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BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of chimeric antigen receptor (CAR) T-cell therapy in patients with glioblastoma have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T-cell therapy also remain limited.MethodsWe profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR- T cells from patients with peripheral blood of glioblastoma and diffuse midline glioma and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control.ResultsHere we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite patients with glioblastoma having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an interleukin-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors.ConclusionsTargeting GD2 using a clinically deployed CAR-T-cell therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.

While the two mammalian sphingosine kinases, SK1 and SK2, both catalyze the generation of pro-survival sphingosine 1-phosphate (S1P), their roles vary dependent on their different subcellular localization. SK1 is generally found in the cytoplasm or at the plasma membrane where it can promote cell proliferation and survival. SK2 can be present at the plasma membrane where it appears to have a similar function to SK1, but can also be localized to the nucleus, endoplasmic reticulum or mitochondria where it mediates cell death. Although SK2 has been implicated in cancer initiation and progression, the mechanisms regulating SK2 subcellular localization are undefined. Here, we report that SK2 interacts with the intermediate chain subunits of the retrograde-directed transport motor complex, cytoplasmic dynein 1 (DYNC1I1 and -2), and we show that this interaction, particularly with DYNC1I1, facilitates the transport of SK2 away from the plasma membrane. DYNC1I1 is dramatically downregulated in patient samples of glioblastoma (GBM), where lower expression of DYNC1I1 correlates with poorer patient survival. Notably, low DYNC1I1 expression in GBM cells coincided with more SK2 localized to the plasma membrane, where it has been recently implicated in oncogenesis. Re-expression of DYNC1I1 reduced plasma membrane-localized SK2 and extracellular S1P formation, and decreased GBM tumor growth and tumor-associated angiogenesis in vivo. Consistent with this, chemical inhibition of SK2 reduced the viability of patient-derived GBM cells in vitro and decreased GBM tumor growth in vivo. Thus, these findings demonstrate a tumor-suppressive function of DYNC1I1, and uncover new mechanistic insights into SK2 regulation which may have implications in targeting this enzyme as a therapeutic strategy in GBM.

CIB1 (calcium and integrin binding protein 1) is a small intracellular protein with numerous interacting partners, and hence has been implicated in various cellular functions. Recent studies have revealed emerging roles of CIB1 in regulating cancer cell survival and angiogenesis, although the mechanisms involved have remained largely undefined. In investigating the oncogenic function of CIB1, we initially found that CIB1 is widely up-regulated across a diverse range of cancers, with this upregulation frequently correlating with oncogenic mutations of KRas. Consistent with this, we found that ectopic expression of oncogenic KRas and HRas in cells resulted in elevated CIB1 expression. We previously described the Ca 2+ -myristoyl switch function of CIB1, and its ability to facilitate agonist-induced plasma membrane localisation of sphingosine kinase 1 (SK1), a location where SK1 is known to elicit oncogenic signalling. Thus, we examined the role this may play in oncogenesis. Consistent with these findings, we demonstrated here that over-expression of CIB1 by itself is sufficient to drive localisation of SK1 to the plasma membrane and enhance the membrane-associated enzymatic activity of SK1, as well as its oncogenic signalling. We subsequently demonstrated that elevated levels of CIB1 resulted in full neoplastic transformation, in a manner dependent on SK1. In agreement with our previous findings that SK1 is a downstream mediator of oncogenic signalling by Ras, we found that targeting CIB1 also inhibited neoplastic growth of cells induced by oncogenic Ras, suggesting an important pro-tumorigenic role for CIB1. Thus, we have demonstrated for the first time a role for CIB1 in neoplastic transformation, and revealed a novel mechanism facilitating oncogenic signalling by Ras and SK1.

Aggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited. We profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control. Here we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity. Targeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors. GD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors. This is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing. The results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.

Epithelia must eliminate apoptotic cells to preserve tissue barriers and prevent inflammation [1]. Several different mechanisms exist for apoptotic clearance, including efferocytosis [2, 3] and apical extrusion [4, 5]. We found that extrusion was the first-line response to apoptosis in cultured monolayers and in zebrafish epidermis. During extrusion, the apoptotic cell elicited active lamellipodial protrusions and assembly of a contractile extrusion ring in its neighbours. Depleting E-cadherin compromised both the contractile ring and extrusion, implying that a cadherin-dependent pathway allows apoptotic cells to engage their neighbours for extrusion. We identify RhoA as the cadherin-dependent signal in the neighbour cells and show that it is activated in response to contractile tension from the apoptotic cell. This mechanical stimulus is conveyed by a Myosin VI-dependent mechanotransduction pathway that is necessary both for extrusion and to preserve the epithelial barrier when apoptosis was stimulated. Earlier studies suggested that release of sphingosine-1-phosphate (S1P) from apoptotic cells might define where RhoA was activated. However, we found that although S1P is necessary for extrusion, its contribution does not require a localized source of S1P in the epithelium. We therefore propose a unified view of how RhoA is stimulated to engage neighbour cells for apoptotic extrusion. Here, tension-sensitive mechanotransduction is the proximate mechanism that activates RhoA specifically in the immediate neighbours of apoptotic cells, but this also must be primed by S1P in the tissue environment. Together, these elements provide a coincidence detection system that confers robustness on the extrusion response.