Protein ubiquitination in eukaryotic cells is mediated by diverse E3 ligase enzymes that each target specific substrates. The cullin E3 ligase complexes are the most abundant class of E3 ligases; they contain various cullin components that serve as scaffolds for interaction with substrate-recruiting adaptor proteins. SPOP is a BTB-domain adaptor of the cullin-3 E3 ligase complexes; it selectively recruits substrates via its N-terminal MATH domain, whereas its BTB domain mediates dimerization and interactions with cullin-3. It has recently been recognized that the high-order oligomerization of SPOP enhances the ubiquitination of substrates. Here, a dimerization interface in the SPOP C-terminus is identified and it is shown that the dimerization interfaces of the BTB domain and of the C-terminus act independently and in tandem to generate high-order SPOP oligomers. The crystal structure of the dimeric SPOP C-terminal domain is reported at 1.5 Å resolution and it is shown that Tyr353 plays a critical role in high-order oligomerization. A model of the high-order SPOP oligomer is presented that depicts a helical organization that could enhance the efficiency of substrate ubiquitination.

Summary Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To understand this problem more clearly we used a combination of natural killer (NK) cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment. In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem‐like cells, a source of post‐treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM‐infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of transforming growth factor (TGF)‐β‐mediated inhibition. This NK cell inhibition is accompanied by expression of multiple immune checkpoint molecules on T cells. Single‐cell transcriptomics demonstrated that both tumour and haematopoietic‐derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co‐expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognized by the immune system but that anti‐tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more probably benefit from combination immunotherapies directed against multiple immunosuppressive pathways.

Glioblastoma (GBM) is an aggressive cancer with a very poor prognosis. Generally viewed as weakly immunogenic, GBM responds poorly to current immunotherapies. To better understand this problem we used a combination of NK cell functional assays together with gene and protein expression profiling to define the NK cell response to GBM and explore immunosuppression in the GBM microenvironment.In addition, we used transcriptome data from patient cohorts to classify GBM according to immunological profiles. We show that glioma stem-like cells, a source of post-treatment tumour recurrence, express multiple immunomodulatory cell surface molecules and are targeted in preference to normal neural progenitor cells by natural killer (NK) cells ex vivo. In contrast, GBM-infiltrating NK cells express reduced levels of activation receptors within the tumour microenvironment, with hallmarks of TGF-b mediated inhibition. This NK cell inhibition is accompanied by expression of mutiple immune checkpoint molecules on T cells. Single cell transcriptomics demonstrated that both tumour and haematopoietic-derived cells in GBM express multiple, diverse mediators of immune evasion. Despite this, immunome analysis across a patient cohort identifies a spectrum of immunological activity in GBM, with active immunity marked by co-expression of immune effector molecules and feedback inhibitory mechanisms. Our data show that GBM is recognised by the immune system but that anti-tumour immunity is restrained by multiple immunosuppressive pathways, some of which operate in the healthy brain. The presence of immune activity in a subset of patients suggests that these patients will more likely benefit from combination immunotherapies directed against multiple immunosuppressive pathways.