The phosphoinositide 3-kinase (PI3K) signalling pathway is highly dysregulated in cancer, leading to elevated PI3K signalling and altered cellular processes that contribute to tumour development. The pathway is normally orchestrated by class I PI3K enzymes and negatively regulated by the phosphatase and tensin homologue, PTEN. Endometrial carcinomas harbour frequent alterations in components of the pathway, including changes in gene copy number and mutations, in particular in the oncogene PIK3CA, the gene encoding the PI3K catalytic subunit p110α, and the tumour suppressor PTEN. PIK3CB, encoding the other ubiquitously expressed class I isoform p110β, is less frequently altered but the few mutations identified to date are oncogenic. This isoform has received more research interest in recent years, particularly since PTEN-deficient tumours were found to be reliant on p110β activity to sustain transformation. In this review, we describe the current understanding of the common and distinct biochemical properties of the p110α and p110β isoforms, summarise their mutations and highlight how they are targeted in clinical trials in endometrial cancer.

Polyphosphoinositides (PPIns) play essential roles as lipid signaling molecules, and many of their functions have been elucidated in the cytoplasm. However, PPIns are also intranuclear where they contribute to chromatin remodeling, transcription, and mRNA splicing. The PPIn, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), has been mapped to the nucleus and nucleoli, but its role remains unclear in this subcellular compartment. To gain further insights into the nuclear functions of PtdIns(3,4,5)P3, we applied a previously developed quantitative MS-based approach to identify the targets of PtdIns(3,4,5)P3 from isolated nuclei. We identified 179 potential PtdIns(3,4,5)P3-interacting partners, and gene ontology analysis for the biological functions of this dataset revealed an enrichment in RNA processing/splicing, cytokinesis, protein folding, and DNA repair. Interestingly, about half of these interactors were common to nucleolar protein datasets, some of which had dual functions in rRNA processes and DNA repair, including poly(ADP-ribose) polymerase 1 (PARP1, now referred as ADP-ribosyltransferase 1). PARP1 was found to interact directly with PPIn via three polybasic regions in the DNA-binding domain and the linker located N-terminal of the catalytic region. PARP1 was shown to bind to PtdIns(3,4,5)P3 as well as phosphatidylinositol 3,4-bisphosphate in vitro and to colocalize with PtdIns(3,4,5)P3 in the nucleolus and with phosphatidylinositol 3,4-bisphosphate in nucleoplasmic foci. In conclusion, the PtdIns(3,4,5)P3 interactome reported here will serve as a resource to further investigate the molecular mechanisms underlying PtdIns(3,4,5)P3-mediated interactions in the nucleus and nucleolus.

The class I phosphoinositide 3-kinase (PI3K) catalytic subunits p110α and p110β are ubiquitously expressed but differently targeted in tumours. In cancer, PIK3CB (encoding p110β) is seldom mutated compared with PIK3CA (encoding p110α) but can contribute to tumorigenesis in certain PTEN-deficient tumours. The underlying molecular mechanisms are, however, unclear. We have previously reported that p110β is highly expressed in endometrial cancer (EC) cell lines and at the mRNA level in primary patient tumours. Here, we show that p110β protein levels are high in both the cytoplasmic and nuclear compartments in EC cells. Moreover, high nuclear:cytoplasmic staining ratios were detected in high-grade primary tumours. High levels of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] were measured in the nucleus of EC cells, and pharmacological and genetic approaches showed that its production was partly dependent upon p110β activity. Using immunofluorescence staining, p110β and PtdIns(3,4,5)P3 were localised in the nucleolus, which correlated with high levels of 47S pre-rRNA. p110β inhibition led to a decrease in both 47S rRNA levels and cell proliferation. In conclusion, these results present a nucleolar role for p110β that may contribute to tumorigenesis in EC. This article has an associated First Person interview with Fatemeh Mazloumi Gavgani, joint first author of the paper.

Glioblastoma (GBM) is the most prevalent, aggressive primary brain tumour with a dismal prognosis. Treatment at diagnosis has limited efficacy and there is no standardised treatment at recurrence. New, personalised treatment options are under investigation, although challenges persist for heterogenous tumours such as GBM. Gene editing technologies are a game changer, enabling design of novel molecular-immunological treatments to be used in combination with chemoradiation, to achieve long lasting survival benefits for patients. Here, we review the literature on how cutting-edge molecular gene editing technologies can be applied to known and emerging tumour-associated antigens to enhance chimeric antigen receptor T and NK cell therapies for GBM. A tight balance of limiting neurotoxicity, avoiding tumour antigen loss and therapy resistance, while simultaneously promoting long-term persistence of the adoptively transferred cells must be maintained to significantly improve patient survival. We discuss the opportunities and challenges posed by the brain contexture to the administration of the treatments and achieving sustained clinical responses.

Genes encoding for components of the phosphoinositide 3-kinase (PI3K) pathway are frequently mutated in cancer, including inactivating mutations ofPTENand activating mutations ofPIK3CA, encoding the PI3K catalytic subunit p110α.PIK3CB, encoding p110β, is rarely mutated, but can contribute to tumourigenesis in some PTEN-deficient tumours. The underlying molecular mechanisms are however poorly understood. By analysing cell lines and annotated clinical samples, we have previously found that p110β is highly expressed in endometrial cancer (EC) cell lines and thatPIK3CBmRNA levels increase early in primary tumours correlating with lower survival. Selective inhibition of p110α and p110β led to different effects on cell signalling and cell function, p110α activity being correlated to cell survival inPIK3CAmutant cells and p110β with cell proliferation in PTEN-deficient cells. To understand the mechanisms governing the differential roles of these isoforms, we assessed their sub-cellular localisation. p110α was cytoplasmic whereas p110β was both cytoplasmic and nuclear with increased levels in both compartments in cancer cells. Immunohistochemistry of p110β in clinically annotated patient tumour sections revealed high nuclear/cytoplasmic staining ratio, which correlated significantly with higher grades. Consistently, the presence of high levels of p110β in the nuclei of EC cells, correlated with high levels of its product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) in the nucleus. Using immunofluorescence labelling, we observed both p110β and PtdIns(3,4,5)P₃in the nucleoli of EC cell lines. The production of nucleolar PtdIns(3,4,5)P₃was dependent upon p110β activity. EC cells with high levels of nuclear PtdIns(3,4,5)P₃and p110β showed elevated nucleolar activity as assessed by the increase in 47S pre-rRNA transcriptional levels in a p110β-dependent manner. Altogether, these results present a nucleolar role for the PI3K pathway that may contribute to tumour progression in endometrial cancer.

Introduction: Glioblastoma (GBM) is the most frequent treatment resistant and poor prognosis primary brain tumor. Age-adjusted incidence among Caucasians is approx. twice that of Africans. Genetic changes in ethnicity may represent druggable targets for development of new treatments for GBM. Chondroitin sulfate proteoglycan 4 (CSPG4) with sequence homology to neuron-glial-2 (NG2), henceforth CSPG4/NG2, is a transmembrane proteoglycan that is upregulated in GBM, resulting in leaky neovasculature and high cell proliferation, and is independently prognostic for poor survival. Mechanisms regulating CSPG4/NG2 expression remain to be elucidated. Experiments: Tumor-derived DNA whole exome sequences (WES) from mixed American GBM patients (n=300) collected into the TCGA database and blood-derived DNA WES from controls (n = 2504) in the 1000 Genome database were analyzed for mutations in the CSPG4 gene by GATK-3.5. We used prime editing (PE) to introduce a 13-base pair (bp) deletion through design of PE guide RNA (n=27), screening GBM cells (n=14) for CSPG4/NG2 expression, transfecting and expanding PE cells, confirming editing using Sanger sequencing. We functionally characterized the mutation in PE cells and transplanted in orthotopic PDX mouse models compared to wild type (WT) and Cas9 control cells. Western blotting, BrdU proliferation assays, clonogenic- and wound healing assays were used to investigate function. Gelatin zymography assessed CSPG4/NG2 matrix metalloproteinase (MMP) cross-talk during invasion on mixed extracellular matrix- or collagen IV- coated transwells. Results: A novel 13bp frameshift deletion in exon 3 of the CSPG4 gene was identified from WES of GBM samples deposited in TCGA. The mutation was cancer specific (p<0.001). Comparison of frequency in various ethnicities revealed the mutation to be more frequent among African American GBM patients (p=0.019). We generated 3 clones heterozygous for the CSPG4/NG2 deletion in patient derived GBMs. A homozygous deletion was lethal to the cells. The 13bp deletion reduced CSPG4/NG2 expression by approx. 60%, slowed tumor cell proliferation compared to both WT (p<0.001) and Cas9 cells (p<0.0001). PE cells had significantly reduced colony formation (p<0.001), scratch wound healing (p<0.001), had a weaker invasive phenotype when challenged with collagen IV (p<0.001), and reduced cleavage of collagen I by MMP2 that was corroborated by RNAseq gene expression. Reduced growth of PE cells in vivo was verified by multiplex immunohistochemistry on ex vivo brain sections and identified biomarkers for differential tumor development. Tumor growth was reduced in vivo in mice. Conclusions: We identified a novel 13bp deletion in CSPG4/NG2 with phenotypic effect that could be used as a future molecular target in GBM treatment, and present the first successful application of PE in human GBM cells. Citation Format: Victoria Smith Arnesen, Susina Suntharalingam, Żaneta Matuszek, Shahin Sarowar, Stian Knappskog, Stein Atle Lie, David R. Liu, Mohummad Aminur Rahman, Martha Chekenya. A novel 13-basepair deletion in CSPG4/NG2 abrogates protein expression, glioblastoma proliferation and invasion in vitro and in vivo in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3996.