Pharmacological Blockade Research Articles

Sorafenib enhanced the function of myeloid-derived suppressor cells in hepatocellular carcinoma by facilitating PPARα-mediated fatty acid oxidation

BackgroundSorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC), faces resistance issues, partly due to myeloid-derived suppressor cells (MDSCs) that enhance immunosuppression in the tumor microenvironment (TME).MethodsVarious murine HCC cell lines and MDSCs were used in a series of in vitro and in vivo experiments. These included subcutaneous tumor models, cell viability assays, flow cytometry, immunohistochemistry, and RNA sequencing. MDSCs were analyzed for chemotaxis, immunosuppressive functions, fatty acid oxidation (FAO), and PPARα expression. The impact of sorafenib on tumor growth, MDSC infiltration, differentiation, and immunosuppressive function was assessed, alongside the modulation of these processes by PPARα.ResultsHere, we revealed increased infiltration and enhanced function of MDSCs in TME after treatment with sorafenib. Moreover, our results indicated that sorafenib induced the accumulation of MDSCs mediated by CCR2, and pharmacological blockade of CCR2 markedly reduced MDSCs migration and tumor growth. Mechanistically, sorafenib promoted the effect and fatty acid uptake ability of MDSCs and modulated peroxisome proliferator-activated receptor α (PPARα)-mediated fatty acid oxidation (FAO). In addition, tumor-bearing mice fed a high-fat diet (HFD) at the beginning of sorafenib administration had worse outcomes than mice fed a regular diet. Genetic deficiency of PPARα weakens the effect of sorafenib on MDSCs in mice with HCC. Pharmacological inhibition of PPARα has a synergistic anti-tumor effect with sorafenib, which is attenuated by the inhibition of MDSCs. Mechanistically, sorafenib significantly inhibited the differentiation of macrophages by upregulating PPARα expression and suppressing the PU.1-CSF1R pathway.ConclusionOverall, our study demonstrated that sorafenib enhanced the function of MDSCs by facilitating PPARα-mediated FAO and further augmenting sorafenib resistance, which sheds light on dietary management and improves the therapeutic response in HCC.

Infiltrating peripheral monocyte TREM-1 mediates dopaminergic neuron injury in substantia nigra of Parkinson’s disease model mice

Neuroinflammation is a key factor in the pathogenesis of Parkinson’s disease (PD). Activated microglia in the central nervous system (CNS) and infiltration of peripheral immune cells contribute to dopaminergic neuron loss. However, the role of peripheral immune responses, particularly triggering receptor expressed on myeloid cells-1 (TREM-1), in PD remains unclear. Using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced PD mouse model, we examined TREM-1 expression and monocyte infiltration in the substantia nigra pars compacta (SNpc). We found that MPTP increased peripheral monocytes, and deletion of peripheral monocytes protected against MPTP neurotoxicity in the SNpc. TREM-1 inhibition, both genetically and pharmacologically, reduced monocyte infiltration, alleviated neuroinflammation, and preserved dopaminergic neurons, resulting in improved motor function. Furthermore, adoptive transfer of TREM-1-expressing monocytes from PD model mice to naive mice induced neuronal damage and motor deficits. These results underscore the critical role of peripheral monocytes and TREM-1 in PD progression, suggesting that targeting TREM-1 could be a promising therapeutic approach to prevent dopaminergic neurodegeneration and motor dysfunction in PD.Schematic diagram of monocyte TREM-1-mediated dopaminergic neuron damage. The figure illustrates that in experimental MPTP-induced PD model mice, the number of inflammatory monocytes in the peripheral blood increases, after which the monocytes infiltrate the CNS through the Blood-Brain Barrier(BBB). These infiltrating monocytes increase the release of inflammatory cytokines and eventually cause neuronal injury. TREM-1 gene deletion and pharmacological blockade limit inflammatory monocyte recruitment into the SNpc and ameliorate neuroinflammatory events and the loss of dopaminergic neurons.

ZBP1-driven cell death in severe influenza.

Influenza A virus (IAV) infections can cause life-threatening illness in humans. The severity of disease is directly linked to virus replication in the alveoli of the lower respiratory tract. In particular, the lytic death of infected alveolar epithelial cells (AECs) is a major driver of influenza severity. Recent studies have begun to define the molecular mechanisms by which IAV triggers lytic cell death. Z-form nucleic-acid-binding protein 1 (ZBP1) senses replicating IAV and drives programmed cell death (PCD) in infected cells, including apoptosis and necroptosis in AECs and pyroptosis in myeloid cells. Necroptosis and pyroptosis, both lytic forms of death, contribute to pathogenesis during severe infections. Pharmacological blockade of necroptosis shows strong therapeutic potential in mouse models of lethal influenza. We suggest that targeting ZBP1-initiated necroinflammatory cell lysis, either alone or in combination antiviral drugs, will provide clinical benefit in severe influenza.

Peripheral inflammation enhances opioid-induced gastrointestinal motility inhibition via up-regulating spinal mu opioid receptor.

Opioids are potent analgesics in clinical pain management but exert variable analgesia in different pain types. Opioid-induced constipation is a common side effect of opioid therapy, and whether opioids induce different gastrointestinal motility inhibitions in different pain types is unknown. In this study, we evaluated the antinociceptive effects and inhibition of upper gastrointestinal transit and colonic bead expulsion of morphine, DAMGO, and Deltorphin in mouse CFA chronic inflammatory pain, SNI chronic neuropathic pain, and carrageenan chronic inflammatory pain models. Furthermore, quantitative PCR and immunofluorescence were used to investigate the mechanisms underlying the altered inhibition. Results showed that intrathecal administration of morphine, DAMGO, and Deltorphin produced higher antinociceptive effects in the CFA and carrageenan groups than in the SNI group. Upper gastrointestinal transit inhibition was significantly enhanced in the carrageenan group by morphine and DAMGO; colonic bead expulsion inhibition was also enhanced in the CFA and carrageenan groups by morphine and DAMGO, but not in Deltorphin treatment. Additionally, mu (MOR) opioid receptor mRNA and MOR-expressing cell density in the lumbar spinal cord of CFA and carrageenan mice were increased, whereas delta opioid receptor expression remained unchanged in these groups. Finally, the pharmacological blockade of MOR completely prevented the enhanced upper gastrointestinal transit inhibition in the carrageenan group by morphine and DAMGO. Altogether, our results indicate that gastrointestinal motility inhibition induced by MOR agonists can be enhanced with upregulated spinal MOR expression in chronic inflammatory pain.

Plasticity of Mouse Dorsal Root Ganglion Neurons by Innate Immune Activation Is Influenced by Electrophysiological Activity.

The complex relationship between inflammation, its effects on neuronal excitability and the ensuing plasticity of dorsal root ganglion (DRG) sensory neurons remains to be fully explored. In this study, we have employed a system of experiments assessing the impact of inflammatory conditioned media derived from activated immune cells on the excitability and activity of DRG neurons and how this relates to subsequent growth responses of these cells. We show here that an early phase of increased neuronal activity in response to inflammatory conditioned media is critical for the engagement of plastic processes and that neuronal excitability profiles are linked through time to the structural phenotype of individual neurons. Pharmacological blockade of neuronal activity was able to abolish the growth-promoting effects of inflammatory media. Our results suggest that targeting the activity of DRG neurons may provide a novel therapeutic avenue to manipulate their growth status and potential for plasticity in response to inflammation. Importantly, the same pharmacological blockade invivo abolished pain responses in a mouse model of multiple sclerosis. While further studies are needed to fully elucidate the underlying mechanisms of the relationship between neural activity and growth status, a more complete understanding of this relationship may ultimately lead to the development of new treatments for neuropathic pain in disorders associated with heightened immune responses such as rheumatoid arthritis and multiple sclerosis.

Pharmacological blockade of infection chronification modulates oxy-inflammation and prevents the activation of stress-induced premature senescence markers in schistosomiasis.

Chronic inflammation, oxidative stress, and DNA damage are observed in schistosomiasis and premature aging. However, the potential of these events to trigger stress-induced premature senescence (SIPS) throughout schistosomiasis progression remains overlooked, especially in response to the first-line pharmacological treatment. Thus, we investigated the relationship between oxidative stress and SIPS sentinel markers in untreated Schistosoma mansoni-infected mice and those receiving praziquantel (Pz)-based reference treatment. Swiss mice were randomized into 5 groups: uninfected (followed by 60- and 180-days post-infection), acutely (60 days) and chronically (180 days) infected untreated, and infected treated with Pz followed until 180 days. Our results indicated that infection chronification was accompanied by the worsening of hepatic granulomatous inflammation, increased number of granulomas, IL-4, TGF-β, reactive oxygen species (ROS) levels, fibrosis, hepatocytes DNA damage, upregulation in SA-β-gal activity, p16 and p21 gene expression, and hepatocytes proliferation down-regulation in the absence of telomeric shortening. These abnormalities were blocked by Pz treatment, which prevented infection chronification and the decline in hepatocytes proliferative potential, stimulating granulomatous inflammation resolution. Taken together, our findings provide the evidence that progressive fibrosis, sustained production of high ROS levels, marked DNA damage and decline in p16 and p21 expression are associated with hepatocytes replication attenuation in the chronic phase of S. mansoni infection. Thus, pharmacological blockade of infection and granulomatous inflammation is essential to prevent these premature senescence markers associated with hepatocytes replicative disorders, stimulating liver regeneration in schistosomiasis mansoni.

Contribution of glutamatergic projections to neurons in the nonhuman primate lateral substantia nigra pars reticulata for the reactive inhibition.

The basal ganglia play a crucial role in action selection by facilitating desired movements and suppressing unwanted ones. The substantia nigra pars reticulata (SNr), a key output nucleus, facilitates movement through disinhibition of the superior colliculus (SC). However, its role in action suppression, particularly in primates, remains less clear. We investigated whether individual SNr neurons in three male macaque monkeys bidirectionally modulate their activity to both facilitate and suppress actions and examined the role of glutamatergic inputs in suppression. Monkeys performed a sequential choice task, selecting or rejecting visually presented targets. Electrophysiological recordings showed SNr neurons decreased firing rates during target selection and increased firing rates during rejection, demonstrating bidirectional modulation. Pharmacological blockade of glutamatergic inputs to the lateral SNr disrupted saccadic control and impaired suppression of reflexive saccades, providing causal evidence for the role of excitatory input in behavioral inhibition. These findings suggest that glutamatergic projections, most likely from the subthalamic nucleus, drive the increased SNr activity during action suppression. Our results highlight conserved basal ganglia mechanisms across species and offer insights into the neural substrates of action selection and suppression in primates, with implications for understanding disorders such as Parkinson's disease.

Pharmacological Blockade of Group II Metabotropic Glutamate Receptors Reduces the Incidence of Brain Tumors Induced by Prenatal Exposure to N-ethyl-N-nitrosourea in Rats.

The study demonstrates that pharmacological blockade of type 3 metabotropic glutamate (mGlu3) receptors at the time of tumor induction significantly reduces the incidence of brain gliomas in rats. The overall survival of patients with high-grade brain gliomas is 14-20 months after current multimodal therapy, including surgery, radiotherapy, and adjuvant chemotherapy. To demonstrate in this experimental model that pharmacological blockade of group II metabotropic glutamate receptors reduces the incidence of brain tumors induced by prenatal exposure to N- ethyl-N-nitrosourea (ENU) in rats. Dams received a single injection of ENU (40 mg/kg, e.v.) at day 20 of pregnancy, combined with 5 daily injections of either saline or the mGlu2/3 receptor antagonist, LY341495 (10 mg/kg) (from day 15 to day 21 of pregnancy). Assessment of brain tumors in the offspring at 5 months of age showed the presence of mixed gliomas (astrocytomas/oligodendrogliomas) in 70% of the ENU + saline group of rats and only in 30% of the ENU + LY341495 group. Tumors in both groups of rats showed a moderate/high expression of the astrocyte marker, GFAP, and the oligodendrocyte marker, OLIG-2, and a low expression of the proliferation marker, Ki-67. However, tumors of the ENU + LY341495 group showed a reduced density of Iba-1+ cells, suggesting a lower extent of neuroinflammation in the tumor microenvironment. These findings strengthen the hypothesis that mGlu3 receptors are candidate drug targets for the treatment of malignant gliomas.

Inhibiting triggering receptor expressed on myeloid cells 1 signaling to ameliorate skin fibrosis.

Systemic sclerosis (SSc) is characterized by immune system failure, vascular insult, autoimmunity, and tissue fibrosis. TGF-β is a crucial mediator of persistent myofibroblast activation and aberrant extracellular matrix production in SSc. The factors responsible for this are unknown. By amplifying pattern recognition receptor signaling, triggering receptor expressed on myeloid cells 1 (TREM-1) is implicated in multiple inflammatory conditions. In this study, we used potentially novel ligand-independent TREM-1 inhibitors in preclinical models of fibrosis and explanted SSc skin fibroblasts in order to investigate the pathogenic role of TREM-1 in SSc. Selective pharmacological TREM-1 blockade prevented and reversed skin fibrosis induced by bleomycin in mice and mitigated constitutive collagen synthesis and myofibroblast features in SSc fibroblasts in vitro. Our results implicate aberrantly activated TREM-1 signaling in SSc pathogenesis, identify a unique approach to TREM-1 blockade, and suggest a potential therapeutic benefit for TREM-1 inhibition.

Conjugated fatty acids drive ferroptosis through chaperone-mediated autophagic degradation of GPX4 by targeting mitochondria

Conjugated fatty acids (CFAs) have been known for their anti-tumor activity. However, the mechanism of action remains unclear. Here, we identify CFAs as inducers of glutathione peroxidase 4 (GPX4) degradation through chaperone-mediated autophagy (CMA). CFAs, such as (10E,12Z)-octadecadienoic acid and α-eleostearic acid (ESA), induced GPX4 degradation, generation of mitochondrial reactive oxygen species (ROS) and lipid peroxides, and ultimately ferroptosis in cancer cell lines, including HT1080 and A549 cells, which were suppressed by either pharmacological blockade of CMA or genetic deletion of LAMP2A, a crucial molecule for CMA. Mitochondrial ROS were sufficient and necessary for CMA-dependent GPX4 degradation. Oral administration of an ESA-rich oil attenuated xenograft tumor growth of wild-type, but not that of LAMP2A-deficient HT1080 cells, accompanied by increased lipid peroxidation, GPX4 degradation and cell death. Our study establishes mitochondria as the key target of CFAs to trigger lipid peroxidation and GPX4 degradation, providing insight into ferroptosis-based cancer therapy.

SGK1-Mediated Vascular Smooth Muscle Cell Phenotypic Transformation Promotes Thoracic Aortic Dissection Progression.

The occurrence of thoracic aortic dissection (TAD) is closely related to the transformation of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic phenotype. The role of SGK1 (serum- and glucocorticoid-regulated kinase 1) in VSMC phenotypic transformation and TAD occurrence is unclear. Four-week-old male Sgk1F/F (Sgk1 floxed) and Sgk1F/F;TaglnCre (smooth muscle cell-specific Sgk1 knockout) mice were administered β-aminopropionitrile monofumarate for 4 weeks to model TAD. The SGK1 inhibitor GSK650394 was administered daily via intraperitoneal injection to treat the mouse model of TAD. Immunopurification and mass spectrometry were used to identify proteins that interact with SGK1. Immunoprecipitation, immunofluorescence colocalization, and GST (glutathione S-transferase) pull-down were used to detect molecular interactions between SGK1 and SIRT6 (sirtuin 6). RNA-sequencing analysis was performed to evaluate changes in the SIRT6 transcriptome. Quantitative chromatin immunoprecipitation was used to determine the target genes regulated by SIRT6. Functional experiments were also conducted to investigate the role of SGK1-SIRT6-MMP9 (matrix metalloproteinase 9) in VSMC phenotypic transformation. The effect of SGK1 regulation on target genes was evaluated in human and mouse TAD samples. Sgk1F/F;TaglnCre or pharmacological blockade of Sgk1 inhibited the formation and rupture of β-aminopropionitrile monofumarate-induced TADs in mice and reduced the degradation of the ECM (extracellular matrix) in vessels. Mechanistically, SGK1 promoted the ubiquitination and degradation of SIRT6 by phosphorylating SIRT6 at Ser338, thereby reducing the expression of the SIRT6 protein. Furthermore, SIRT6 transcriptionally inhibits the expression of MMP9 through epigenetic modification, forming the SGK1-SIRT6-MMP9 regulatory axis, which participates in the ECM signaling pathway. Additionally, our data showed that the lack of SGK1-mediated inhibition of ECM degradation and VSMC phenotypic transformation is partially dependent on the regulatory effect of SIRT6-MMP9. These findings highlight the key role of SGK1 in the pathogenesis of TAD. A lack of SGK1 inhibits VSMC phenotypic transformation by regulating the SIRT6-MMP9 axis, providing insights into potential epigenetic strategies for TAD treatment.

Acid ceramidase controls proteasome inhibitor resistance and is a novel therapeutic target for the treatment of relapsed / refractory multiple myeloma.

Multiple myeloma (MM) patients are often refractory to targeted therapies including proteasome inhibitors (PIs). Here, analysis of RNA sequencing data derived from 672 patients with newly diagnosed or relapsed/refractory disease identified the acid ceramidase, ASAH1, as a key regulator of PI resistance. Genetic or pharmacological blockade of ASAH1 remarkably restored PI sensitivity and protected mice from resistant MM progression in vivo. Mechanistically, ASAH1 depletion of ceramide promoted SET inhibition of PP2A phosphatase activity thus facilitating the increased expression and activity of the pro-survival proteins, MCL-1, and BCL-2. We corroborated these findings in human MM datasets, and in ex vivo patient MM cells. These preclinical studies suggest that ASAH1 may be a potential therapeutic target for the treatment of relapsed/refractory MM (RRMM).

GluN2D-containing NMDA receptors in parvalbumin neurons in the nucleus accumbens regulate nocifensive responses in neuropathic pain.

Neuropathic pain presents a significant challenge, with its underlying mechanisms still not fully understood. Here, we investigated the role of GluN2C- and GluN2D-containing NMDA receptors in the development of neuropathic pain induced by cisplatin, a widely used chemotherapeutic agent. Through genetic and pharmacological strategies, we found that GluN2D-containing NMDA receptors play a targeted role in regulating cisplatin-induced neuropathic pain (CINP), while sparing inflammatory or acute pain responses. Specifically, both GluN2D knockout (KO) mice and pharmacological blockade of GluN2D-containing receptors produced robust reduction in mechanical nocifensive response in CINP. In contrast, GluN2C KO mice behaved similar to wildtype mice in CINP but showed reduced mechanical hypersensitivity in inflammatory pain. Using conditional KO strategy, we addressed the region- and cell-type involved in GluN2D-mediated changes in CINP. Animals with conditional deletion of GluN2D receptors from parvalbumin interneurons (PVIs) or local ablation of GluN2D from nucleus accumbens (NAc) displayed reduced mechanical hypersensitivity in CINP, underscoring the pivotal role of accumbal GluN2D in PVIs in neuropathic pain. Furthermore, CINP increased excitatory neurotransmission in the NAc in wildtype mice and this effect is dampened in PV-GluN2D KO mice. Other changes in CINP in NAc included an increase in vGluT1 and c-fos labeled neurons in wildtype which were absent in PV-GluN2D KO mice. GiDREADD-induced inhibition of PVIs in the NAc produced reduction in mechanical hypersensitivity in CINP. These findings unveil a novel cell-type and region-specific role of GluN2D-containing NMDA receptors in neuropathic pain and identify PVIs in NAc as a novel mediator of pain behaviors.

The IL-33/ST2 signaling axis drives pathogenesis in acute SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), remains a significant threat to global public health. Immunopathological damage plays a role in driving pneumonia, acute respiratory distress syndrome (ARDS), and multiorgan failure in severe COVID-19. Therefore, dissecting the pulmonary immune response to SARS-CoV-2 infection is critical to understand disease pathogenesis and identify immune pathways targetable by therapeutic intervention. Considering that the type 2 cytokine IL-13 enhances COVID-19 disease severity, therapeutic targeting of upstream signals that drive type 2 immunity may confer further protection. In this study, we investigate the role of the IL-33/ST2 signaling axis, a potent inducer of type 2 immunity in the lung, in a mouse model of COVID-19. Upon infection with mouse-adapted SARS-CoV-2 MA10, ST2 -/- mice had significantly improved weight loss and survival (69.2% vs 13.3% survival; P = 0.0005), as compared to wild-type mice. In a complementary pharmacologic approach, IL-33/ST2 signaling was inhibited using HpBARI_Hom2, a helminth derived protein that binds to mouse ST2 and blocks IL-33 signaling. In SARS-CoV-2 MA10 infection, HpBARI_Hom2-treated mice had significantly improved weight loss and survival (60% vs 10% survival; P = 0.0035), as compared to inert control-treated mice. These data demonstrate that loss of IL-33/ST2 signaling confers protection during acute SARS-CoV-2 MA10 infection, implicating the IL-33/ST2 signaling axis as an enhancer of COVID-19 disease severity. The protection conferred by pharmacologic blockade of IL-33/ST2 signaling was independent of viral control, as HpBARI_Hom2-treated mice had no reduction in viral titers. This finding suggests an immunopathogenic role for IL-33/ST2 signaling. One potential mechanism through which IL-33/ST2 signaling may drive severe disease is through enhancement of type 2 immune pathways including IL-5 production, as pulmonary IL-5 concentrations were found to depend on IL-33/ST2 signaling in acute SARS-CoV-2 MA10 infection.

Role of stretch-activated channels in light-generated action potentials mediated by an intramembrane molecular photoswitch

BackgroundThe use of light to control the activity of living cells is a promising approach in cardiac research due to its unparalleled spatio-temporal selectivity and minimal invasiveness. Ziapin2, a newly synthesized azobenzene compound, has recently been reported as an efficient tool for light-driven modulation of excitation-contraction coupling (ECC) in human-induced pluripotent stem cells–derived cardiomyocytes. However, the exact biophysical mechanism of this process remains incompletely understood.MethodsTo address this, we performed a detailed electrophysiological characterization in a more mature cardiac model, specifically adult mouse ventricular myocytes (AMVMs).ResultsOur in vitro results demonstrate that Ziapin2 can photomodulate cardiac ECC in mature AMVMs without affecting the main transporters and receptors located within the sarcolemma. We established a connection between Ziapin2-induced membrane thickness modulation and light-generated action potentials by showcasing the pivotal role of stretch-activated channels (SACs). Notably, our experimental findings, through pharmacological blockade, suggest that non-selective SACs might serve as the biological culprit responsible for the effect.ConclusionsTaken together, these findings elucidate the intricacies of Ziapin2-mediated photostimulation mechanism and open new perspectives for its application in cardiac research.

PD-1 and CD73 on naive CD4+ T cells synergistically limit responses to self.

Vaccination with self- and foreign peptides induces weak and strong expansion of antigen-specific CD4+ T cells, respectively, but the mechanism is not known. In the present study, we used computational analysis of the entire mouse major histocompatibility complex class II peptidome to test how much of the naive CD4+ T cell repertoire specific for self-antigens was shaped by negative selection in the thymus and found that negative selection only partially explained the difference between responses to self and foreign. In naive uninfected and unimmunized mice, we identified higher expression of programmed cell death protein 1 (PD-1) and CD73 mRNA and protein on self-specific CD4+ T cells compared with foreign-specific CD4+ T cells. Pharmacological or genetic blockade of PD-1 and CD73 significantly increased the vaccine-induced expansion of self-specific CD4+ T cells and their transcriptomes were similar to those of foreign-specific CD4+ T cells. We concluded that PD-1 and CD73 synergistically limited CD4+ T cell responses to self. These observations have implications for the development of tolerogenic vaccines and cancer immunotherapy.

Actin Dysregulation Induces Neuroendocrine Plasticity and Immune Evasion: A Vulnerability of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is aggressive with limited therapeutic options. Despite recent advances in targeted therapies and immunotherapies, therapy resistance is a recurring issue, which might be partly due to tumor cell plasticity, a change in cell fate. Nonetheless, the mechanisms underlying tumor cell plasticity and immune evasion in SCLC remain elusive. CRACD, a capping protein inhibitor that promotes actin polymerization, is frequently inactivated in SCLC. Cracd knockout (KO) transforms preneoplastic cells into SCLC tumor-like cells and promotes in vivo SCLC development driven by Rb1, Trp53, and Rbl2 triple KO. Cracd KO induces neuroendocrine (NE) plasticity and increases tumor cell heterogeneity of SCLC tumor cells via dysregulated NOTCH1 signaling by actin cytoskeleton disruption. CRACD depletion also reduces nuclear actin and induces EZH2-mediated H3K27 methylation. This nuclear event suppresses the MHC-I genes and thereby depletes intratumoral CD8+ T cells for accelerated SCLC tumorigenesis. Pharmacological blockade of EZH2 inhibits CRACD-negative SCLC tumorigenesis by restoring MHC-I expression and immune surveillance. Unsupervised single-cell transcriptomics identifies SCLC patient tumors with concomitant inactivation of CRACD and downregulated MHC-I pathway. This study defines CRACD, an actin regulator, as a tumor suppressor that limits cell plasticity and immune evasion and proposes EZH2 blockade as a viable therapeutic option for CRACD-negative SCLC.

ANGI-03. GLIOMA STEM CELLS INDUCE VASECTASIA – A NON-ANGIOGENIC BRAIN TUMOUR NEOVASCULARIZATION PROCESS DRIVEN BY INTERCELLULAR EGFR TRANSFER THROUGH EXTRACELLULAR VESICLES

Abstract Tumour neovascularization in glioblastoma (GBM) is abundant, abnormal and poorly understood, and it likely involves multiple co-existing mechnanisms with distinct modes of regulation. Since glioma stem cells (GSCs) engage in bidirectional interactions with the vasculature we dissected the resulting vascular responses elicited by either proneural (PN), or mesenchymal (MES) GSC subtypes established from patients isolates and able to drive progression of orthotopic intracranial xenografts in mice. We report here that while PN-GSCs elicited mostly angiogenesis-type vascular capillary growth processes, their MES-GSC counterparts elicited non-angiogenic growth of enlarged vessels (vasectesia), paradoxically associated with reduced blood vessel density. Vasectasia was dependent on the expression of oncogenic EGFR/EGFRvIII by MES-GSCs, and on the related kinase/signalling activity, as EGFR/EGFRvIII gene disruption and Dacomitinib treatment suppressed formation of large blood vessels in vivo and resulted in a preponderance of a small calliber (angiogenic) vasculature throughout xenografts. Notably, the analysis of spatial transcriptomes and single cell RNA sequencing revealed distinct molecular traits of endothelial cells asociated with vasectasia and angiogesis, the former enriched in Birc5, but depleted for angiogenic tip cell markers (Vegfr2, Apln). Mechnaistically, vasectasia was linked to the intercellular transfer of EGFR from MES-GSCs to endothelial cells resulting in ectopic activation of EGFR signalling in the endothelial cell bacground, both in vitro and in a model of in vivo blood vessel recruitment into implanted matrigel pellets. Simultaneous pharmacological blockade of both, angiogenesis (anti-VEGR2) and vasectasia (Dacomitinib) lead to a marked increase in survival of mice with aggressive GBM lesions initiated by MES-GSCs. We suggest that, vasectasia driven by intercellular transfer of oncogenic EGFR may represent a new, subtype-specific mechanism of GBM neovascularization, and a plausible target for agents aiming to modify vascular tumour microenvironment.

CB1 receptors in NG2 cells mediate cannabinoid-evoked functional myelin regeneration

Defects in myelin homeostasis have been reported in many neuropathological conditions. Cannabinoid compounds have been shown to efficiently promote myelin regeneration in animal models of demyelination. However, it is still unknown whether this action relies mostly on a cell autonomous effect on oligodendroglial-lineage-NG2 cells. By using conditional genetic mouse models, here we found that cannabinoid CB1 receptors located on NG2 cells are required for oligodendroglial differentiation and myelin regeneration after demyelination. Selective CB1 receptor gene depletion in NG2 cells following toxin-induced demyelination disrupted oligodendrocyte regeneration and functional remyelination and exacerbated axonal damage. These deficits were rescued by pharmacological blockade of the RhoA/ROCK/Cofilin pathway. Conversely, tetrahydrocannabinol administration promoted oligodendrocyte regeneration and functional remyelination in wild-type but not Ng2-CB1-deficient mice. Overall, this study identifies CB1 receptors as essential modulators of remyelination and support the therapeutic potential of cannabinoids for promoting remyelination in neurological disorders.

Mineralocorticoid Receptor in Endothelial Cells Contributes to Vascular Endothelial Growth Factor Receptor Inhibitor-Induced Vascular and Kidney Damage.

Vascular endothelial growth factor receptor inhibitors (VEGFRis) improve cancer patient survival by inhibiting tumor angiogenesis. However, VEGFRis induce treatment-limiting hypertension which has been associated with impaired vascular endothelial cell (EC) function and kidney damage. The mineralocorticoid receptor (MR) regulates blood pressure (BP) via its effects on the vasculature and the kidney. Thus, we interrogated the role of the MR in EC dysfunction, renal impairment, and hypertension in a mouse model of VEGFRi-induced hypertension using sorafenib. EC dysfunction in mesenteric arterioles was assessed by immunoblotting for phosphorylation of endothelial nitric oxide synthase (eNOS) at serine 1177. Renal damage was measured by assessing glomerular endotheliosis histologically. BP was measured using implanted radiotelemetry. Six days of sorafenib treatment significantly impaired mesenteric resistance vessel EC function, induced renal damage, and increased BP. Pharmacologic MR blockade with spironolactone prevented the sorafenib-induced decline in eNOS phosphorylation and renal glomerular endotheliosis, without affecting systolic BP (SBP) or diastolic BP. Mice with the MR knocked out specifically in ECs (EC-MR-KO) were protected from sorafenib-induced EC dysfunction and glomerular endotheliosis, whereas smooth muscle cell-specific MR (SMC-MR) knockout mice were not. Neither EC-MR nor SMC-MR knockout affected the degree to which sorafenib increased SBP or diastolic BP. These results reveal that the MR, specifically in EC but not in SMCs, is necessary for VEGFRi-induced renal and vascular injury. While ineffective at lowering SBP, these data suggest potential therapeutic benefits of MR antagonists, like spironolactone, to protect the vasculature and the kidneys from VEGFRi-induced injury.