Biguanides, such as the diabetes therapeutics metformin and phenformin, have shown antitumor activity both in vitro and in vivo. However, their potential effects on the tumor microenvironment are largely unknown. Here we report that phenformin selectively inhibits granulocytic myeloid-derived suppressor cells in spleens of tumor-bearing mice and ex vivo. Phenformin induces production of reactive oxygen species in granulocytic myeloid-derived suppressor cells, whereas the antioxidant N-acetylcysteine attenuates the inhibitory effects of phenformin. Co-treatment of phenformin enhances the effect of anti-PD-1 antibody therapy on inhibiting tumor growth in the BRAF V600E/PTEN-null melanoma mouse model. Combination of phenformin and anti PD-1 cooperatively induces CD8 T-cell infiltration and decreases levels of proteins that are critical for immune suppressive activities of myeloid-derived suppressor cells. Our findings show a selective, inhibitory effect of phenformin on granulocytic myeloid-derived suppressor cell-driven immune suppression and support that phenformin improves the anti-tumor activity of PD-1 blockade immunotherapy in melanoma.

Metabolic rearrangements subsequent to malignant transformation are not well characterized in endometrial cancer. Identification of altered metabolites could facilitate imaging-guided diagnosis, treatment surveillance, and help to identify new therapeutic options. Here, we used high-resolution magic angle spinning magnetic resonance mass spectroscopy on endometrial cancer surgical specimens and normal endometrial tissue to investigate the key modulators that might explain metabolic changes, incorporating additional investigations using qRT-PCR, Western blotting, tissue microarrays (TMA), and uptake assays of [ 3 H]-labeled choline. Lipid metabolism was severely dysregulated in endometrial cancer with various amino acids, inositols, nucleobases, and glutathione also altered. Among the most important lipid-related alterations were increased phosphocholine levels (increased 70% in endometrial cancer). Mechanistic investigations revealed that changes were not due to altered choline transporter expression, but rather due to increased expression of choline kinase a (CHKA) and an activated deacylation pathway, as indicated by upregulated expression of the catabolic enzymes LYPLA1, LYPLA2, and GPCPD1. We confirmed the significance of CHKA overexpression on a TMA, including a large series of endometrial hyperplasia, atypical hyperplasia, and adenocarcinoma tissues, supporting a role for CHKA in malignant transformation.

Background:This study investigates whether a histone deacetylase subtype 6 (HDAC6) inhibitor could be used in the treatment of solid tumours.Methods:We evaluated the effect of a novel inhibitor, C1A, on HDAC6 biochemical activity and cell growth. We further examined potential of early noninvasive imaging of cell proliferation by [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to detect therapy response.Results:C1A induced sustained acetylation of HDAC6 substrates, α-tubulin and HSP90, compared with current clinically approved HDAC inhibitor SAHA. C1A induced apoptosis and inhibited proliferation of a panel of human tumour cell lines from different origins in the low micromolar range. Systemic administration of the drug inhibited the growth of colon tumours in vivo by 78%. The drug showed restricted activity on gene expression with <0.065% of genes modulated during 24 h of treatment. C1A treatment reduced tumour [18F]FLT uptake by 1.7-fold at 48 h, suggesting that molecular imaging could provide value in future studies of this compound.Conclusion:C1A preferentially inhibits HDAC6 and modulates HDAC6 downstream targets leading to growth inhibition of a diverse set of cancer cell lines. This property together with the favourable pharmacokinetics and efficacy in vivo makes it a candidate for further pre-clinical and clinical development.

Background Aberrant activation of Axl is implicated in the progression of hepatocellular carcinoma (HCC). We explored the biologic significance and preclinical efficacy of Axl inhibition as a therapeutic strategy in sorafenib-naive and resistant HCC. Methods We evaluated Axl expression in sorafenib-naive and resistant (SR) clones of epithelial (HuH7) and mesenchymal origin (SKHep-1) using antibody arrays and confirmed tissue expression. We tested the effect of Axl inhibition with RNA-interference and pharmacologically with R428 on a number of phenotypic assays. Results Axl mRNA overexpression in cell lines ( n = 28) and RNA-seq tissue datasets ( n = 373) correlated with epithelial-to-mesenchymal transition (EMT). Axl was overexpressed in HCC compared to cirrhosis and normal liver. We confirmed sorafenib resistance to be associated with EMT and enhanced motility in both HuH7-SR and SKHep-1-SR cells documenting a 4-fold increase in Axl phosphorylation as an adaptive feature of chronic sorafenib treatment in SKHep-1-SR cells. Axl inhibition reduced motility and enhanced sensitivity to sorafenib in SKHep-1SR cells. In patients treated with sorafenib ( n = 40), circulating Axl levels correlated with shorter survival. Conclusions Suppression of Axl-dependent signalling influences the transformed phenotype in HCC cells and contributes to adaptive resistance to sorafenib, providing a pre-clinical rationale for the development of Axl inhibitors as a measure to overcome sorafenib resistance.

The protein kinase V-Raf murine sarcoma viral oncogene homolog B (BRAF) is an oncogenic driver and therapeutic target in melanoma. Inhibitors of BRAF (BRAFi) have shown high response rates and extended survival in melanoma patients bearing tumors that express BRAF Val600 mutations, but a vast majority of these patients develop drug resistance. Here we show that loss of Stromal antigen 2 or 3 (STAG2 or STAG3), which encode subunits of the cohesin complex, in melanoma cells results in resistance to BRAFi. We identified loss-of-function mutations in STAG2 as well as decreased expression of STAG2 or STAG3 proteins in several tumor samples from patients with acquired resistance to BRAFi and in BRAFi-resistant melanoma cell lines. Knockdown of STAG2 or STAG3 decreased sensitivity of Val600Glu BRAF-mutant melanoma cells and xenograft tumors to BRAFi. Loss of STAG2 inhibited CCCTC-binding factor (CTCF)-mediated expression of dual specificity phosphatase 6 (DUSP6), leading to reactivation of ERK signaling. Our studies unveil a previously unknown genetic mechanism of BRAFi resistance and provide new insights into the tumor suppressor function of STAG2 and STAG3.

The hypoxic response underlies the pathogenesis and malignant behavior of PCC/PGL. Regulation of PD-1 receptor-ligand signaling, a therapeutically actionable driver of the anti-tumor immune response, is a hypoxic-driven trait across malignancies. We evaluated the prognostic role of PD ligands in association with biomarkers of hypoxia and angiogenesis in patients with PCC/PGL.Tissue microarrays sections including consecutive cases diagnosed between 1983–2011 were stained for PD-L1 and 2, hypoxia inducible factor 1a (Hif-1a), Carbonic Anhydrase IX (CaIX), Vascular Endothelial Growth Factor-A (VEGF-A). We explored the biologic significance of PD ligands expression using gene set enrichment analysis (GSEA) on The Cancer Genome Atlas (TCGA) for PCC/PGL (n = 184).In total, 100 patients, 10% malignant, 64% PCC, 29% familial with median tumor size of 4.7 cm (range 1–14) were included. Median follow-up was 4.7 y. We found PD-L1 expression in 18% of PCC/PGL, which was independent of adverse pathological features including capsular (CI), vascular invasion (VI), necrosis (N) and expression of biomarkers of hypoxia. PD-L2 expression (16%) strongly correlated with CI, VI, N and malignant behavior (p < 0.05) and was associated with stronger Hif-1a and CaIX immunolabeling (p < 0.01). PD-L2 was predictive of shorter survival (162 versus 309 months, HR 3.1 95%CI 1.1–9.2, p = 0.02). GSEA on TGCA samples confirmed enrichment of transcripts involved in hypoxia and anti-cancer immunity.We report for the first time PD ligands expression in PCC/PGL with a distinctive prognostic, clinico-pathologic and immuno-biologic role. These findings support a potential therapeutic role for PD-1/PD-L1 targeted checkpoint inhibitors in these tumors.KEY MESSAGEThe molecular mechanisms underlying immune evasion in malignant phaeochromocytomas and paragangliomas (PCC/PGL) are poorly understood. This study demonstrates for the first time a distinctive immune-biologic and prognostic role of programmed death ligands 1 and 2 (PD-L1, PD-L2), two actionable drivers of the anti-cancer immune response. RNA-sequencing of tumor tissues reveals enrichment of transcripts relating to hypoxia and immune-exhaustion to explain the adverse clinical course observed in PD-L2 overexpressing tumors. These findings provide a rationale for the development of anti PD-1 therapies in malignant PCC/PGL.

In humans, C–X–C chemokine receptor type 4 (CXCR4) is a protein that is encoded by the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4–CXCL12 interaction in cancer elicits biological activities that result in tumor progression and has accordingly been the subject of significant investigation for detection and treatment of the disease. Peptidic antagonists have been labeled with a variety of radioisotopes for the detection of CXCR4, but the methodology utilizing small molecules has predominantly used radiometals. We report here the development of a 18F-radiolabeled cyclam-based small molecule radioprobe, [18F]MCFB, for imaging CXCR4 expression. The IC50 value of [19F]MCFB for CXCR4 was similar to that of AMD3465 (111.3 and 89.8 nM, respectively). In vitro binding assays show that the tracer depicted a differential CXCR4 expression, which was blocked in the presence of AMD3465, demonstrating the specificity of [18F]MCFB. Positron emission tomography (PET) imaging studies showed a distinct uptake of the radioprobe in lymphoma and breast cancer xenografts. High liver and kidney uptakes were seen with [18F]MCFB, leading us to further examine the basis of its pharmacokinetics in relation to the tracer’s cationic nature and thus the role of organic cation transporters (OCTs). Substrate competition following the intravenous injection of metformin led to a marked decrease in the urinary excretion of [18F]MCFB, with moderate changes observed in other organs, including the liver. Our results suggest involvement of OCTs in the renal elimination of the tracer. In conclusion, the 18F-radiolabeled monocyclam, [18F]MCFB, has potential to detect tumor CXCR4 in nonhepatic tissues.

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2–2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids.