Background Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with multiple roles in tumour growth, cell invasion and metastasis. We have previously established GSK-3 as an upstream regulator of PD-1 gene expression in CD8 + T cells and demonstrated that GSK-3 inhibition is as effective as anti-PD-1 mAb blockade in controlling tumour growth. Elraglusib (9-ING-41) is a specific small-molecule inhibitor of GSK-3β with clinical activity in patients with advanced cancers, including a patient with refractory melanoma whose response provided the rationale for the current study. Methods The B16 melanoma mouse model was used to observe the effect of elraglusib on tumour growth either as a single agent or in combination (simultaneously and sequentially) with anti-PD-1 mAb treatment. B16 tumour cells were implanted in either the flank, brain or both locations, and Kaplan–Meier plots were used to depict survival and significance determined using log rank tests. Expression of the immune checkpoint molecules, TIGIT, LAG-3 and PD-1, was evaluated using flow cytometry alongside expression of the chemokine receptor, CXCR3. Further evaluation of PD-1 expression was determined through RT-qPCR and immunohistochemistry. Results We demonstrated that elraglusib has a suppressive effect against melanoma as a single agent and enhanced anti-PD-1 therapy. There was a synergistic effect when elraglusib was used in combination with anti-PD-1 mAb, and an even greater effect when used as sequential therapy. Suppression of tumour growth was associated with a reduced expression of immune checkpoint molecules, PD-1, TIGIT and LAG-3 with upregulation of CXCR3 expression. Conclusions These data highlight the potential of elraglusib as an immune-modulatory agent and demonstrate the benefit of a sequential approach with immune checkpoint inhibition followed by GSK-3β inhibition in melanoma and provide a rationale for clinical investigation of elraglusib combined with immune checkpoint inhibitory molecules, including those targeting PD-1, TIGIT and LAG-3. This has several potential implications for current immunotherapy regimes, including possibly reducing the intensity of anti-PD-1 mAb treatment needed for response in patients receiving elraglusib, especially given the benign adverse event profile of elraglusib observed to date. Based on these data, a clinical study of elraglusib, an anti-PD-1 mAb and chemotherapy is ongoing (NCT NCT05239182).

The overall survival for patients with primary glioblastoma is very poor. Glioblastoma contains a subpopulation of glioma stem cells (GSC) that are responsible for tumour initiation, treatment resistance and recurrence. PPARα is a transcription factor involved in the control of lipid, carbohydrate and amino acid metabolism. We have recently shown that PPARα gene and protein expression is increased in glioblastoma and has independent clinical prognostic significance in multivariate analyses. In this work, we report that PPARα is overexpressed in GSC compared to foetal neural stem cells. To investigate the role of PPARα in GSC, we knocked down its expression using lentiviral transduction with short hairpin RNA (shRNA). Transduced GSC were tagged with luciferase and stereotactically xenografted into the striatum of NOD‐SCID mice. Bioluminescent and magnetic resonance imaging showed that knockdown (KD) of PPARα reduced the tumourigenicity of GSC in vivo . PPARα‐expressing control GSC xenografts formed invasive histological phenocopies of human glioblastoma, whereas PPARα KD GSC xenografts failed to establish viable intracranial tumours. PPARα KD GSC showed significantly reduced proliferative capacity and clonogenic potential in vitro with an increase in cellular senescence. In addition, PPARα KD resulted in significant downregulation of the stem cell factors c‐Myc, nestin and SOX2. This was accompanied by downregulation of the PPARα‐target genes and key regulators of fatty acid oxygenation ACOX1 and CPT1A , with no compensatory increase in glycolytic flux. These data establish the aberrant overexpression of PPARα in GSC and demonstrate that this expression functions as an important regulator of tumourigenesis, linking self‐renewal and the malignant phenotype in this aggressive cancer stem cell subpopulation. We conclude that targeting GSC PPARα expression may be a therapeutically beneficial strategy with translational potential as an adjuvant treatment. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Anti-1-amino-3-18fluorine-fluorocyclobutane-1-carboxylic acid (18F-fluciclovine) positron emission tomography (PET) shows preferential glioma uptake but there is little data on how uptake correlates with post-contrast T1-weighted (Gd-T1) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) activity during adjuvant treatment. This pilot study aimed to compare 18F-fluciclovine PET, DCE-MRI and Gd-T1 in patients undergoing chemoradiotherapy for glioblastoma (GBM), and in a parallel pre-clinical GBM model, to investigate correlation between 18F-fluciclovine uptake, MRI findings, and tumour biology. 18F-fluciclovine-PET-computed tomography (PET-CT) and MRI including DCE-MRI were acquired before, during and after adjuvant chemoradiotherapy (60 Gy in 30 fractions with temozolomide) in GBM patients. MRI volumes were manually contoured; PET volumes were defined using semi-automatic thresholding. The similarity of the PET and DCE-MRI volumes outside the Gd-T1 volume boundary was measured using the Dice similarity coefficient (DSC). CT-2A tumour-bearing mice underwent MRI and 18F-fluciclovine PET-CT. Post-mortem mice brains underwent immunohistochemistry staining for ASCT2 (amino acid transporter), nestin (stemness) and Ki-67 (proliferation) to assess for biologically active tumour. 6 patients were recruited (GBM 1–6) and grouped according to overall survival (OS)—short survival (GBM-SS, median OS 249 days) and long survival (GBM-LS, median 903 days). For GBM-SS, PET tumour volumes were greater than DCE-MRI, in turn greater than Gd-T1. For GBM-LS, Gd-T1 and DCE-MRI were greater than PET. Tumour-specific 18F-fluciclovine uptake on pre-clinical PET-CT corresponded to immunostaining for Ki-67, nestin and ASCT2. Results suggest volumes of 18F-fluciclovine-PET activity beyond that depicted by DCE-MRI and Gd-T1 are associated with poorer prognosis in patients undergoing chemoradiotherapy for GBM. The pre-clinical model confirmed 18F-fluciclovine uptake reflected biologically active tumour.