to treatment as these neoplasms are more likely to recur, exhibit invasion of brain parenchyma, and result in decreased long term survival. Recent works demonstrate the epigenetic modifier, EZH2, to be increased in higher grade meningiomas, and to correlate with poor survival. The selective targeting of EZH2 has shown promise in other malignancies with overexpression of or gain-of-function mutations in EZH2, leading to promising findings. Here we sought to investigate the effect of EZH2 inhibition on meningioma cell growth and viability, using a panel of in vitro models. METHODS Human immortalized meningioma cell lines, mouse meningioma cell lines and a panel of human primary cell cultures with varying baseline protein expression of NF2, SMARCB1, and EZH2, were selected for investigation. Cells were treated with several selective small molecule inhibitors of EZH2. Cell proliferation, cell cycle and cell death processes were assessed. RNAseq was used to assess transcriptional changes in response to EZH2 inhibition. Western blots were used to assess protein changes in response to EZH2 inhibition. RESULTS EZH2 inhibition significantly impairs meningioma cell proliferation, at varying IC50s, in all cell lines and primary cultures tested, utilizing a panel of EZH2 inhibitors. EZH2 inhibition resulted in significantly decreased colony formation in a high-grade cell line. Furthermore, EZH2 inhibition results in a reduction of Ki67+ cells, induction of cell cycle arrest, and apoptosis. Similar grouping of differential gene expression in immortalized cell lines and primary cell cultures is observed across cell lines and primary cell cultures, after EZH2 inhibition. CONCLUSION Our work demonstrates that EZH2 inhibition results in growth inhibition, cell cycle arrest, and cell death, across all cell lines, and cell cultures tested. Futhermore, modulation of protein levels of the EZH2 target H3K27 is observed upon treatment. RNA changes are broad and involve cell proliferative and migratory processes. This work provides the framework for further investigation of EZH2 inhibition in meningioma.

USP15 regulates radiation-induced DNA damage and intestinal injury through K48-linked deubiquitination and stabilisation of ATM

BackgroundRadiation-induced intestinal injury (RIII) interrupts the scheduled processes of abdominal and pelvic radiotherapy (RT) and compromises the quality of life of cancer survivors. However, the specific regulators and mechanisms underlying the effects of RIII remain unknown. The biological effects of RT are caused primarily by DNA damage, and ataxia telangiectasia mutated (ATM) is a core protein of the DNA damage response (DDR). However, whether ATM is regulated by deubiquitination signaling remains unclear.MethodsWe established animal and cellular models of RIII. The effects of ubiquitin-specific protease 15 (USP15) on DNA damage and radion-induced intestinal injury were evaluated. Mass spectrometry analysis, truncation tests, and immunoprecipitation were used to identify USP15 as a binding partner of ATM and to investigate the ubiquitination of ATM. Finally, the relationship between the USP15/ATM axes was further determined via subsequent experiments.ResultsIn this study, we identified the deubiquitylating enzyme USP15 as a regulator of DNA damage and the pathological progression of RIII. Irradiation upregulates the expression of USP15, whereas pharmacological inhibition of USP15 exacerbates radiation-induced DNA damage and RIII both in vivo and in vitro. Mechanistically, USP15 interacts with, deubiquitinates, and stabilises ATM via K48-linked deubiquitination. Notably, ATM overexpression blocks the effect of USP15 genetic inhibition on DNA damage and RIII progression.ConclusionsThese findings describe ATM as a novel deubiquitination target of USP15 upon radiation-induced DNA damage and intestinal injury, and provides experimental support for USP15/ATM axis as a potential target for developing strategies that mitigate RIII.

Platelet-Function-Monitoring-Guided Therapy After Emergent Carotid Artery Stenting

Background/Objectives: Antiplatelet therapy after emergent carotid stenting (eCAS) represents a challenge in balancing the risk of intracerebral hemorrhages (ICHs) and in-stent thrombosis (IST). Post-procedural platelet function monitoring may guide antiplatelet therapy and could potentially improve outcomes due to fewer post-procedural complications. Methods: Consecutive eCAS patients (2019–2021) were included in a single-center retrospective observational study. Patients treated with eCAS received peri-procedural eptifibatide followed by dual antiplatelet treatment with aspirin and clopidogrel. The effect of platelet ADP inhibition by clopidogrel was monitored using the Multiplate® Analyzer (Roche). Clopidogrel non-responders were changed to ticagrelor treatment. The primary outcome was defined as a favorable outcome at 90 days using the modified Rankin Scale (mRS) of 0–2 versus 3–6. Safety outcomes included ICH, IST, and mortality. Data were analyzed and compared in clopidogrel- and ticagrelor-treated patients using Fischer’s exact test and multivariate logistic regression. Results: A total of 105 patients had eCAS, and 28 patients (27%) were clopidogrel non-responders and were changed to treatment with ticagrelor. The favorable outcome was more frequent in ticagrelor-treated patients, 23 (82%), than in clopidogrel-treated patients, 44 (57%), p = 0.036. Numerically, ICH, IST, and mortality were more frequent in clopidogrel-treated patients, but none of the differences were statistically significant. In multivariate analyses, ticagrelor treatment was significantly associated with the favorable outcome, OR = 3.89 (95% CI: 1.09–13.86), p = 0.036. Conclusions: One in four eCAS patients were clopidogrel non-responders. This study suggests that personalized antiplatelet treatment therapy was safe, and that changing treatment to ticagrelor in clopidogrel non-responders was associated with better outcomes in eCAS patients.

Effects of immunoproteasome inhibition on acute respiratory infection with murine hepatitis virus strain 1.

Inflammatory responses triggered by acute infection by respiratory viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drive morbidity and mortality. Infection of mice with murine hepatitis virus strain 1 (MHV-1), a mouse coronavirus, is a useful model to study the pathogenesis of coronavirus respiratory infections. The immunoproteasome is an inducible component of the ubiquitin proteasome system that is poised to contribute to multiple aspects of immune function, inflammation, and protein homeostasis during an infection. We used the MHV-1 model to define the role of the immunoproteasome in coronavirus pathogenesis. We found that immunoproteasome subunit expression increases in the lungs and the liver during acute MHV-1 respiratory infection. Inhibition of immunoproteasome activity did not affect MHV-1 replication but increased MHV-1-induced weight loss, mortality, and inflammation in lungs and livers. Thus, our findings indicate that the immunoproteasome is a critical protective host factor during coronavirus respiratory infection.

Molecular Imaging Reveals Antineuroinflammatory Effects of HDAC6 Inhibition in Stroke Models.

Ischemic stroke is a devastating disease that causes neuronal death, neuroinflammation, and other cerebral damage. However, effective therapeutic strategies for ischemic stroke are still lacking. Histone deacetylase 6 (HDAC6) has been implicated in the pathogenesis of ischemic stroke, and the pharmacological inhibition of HDAC6 has shown promising neuroprotective effects. In this study, we utilized positron emission tomography (PET) imaging with the HDAC6-specific radioligand [18F]PB118 to investigate the dynamic changes of HDAC6 expression in the brain after ischemic injury. The results revealed a significant decline in [18F]PB118 uptake in the ipsilateral hemisphere on the first day after ischemia, followed by a gradual increase on days 4 and 7. To evaluate the therapeutic potential of HDAC6 inhibitors, we developed a novel brain-permeable and potent HDAC6 inhibitor, PB131, and assessed its neuroprotective effects in an ischemic stroke mouse model. PET imaging studies demonstrated that PB131 treatment alleviated the decline in [18F]PB118 uptake and reduced the infarct size in middle cerebral artery occlusion mice. Furthermore, PET imaging with the TSPO-specific radioligand [18F]FEPPA revealed that PB131 significantly suppressed neuroinflammation in the ischemic brain. These findings provide insights into the dynamic changes of HDAC6 in ischemic stroke and the potential of HDAC6 inhibitors as novel therapeutic agents for this condition.

Modeling Lymphoma Angiogenesis, Lymphangiogenesis, and Vessel Co-Option, and the Effects of Inhibition of Lymphoma–Vessel Interactions with an αCD20-EndoP125A Antibody Fusion Protein

Lymphoma growth, progression, and dissemination require tumor cell interaction with supporting vessels and are facilitated through tumor-promoted angiogenesis, lymphangiogenesis, and/or lymphoma vessel co-option. Vessel co-option has been shown to be responsible for tumor initiation, metastasis, and resistance to anti-angiogenic treatment but is largely uncharacterized in the setting of lymphoma. We developed an in vitro model to study lymphoma–vessel interactions and found that mantle cell lymphoma (MCL) cells co-cultured on Matrigel with human umbilical vein (HUVEC) or human lymphatic (HLEC) endothelial cells migrate to and anneal with newly formed capillary-like (CLS) or lymphatic-like (LLS) structures, consistent with lymphoma–vessel co-option. To inhibit this interaction, we constructed an antibody fusion protein, αCD20-EndoP125A, linking mutant anti-angiogenic endostatin (EndoP125A) to an αCD20-IgG1-targeting antibody. αCD20-EndoP125A inhibited both CLS and LLS formation, as well as MCL migration and vessel co-option. Lymphoma vessel co-option requires cell migration, which is regulated by chemokine–chemokine receptor interactions. CXCL12 and its receptor, CXCR4, are highly expressed by both endothelial cells forming CLS and by MCL cells during vessel co-option. αCD20-EndoP125A suppressed expression of both CXCL12 and CXCR4, which were required to facilitate CLS assembly and vessel co-option. We also tested αCD20-EndoP125A effects in vivo using an aggressive murine B cell lymphoma model, 38c13-hCD20, which demonstrated rapid growth and dissemination to tumor-draining lymph nodes (TDLNs) and the spleen, lung, and brain. The pattern of lymphoma distribution and growth within the lung was consistent with vessel co-option. As predicted by our in vitro model, αCD20-EndoP125A treatment inhibited primary tumor growth, angiogenesis, and lymphangiogenesis, and markedly reduced the number of circulating tumor cells and lymphoma dissemination to TDLNs and the lungs, spleen, and brain. αCD20-EndoP125A inhibited lymphoma vessel co-option within the lung. Marked inhibition of MCL primary tumor growth and dissemination were also seen using an MCL xenograft model. The ability of αCD20-EndoP125A to inhibit angiogenesis, lymphangiogenesis, and lymphoma vessel co-option provides a novel therapeutic approach for inhibition of lymphoma progression and dissemination.

Inhibition of Hedgehog Signaling Ameliorates Severity of Chronic Pancreatitis in Experimental Mouse Models.

Chronic pancreatitis (CP) is a fibro-inflammatory disease of the pancreas with no specific cure. Research highlighting the pathogenesis and especially the therapeutic aspect remains limited. Aberrant activation of developmental pathways in adults have been implicated in several diseases. Hedgehog pathway is a notable embryonic signaling pathway, known to promote fibrosis of various organs when over-activated. The aim of this study is to explore the role of hedgehog pathway in the progression of CP and evaluate its inhibition as a novel therapeutic strategy against CP. CP was induced in mice by repeated injections of L-arginine or Caerulein in two separate models. Mice were administered with the FDA approved pharmacological hedgehog pathway inhibitor, Vismodegib during or after establishing the disease condition to inhibit hedgehog signaling. Various parameters of CP were analyzed to determine the effect of hedgehog pathway inhibition on the severity and progression of the disease. Our study shows that hedgehog signaling was over-activated during CP and its inhibition was effective in improving the histopathological parameters associated with CP. Vismodegib administration not only halted the progression of CP but was also able to resolve already established fibrosis. Additionally, inhibition of hedgehog signaling resulted in reversal of pancreatic stellate cell activation ex vivo. Conclusions: Findings from our study justify conducting clinical trials using Vismodegib against CP and thus, could lead to development of novel therapeutic strategy for the treatment of CP.